Acute herpetic stomatitis in children. Etiology, pathogenesis, clinical manifestations, diagnosis, treatment and prevention.
Herpetic stomatitis is a viral infection of the mouth that causes ulcers and inflammation. These mouth ulcers are not the same as canker sores, which are caused by a different virus.
Causes
Herpetic stomatitis is a contagious viral illness caused by Herpes virus hominis (also herpes simplex virus, HSV). It is seen mainly in young children. This condition is probably a child’s first exposure to the herpes virus.
An adult member of the family may have a cold sore at the time the child develops herpetic stomatitis. More likely, no source for the infection will be discovered.
Symptoms
Blisters in the mouth, often on the tongue, cheeks, palate, gums, and a border between the lip (red colored) and the normal skiext to it
Decrease in food intake, even if the patient is hungry
Difficulty swallowing (dysphagia)
Drooling
Fever (often as high as 104 °Fahrenheit) may occur 1 – 2 days before blisters and ulcers appear
Irritability
Pain in mouth
Swollen gums
Ulcers in the mouth, often on the tongue or cheeks — these form after the blisters pop
Exams and Tests
Your health care provider can usually diagnose this condition by looking at the mouth sores. Further tests are not usually done.
Sometimes, special laboratory tests can help confirm the diagnosis.
Treatment
Treatment involves:
Acyclovir, which fights the virus causing the infection
A mostly liquid diet of cool/cold nonacidic drinks
Numbing medicine (viscous lidocaine) applied to the mouth if there is severe pain
Lidocaine must be used with care because it can kill all feeling in the mouth. This may interfere with swallowing, and may lead to burns in the mouth or throat, or choking. There have been rare reports of death from overdose or misuse of lidocaine.
Outlook (Prognosis)
The child should recover completely within 10 days without medical treatment. Acyclovir taken by mouth may speed up recovery.
Possible Complications
Herpetic keratoconjunctivitis, a secondary herpes infection in the eye, may develop. This is an emergency and can lead to blindness. Dehydration may develop if the child refuses to eat and drink enough because of a sore mouth.
When to Contact a Medical Professional
Call your health care provider if your child develops a fever followed by a sore mouth, especially if they begin eating poorly (dehydration can develop rapidly in children).
Prevention
Approximately 90% of the population carries herpes simplex virus. It is difficult to prevent children from picking up the virus at some time during their childhood.
Children should strictly avoid close contact with people who have cold sores (for example, no kissing parents who have active cold sores). Children should also avoid other children with herpetic stomatitis. They should not share utensils, glasses, or food with actively infected people.
Herpetic Stomatitis
This is a contagious viral infection, which produces ulceration and inflammation of the gums.
Causes
Herpetic stomatitis is caused by Herpes Simplex Virus and is seen in children.
Incidence
Infection within the first 6 months is rare due to passive protection from antibody transferred across the placenta. After this period the infant is susceptible and subclinical infection is very common.
The primary infection occurs between 9 months and 5 years and may result in an acute gingivostomatitis.
Primary infections can be seen later in childhood.
Symptoms
Clinical features include:
Irritability and refusal of food due to difficulty in swallowing.
High fever.
Vesicles on the tongue, buccal mucosa, gums and skin around the mouth.The ulcers are very painful.
The mucosa becomes red, swollen and bleeds easily.
The vesicles breakdown to form ulcers.
Secondary bacterial infection may occur with enlarged lymphnodes and difficulty in swallowing.
It is self-limiting and lasts between 7 to 10 days.
Prevention
Approximately 80% of the population carry the HSV which makes it difficult to prevent children contracting the virus. Parents should avoid kissing their children when they have a cold sore. Also avoid sharing glasses, food and utensils.
Test
Can be diagnosed by its appearance.
Treatment
The patient can recover without any medication within 10 days.
Acyclovir may be used. Topical lidocaine is suitable for severe pain.
As the childs mouth will be sore, a liquid diet will be needed.
Primary herpetic gingivostomatitis is a highly contagious infection of the oral cavity which is caused by the herpes simplex virus. It is prevalent in children and young adolescents and sometimes can cause uncomfortable symptoms including eating and drinking difficulties and even life-threatening inflammation of the brain (encephalitis).
Background
Primary herpetic gingivostomatitis (PHG) is an infection of the oral cavity which is caused by the herpes simplex virus (HSV). It is highly contagious, typically affects children but can also occur in adults, and has a high rate of recurrence of infection (Chauvin 2002; Kolokotronis 2006). Symptoms may vary widely from mild discomfort to life-threatening encephalitis (Amir 2001). Only about 5% to 10% of patients initially infected with the herpes simplex virus develop clinical lesions. This is referred to as primary herpetic gingivostomatitis.
Aetiology and prevalence
Many patients may remain undiagnosed because they are either asymptomatic or have mild symptoms (Amir 2001). Ninety per cent of Americans are said to have HSV-1 serumantibodies (Siegel 2002). The herpes simplex virus is a double-strandedDNA virus of which the HSV-1 type is responsible for oral, facial and ocular infections including primary herpetic gingivostomatitis. Although HSV-2 is primarily responsible for most genital and cutaneous lower body herpetic lesions, it can also be the cause of primary herpetic gingivostomatitis. The peak periods of PHG incidence are between the ages of 6 months and 5 years, and in young adults who are in their early 20s (Amir 1997a). Although the virus is short lived on external surfaces it can readily disrupt the integrity of skin or mucous membranes and viral replication occurs as soon as it penetrates the epithelial cell. It is at a later stage that the virus travels along the sensory nerve endings to the corresponding nerve ganglion (i.e. trigeminal ganglion) where it enters a latent phase and can remain dormant until it is reactivated either spontaneously or by one or another of a number of stimulants (e.g. infection, ultraviolet light, fever, cold) (Chauvin Acyclovir for treating primary herpetic gingivostomatitis
As the infection is highly contagious most patients acquire it through direct contact (e.g. skin or via infected secretions (e.g. saliva)). It can spread rapidly in a closed community setting such as a daycare nursery or orphanage. Therefore, factors such as location and socio-economic status can influence the rate of HSV- 1 infection and it has beeoted that individuals in developing countries and from lower socio-economic groups become HSV-1 seropositive at an earlier age than individuals from developed countries (Amir 2001; Chauvin 2002; Kolokotronis 2006)
Symptoms
Primary herpetic gingivostomatitis is characterised by a sudden onset and with the severity of symptoms related to the virulence of the HSV and the host’s immune response (Chauvin 2002).
Non-specific symptoms may include cervical lymphadenopathy, malaise and low grade fever, and can occur in the absence of any discrete clinical lesions. The general course of PHG infection is 10 to 14 days which is usually preceded by an incubation period of 1 to 26 days (Faden 2006; Kolokotronis 2006).
Primary herpetic gingivostomatitis can include oral as well as extraoral lesions, swollen and bleeding gums, and symptoms such as pain, fever, irritability, malaise, headache and upper respiratory tract infection.
The oral lesions in PHG may start as vesicles on the tongue and the buccal and gingival mucosa, and which rapidly rupture to become ulcers. The ulcers, which are usually 1 to
may subsequently enlarge to form a large ulcerated area covered by a yellowish-greymembrane. In some patients, especially adults, the gingivae (gums) may also become swollen. Healing generally occurs without scarring.
During the acute phase of PHG many children may refuse to eat or drink because of the discomfort and pain from these lesions and consequently become rapidly dehydrated. Extraoral lesions (herpes labialis), which appear as erythematous papules on the vermilion border and adjacent skin of the lips, may accompany PHG (Kolokotronis 2006).
Complications are rare but do occur and usually as a result of viral shedding, and it is at this stage that HSV infection becomes readily transmittable. During this phase with its high risk of direct transmission, eye infections (ocular herpes or herpetic keratoconjunctivitis) and infections of the digits (herpetic whitlow) are not infrequent complications. After the primary infection, the virus may become dormant but can be readily reactivated producing episodic bouts of recurrent infection which are generally considered to be less severe than the primary infection.
Diagnosis
This is usually made by clinical presentation and history. In addition, the diagnosis may be confirmed via laboratory tests: serological assays (anti-HSV IgM and IgG), the Tzanck test and immunofluorescence, but the culture of viral isolates is still considered to be the gold standard (Neville 2002).
The differential diagnosis of primary herpetic gingivostomatitis includes acute necrotizing ulcerative gingivitis, herpangina, aphthous stomatitis, candidiasis of the mouth, Steven-Johnson syndrome and hand, foot and mouth disease (Amir 2001; Chauvin 2002).
Treatment options
Individuals with symptoms such as pain, fever, and dehydration may seek treatment (Amir 2001) including rehydration, analgesics and oral lavage. Systemic analgesics (acetaminophen) may be adequate to manage the associated pain but topical applications of diphenhydramine and Maalox, Kaopectate, viscous lidocaine are
also frequently prescribed.
Reduction of viral replication by using antivirals may shorten the acute phase of the illness, relieve symptoms, stop the virus from going into the latent phase and possibly prevent future recurrence (Siegel 2002). A known selective inhibitor of replicating HSV is acyclovir which is widely used for different forms of HSV infections.
Antiviral therapy and acyclovir
Acyclovir, in either oral or topical form, is used fairly routinely for HSV infections (e.g. herpes encephalitis, neonatal herpes, primary herpes genitalis and recurrent herpes labialis) (Amir 2001).One of the limitations of acyclovir is its poor gastrointestinal absorption and bioavailability and therefore derivatives (e.g. valacyclovir and famciclovir) have been developed with an apparently increased bioavailability but these are not currently available as paediatric suspensions.
A number of studies on the efficacy of acyclovir for treating primary herpetic gingivostomatitis have shown promising results and this systematic review sought to assess the available evidence.
Objectives
The objective of this review was to provide reliable evidence regarding the effectiveness of systemic acyclovir versus placebo or no treatment for primary herpetic gingivostomatitis.
The following null hypothesis was tested: there is no difference between acyclovir and placebo or no intervention for the treatment of primary herpetic gingivostomatitis against the alternative hypothesis of a difference.
Discussion
Primary herpetic gingivostomatitis (PHG) is the most commonly observed clinical manifestation of primary herpes simplex infection and can produce a range of symptoms (e.g. pain, drooling) in addition to difficulties with eating and drinking. Acyclovir is frequently prescribed for PHG and is presumed to be effective at decreasing symptoms as a result of its capability of reducing viral shedding.
This review sought high level evidence for the effectiveness of systemic acyclovir versus placebo or no treatment for primary herpetic gingivostomatitis but found only two eligible studies, one of which (Ducoulombier 1988) provided very limited data on the outcomes of interest and was not consistent in the reporting of its results. It was also unclear to what extent the participants adhered to the treatment reported in the study.
Although some of the limitations and side effects of acyclovir (i.e. poor gastrointestinal absorption and bioavailability), have been previously reported, none of the included studies in this review illustrated any significant side effects.Mild gastrointestinal symptoms were reported in one of the included studies (Amir 1997b) but these resolved spontaneously after 48 hours.
Whilst recognising the clinical heterogeneity, the methodological limitations, the incompleteness of data and the likelihood of bias and uncertainties in respect of outcomes assessment in both of these studies, we have nevertheless chosen to include them but advise caution in the interpretation of their results.
Author’s Conclusions
Implications for practice
Out of the two trials in this systematic review, only one was able to provide some weak evidence that acyclovir is an effective treatment in reducing the number of oral lesions, preventing the development of new extraoral lesions, decreasing the number of individuals with difficulties experienced in eating and drinking and of those who are admitted to hospital for children under the age of 6 with primary herpetic gingivostomatitis.
Implications for research
The results of this systematic review confirm the necessity for further larger sample, methodologically sound trials that are reported according to the Consolidated Standards of Reporting Trials (CONSORT) statement (www.consort-statement.org/) and provide means and standard deviations for important outcomes measurements.
To helpminimise the effects of systematic bias in outcomes assessment, it would be prudent if in future trials the trialists are not included as evaluators of outcomes and that appropriate training is given to independent assessors to ensure standardisation of criteria to be used in any outcomes assessments.
The two included studies only provided data on children under 6 years of age and there is therefore a requirement for trials to be conducted on patients in other age groups. Moreover, to enable patients and carers to make better healthcare decisions it would be beneficial if patient-reported outcomes (i.e. quality of life and patient satisfaction) were included in future trials.
This image is what the initial infection may look like when a child, or young adult is first infected with the Herpes Simplex virus. While most young children and adolescents experience a subclinical infection (no outward signs of the infection), a small percentage will develop “Primary Herpes stomatitis“. As you can see, it can look quite severe with blisters both inside and outside the mouth. (“Stomatitis” means inflammation of the entire mouth.) It starts out with “prodromal” symptoms including fever, malaise, headache and irritability, and then progresses after several days into severe gingival (gums) inflammation followed by the outbreak of numerous small blisters both inside and around the mouth. The blisters break leaving behind very painful sores with yellowish centers and red borders. The sores in the mouth are generally accompanied by severe pain, foul odor and increased salivation. Often the sores extend into the throat (pharyngitis). On rare occasions, the primary herpes infection can be confined to the throat. In any case, the patient is quite sick, but this primary infection will disappear after 10-14 days with rest, Tylenol®l and lots of fluids. In healthy people, this infection happens only once in a lifetime. Later in life, the presence of the virus only becomes apparent whenever an “ordinary” cold sore appears. – See more at: http://doctorspiller.com/Oral_disease_Processes/Oral_Viral_Infections.html#sthash.biW5WMDC.dpuf
Herpes Simplex (type I) is the virus that causes cold sores (herpes labialis) in normal, healthy adults. The image at the right shows a recurrent herpetic infection, in other words, a typical cold sore, sometimes called a fever blister due to its propensity to appear when the patient has a cold or other febrile (fever causing) illness. This is another bug that, like Shingles, tends to “hang out” in a nerve root for the life of the patient after the initial infection, which often occurs in childhood. Once infected, the patient remains infected for life. The virus remains dormant inside the nerve root most of the time until the patient suffers an illness or other problem which lowers his immune response. The virus takes advantage of the drop in immune response to flare up in the typical cold sore seen in this image. Click the image above for more information on recurrent herpes labialis and many more images of herpes infections. – See more at: http://doctorspiller.com/Oral_disease_Processes/Oral_Viral_Infections.html#sthash.biW5WMDC.dpuf
Herpes Labialis (Cold Sore)
These sores can be brutally painful. They are caused by the Herpes Simplex virus, (HSV) and when they occur, they are often accompanied by the symptoms one associates with the flu; nausea, fever, chills, muscle aches and malaise. Once a person is infected with the virus, generally early in life, he or she will suffer reoccurrences on a fairly regular basis, depending on the state of their mental or physical health.
The virus is an opportunistic invader, taking advantage of a depressed immune responses in a patient who is under psychological or physical distress. They are called “cold sores” or “fever blisters” because they tend to happen when the patient is physically burdened with another viral infection such as rhinovirus (the cold virus). The image above is unusual because it was taken before the blister actually broke. All cold sores start as blisters, but the blister rarely survives for long enough to actually get a picture of it.
Herpes Labialis starts with prodromal symptoms such as burning, tingling, soreness or swelling on the lip and is followed by the formation of tiny blisters, called vesicles, which coalesce and break forming a crusty sore. In healthy people, these will heal without scaring in seven to fourteen days.
New antibiotics like acyclovir (Zovirax®), famciclovir (Famvir®) or valacyclovir (Valtrex®) are effective in suppressing the Herpes virus and will generally alleviate the symptoms within a fairly short time. Systemic drugs like these are generally used in very severe cases in immunocompromised patients, or in herpes infections of the eye. In patients exposed to a lot of sun who are prone to outbreaks of recurrent herpes labialis, a prophylactic regimen of 400 mg of acyclovir twice a day may prevent the outbreaks Unfortunately, the antibiotics do not often “cure” the disease since the virus continues to remain in an inactive form inside a nerve root waiting for another chance to cause an outbreak.
Recently, the Food and Drug Administration has approved penciclovir 1% topical cream for the treatment of herpes labialis. This is applied every two hours while awake, and will help to shorten the duration and severity of the cold sore. Acyclovir cream (Zovirax®) is less effective
Lysine (available at most drug stores) has been reported to reduce the severity of recurrent outbreaks if taken in high enough doses (2-
Herpes labialis, (also called cold sores,[1] herpes simplex labialis, recurrent herpes labialis, or orolabial herpes),:368 is a type of herpes simplexoccurring on the lip, i.e. an infection caused by herpes simplex virus (HSV). An outbreak typically causes small blisters or sores on or around the mouth commonly known as cold sores or fever blisters. The sores typically heal within 2–3 weeks, but the herpes virus remains dormant in the facial nerves, following orofacial infection, periodically reactivating (in symptomatic people) to create sores in the same area of the mouth or face at the site of the original infection.
Cold sore has a rate of frequency that varies from rare episodes to 12 or more recurrences per year. People with the condition typically experience one to three attacks annually. The frequency and severity of outbreaks generally decreases over time.
Definitions
In medical contexts, “labia” is a general term for “lip”; “herpes labialis” does not refer to the labia of the genitals, though the etymology is the same. When the viral infection affects both face and mouth, the broader term “orofacial herpes” is used to describe the condition, whereas the term “herpetic stomatitis” is used to specifically describe infection of the mouth; “stomatitis” is derived from the Greek wordstoma that means “mouth”.
Signs and symptoms
Herpes infections usually show no symptoms; when symptoms do appear they typically resolve within two weeks. The main symptom of oral infection is inflammation of the mucosa of the cheek and gums—known as acute herpetic gingivostomatitis—which occurs within 5–10 days of infection. Other symptoms may also develop, including headache, nausea, dizziness and painful ulcers—sometimes confused with canker sores—fever, and sore throat.
Primary HSV infection in adolescents frequently manifests as severe pharyngitis with lesions developing on the cheek and gums. Some individuals develop difficulty in swallowing (dysphagia) and swollenlymph nodes (lymphadenopathy).[5] Primary HSV infections in adults often results in pharyngitis similar to that observed in glandular fever (infectious mononucleosis), but gingivostomatitis is less likely.
Recurrent oral infection is more common with HSV-1 infections than with HSV-2. Symptoms typically progress in a series of eight stages (see viral life cycle):
1. Latent (weeks to months incident-free): The remission period; After initial infection, the viruses move to sensory nerve ganglia (Trigeminal ganglion),[6] where they reside as lifelong, latent viruses. Asymptomatic shedding of contagious virus particles can occur during this stage.
2. Prodromal (day 0–1): Symptoms often precede a recurrence. Symptoms typically begin with tingling (itching) and reddening of the skin around the infected site. This stage can last from a few days to a few hours preceding the physical manifestation of an infection and is the best time to start treatment.
3. Inflammation (day 1): Virus begins reproducing and infecting cells at the end of the nerve. The healthy cells react to the invasion with swelling and redness displayed as symptoms of infection.
4. Pre-sore (day 2–3): This stage is defined by the appearance of tiny, hard, inflamed papules and vesicles that may itch and are painfully sensitive to touch. In time, these fluid-filled blisters form a cluster on the lip (labial) tissue, the area between the lip and skin (vermilion border), and can occur on the nose, chin, and cheeks.
5. Open lesion (day 4): This is the most painful and contagious of the stages. All the tiny vesicles break open and merge to create one big, open, weeping ulcer. Fluids are slowly discharged from blood vessels and inflamed tissue. This watery discharge is teeming with active viral particles and is highly contagious. Depending on the severity, one may develop a fever and swollen lymph glands under the jaw.[7]
6. Crusting (day 5–8): A honey/golden crust starts to form from the syrupy exudate. This yellowish or brown crust or scab is not made of active virus but from blood serum containing useful proteins such as immunoglobulins. This appears as the healing process begins. The sore is still painful at this stage, but, more painful, however, is the constant cracking of the scab as one moves or stretches their lips, as in smiling or eating. Virus-filled fluid will still ooze out of the sore through any cracks.
7. Healing (day 9–14): New skin begins to form underneath the scab as the virus retreats into latency. A series of scabs will form over the sore (called Meier Complex), each one smaller than the last. During this phase irritation, itching, and some pain are common.
8. Post-scab (12–14 days): A reddish area may linger at the site of viral infection as the destroyed cells are regenerated. Virus shedding can still occur during this stage.
The recurrent infection is thus often called herpes simplex labialis. Rare reinfections occur inside the mouth (intraoral HSV stomatitis) affecting the gums, alveolar ridge, hard palate, and the back of the tongue, possibly accompanied by herpes labialis.]
A lesion caused by herpes simplex can occur in the corner of the mouth and be mistaken for angular cheilitis of another cause. Sometimes termed “angular herpes simplex”.[ A cold sore at the corner of the mouth behaves similarly to elsewhere on the lips. Rather than utilizing antifungal creams, angular herpes simplex is treated in the same way as a cold sore, with topical antiviral drugs.
Causes
Herpes labialis infection occurs when the herpes simplex virus comes into contact with oral mucosal tissue or abraded skin of the mouth. Infection by the type 1 strain of herpes simplex virus (HSV-1) is most common; however, cases of oral infection by the type 2 strain are increasing. Specifically, type 2 has been implicated as causing 10–15% of oral infections.
Cold sores are the result of the virus’s reactivating in the body. Once HSV-1 has entered the body, it never leaves. The virus moves from the mouth to remain latent in the central nervous system. In approximately one-third of people, the virus can “wake up” or reactivate to cause disease. When reactivation occurs, the virus travels down the nerves to the skin where it may cause blisters (cold sores) around the lips, in the mouth or, in about 10% of cases, on the nose, chin, or cheeks.
Cold sore outbreaks may be influenced by stress, menstruation, sunlight, sunburn, fever, dehydration, or local skin trauma. Surgical procedures such as dental or neural surgery, lip tattooing, or dermabrasion are also common triggers. HSV-1 can in rare cases be transmitted to newborn babies by family members or hospital staff who have cold sores; this can cause a severe disease called Neonatal herpes simplex.
The colloquial term for this condition, “cold sore” comes from the fact that herpes labialis is often triggered by fever, for example, as may occur during an upper respiratory tract infections (i.e. a cold).
People can transfer the virus from their cold sores to other areas of the body, such as the eye, skin, or fingers; this is called autoinoculation. Eye infection, in the form of conjunctivitis or keratitis, can happen when the eyes are rubbed after touching the lesion. Finger infection (herpetic whitlow) can occur when a child with cold sores or primary HSV-1 infection sucks his fingers.
Blood tests for herpes may differentiate between type 1 and type 2. When a person is not experiencing any symptoms, a blood test alone does not reveal the site of infection. Genital herpes infections occurred with almost equal frequency as type 1 or
Treatment
Docosanol, a saturated fatty alcohol, is a safe and effective topical application that has been approved by the United States Food and Drug Administration for herpes labialis in adults with properly functioningimmune systems. It is comparable in effectiveness to prescription topical antiviral agents. Due to its mechanism of action, there is little risk of drug resistance.[10] The duration of symptoms can be reduced by a small amount if an antiviral, anaesthetic or non-treatment cream (such as zinc oxide or zinc sulfate) is applied promptly.
Effective antiviral medications include acyclovir and penciclovir, which can speed healing by as much as 10%. Famciclovir or valaciclovir, taken in pill form, can be effective using a single day, high-dose application and is more cost effective and convenient than the traditional treatment of lower doses for 5–7 days.
Lysine has been suggested as a treatment for herpes labialis based on in vitro studies, but the evidence is inconclusive in humans.
Prevention
Avoiding touching an active outbreak site, washing hands frequently while the outbreak is occurring, not sharing items that come in contact with the mouth, and not coming into contact with others (by avoiding kissing, oral sex, or contact sports) can reduce the likelihood of the infection being spread to others.
Because the onset of an infection is difficult to predict, lasts a short period of time and heals rapidly, it is difficult to conduct research on cold sores. Though famciclovir improves lesion healing time, it is not effective in preventing lesions; valaciclovir and a mixture of acyclovir and hydrocortisone are similarly useful in treating outbreaks but may also help prevent them.
Oral acyclovir (800-1,600 mg daily) and valacyclovir (500 mg daily for 4 months) are effective in preventing recurrent herpes labialis if taken prior to the onset of any symptoms or exposure to any triggers.
Epidemiology
Herpes labialis is endemic throughout the world. A large survey of young adults on six continents reported that 33.2% of males and 28% of females had herpes labialis on two or more occasions during the year before study. The lifetime prevalence in the United States of America is estimated at 20-45% of the adult population. 100 million episodes Lifetime prevalence in France was reported by one study as 32.4% in males and 42.1% in females. In Germany, the prevalence was reported at 31.7% in people aged between 35 and 44 years, and 20% in those aged 65–74. In Jordan, another study reported a lifetime prevalence of 26.4%.
Abstract
Oral infections caused by herpes simplex type 1 are widespread, even among otherwise healthy people. While most of these herpetic infections are asymptomatic, young children are at risk for developing extensive oropharyngeal vesicular eruptions when first infected with the virus. This initial outbreak is known as primary herpetic gingivostomatitis. Although a self-limiting disease, this oral infection can cause significant mouth discomfort, fever, lymphadenopathy, and difficulty with eating and drinking. Symptoms may persist for 2 weeks. Diagnosis can be made clinically and confirmed by laboratory tests. Some young children require hospitalization for management of dehydration and pain control. Pediatric nurses working in both primary and acute care settings will encounter this oral infection in young children. Antiviral therapy with acyclovir has proven effective in the management of primary herpetic gingivostomatitis. Providing supportive care and educating parents about transmission of the virus are important aspects of nursing care.
Introduction
The herpes virus is ubiquitous, passing from person to person through contaminated secretions or lesions. At least 70% of the American population has been infected, likely occurring in early childhood (Murph & Grose, 1999). Worldwide, the rate of infection is over 85% (Rosen & Ablon, 1997). After levels of passively acquired maternal antibodies have diminished, infants and young children are at increased risk for acquiring infections. Sealander and Kerr (1989) reported that children ages 2 to 4 are most susceptible to herpes simplex virus (HSV) infections.
The human herpes virus family consists of eight currently known pathogens that represent the most frequent isolates in general laboratories (Koneman, Allen, Janda, Schreckenberger, & Winn, 1997). The group includes HSV (types 1 and 2), cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and human herpes viruses-6, 7, and 8. The HSV is a large, DNA-containing virus that has two serotypes: type 1 (HSV-1) and type 2 (HSV-2). HSV-1 usually affects the oral cavity, whereas HSV-2 infects the genital area; however, there are exceptions to the rule. HSV-1 infections are extremely common.
The majority of primary HSV-1 infections are asymptomatic or mild enough to go unrecognized (Amir, Harel, Smetana, & Varsano, 1997; White, 1998). Mild cases may be confused with teething or other poorly defined illness. When clinically evident, however, the most frequent manifestation of initial HSV-1 disease in young children is primary herpetic gingivostomatitis (PHGS) (Amir et al., 1997; Murph & Grose, 1999). This occurs in 25-30% of affected children (Amir et al., 1997). Pediatric nurses working in both acute and primary care settings will encounter this condition in otherwise healthy young children, yet information about this pediatric infection is extremely limited in the nursing literature. Sheff (2000) highlights HSV as a “microbe of the month,” briefly describing type-1 oral infections. Klotz (1990) explores transmission of HSV-1 infections from clients to nurse anesthetists. As an aid to pediatric clinical care, assessment findings, diagnosis, medical management, and nursing implications for PHGS in young children will be examined.
PHGS is a blistering disease of the mouth, easily transmitted when the shedding herpes virus comes in direct contact with mucous membranes or broken skin. Most primary infections are contracted from others who are shedding HSV-1, yet may be free of evident lesions (White, 1998). As is true of other infectious childhood diseases, infection rates for HSV-1 are higher among young children who attend day care centers (Chandrasekar, 1999; Murph, 1999). Close proximity during activities and play increases the likelihood of exchanging oral secretions and viral organisms. Examples of transmission-potential behaviors seen in young children include mouth touching; sharing utensils, cups, and bottles; thumb sucking; and mouthing toys. The virus invades epithelial cells lining the oral cavity of a susceptible host and replicates. Epithelial cells full of the virus break open, spilling their contents. The virus is then free to invade neighboring epithelial cells, be transported to new locations, and infect other people (Crawford, 2000).
Although PHGS is a self-limiting disease, the virus is transported to the trigeminal ganglia, where a latent or dormant infection is established that remains for life (Chandrasekar, 1999; White, 1998). Reactivation of the dormant virus can occur, often precipitated by factors such as sun exposure, stress, illness, menses, fever, or immune suppression. When this happens, the virus travels back along the neural system at or near the original site causing infection again. Most recurrent infections are asymptomatic, yet viral shedding occurs (White, 1998). When clinically evident, these recurrent infections are rarely of the same magnitude as the primary infection. The antibodies specific to HSV-1 will remain in circulation, weakening subsequent infections. Recurrent infections generally cause only single labial lesions (herpes labialis) widely known as fever blisters or cold sores. Repeated intraoral lesions can occur.
Treatment of herpes simplex gingivostomatitis with aciclovir in children: a randomised double blind placebo controlled study
Introduction
Herpetic gingivostomatitis is the most common clinical manifestation of primary herpes simplex virus infection in young children. Although it is a self limiting disease, the general course is 10-14 days, and the children experience extreme discomfort and refuse to eat. If they also refuse to drink they often have to be admitted to hospital for rehydration.
Parenteral aciclovir has been shown to be effective in herpes simplex virus infections such as encephalitis,1 primary genital herpes,and herpes neonatorum.Oral aciclovir has been used successfully to treat genital herpes and recurrent herpes labialis.No study has shown definitively, however, that oral aciclovir is effective for primary herpes gingivostomatitis. Encouraging results have been reported in a few open studies,a small controlled study, and a prophylactic trial during an outbreak in a closed community.
This randomised, double blind, placebo controlled study was designed to examine the efficacy of oral aciclovir suspension for treating herpetic gingivostomatitis in young children.
Subjects and methods
Subjects
All children aged 1-6 years of age with clinical manifestation of gingivostomatitis lasting less than 72 hours were identified by their primary pediatrician and referred to the paediatric day care unit of Hasharon Hospital, Petah Tiqva, Israel. Children seen in the emergency room of Hasharon Hospital and the Schneider Children’s Medical Centre were also recruited.
After a swab from the oral lesions for viral cultures and blood for serological tests for herpes simplex virus were obtained, the children were assigned the next available study number by using a randomised number table with a block size of eight. The same numbers were on the study treatments–aciclovir suspension or placebo suspension. The placebo bottles were identical to the aciclovir bottles, and the suspension looked exactly the same with a similar smell. The aciclovir was given in a dose of 15 mg/kg (0.375 ml/kg), five times a day (up to a maximum of 200 mg per dose) for a period of seven days, and the placebo was given in the same volume, five times a day.
Written informed consent was obtained from the parents, and the study was approved by the hospital’s ethics committee.
Assessment
On enrolment (day 0), a medical history was taken and physical examination performed. On clinical evaluation, fever, severity of the oral lesions, presence of extraoral skin lesions (lesions around the mouth but outside the oral cavity), drooling, and drinking and eating difficulties were noted. The oral lesions were categorised as mild (up to 10 lesions on the tongue or oral mucous membrane), moderate (11 to 20 lesions with swelling of the gums), or severe (more than 20 tongue or oral lesions and gum lesions). Drinking and eating abilities were categorised as normal, less thaormal, and total inability to drink or eat.
The clinical examination was repeated on days 3, 6, and 8, the day after ending the treatment, and thereafter every two to three days if symptoms persisted. The parents recorded the child’s symptoms, and the rectal temperature was measured daily until a normal reading (<
Laboratory investigations
On day
Culture of herpes simplex virus–The culture swabs were placed into 2 ml of transport medium,11 and the specimens were either sent immediately on ice to the Central Virology Laboratory, Tel Hashomer, or kept refrigerated overnight and sent the next day. The herpes simplex virus was isolated as described previously.12
Serotyping of herpes simplex virus with monoclonal antibodies–All isolates of herpes simplex virus were typed by fluorescence conjugate monoclonal antibodies (Syva Microtak HSV-1, HSV-2; Palo Alto, CA), according to the manufacturer’s instructions.12
Serology assays–An indirect immunofluorescence antibody test was performed as described previously.13
Statistical analysis
The sample size was calculated on the assumption that the clinical symptoms of herpes simplex virus gingivostomatitis may last 5-15 days with a standard deviation of five days. On the basis of these data, we estimated that a sample size of 60 children with proved herpes simplex virus would be needed to detect a difference of 2.5-3.0 days between the treatment groups with a power of 0.80 and a significance level of 0.05. The times of disappearance of symptoms (mouth lesions, fever, external lesions, drooling, and eating and drinking difficulties) were compared by the Mann-Whitney non-parametric test. The severity scores of eating and drinking difficulties were compared by the χ2 test.
The t test was applied to compare the groups with respect to the continuous variables–that is, the difference in maximal temperature, the laboratory results (haemoglobin, polymorphonuclear cells, and lymphocytes), and the number of days to the last positive culture and to the first negative culture. Finally, Fisher’s exact test was used to determine differences in the level of compliance and admission between the groups. Only children with laboratory evidence of herpes simplex virus infection (positive culture or serological result) were included in the statistical analysis for efficacy outcome, according to the protocol.
Results
Altogether, 72 children were enrolled between December 1993 and February 1995. Thirty six children were randomly allocated to receive aciclovir and 36 to receive the placebo. Ten children whose viral cultures were negative for herpes simplex virus and whose serological results remained negative during convalescence were excluded from the clinical evaluation, as was one child whose parents refused to continue with the follow up after day 2. Thus, the final study population comprised 61 children with positive cultures for herpes simplex virus; 31 children were in the group receiving aciclovir and 30 were in the placebo group.
On enrolment both groups were similar regarding demographic variables and severity of clinical symptomatology (table 1). Two children in the aciclovir group and three in the placebo group were admitted for rehydration at enrolment.
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Table 1
Demographic, clinical, and haematological variables at admission in 61 children in ambulatory care unit of a tertiary paediatric hospital
The children in the aciclovir group had significantly more blood lymphocytes than those in the placebo group (table 1). The other haematological variables were similar.
Efficacy outcome
Oral lesions–The oral lesions persisted for a significantly shorter time in the children receiving aciclovir than in those receiving placebo (median 4 (range 2-12) days v 10 (3-15) (table 2). At the end of treatment on day 8, two out of 31 children in the aciclovir group had oral lesions compared with 21 out of
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Table 2
Median (range) duration (in days) of clinical variables in 61 children with confirmed herpes simplex virus gingivostomatitis
Fever–The fever disappeared significantly earlier in the children in the aciclovir group than in those in the placebo group (median 1 day v 3 days (table 2).
Extraoral lesions–On day 0 about one third of the children in each group had extraoral herpetic lesions (table 1). Children in the aciclovir group did not develop new lesions after treatment had been started. Twelve of those in the placebo group, however, continued to develop extraoral lesions after the treatment had been started. The duration of the lesion was significantly shorter in the aciclovir group (median 0.0v 5.5 days (table 2).
Eating and drinking ability–On enrolment all the children had eating and drinking difficulties. On day 8 of the treatment, in the aciclovir group two children had eating difficulties and one child had drinking difficulties, compared with 14 and 9 children respectively in the placebo group. The median duration of the eating difficulties was 4 v 7 days respectively and of drinking difficulties was 3 v 6 days respectively (table2).
Duration of clinical variables–Table 3) represents the clinical data of all the children randomised in the study except the one child who left the study on day 2. Ten of the 71 children showed no evidence of herpes simplex virus infection. In the intention to treat analysis, the duration of all measured clinical variables was still significantly shorter in the aciclovir group than in the placebo group (table 3).
View this table:
Table 3
Median (range) duration (in days) of clinical variables of all enrolled children (n=72)
Hospital admission–Five children were admitted before inclusion in the study (table1). Among the childreot admitted, none treated with aciclovir was admitted after treatment was started, while three children in the placebo group were admitted for two to three days for rehydration (P=0.11).
Recurrences–Telephone screening of the enrolled children 16 months after the start of the study showed only one case of herpes labialis (in a child in the placebo group).
Viral cultures–All the cultures (positive for herpes simplex virus in all the children included in the study) were identified as type 1. The cultures were obtained every two to three days, so if a child had a positive culture on day 0 and on day 3, we assumed that he or she was positive also on days 1 and 2. According to this evaluation, the children in the aciclovir group had a significantly shorter period of positive viral culture than those in the placebo group (median (range 1-3) 1 v 5 (1-10) days) respectively (difference 4 days, 95% confidence interval 2.9 to 5.1) (table 2).
Serological examination–Serology samples were taken during the acute and convalescence phases of the infection were available for 41 children. In those with culture negative for herpes simplex virus (10 cases), the serological findings for the convalescence samples were negative. Of those included in the efficacy calculation, 18 were from the aciclovir group and 13 from the placebo group. No differences between groups in seroconversion or maximal titres of immunofluorescence antibody were observed. All children had seroconversion with titres of >64.
Compliance–Compliance was good in both groups: 29 children in the aciclovir group and
Side effects–No significant side effects were recorded in either group. Two children in each group had mild gastrointestinal symptoms that resolved spontaneously after 24 to 48 hours without a change in the study treatment.
Discussion
Treatment with oral aciclovir suspension–started during the first three days of the appearance of herpetic gingivostomatitis and continued for seven days–was shown to be significantly more effective than placebo in reducing the severity of the clinical symptoms and shortening the period of infectivity as a result of viral shedding. The beneficial effect of aciclovir was evident in all clinical variables evaluated, the healing rate of the oral and extraoral lesions, duration of the fever, and the duration of the eating and drinking difficulties. In an intention to treat analysis of all enrolled children, including those without proved herpes simplex virus infection, the difference between the treatment groups remained highly significant (table 3). No child in the aciclovir group was admitted after treatment had been started, compared with three children in the placebo group who were admitted for rehydration.
The beneficial effect of aciclovir treatment has been previously reported in an open study.7 The study showed that in children with herpetic gingivostomatitis who were treated with aciclovir, fever disappeared after the third day of treatment in all cases, concomitant with marked improvement in the oral lesions. Of the 33 children, only about 10% had oral or extraoral lesions after six days of treatment.
Herpes gingivostomatitis is a contagious disease, especially among children in closed communities or day care centres.14 15 Data regarding intrafamilial transmission are unclear.
In young children herpes simplex virus is transmitted primarily by contact with infected oral secretions. In the children treated with aciclovir, the period of viral shedding was significantly shorter than in those receiving placebo. After three days of aciclovir treatment all viral cultures became negative, compared with almost 50% positive cultures on day
Recurrences of gingivostomatitis are unusual iormal hosts and are most probably related to immunity after infections. One important question is the effect of aciclovir treatment on long term immunity against herpes simplex virus. The serological data in this study, although available for only half the children, showed no difference between the aciclovir and placebo groups in the humoral immune response to the virus. The influence of the treatment on local recurrences needs long term follow up.
Concern has been expressed over the possible selection of resistant strains once aciclovir is being used for such relatively common disorders as herpetic gingivostomatitis. Most clinical isolates resistant to aciclovir have been recovered from immunocompromised patients receiving multiple treatment courses. A seven day treatment of aciclovir iormal children is unlikely therefore to create a problem. Aciclovir has been used to prevent recurrent genital herpes for more than six years, and no resistant strains have been isolated.16
The clinical manifestation of herpetic gingivostomatitis varies from a mild illness to a severe course with admission to hospital. In the placebo group (n=30), which represented the natural course of herpetic gingivostomatitis, oral lesions were found in 25 children for seven days or more, and eating and drinking difficulties in 16 (data not shown). During this period, the sick children were unable to attend day care or kindergarten. Although this study did not attempt to address the economic issue of aciclovir treatment in gingivostomatitis, the significant reduction in the duration of illness and the prevention of admission are likely to allow children and parents to return to their normal life earlier, a benefit that may balance the price of the treatment.
ORAL HERPES AND COLD SORES IN CHILDREN
One of the most common causes of painful blisters around the mouth is oral herpes. Herpes simplex virus (HSV) is responsible for most of the viral infections of the mouth. Herpes is also one of the most difficult viruses to control.
Oral herpes is easily transmitted, and is the most common form of the herpes simplex virus. Oral herpes affects people of all ages, races, and social groups. The highest incidence of initial infection occurs in children between six months and three years of age. By adolescence, 62% of Americans have been infected with herpes simplex virus-1 (HSV-1). Serum antibodies to HSV-1 can be found in up to 90% of Americans.
DESCRIPTION OF ORAL HERPES SIMPLEX:
- Oral herpes in an infection caused by the herpes simplex virus. This virus can cause painful sores on the lips, tongue, roof of the mouth, and face. Herpes simplex virus-1 causes 80% of oral herpes infections, and herpes simplex virus–2 causes the rest. The herpes family of viruses contain a DNA core (double-stranded linear virus) which is surrounded by a capsid and lipid envelope.
- The first infection a child will experience is termed primary herpetic gingivostomatitis. During this initial infection, infants and toddlers can develop very painful blisters on their tongue, and on the roof of the mouth. The gums will have a fiery red color, and the toddler may have a fever.
- Between 20% to 40% of initial oral HSV-1 infections recur within one year. Recurrences are milder than the primary infection, and are commonly known as cold sores or fever blisters. Fever blisters on the lips and face is called herpes simplex labialis. This condition lasts about 7-10 days in healthy, immunocompetent children.
- Oral herpes infections should not be confused with canker sores (aphthous ulcers). Aphthous ulcers are not caused by viruses, and appear as small white or grayish ulcers on the movable (unattached) gingival tissue.
- THE DISEASE PROCESS OF ORAL HERPES:
Immediately after a child has been infected with the oral herpes virus, the infection proceeds to three distinct stages: primary herpes infection, dormancy, and recurrent herpes infection. - PRIMARY INFECTION:
This is the first infection a child will experience with the HSV-1 virus. The virus enters through abraded skin or normal intraoral tissue (mucous membrane). An oral infection called primary herpetic gingivostomatitis can develop at this time. In most cases, the virus never causes symptoms during this primary infection, however. - LATENCY (DOMRANCY):
After the primary infection, the viral particles move from the skin through branches of nerve cells that end at the trigeminal nerve ganglia, facial nerve ganglia, or dorsal root spinal ganglia. While residing in the ganglia, the herpes virus reproduces, and then becomes inactive (dormant). It is also possible for the virus to become dormant in the lips. - RECURRENT ORAL HERPES (HERPES SIMPLEX LABIALIS):
Approximately 30 to 40 percent of children who have been exposed to HSV will develop recurrent infections. These recurrent infections are a reactivation of the herpes simplex virus. The first symptoms may include a tingling or itching sensation on the lips or face. Recurrent herpes blisters usually appear around the lips within 12-36 hours after the first symptoms (the prodrome) appear. The blisters (vesicles) rupture quickly, and coalesce into larger lesions that have a crusted surface. In healthy children, these recurrent lesions on the lips and face will heal without scarring within seven to fourteen days.Recurrent erythema multiforme has been linked to a previous HSV infection, and is called herpes-associated erythema multiforme.
The body normally produces an immune response to HSV, but the immune system cannot eradicate the virus completely. For patients with a depressed immune system, a herpes virus infection can have life-threatening consequences. Occasionally during the primary infection in infants, the virus can spread to the brain and cause aseptic meningitis, or disseminated neonatal HSV infection.
- SIGNS AND SYMPTOMS OF AN ORAL HERPES INFECTION:
- PRIMARY HERPTIC GINGIVOSTOMATITIS:
The symptoms of this intial infection are painful for infants and toddlers. Blisters form on the tongue and palate. The child’s gums (gingiva) appear fiery red and are painful to the touch. Infected children easily become dehydrated and weak, due to the pain associated with eating and drinking. Toddlers with primary herpes experience fever, malaise, loss of appetite, and severe intraoral pain. Increased salivation and foul breath may also be present. - RECURRENT HERPES LABIALIS:
Tingling or itching (prodromal signs) occurs at the infection site before the lip blisters actually appear. Then, clusters of blisters (vesicles) erupt. These blisters break down rapidly and appear as tiny gray ulcers on a red base. Within one or two days, they form a crust, and appear more yellow. It can take up to 14 days for the lip lesions to heal.Sores may also appear inside of the mouth, along with swollen and red gums ( edematous and erythematous gingival tissue). These occasional intraoral sores may occur on the tongue, roof of the mouth, inside of the cheeks, and throat. The lymph nodes of the neck may swell and become painful.
- DIAGNOSIS OF ORAL HERPES:
- The child’s medical history and physical examination are usually sufficient to diagnose an HSV-1 infection. Any time an infant or toddler presents with: intraoral blisters, fiery red gingiva, fever, and intraoral pain – the diagnosis is likely to be primary herpetic gingivostomatisis.
- Children older than 4 who experience painful lip or perioral lesions likely suffer from recurrent herpes labialis. The appearance of the lip lesions are very typical. Fever is usually not present during recurrent RHL.
- LABORATORY TESTS USED IN DIAGNOSING ORAL HERPES:
- The laboratory tests used to diagnose herpes viruses include:
the direct fluorescent assay, microscopic Tzank smear, viral culture analysis, immunologic tests, and polymerase chain reaction. - In the direct fluorescent assay, a monoclonal antibody labeled with fluorescein isothyocyanate is incubated with virally infected cells which have been placed on a glass slide. The infected cells appear fluorescent green when examined under a fluorescent microscope. The overall sensitivity of the DFA technique to detect HSV is 80% and the specificity is 98%-100%.
- In the Tzank test, the base of the oral lesion must be scraped to obtain a sample of infected cells. The scraping are stained and examined microscopically. Finding specific giant cells with nuclei that carry the virus (inclusion bodies) suggests an HSV infection. Unfortunately, the test is accurate in only 50% to 70% of cases.
- In the viral culture analysis, vigorous swabbing of the lesion with a special Dacron swab is needed to produce a usable fluid sample. The fluid containing the infected cells must be placed into a special viral transport medium, and must be kept refrigerated during transport to the laboratory. Viral cultures are almost 100% accurate if the lesions are still in the blister stage. A viral culture is the “gold standard” for the diagnosis of HSV-1.
- In immunologic tests, a blood sample is taken for antibody studies. These test are most accurate when administered 12 to 16 weeks after exposure to the virus, however.
The three immunlolgic tests are the Western Blot, Herpes Select, and POCkit tests. These serologic tests are not always useful, however, since fewer than 5% of patients with recurrent HSV have a significant rise in their antibody levels.
- During the polymerase chain reaction (PCR) assay, segments of the DNA of the herpes virus are isolated – and then replicated millions of times until the virus is detectable. It is a very expensive test.
- TRANSMISSION OF ORAL HERPES VIRUS TO CHILDREN:
- Most people have been infected by oral herpes before they reach adulthood. The oral herpes virus has been detected in the blood and saliva of children who have an active herpes infection. Children contract oral herpes having by having infected saliva touch their oral soft tissue or skin wounds.
Transmission may occur by:
kissing an infected child or adult, eating with contaminated utensils, mouthing or playing with contaminated toys, using a contaminated toothbrush, and exposure to another child with who has an oral herpes infection. Many children contract oral herpes by sucking on contaminated toys that have been used by infected children. Day care is notorious for spreading the oral herpes virus. - DENTAL TREATMENT AND ORAL HERPES INFECTION:
- All stages of a herpes virus infection can be contagious, and the dentist must decide whether it is prudent to treat a child with an active infection. If a dentist decides to treat a child with an oral herpes infection – then gloves, mask, and eye protection are mandatory. The dentist must avoid spreading the virus to other areas of the child’s mouth and face. Fluid-filled vesicles are much more infectious than other stages of the herpes infection.
Dental treatment can cause reactivation of oral herpes within 3 days of major dental treatment, such as root canal treatment or dental surgery. Dental treatment may also cause introaoral recurrent herpes in the oral soft tissue (mucosa) adjacent to the teeth. Injections into the trigeminal or facial ganglions could cause massive outbreaks of the herpes infection.
- PREVENTING RECURRENT ORAL HERPES INFECTION:
- By avoiding certain triggers, it is possible to prevent recurrent herpes labialis infections (cold sores). Certain stimuli or triggers can activate a dormant (latent) herpes virus. The reactivation triggers include: UVB radiation from sunlight, oral or lip tissue injury, physical or emotional stress, common cold or influenza, fever, and malnutrition.
- Using sunscreen is a very effective preventive measure for individuals whose recurrent herpes infections are triggered by ultraviolet light.
- Colds and influenza are physically stressful conditions that affect the immune system. To reduce the risk of a recurrent herpes infection, sick children should get adequate rest, drink plenty of fluids, and maintain a healthy diet.
- Individuals with 6 or more recurrences of herpes per year, those whose immune systems are suppressed, and children who suffer from erythema multiforme – require antiviral drugs to suppress the herpes virus.
- It is almost impossible to prevent primary herpes in infants and toddlers, however.
- TREATMENT FOR ORAL HERPES SIMPLEX:
- No drug can actually cure or eliminate the herpes simplex virus. There are some drugs, however, that can improve healing time and reduce symptoms. The drugs developed against herpes are antiviral agents called nucleosides and nucleoside analogues – which block virus reproduction. Penciclovir 1% cream, acyclovir, and valacyclovir are prescription antiviral drugs which have been approved by the Federal Drug Administration for the treatment of recurrent herpes labialis (cold sores).
- Penciclovir 1% cream (Denavir) is applied every 2 hours for 4 days. It reduces the duration of pain caused by recurrent herpes labialis.
- Oral acyclovir (Zovirax) is prescribed at 20 mg/kg per day for children. Acyclovir may also be prescribed for a severe primary attack of HSV-1 in infants and toddlers. The oral and IV forms of acyclovir speed the healing of oral sores, and suppress viral shedding if taken within 24 hours of the first symptoms (the prodrome). Acyclovir is a competitive inhibitor of viral DNA polymerase. It does not kill the herpes virus, but limits the replication and spread of the virus to other cells.
- Valacyclovir (Valtrex) is converted to acyclovir in the liver and intestine. It provides a higher concentration of acyclovir in the bloodstream, and requires less frequent dosing than with acyclovir.
- Docosanol cream 10% (Abreva) is an OTC agent for oral herpes. It works by altering the healthy cell membranes to prevent viral entry.
- Oral or IV antiviral medications are used only for children who : are younger than 6 months, have a weakened immune system, or have a severe infection.
- TIPS FOR PARENTS:
- For a primary oral herpes infection in an infant or toddler, ask your dentist or physician to prescribe a mouth rinse containing Benadryl and Kaopectate. This mixture can then be applied to the painful oral tissue using a couple of cotton swabs.
- For recurrent herpes infection, consider using an OTC product such as docosanol cream (Abreva) – which will decrease pain and healing time.
- For any herpes infection, discourage your child from touching oral sores. Wash your child’s hands frequently to prevent infecting the eyes or other parts of the body.
- Do not let an infected child kiss siblings or other loved-ones.
- Blisters and cold sores should be kept dry. Use tissue or apply some cornstarch.
- Eating utensils should be thoroughly washed either by a dishwasher, or by hand – using hot, soapy water. Infected children must not share spoons, forks, or small toys.
- Provide children’s Tylenol for fever and muscle aches.
- Provide plenty of fluids to prevent dehydration.
If your child’s immune system is weakened, you should call your physician as soon as the cold sores appear. If your child is younger than 6 months, notify your physician when oral sores appear. In addition to affecting the mouth, the herpes virus can travel to the brain and cause encephalitis.
Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known as Human herpes virus 1 and 2 (HHV-1 and -2), are two members of the herpes virus family,Herpesviridae, that infect humans.Both HSV-1 (which produces most cold sores) and HSV-2 (which produces most genital herpes) are ubiquitous andcontagious. They can be spread when an infected person is producing and shedding the virus. Herpes Simplex can be spread through contact with saliva, such as sharing drinks.
Symptoms of herpes simplex virus infection include watery blisters in the skin or mucous membranes of the mouth, lips or genitals.[1] Lesions heal with a scabcharacteristic of herpetic disease. Sometimes, the viruses cause very mild or atypical symptoms during outbreaks. However, as neurotropic and neuroinvasive viruses, HSV-1 and -2 persist in the body by becoming latent and hiding from the immune system in the cell bodies of neurons. After the initial or primaryinfection, some infected people experience sporadic episodes of viral reactivation or outbreaks. In an outbreak, the virus in a nerve cell becomes active and is transported via the neuron’s axon to the skin, where virus replication and shedding occur and cause new sores.
Transmission
HSV-1 and -2 are transmitted by contact with an infected area of the skin during re-activations of the virus. Although less likely, the herpes viruses can be transmitted during latency. Transmission is likely to occur during symptomatic re-activation of the virus that causes visible and typical skin sores. Asymptomatic reactivation means that the virus causes atypical, subtle or hard to notice symptoms that are not identified as an active herpes infection. Daily genital swab samples show that HSV-2 is found in a median of 12-28% of days among those who have had an outbreak and 10% of days among those suffering from asymptomatic infection, with many of these episodes occurring without visible outbreak (“subclinical shedding”).For HSV-2, subclinical shedding may account for most of the transmission, and one study found that infection occurred after a median of 40 sex acts.Atypical symptoms are often attributed to other causes such as a yeast infection. HSV-1 is often acquired orally during childhood. It may also be sexually transmitted, including contact with saliva, such as kissing and mouth-to-genital contact (oral sex). HSV-2 is primarily a sexually transmitted infection but rates of HSV-1 genital infections are increasing.
Both viruses may also be transmitted vertically during childbirth, although the real risk is very low. The risk of infection is minimal if the mother has no symptoms or exposed blisters during delivery. The risk is considerable when the mother gets the virus for the first time during late pregnancy.
Herpes simplex viruses can affect areas of skin exposed to contact with an infected person. An example of this is herpetic whitlow which is a herpes infection on the fingers. This was a common affliction of dental surgeons prior to the routine use of gloves when conducting treatment on patients.
Microbiology
Viral structure
Animal herpes viruses all share some common properties. The structure of herpes viruses consists of a relatively large double-stranded, linear DNA genome encased within an icosahedral protein cage called the capsid, which is wrapped in a lipid bilayer called the envelope. The envelope is joined to the capsid by means of a tegument. This complete particle is known as the virion. HSV-1 and HSV-2 each contain at least 74 genes (or open reading frames, ORFs) within their genomes, although speculation over gene crowding allows as many as 84 unique protein coding genes by 94 putative ORFs. These genes encode a variety of proteins involved in forming the capsid, tegument and envelope of the virus, as well as controlling the replication and infectivity of the virus. These genes and their functions are summarized in the table below.
The genomes of HSV1 and HSV2 are complex and contain two unique regions called the long unique region (UL) and the short unique region (US). Of the 74 known ORFs, UL contains 56 viral genes, whereas US contains only 12. Transcription of HSV genes is catalyzed by RNA polymerase II of the infected host. Immediate early genes, which encode proteins that regulate the expression of early and late viral genes, are the first to be expressed following infection. Early gene expression follows, to allow the synthesis of enzymes involved in DNA replication and the production of certain envelope glycoproteins. Expression of late genes occurs last; this group of genes predominantly encode proteins that form the virion particle.
Five proteins from (UL) form the viral capsid; UL6, UL18, UL35, UL38 and the major capsid protein UL19.[9]
Cellular entry
Entry of HSV into the host cell involves interactions of several glycoproteins on the surface of the enveloped virus, with receptors on the surface of the host cell. The envelope covering the virus particle, when bound to specific receptors on the cell surface, will fuse with the host cell membrane and create an opening, or pore, through which the virus enters the host cell.
The sequential stages of HSV entry are analogous to those of other viruses. At first, complementary receptors on the virus and the cell surface bring the viral and cell membranes into proximity. In an intermediate state, the two membranes begin to merge, forming a hemifusion state.Finally, a stable entry pore is formed through which the viral envelope contents are introduced to the host cell. In the case of a herpes virus, initial interactions occur when a viral envelope glycoprotein called glycoprotein C (gC) binds to a cell surface particle called heparan sulfate. A second glycoprotein, glycoprotein D (gD), binds specifically to at least one of three known entry receptors. These include herpesvirus entry mediator(HVEM), nectin-1 and 3-O sulfated heparan sulfate. The receptor provides a strong, fixed attachment to the host cell. These interactions bring the membrane surfaces into mutual proximity and allow for other glycoproteins embedded in the viral envelope to interact with other cell surface molecules. Once bound to the HVEM, gD changes its conformation and interacts with viral glycoproteins H (gH) and L (gL), which form a complex. The interaction of these membrane proteins results in the hemifusion state. Afterward, gB interaction with the gH/gL complex creates an entry pore for the viral capsid. Glycoprotein B interacts with glycosaminoglycans on the surface of the host cell.
Genetic inoculation
After the viral capsid enters the cellular cytoplasm, it is transported to the cell nucleus. Once attached to the nucleus at a nuclear entry pore, the capsid ejects its DNA contents via the capsid portal. The capsid portal is formed by twelve copies of portal protein, UL6, arranged as a ring; the proteins contain a leucine zipper sequence of amino acids which allow them to adhere to each other. Each icosahedral capsid contains a single portal, located in one vertex. The DNA exits the capsid in a single linear segment.[16]
Immune evasion
HSV evades the immune system through interference with MHC class I presentation of antigen on the cell surface. It achieves this through blockade of the transporter associated with antigen processing (TAP) induced by the secretion of ICP-47 by HSV.[17] In the host cell, TAP transports digested viral antigen epitopes from the cytosol to the endoplasmic reticulum, allowing these epitopes to be combined with MHC class I molecules and presented on the surface of the cell. Viral epitope presentation with MHC class I is a requirement for activation of cytotoxic T-lymphocytes (CTLs), the major effectors of the cell-mediated immune response against virally-infected cells. ICP-47 prevents initiation of a CTL-response against HSV, allowing the virus to survive for a protracted period in the host.
Replication
Micrograph showing the viral cytopathic effect of HSV (multi-nucleation, ground glass chromatin).
Following infection of a cell, a cascade of herpes virus proteins, called immediate-early, early, and late, are produced. Research using flow cytometry on another member of the herpes virus family, Kaposi’s sarcoma-associated herpesvirus, indicates the possibility of an additional lytic stage, delayed-late. These stages of lytic infection, particularly late lytic, are distinct from the latency stage. In the case of HSV-1, no protein products are detected during latency, whereas they are detected during the lytic cycle.
The early proteins transcribed are used in the regulation of genetic replication of the virus. On entering the cell, an α-TIF protein joins the viral particle and aids in immediate-early transcription. The virion host shutoff protein (VHS or UL41) is very important to viral replication.[19] This enzyme shuts off protein synthesis in the host, degrades host mRNA, helps in viral replication, and regulates gene expression of viral proteins. The viral genome immediately travels to the nucleus but the VHS protein remains in the cytoplasm.
The late proteins are used in to form the capsid and the receptors on the surface of the virus. Packaging of the viral particles — including the genome, core and thecapsid – occurs in the nucleus of the cell. Here, concatemers of the viral genome are separated by cleavage and are placed into pre-formed capsids. HSV-1 undergoes a process of primary and secondary envelopment. The primary envelope is acquired by budding into the inner nuclear membrane of the cell. This then fuses with the outer nuclear membrane releasing a naked capsid into the cytoplasm. The virus acquires its final envelope by budding into cytoplasmic vesicles.
Latent infection
HSVs may persist in a quiescent but persistent form known as latent infection, notably in neural ganglia.[1] HSV-1 tends to reside in the trigeminal ganglia, while HSV-2 tends to reside in the sacral ganglia, but note that these are tendencies only, not fixed behavior. During such latent infection of a cell, HSVs express Latency Associated Transcript (LAT) RNA. LAT is known to regulate the host cell genome and interferes with natural cell death mechanisms. By maintaining the host cells, LAT expression preserves a reservoir of the virus, which allows subsequent, usually symptomatic, periodic recurrences or “outbreaks” characteristic of non-latency. Whether or not recurrences are noticeable (symptomatic), viral shedding occurs to produce further infections (usually in a new host, if any). A protein found in neuronsmay bind to herpes virus DNA and regulate latency. Herpes virus DNA contains a gene for a protein called ICP4, which is an important transactivator of genes associated with lytic infection in HSV-1.[23]Elements surrounding the gene for ICP4 bind a protein known as the humaeuronal protein Neuronal Restrictive Silencing Factor (NRSF) or human Repressor Element Silencing Transcription Factor (REST). When bound to the viral DNA elements, histone deacetylation occurs atop the ICP4 gene sequence to prevent initiation of transcription from this gene, thereby preventing transcription of other viral genes involved in the lytic cycle. Another HSV protein reverses the inhibition of ICP4 protein synthesis. ICP0 dissociates NRSF from the ICP4 gene and thus prevents silencing of the viral DNA.
The virus can be reactivated by illnesses such as colds and influenza, eczema, emotional and physical stress, gastric upset, fatigue or injury, by menstruation and possibly exposure to bright sunlight.
Viral genome
|
Gene |
Protein |
Function/description |
Gene |
Protein |
Function/description |
|
Surface and membrane |
UL38; VP19C [2] |
Capsid assembly and DNA maturation |
|||
|
UL2 [3] |
UL39 [4] |
Ribonucleotide reductase (Large subunit) |
|||
|
UL3 [5] |
unknown |
UL40 [6] |
Ribonucleotide reductase (Small subunit) |
||
|
UL4 [7] |
unknown |
UL41; VHS [8] |
Tegument protein; Virion host shutoff[19] |
||
|
UL5 [9] |
UL42 [10] |
DNA polymerase processivity factor |
|||
|
UL6 |
Twelve of these proteins constitute the capsid portal ring through which DNA enters and exits the capsid.[13][14][15] |
UL43 [11] |
Membrane protein |
||
|
UL7 [12] |
Virion maturation |
Glycoprotein C [13] |
Surface and membrane |
||
|
UL8 [14] |
DNA helicase/primase complex-associated protein |
UL45 [15] |
Membrane protein; C-type lectin[27] |
||
|
UL9 [16] |
Replication origin-binding protein |
VP11/12 [17] |
Tegument proteins |
||
|
Glycoprotein M [18] |
Surface and membrane |
UL47; VP13/14 [19] |
Tegument protein |
||
|
UL11 [20] |
virion exit and secondary envelopment |
VP16 (Alpha-TIF) [21] |
Virion maturation; activate IE genes by interacting with the cellular transcription factors Oct-1 and HCF. Binds to the sequence 5′TAATGARAT3′. |
||
|
UL12 [22] |
UL49A [23] |
Envelope protein |
|||
|
UL13 [24] |
UL50 [25] |
||||
|
UL14 [26] |
Tegument protein |
UL51 [27] |
Tegument protein |
||
|
Processing and packaging of DNA |
UL52 [29] |
DNA helicase/primase complex protein |
|||
|
UL16 [30] |
Tegument protein |
Glycoprotein K [31] |
Surface and membrane |
||
|
UL17 [32] |
Processing and packaging DNA |
IE63; ICP27 [33] |
Transcriptional regulation |
||
|
VP23 [34] |
Capsid protein |
UL55 [35] |
Unknown |
||
|
VP5 [36] |
Major capsid protein |
UL56 [37] |
Unknown |
||
|
UL20 [38] |
Membrane protein |
ICP22; IE68 [39] |
Viral replication |
||
|
UL21 [40] |
Tegument protein[28] |
US2 [41] |
Unknown |
||
|
Glycoprotein H [42] |
Surface and membrane |
US3 [43] |
Serine/threonine-protein kinase |
||
|
Peripheral to DNA replication |
Glycoprotein G [45] |
Surface and membrane |
|||
|
UL24 [46] |
unknown |
Glycoprotein J [47] |
Surface and membrane |
||
|
UL25 [48] |
Processing and packaging DNA |
Glycoprotein D [49] |
Surface and membrane |
||
|
P40; VP24; VP22A [50] |
Capsid protein |
Glycoprotein I [51] |
Surface and membrane |
||
|
Surface and membrane |
Glycoprotein E [53] |
Surface and membrane |
|||
|
ICP18.5 [54] |
Processing and packaging DNA |
US9 [55] |
Tegument protein |
||
|
UL29; ICP8 [56] |
Major DNA-binding protein |
US10 [57] |
Capsid/Tegument protein |
||
|
DNA replication |
US11; Vmw21 [59] |
Binds DNA and RNA |
|||
|
UL31 [60] |
Nuclear matrix protein |
ICP47; IE12 [61] |
Inhibits MHC class I pathway by preventing binding of antigen to TAP |
||
|
UL32 [62] |
RS1 |
Major transcriptional activator. Essential for progression beyond the immediate-early phase of infection. IEG transcription repressor. |
|||
|
UL33 [64] |
Processing and packaging DNA |
ICP0; IE110; α0 [65] |
E3 ubiquitin ligase that activates viral gene transcription by opposing chromatinization of the viral genome and counteracts intrinsic- andinterferon-based antiviral responses.[29] |
||
|
UL34 [66] |
Inner nuclear membrane protein |
LRP1 [67] |
Latency-related protein |
||
|
VP26 [68] |
Capsid protein |
LRP2 [69] |
Latency-related protein |
||
|
UL36 [70] |
Large tegument protein |
RL1; ICP34.5 [71] |
Neurovirulence factor. Antagonizes PKR by de-phosphorylating eIF4a. Binds to BECN1 and inactivates autophagy. |
||
|
UL37 [72] |
Capsid assembly |
none [73] |
Latency-associated transcript |
||
Evolution
The Herpes simplex 1 genomes can be classified into six clades. Four of these occur in East Africa with one clade in East Asian and another in Europe/North America. This suggests that this virus may have originated in East Africa. The most recent common ancestor of the Eurasian strains appears to have evolved ~60,000 years ago.The East Asian HSV-1 isolates have an unusual pattern that is currently best explained by the two waves of migration responsible for the peopling of Japan.
The mutation rate has been estimated to be ~1.38×10-7 subsitutions/site/year.
Treatment and vaccine development
Herpes viruses establish lifelong infections, and the virus cannot yet be eradicated from the body. Treatment usually involves general-purpose antiviral drugs that interfere with viral replication, reduce the physical severity of outbreak-associated lesions, and lower the chance of transmission to others. Studies of vulnerable patient populations have indicated that daily use of antivirals such as acyclovir andvalacyclovir can reduce reactivation rates.
Connection between facial sores and Alzheimer’s disease
In the presence of a certain gene variation (APOE-epsilon4 allele carriers), a possible link between HSV-1 (i.e., the virus that causes cold sores or oral herpes) and Alzheimer’s disease was reported in 1979. HSV-1 appears to be particularly damaging to the nervous system and increases one’s risk of developing Alzheimer’s disease. The virus interacts with the components and receptors of lipoproteins, which may lead to the development of Alzheimer’s disease.This research identifies HSVs as the pathogen most clearly linked to the establishment of Alzheimer’s. According to a study done in 1997, without the presence of the gene allele, HSV-1 does not appear to cause any neurological damage or increase the risk of Alzheimer’s.However, a more recent prospective study from 2008 with a cohort of several thousand people showed a high correlation between seropositivity for HSV and Alzheimer’s disease, without direct correlation to the APOE-epsilon4 allele. In 2011 Manchester University scientists showed that treating HSV1-infected cells with antiviral agents decreased the accumulation of ß-amyloid and P-tau, and also decreased HSV1 replication as expected.
Multiplicity reactivation
Multiplicity reactivation (MR) is the process by which viral genomes containing inactivating damage interact within an infected cell to form a viable viral genome. MR was originally discovered with the bacterial virus bacteriophage T4, but was subsequently also found with pathogenic viruses including influenza virus, HIV-1, adenovirus simian virus 40, vaccinia virus, reovirus, poliovirus and herpes simplex virus.
When HSV particles are exposed to doses of a DNA damaging agent that would be lethal in single infections, but are then allowed to undergo multiple infection (i.e. two or more viruses per host cell), MR is observed. Enhanced survival of HSV-1 due to MR occurs upon exposure to different DNA damaging agents, including methyl methanesulfonate, trimethylpsoralen (which causes inter-strand DNA cross-links, and UV light. After treatment of genetically marked HIV with trimethylpsoralen, recombination between the marked viruses increases, suggesting that trimethylpsoralen damages stimulate recombination. MR of HSV appears to partially depend on the host cell recombinational repair machinery since skin fibroblast cells defective in a component of this machinery (i.e. cells from Bloom’s syndrome patients) are deficient in MR. These observations suggest that MR in HSV infections involves genetic recombination between damaged viral genomes resulting in production of viable progeny viruses. HSV-1, upon infecting host cells, induces inflammation and oxidative stress.[ Thus it appears that the HSV genome may be subjected to oxidative DNA damage during infection, and that MR may enhance viral survival and virulence under these conditions.
Adenovirus Family
Adenoviruses consist of 2 genera, one that infects birds and another that infects mammals. Human adenoviruses are divided into 5 groups (A-E) based on their physical, chemical, and biologic properties. There are at least 37 antigenic types of human adenoviruses that may produce subclinical infection, respiratory tract or eye diseases, and occasionally other disorders. A few types serve as models for cancer induction in animals.
Properties of the Virus. Structure: Infective virions, 70-90 nm in diameter, are icosahedrons with capsids composed of 252 capsomeres. Three structural proteins, produced in large excess, constitute “soluble antigens” A, B, and C (Table 1). There is no envelope. The DNA is linear and double-stranded (MW 20-30 x 106). The guanine-cytosine (G + C) content of the DNA is lowest (48-49%) in group A (types 12, 18, and 31), which are the most strongly oncogenic types. The DNA can be isolated in an infectious form capable of transforming cells in culture.
Table 1.
Comparative data on adenovirus type 2 morphologic and antigenic subunits and protein components
Dodecon: Hemagglutinin made up of 12 pentons
with their fibers.
Animal Susceptibility and Transformation of Cells. Most laboratory animals are not readily infected with adenoviruses. Newborn hamsters sustain a fatal infection with type 5 and develop malignant tumours when inoculated with any of 8 or more types, including types 12, 18, and 31. Adenovirus cannot be recovered from these tumours, but in the tumour a new antigen can be detected by complement fixation or immunofluorescence. This tumour, or T, antigen also develops in hamster cells that are infected or transformed by oncogenic adenovirus types. Transformed cells produce tumours when inoculated into adult hamsters but do not contain infectious virus. Only a small part (< 10%) of the adenovirus genome is present in many transformed cells. This explains the inability to recover infectious virus from such cells.
Adenovirus messenger RNA (mRNA) can be detected in transformed or tumour cells. Different types of adenovirus result in different mRNA in transformed cells.
In human tumours, adenovirus DNA or mRNA has never been found.
Antigenic Properties. All adenoviruses contain a common complement-fixing antigen mat persists in suspensions of virus treated with heat or formalin to inactivate infectivity. At least 47 antigenic types have been isolated from humans and many additional ones from various animals. They are typed by cross-neutralization tests or hemagglutination-inhibition.
The major antigens, their size, and their structural position in the virion are shown in Table 1. Group-reactive complement-fixing antigens are hexons that form a majority of capsomeres and are 8 nm in diameter. Pentons have a similar size, occur at the 12 vertices of the capsid, and have a fiber protruding from them. The penton base carries a toxinlike activity that results in detachment of cells from the surface on which they are growing. Pentons and fibers are associated with hemagglutinating activity.
Group B adenoviruses (types 3,7,11, 14, 16,21, 34, 35) clump rhesus but not rat erythrocytes: group D (types 8, 9, 10, 13, 15, 17, 19. 22, 23, 24, 26, 27, 29, 30, 32, 33, 36, 37) clump rat but not rhesus erythrocytes; groups C (types 1, 2, 5, 6) and D (type 4) only partly clump rat cells. Types 20, 25, and 28 are atypical in that they have the physical and chemical properties of group D but agglutinate only rhesus cells. Group A (oncogenic types 12, 18, and 31) adenoviruses usually fail to hemagglutinate. Inhibition of hemagglutination by type-specific sera can be used for typing isolates. Some cross-reactions, however, do occur.
Virus Growth in Cell Culture. Adenoviruses are cytopathic for human cell cultures, particularly primary kidney and continuous epithelial cells. Growth of virus in tissue culture is associated with a stimulation of acid production (increased glycolysis) in the early stages of infection. The cytopathic effect usually consists of marked rounding and aggregation of affected cells into grapelike clusters. The infected cells do not lyse even though they round up and leave the glass surface on which they have been grown.
In HeLa cells infected with adenovirus types 3,4, and 7, rounded intranuclear inclusions containing DNA are seen. The virus particles develop in the nucleus and frequently exhibit crystalline arrangement. Many cells infected with type 5 virus also contain crystals, but these crystals are composed of a protein that has not been clearly identified.
During adenovirus replication in cultures of human cells, at least 12 virus-specific polypeptides are synthesized. These peptides are cataloged and their relationship to the virus structure is shown in Table 1.
Adenovirus DNA replication occurs in the nucleus and requires host cell DNA polymerase. Adenovirus mRNA is also made in the nucleus in a complex sequence that requires first the synthesis of larger molecules which are broken up and some sections of which are respliced by special enzymes. The spliced mRNA is translated into virus proteins.
Adenovirus-specific proteins are synthesized in the cytoplasm of infected cells and then move rapidly into the nucleus, where viral maturation occurs. In the adenovirus growth cycle in human epithelial cells, new virus particles can be detected about 16-20 hours after inoculation and continue to be formed at a uniform rate for the next 24 hours. About 7000 virus particles are produced per infected cell, and most of them remain intracellular. Particles having a density of 1.34 are infectious (one particle in 5 is infectious), whereas those having densities of less than 1.30 are noninfectious, since they lack the DNA core. Crude infected cell lysates show huge quantities of capsomeres, sometimes partially assembled into viral components.
When infecting cells derived from species other than humans, the human adenoviruses undergo an abortive replication cycle. Adenovirus tumour antigen, mRNA, and DNA are all synthesized, but no capsid proteins or infectious progeny are produced.
Adenovirus-SV40 “Hybrids”: Certain adenoviruses grown in monkey kidney cell cultures have become “contaminated” with the monkey virus SV40. While some of it was free in the mixture, other SV40 genomes became covalently linked to the adenovirus, so that stable “hybrids” were formed. Two types of hybrids have been identified. One is a defective adenovirus-SV40 genome encased in an adenovirus capsid. The other consists of nondefective (i.e., self-replicating) adenovirus type 2 that carries 5-40% of the SV40 genome. These hybrids have been used in genetic analysis but have no manifest medical relevance.
Adenoassociated Virus (AAV): In some adenovirus preparations, small 20-nm particles were found. These proved to be small viruses that could not replicate unless adenovirus (or sometimes herpesvirus) was present as a helper. AAV contains single-stranded DNA (MW 1.6 x 106) and is serologically unrelated to adenovirus. Four antigenic types of AAV are known, 3 of which infect humans but do not seem to produce disease. AAV can infect cells in the absence of an adenovirus helper and induce a latent infection. AAV enters the cell nucleus and is uncoated there, but no mRNA synthesis occurs. Upon addition of an adenovirus, AAV is “rescued” and replication occurs
Pathogenesis. Adenoviruses infect epithelial cells of mucous membranes, the cornea, and other organ systems. They can be isolated from such structures during acute illness and may persist for long periods. Types 1,2,5 and 6 can be isolated from surgically removed adenoids or tonsils of most children by growing the epithelium in vitro. Gradual removal of antibody during long culture in vitro permits the viruses to grow, as they cannot be isolated directly from suspensions of such tissues.
Most human adenoviruses grow in intestinal epithelium after ingestion but usually do not produce symptoms or lesions.
Clinical Findings. Adenovirus diseases include syndromes designated as undifferentiated acute respiratory disease, pharyngoconjunctival fever, nonstreptococcal exudative pharyngitis, and primary atypical pneumonia not associated with the development of cold agglutinins.
Pharyngoconjunctival fever may be caused by several adenovirus types. It is characterized by fever, conjunctivitis, pharyngitis, malaise, and cervical lymphadenopathy. The conjunctivitis is readily reproduced when any adenovirus is swabbed onto the eyes of volunteers, However, under natural conditions, only types 3 and 7 regularly cause outbreaks in which conjunctivitis is a predominant symptom. Types 1, 2, 5, 6, 37, and many others have produced sporadic cases of conjunctivitis.
Types 8 and 19 cause epidemic keratoconjunctivitis (shipyard eye). The disease is characterized by an acute conjunctivitis, with enlarged, tender preauricular nodes, followed by keratitis that leaves round, subepithelial opacities in the cornea for up to 2 years. Type 8 infections have been characterized by their lack of associated systemic symptoms except in infants. Intussusception of infancy has been ascribed to adenoviruses 1, 2, 3, and 5.
Types 11 and 21 may be a cause of acute hemorrhagic cystitis in children. Virus commonly occurs in the urine of such patients- Type 37 occurs in cervical lesions and in male urethritis and may be sexually transmitted.
A newly discovered serotype has been associated with infantile gastroenteritis. The virus is abundantly present in stools but has not been grown in cell culture.
Laboratory Diagnosis. Recovery of Virus. The viruses are isolated by inoculation of tissue cultures of human cells in which characteristic cytopathic changes are produced.
The viruses have been recovered from throat swabs, conjunctival swabs, rectal swabs, stools of patients with acute pharyngitis and conjunctivitis, and urine of patients with acute hemorrhagic cystitis. Virus isolations from the eye are obtained mainly from patients with conjunctivitis.
A new serotype that has not been isolated in cell cultures can be detected by direct examination of fecal extracts by electron microscopy or by enzyme-linked immunosorbent assay.
Serology. In most cases, the neutralizing antibody titer of infected persons shows a 4-fold or greater rise against the type recovered from the patient and a lesser response to other types. Neutralizing antibodies are measured in human cell cultures using the cytopathic end point in tube cultures or the colour test in panel cups. The latter test depends upon the phenomenon that adenovirus growing in HeLa cell cultures produces an excess of acid over that of uninfected control cultures. This viral lowering of pH can be prevented by immune serum. The pH is measured by incorporating phenol red into the medium and observing the colour changes after 3 days of incubation. Serum and cell control cultures reach a pH of 7.4; virus activity is indicated by a pH of 7.0; and neutralization is presumed to have occurred when the pH is 0.2 unit above that of the virus control.
Infection of humans with any adenovirus type stimulates a rise in complement-fixing antibodies to adenovirus antigens of all types. The CF test, using the common antigen, is an easily applied method for detecting infection by any member of the group.
A sensitive radioimmunoassay can measure serum antibody to type 5 fiber antigen. In response to vaccination with the fiber subunit, volunteers exhibited a 54-fold increase in antifiber antibody.
Immunity. Studies in volunteers revealed that type-specific neutralizing antibodies protect against the disease but not always against reinfection. Infections with the viruses were frequently induced without the production of overt illness.
Neutralizing antibodies against one or more types may be present in over 50% of infants 6-11 months old. Normal healthy adults generally have antibodies to several types. Neutralizing antibodies to types 1 and 2 occur in 55-70% of individuals age 6-15, but antibodies to types 3 and 4 are less prevalent. Neutralizing antibodies probably persist for life.
Infants are usually born without complement-fixing antibodies but develop these by age 6 months. Older individuals with neutralizing antibodies to 4 or more strains frequently give completely negative complement fixation reactions. For military recruits, the incidence of infection (especially due to types 3 and 4) was not influenced by the presence of group complement-fixing antibodies.
Epidemiology. Adenoviruses can readily spread from person to person. Type 1, 2, 5, and 6 infections occur chiefly during the first years of life and are associated with fever and pharyngitis or asymptomatic infection. These are the types most frequently obtained from the adenoids and tonsils.
In children and young adults, types 3 and 7 commonly cause upper respiratory illness, pharyngitis, and conjunctivitis. While the illness is usually mild, occasionally there is high fever, cervical lymphadenitis, and even pneumonitis. Sometimes enteric infection produces gastroenteritis, but more commonly it is asymptomatic. Types 11 and 21 can produce acute hemorrhagic cystitis in children.
In adolescents and young adults, eg, college populations, only 2-5% of respiratory illness is caused by adenoviruses. In sharp contrast, respiratory disease due to types 3, 4, 7, 14, and 21 is common among military recruits. Adenovirus disease causes great morbidity when large numbers of persons are being inducted into the armed forces; consequently, its greatest impact is during periods of mobilization. During a 1-year study, 10% of recruits in basic training were hospitalized for a respiratory illness caused by an adenovirus. During the winter, adenovirus accounted for 72% of all the respiratory illness. However, adenovirus disease is not a problem in seasoned troops.
The follicular conjunctivitis caused by many adenovirus types resembles chlamydial conjunctivitis and is self-limited.
Epidemic keratoconjunctivitis caused by type 8 spread in 1941 from Australia via the Hawaiian Islands to the Pacific Coast. There it spread rapidly through the shipyards and other industries, thence to the East Coast, and finally to the Midwest. A large outbreak caused by type 8 occurred in
Canine hepatitis virus is an adenovirus. Therefore, humans infected with adenoviruses develop group complement-fixing antibodies that also react with canine hepatitis virus.
In prospective family studies, adenovirus infections have been found to be predominantly enteric; they may be abortive or invasive and followed by persistent intermittent excretion of virus. Such excretion is most characteristic of types 1,2,3, and 5, which are usually endemic. Infection rates are highest among infants, but siblings who introduce the infection into a household are more effective in spreading the disease than are infants; similarly, duration of excretion is more important than the mode. In the families studied, neutralizing antibodies provided immunity (85% protective) against homotypic but not heterotypic infection. The contribution of adenoviruses to all infectious illness in the families, based on virus-positive infections only, was 5% in infants and 3% in the 2- to 4-year-old age group.
Prevention & Control. A trivalent vaccine was prepared by growing type 3,4, and 7 viruses in monkey kidney cultures and then inactivating the viruses with formalin. However, when it was found that the vaccine strains were contaminated genetically with SV40 tumour virus determinants, this vaccine was withdrawn from use. Subsequently, it was found that most adenovirus strains do not replicate in monkey cells unless SV40 is present as a helper virus. Thus, a vaccine had to be made from noncontaminated live virus that could be grown in human diploid cells, The vaccine is given orally in a coated capsule to liberate the virus into the intestine. By this route, the live vaccine produces a subclinical infection that confers a high degree of immunity against wild strains. It does not spread from a vaccinated person to contacts. Such live virus vaccines against type 4 and type 7 are licensed and recommended for immunization of military populations. When both are administered simultaneously, vaccines respond with neutralizing antibodies against both virus types.
Rigid asepsis during eye examination is essential in the control of epidemic keratoconjunctivitis.
Herpesvirus Family
All herpesviruses have a core of double-stranded DNA surrounded by a protein coat that exhibits icosahedral symmetry and has 162 capsomeres. The nucleocapsid is surrounded by an envelope. The enveloped form measures 150-200 nm; the “naked” virion, 100 nm. The double-stranded DNA (MW 85-150 x lO6) has a wide range of guanine + cytosine content in different herpesviruses, There is little DNA homology among different herpesviruses, except herpes simplex types 1 and 2.
Various classifications for herpesviruses have been proposed, but individual virus names are generally used. Common and important herpesviruses of humans include herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr (EB) virus, and cytomegalovirus. They have a propensity for subclinical infection, latency following the primary infection, and reactivation thereafter.
Herpesviruses that infect lower animals are B virus of Old World monkeys; herpesviruses saimiri, aotus, and ateles; marmoset herpesvirus of New World monkeys, pseudorabies virus of pigs; virus III of rabbits; infectious bovine rhinotracheitis virus; and many others. Herpesviruses are also known for birds, fish, fungi, and oysters, although the only link between some of these viruses is their appearance in the electron microscope.
Herpesviruses have been linked with malignant diseases in humans and lower animals: herpes simplex virus type 2 with cervical and vulvar carcinoma; EB virus with Burkilt ‘s lymphoma of African children and with nasopharyngeal carcinoma; Lucke virus with renal adenocarcinomas of the frog; Marek’s disease virus with a lymphoma of chickens; Hinze virus with a lymphoma of rabbits; and a number of New World primate herpesviruses with reticulum cell sarcomas and lymphomas in these animals.
HERPES SIMPLEX (Human Herpesvirus 1 & 2) (Herpes Labialis, Herpes Genitalis, & Many Other Syndromes).
Infection with herpes simplex virus (herpesvirus hominis) may take several clinical forms. The infection is most often inapparent. The usual clinical manifestation is a vesicular eruption of the skin or mucous membranes. Infection is sometimes seen as severe keratitis, meningoencephalitis, and a disseminated illness of the newborn.
Properties of the Virus. Morphologically and chemically, herpes simplex virus has been studied in great detail (see Fig. 1). The envelope is derived from the nuclear membrane of the infected cell. It contains lipids, carbohydrate, and protein and is removed by ether treatment. The double-stranded DNA genome is linear (MW 85-106 x 106). Types 1 and 2 show 50% sequence homology. Treatment with restriction endonucleases yields characteristically different cleavage patterns for type 1 and 2 viruses and even for different strains of each type. This “fingerprinting” of strains allows epidemiologic tracing of a given strain, whereas in the past, the ubiquitousness of herpes simplex virus made such investigations impossible.
Animal Susceptibility and Growth of Virus. The virus has a wide host range and can infect rabbits, guinea pigs, mice, hamsters, rats, and the chorioallantois of the embryonated egg.
In rabbits, herpesvirus produces a vesicular eruption in the skin of the inoculated area, sometimes progressing to fatal encephalitis. Corneal inoculation results in dendritic keratitis, which may progress to encephalitis, The virus may remain latent in the brains of survivors, and anaphylactic shock can precipitate an acute relapse of encephalomyelitis. Herpetic keratitis heals, but infective herpesvirus may be recovered from the eye intermittently with or without clinical activity. The virus remains latent in the trigeminal ganglion.
In the chorioallantoic membrane of embryonated eggs the lesions are raised white plaques, each induced by one infectious virus particle. The plaques produced by herpesvirus type 2 are larger than the tiny plaques produced by type 1 virus. The virus grows readily and produces plaques in almost any cell culture. Infected cells develop inclusion bodies and then undergo necrosis (cytopathic effect).
In Chinese hamster cells, which contain 22 chromosomes, the virus causes breaks in region 7 of chromosome No. 1 and in region 3 of the X chromosome. The Y chromosome is unaffected.
Virus Replication. The virus enters the cell either by fusion with the cell membrane or by pinocytosis. It is then uncoated, and the DNA becomes associated with the nucleus. Normal cellular DNA and protein synthesis virtually stop as virus replication begins. The virus induces a number of enzymes, at least 2 of which — thymidine kinase and DNA polymerase — are virus-coded. Thymidine kinases produced by different herpesviruses are serologically different from each other and different from the enzyme in uninfected cells. Phosphonoacetic acid specifically inhibits herpesvirus replication by inhibiting viral DNA polymerase.
Viral proteins are made in a controlled sequence that must proceed stepwise. They are made in the cytoplasm and most are transported to the nucleus, where they take part in virus DNA synthesis and the assembly of nucleocapsids, Maturation occurs by budding of nucleocapsids through the altered inner nuclear membrane. Enveloped virus particles are then released from the cell through tubular structures that are continuous with the outside of the cell or from vacuoles that release their contents at the cell surface.
Defective Interfering Herpesvirions: Serial passage of undiluted herpes simplex virus results in cyclic production of infectious and defective virions. The DNA in defective virions is made up of reiterated sequences of small fragments of the virus DNA. Defective virions interfere with the replication of standard virus and stimulate overproduction of a large polypeptide, which may have a regulatory function. The biologic role of the defective virions is not known.
Antigenic Properties. There are 7-12 precipitating antigens that represent structural and non-structural viral proteins. Some of these antigens are common to both types 1 and 2 and some are specific for one type. A number of tests, eg, fluorescent antibody, complement fixation, virus neutralization, and radioimmunoassay, have been used to detect herpesvirus antigens.
Differentiation of Types 1 and 2. Herpes simplex virus types 1 and 2 cross-react serologically but may be distinguished by a number of tests: (1) The use of type-specific antiserum prepared by adsorption of the viral antiserum with heterotypically infected cells or by inoculation of rabbits with individual type-specific proteins. (2) The greater temperature sensitivity of type 2 infectivity. (3) Preferential growth in different cell species. (4) Restriction enzyme patterns of virus DNA molecules. (5) Differences in the polypeptides produced by type 1 and type 2.
Oncogenic Properties: After inactivation of their lytic capabilities by ultraviolet irradiation or other means, herpesvirus types 1 and 2 can cause transformation of cultured hamster cells, which may induce tumours when inoculated into newborn hamsters. Viral genetic information can be demonstrated in the tumour cells.
Pathogenesis & Pathology. The lesion in the skin involves proliferation, ballooning degeneration, and intranuclear acidophilic inclusions. In fatal cases of herpes encephalitis, there are meningitis, perivascular infiltration, and nerve cell destruction, especially in the cortex. Neonatal generalized herpes infection causes areas of focal necrosis with a mononuclear reaction and formation of intranuclear inclusion bodies in all organs. Survivors may sustain permanent damage.
The fully formed early inclusion (Cowdry type A inclusion body) is rich in DNA and virtually fills the nucleus, compressing the chromatin to the nuclear margin. Later, the inclusion loses its DNA and is separated by a halo from the chromatin at the nuclear margin.
Clinical Findings. Herpesvirus may cause many clinical entities, and the infections may be primary or recurrent. Primary infections occur in persons without antibodies and often result in the virus assuming a latent state in sensory ganglia of the host. Latent infections persist in persons with antibodies, and recurrent lesions are common (eg, recurrent herpes labialis). The primary infection in most individuals is clinically inapparent but is invariably accompanied by antibody production.
The recurrent attacks, in the presence of viral neutralizing antibody, follow non-specific stimuli such as exposure to excess sunlight, fever, menstruation, or emotional stresses.
Herpesvirus Type 1. The clinical entities attributable to herpesvirus type 1 include the following:
1. Acute herpetic gingivostomatitis (aphthous stomatitis, Vincent’s stomatitis). This is the most common clinical entity caused by primary infections with type 1 herpesvirus. It occurs most frequently in small children (1-3 years of age) and includes extensive vesiculoulcerative lesions of the mucous membranes of the mouth, fever, irritability, and local lymphadenopathy. The incubation period is short (about 3-5 days), and the lesions heal in 2-3 weeks.
2. Eczema herpeticum (Kaposi’s varicelliform eruption). This is a primary infection, usually with herpesvirus type
3. Keratoconjunctivitis. The initial infection with herpesvirus may be in the eye, producing severe keratoconjunctivitis. Recurrent lesions of the eye appear as dendritic keratitis or corneal ulcers or as vesicles on the eyelids. With recurrent keratitis, there may be progressive involvement of the corneal stroma, with permanent opacification and blindness.
4. Encephalitis. A severe form of encephalitis may be produced by herpesvirus. In adults, the neurologic manifestations suggest a lesion in the temporal lobe. Pleocytosis (chiefly of lymphocytes) is present in the cerebrospinal fluid; however, definite diagnosis during the illness can usually be made only by isolation of the virus (or by demonstrating viral antigens by immunofluorescence) from brain tissue obtained by biopsy or at post-mortem. The disease carries a high mortality rate, and those who survive often have residual neurologic defects.
5. Herpes labialis (cold sores, herpes febrilis). This is the most common recurrent disease produced by type 1. Clusters of localized vesicles occur, usually at the mucocutaneous junction of the lips. The vesicle ruptures, leaving a painful ulcer that heals without scarring. The lesions may recur, repeatedly and at various intervals of time, in the same location. The permanent site of latent herpes simplex virus is the trigeminal ganglion.
Herpesvirus Type 2. The clinical entities associated with herpesvirus type 2 include the following:
1. Genital herpes (herpes progenitalis). Genital herpes is characterized by vesiculoulcerative lesions of the penis of the male or the cervix, vulva, vagina, and perineum of the female. The lesions are more severe during primary infection and may be associated with fever, malaise, and inguinal lymphadenopathy. In women with herpesvirus antibodies, only the cervix or vagina may be involved, and the disease may therefore be asymptomatic. Recurrence of the lesions is common. Type 2 virus remains latent in lumbar and sacral ganglia. Changing patterns of sexual behaviour are reflected by an increasing number of type 1 virus isolations from genital lesions and of type 2 from facial lesions, presumably as a result of oral-genital sexual activity.
2. Neonatal herpes. Herpesvirus type 2 may be transmitted to the newborn during birth by contact with herpetic lesions in the birth canal. The spectrum of illness produced in the newborn appears to vary from subclinical or local to severe generalized disease with a fatal outcome. Severely affected infants who survive may have permanent brain damage. To avoid infection, delivery by cesarean section has been used in pregnant women with genital herpes lesions. To be effective, cesarean section must be performed before rupture of the membranes.
Severe generalized disease of the newborn can be acquired postnatally by exposure to either type 1 or 2. Efforts should be made to prevent exposure to active lesions among family and especially among hospital personnel.
Transplacental infection of the fetus with types 1 and 2 herpes simplex virus may cause congenital malformations, but this phenomenon is rare.
Miscellaneous. Localized lesions of the skin caused by type 1 or 2 may occur in abrasions that become contaminated with the virus (traumatic herpes), These lesions are seen on the fingers of dentists, hospital personnel (herpetic whitlow), or persons with genital lesions and on the bodies of wrestlers.
Primary and recurrent herpes can occur in the nose (acute herpetic rhinitis).
Mild aseptic meningitis has been attributed to the virus, and recurrent episodes of meningeal irritation have been observed.
Epidemiologic evidence has demonstrated that in most geographic areas, patients with cervical and vulvar cancer have a high frequency of type 2 antibodies. In addition, herpesvirus type 2 non-structural antigens have been detected by immunofluorescence in biopsies of cervical and vulvar carcinomas.
Laboratory Diagnosis
Recovery of Virus. The virus may be isolated from herpetic lesions (skin, cornea, or brain). It may also be found in the throat, saliva, and stools, both during primary infection and during asymptomatic periods. Therefore, the isolation of herpesvirus is not in itself sufficient evidence to indicate that this virus is the causative agent of a disease under investigation.
inoculation of tissue cultures is used for virus isolation. The appearance of typical cytopathic effects in cell culture suggests the presence of herpesvirus in 18-36 hours. The agent is then identified by neutralization test or immunofluorescence staining with specific antiserum.
Scrapings or swabs from the base of early herpetic lesions contain multinucleated giant cells.
Serology. Antibodies may be measured quantitatively by neutralization tests in cell cultures. In the early stage of the primary immune response, neutralizing antibody appears that is detectable only in the presence of fresh complement. This antibody soon is replaced by neutralizing antibody that can function without complement.
Since the only hope for treatment of herpes simplex virus encephalitis lies in early diagnosis, a rapid means of diagnosis is needed. The fluorescent antibody test using brain biopsy material is the method of choice. Passive hemagglutinating antibodies in the cerebrospinal fluid are a better indicator of the presence of infectious virus than are antibody titters in serum.
A soluble complement-fixing antigen of much smaller size than the virus can be prepared from infected chorioallantoic membranes or from tissue culture. This soluble antigen of herpesvirus can detect dermal hypersensitivity in previously infected persons. There is a good correlation between dermal hypersensitivity and the presence of serum antibodies.
Antibodies appear in 4-7 days; can be measured by neutralization, complement fixation, radioimmunoassay, or immunofluorescence; and reach a peak in 2-4 weeks. They persist with minor fluctuations for the life of the host. The majority of adults have antibodies in their blood at all times.
After a primary type I infection, the IgM neutralizing antibody response is type-specific, but after a primary type 2 infection the IgM that develops neutralizes both type 1 and type 2 virus. Subsequently, IgG antibodies react with both type 1 and type 2 antigens, albeit in varying ratios.
There is also some cross-stimulation between herpes simplex and varicella-zoster antigens in patients with pre-existing antibody to the other virus.
Immunity. Many newborns have passively transferred maternal antibodies. This antibody is lost during the first 6 months of life, and the period of greatest susceptibility to primary herpes infection occurs between ages 6 months and 2 years. Type 1 antibodies begin to appear in the population in early childhood; by adolescence they are present in most persons. Antibodies to type 2 (genital herpesvirus) rise during the age of adolescence and sexual activity.
After recovery from a primary infection (inapparent, mild, or severe), the virus is usually carried in a latent state, in the presence of antibodies.
Treatment. Topically applied idoxuridine (5-iodo-2′-deoxy-uridine, IUDR), trifluorothymidine, vidarabine (ade-nine arabinoside, ara-A), acyclovir, and other inhibitors of viral DNA synthesis are effective in herpetic keratitis. These drugs inhibit herpesvirus replication and may suppress clinical manifestations. However, the virus remains latent in the sensory ganglia, and the rate of relapse is similar in drug-treated and untreated individuals. Some drug-resistant virus strains have emerged. Most strains of type 2 herpesvirus are suppressed less effectively than type 1.
For systemic administration, vidarabine (15 mg/kg/d intravenously) is accepted in herpes encephalitis diagnosed by biopsy. Best results are obtained if treatment is begun early in the disease, before coma sets in. Vidarabine also has some effect in disseminated herpes simplex.
Other drugs, especially acyclovir, are undergoing clinical trial, and many new antiherpes compounds are being developed. Acyclovir has low toxicity and has been administered systemically to suppress the activation of a latent herpes infection in immunosuppressed patients.
Epidemiology. The epidemiology of type 1 and type 2 herpesvirus differs. Herpesvirus type 1 is probably more constantly present in humans than any other virus. Primary infection occurs early in life and is often asymptomatic or produces acute gingivostomatitis. Antibodies develop, but the virus is not eliminated from the body; a carrier state is established that lasts throughout life and is punctuated by transient attacks of herpes. If primary infection is avoided in childhood, it may not occur in later life, perhaps because the thicker adult epithelium is less susceptible or because the opportunity for contact with the virus is diminished (less contact with saliva of infected persons).
The highest incidence of type I virus carriage in the oropharynx of healthy persons occurs among children 6 months to 3 years of age. By adulthood, 70-90% of persons have type 1 antibodies.
Type 1 virus is transmitted more readily in families of lower socioeconomic groups; the most obvious explanation is their more crowded living conditions and lower hygienic standards. The virus is spread by direct contact (saliva) or through utensils contaminated with the saliva of a virus shedder. The source of infection for children is usually a parent with an active herpetic lesion.
Type 2 is usually acquired as a sexually transmitted disease, and the age distribution of primary infection is a function of sexual activity. The neonate may acquire type 2 infection from an active lesion in the mother’s birth canal.
Control. Neonates and persons with eczema should be protected from evident active herpetic lesions. Although certain drugs are effective in treatment of herpesvirus infections, once a latent infection is established there has been no known treatment that would prevent recurrences until the recent successful results with acyclovir in immunosuppressed patients.
Little Is known about vaccines. Herpes recurs in the presence of circulating antibody, so a vaccine would be of little use in a person who already had a primary infection. A vaccine currently made in Europe has not been adequately tested.
VARICELLA-ZOSTER VIRUS (Human Herpesvirus 3)
VARICELLA (Chickenpox) ZOSTER (Herpes Zoster, Shingles, Zona)
Varicella (chickenpox) is a mild, highly infectious disease, chiefly of children, characterized clinically by a vesicular eruption of the skin and mucous membranes. However, in immunocompromised children the disease may be severe. The causative agent is indistinguishable from the virus of zoster.
Zoster (shingles) is a sporadic, incapacitating disease of adults (rare in children) that is characterized by an inflammatory reaction of the posterior nerve roots and ganglia, accompanied by crops of vesicles (like those of varicella) over the skin supplied by the affected sensory nerves.
Both diseases are caused by the same virus. Varicella is the acute disease that follows primary contact with the virus, whereas zoster is the response of the partially immune host to a reactivation of varicella virus present in latent form in sensory ganglia.
Properties of the Virus. Varicella-zoster virus is morphologically identical with herpes simplex virus. The virus propagates in cultures of human embryonic tissue and produces typical intranuclear inclusion bodies. Supernatant fluids from such infected cultures contain a complement-fixing antigen but no infective virus. Infectious virus is easily transmitted by infected cells. The virus has not been propagated in laboratory animals. Virus can be isolated from the vesicles of chickenpox or zoster patients or from the cerebrospinal fluid in cases of zoster aseptic meningitis.
Inoculation of vesicle fluid of zoster into children produces vesicles at the site of inoculation in about 10 days. This may be followed by generalized skin lesions of varicella. Generalized varicella may occur in such inoculated children without local vesicle formation. Contacts of such children develop typical varicella after a 2-week incubation period. Children who have recovered from zoster virus-induced infection are resistant to varicella, and those who have had varicella are no longer susceptible to primary zoster virus.
Antibody to varicella-zoster virus can be measured by CF, gel precipitation, neutralization, or indirect immunofluorescence to virus-induced membrane antigens.
The virus has a colchicinelike effect on human cells. Arrest in metaphase, overcontracted chromosomes, chromosome breaks, and formation of micronuclei are often seen.
Pathogenesis & Pathology. Varicella: The route of infection is probably the mucosa of the upper respiratory tract. The virus probably circulates in the blood and localizes in the skin. Swelling of epithelial cells, ballooning degeneration, and the accumulation of tissue fluids result in vesicle formation. Iuclei of infected cells, particularly in the early stages, eosinophilic inclusion bodies are found.
Zoster: In addition to skin lesions — histopathologically identical with those of varicella — there is an inflammatory reaction of the dorsal nerve roots and sensory ganglia. Often only a single ganglion may be involved. As a rule, the distribution of lesions in the skin corresponds closely to the areas of innervation from an individual dorsal root ganglion. There is cellular infiltration, necrosis of nerve cells, and inflammation of the ganglion sheath.
Varicella virus seems able to enter and remain within dorsal root ganglia for long periods. Years later, various insults (eg, pressure on a nerve) may cause a flare-up of the virus along posterior root fibers, whereupon zoster vesicles appear. Thus, varicella-zoster and herpes simplex viruses are similar in their ability to induce latent infections with clinical recurrence of disease in humans. However, zoster rarely occurs more than once.
Clinical Findings. Varicella: The incubation period is usually 14-21 days. Malaise and fever are the earliest symptoms, soon followed by the rash, first on the trunk and then on the face, the limbs, and the buccal and pharyngeal mucosa. Successive fresh vesicles appear in crops during the next 3-4 days, so that all stages of papules, vesicles, and crusts may be seen at one time. The eruption is found together with the fever and is proportionate to its severity. Complications are rare, although encephalitis does at times occur about 5-10 days after the rash. The mortality rate is much less than 1% in uncomplicated cases. Ieonatal varicella (contracted from the mother just before or just after birth), the mortality rate may be 20%. In varicella encephalitis, the mortality rate is about 10%, and another 10% are left with permanent injury to the central nervous system. Primary varicella pneumonia is rare in children but may occur in about 20-30% of adult cases, may produce severe hypoxia, and may be fatal.
Children with immune deficiency disease or those receiving immunosuppressant or cytotoxic drugs are at high risk of development of very severe and sometimes fatal varicella or disseminated zoster.
Zoster: The incubation period is unknown. The disease starts with malaise and fever that are soon followed by severe pain in the area of skin or mucosa supplied by one or more groups of sensory nerves and ganglia. Within a few days after onset, a crop of vesicles appears over the skin supplied by the affected nerves. The eruption is usually unilateral; the trunk, head, and neck are most commonly involved. Lymphocytic pleocytosis in the cerebrospinal fluid may be present.
In patients with localized zoster and no underlying disease, vesicle interferon levels peak early during infection (by the sixth day), whereas those in patients with disseminated infection peak later. Peak interferon levels are followed by clinical improvement within 48 hours. Vesicles pustulate and crust, and dissemination is halted.
Zoster tends to disseminate when there is an underlying disease, especially if the patient is taking immunosuppressive drugs or has lymphoma treated by irradiation.
Laboratory Diagnosis. In stained smears of scrapings or swabs of the base of vesicles, multinucleated giant cells are seen. In similar smears, intracellular viral antigens can be demonstrated by immunofluorescence staining.
Virus can be isolated in cultures of human or other fibroblastic cells in 3-5 days. It does not grow in epithelial cells, in contrast to herpes simplex, and does not infect laboratory animals or eggs. An isolate in fibroblasts is identified by immunofluorescence or neutralization tests with specific antisera.
Herpesviruses can be differentiated from pox-viruses by (1) the morphologic appearance of particles in vesicular fluids examined by electron microscopy, and by (2) the presence of antigen in vesicle fluid or in an extract of crusts as determined by gel diffusion tests with specific antisera to herpes, varicella, or vaccinia viruses, which give visible precipitation lines in 24-48 hours.
A rise in specific antibody titer can be detected in the patient’s serum by CF, Nt (in cell culture), indirect immunofluorescence tests, or enzyme immunoassay, Zoster can occur in the presence of relatively high neutralizing antibody in the blood just prior to onset. The role of cell-mediated immunity is unknown.
Immunity. Varicella and zoster viruses are identical, the 2 diseases being the result of differing host responses. Previous infection with varicella leaves the patient with enduring immunity to varicella. However, zoster may occur in persons who have contracted varicella earlier. This is a reactivation of a varicella virus infection that has been latent for years.
Prophylaxis & Treatment. Gamma globulin of high specific antibody titer prepared from pooled plasma of patients convalescing from herpes zoster (zoster immune globulin) can be used to prevent the development of the illness in immunocompromised children who have been exposed to varicella. Standard immune serum globulin is without value because of the low titer of varicella antibodies.
Zoster immune globulin is available from the American Red Cross Blood Services (through 13 regional blood centres) for prophylaxis of varicella in exposed high-risk immunodeficient or immunosuppressed children. It has no therapeutic value once varicella has started.
Idoxuridine and cytarabine inhibit replication of the viruses in vitro but are not an effective treatment for patients.
Adenine arabinoside (vidarabine, ara-A) has been beneficial in adults with severe varicella pneumonia, immunocompromised children with varicella, and adults with disseminated zoster. Human leukocyte interferon in large doses appears to be similarly beneficial.
Epidemiology. Zoster occurs sporadically, chiefly in adults and without seasonal prevalence. In contrast, varicella is a common epidemic disease of childhood (peak incidence is in children age 2-6 years, although adult cases do occur). It is much more common in winter and spring than in summer. Almost 200,000 cases are reported annually in the USA.
Varicella readily spreads, presumably by droplets as well as by contact with skin. Contact infection is rare in zoster, perhaps because the virus is absent in the upper respiratory tract.
Zoster, whether in children or adults, can be the source of varicella in children and can initiate large outbreaks.
Control. None is available for the general population. Varicella may spread rapidly among patients, especially among children with immunologic dysfunctions or leukemia or in those receiving corticosteroids or cytotoxic drugs. Varicella in such children poses the threat of pneumonia, encephalitis, or death. Efforts should be made to prevent their exposure to varicella. Zoster immune globulin may be used to modify the disease in such children who have been exposed to varicella.
A live attenuated varicella vaccine has been developed in Japan and tested in hospitalized immune-suppressed children who were exposed to varicella. It appeared to prevent spread of chickenpox. The vaccine is being used experimentally for similar high-risk children in the USA.
A number of problems are envisioned for the use of such a vaccine for the general population as opposed to high-risk patients. The vaccine would need to confer immunity comparable to that of natural infections. A short-lasting immunity might result in an increased number of susceptible adults, in whom the disease is more severe. Furthermore, any such vaccine would need to be evaluated for later morbidity due to zoster as compared to that following natural childhood infections with varicella virus.
CYTOMECALOVIRUS (Human Herpesvirus 5) (Cytomegalic Inclusion Disease)
Cytomegalic inclusion disease is a generalized infection of infants caused by intrauterine or early postnatal infection with the cytomegaloviruses. The disease causes severe congenital anomalies in about 10,000 infants in the USA per year. Cytomegalovirus can be found in the cervix of up to 10% of healthy women. Cytomegalic inclusion disease is characterized by large intranuclear inclusions that occur in the salivary glands, lungs, liver, pancreas, kidneys, endocrine glands, and. occasionally, the brain. Most fatalities occur in children under 2 years of age. Inapparent infection is common during childhood and adolescence. Severe cytomegalovirus infections are frequently found in adults receiving immunosuppressive therapy.
Properties of the Virus. Morphologically, cytomegalovirus is indistinguishable from herpes simplex or varicella-zoster virus.
In infected human fibroblasts, virus particles are assembled in the nucleus. The envelope of the virus is derived from the inner nuclear membrane. The growth cycle of the virus is slower, and infectious virus is more cell-associated than herpes simplex virus.
Animal Susceptibility. All attempts to infect animals with human cytomegalovirus have failed. A number of animal cytomegaloviruses exist, all of them species-specific in rats, hamsters, moles, rabbits, and monkeys. The virus isolated from monkeys propagates in cultures of monkey as well as human cells.
Human cytomegalovirus replicates in vitro only in human fibroblasts. although the virus is often isolated from epithelial cells of the host. The virus can transform human and hamster cells in culture, but whether it is oncogenic in vivo is unknown.
Pathogenesis & Pathology. In infants, Cytomegalic inclusion disease is con-genitally acquired, probably as a result of primary infection of the mother during pregnancy. The virus can be isolated from the urine of the mother at the time of birth of the infected baby, and typical cytomegalic cells, 25-40 mcm in size, occur in the chorionic villi of the infected placenta.
Foci of cytomegalic cells are found in fatal cases in the epithelial tissues of the liver, lungs, kidneys, gastrointestinal tract, parotid gland, pancreas, thymus, thyroid, adrenals, and other regions. The cells can be found also in the urine or adenoid tissue of healthy children. The route of infection in older infants, children, and adults is not known.
The isolation of the virus from urine and from tissue cultures of adenoids of healthy children suggests subclinical infections at a young age. The virus may persist in various organs for long periods in a latent state or as a chronic infection. Virus is not recovered from the mouths of adults. Disseminated inclusions in adults occur in association with other severe diseases.
Clinical Findings. Congenital infection may result in death of the fetus in utero or may produce the clinical syndrome of cytomegalic inclusion disease, with signs of prematurity, jaundice with hepatosplenomegaly, thrombocytopenic purpura, pneumonitis, and central nervous system damage (microcephaly, periventricular calcification, chorioretinitis, optic atrophy, and mental or motor retardation).
Infants born with congenital cytomegalic inclusion disease may appear well and live for many years. It has been estimated that one in every 1000 babies born in the USA is seriously retarded as a result of this congenital infection.
Inapparent intrauterine infection seems to occur frequently. Elevated IgM antibody to cytomegalovirus or isolation of the virus from the urine occurs in up to 2% of apparently normal newborns. This high rate occurs in spite of the fact that women may already have cytomegalovirus antibody before becoming pregnant. Such intrauterine infections have been implicated as possible causes of mental retardation and hearing loss.
Many women who have been infected naturally with cytomegalovirus at some time prior to pregnancy begin to excrete the virus from the cervix during the last trimester of pregnancy. At the time of delivery, infants pass through the infected birth canal and become infected, although they possess high titters of maternal antibody acquired transplacentally. These infants begin to excrete the virus in their urine at about 8-12 weeks of age. They continue to excrete the virus for several years but remain healthy.
Acquired infection with cytomegalovirus is common and usually inapparent, In children, acquired infection may result in hepatitis, interstitial pneumonitis, or acquired hemolytic anemia. The virus is shed in the saliva and urine of infected individuals for weeks or months.
Cytomegalovirus can cause an infectious mononucleosis-like disease without heterophil antibodies. “Cytomegalovirus mononucleosis” occurs either spontaneously or after transfusions of fresh blood during surgery (“postperfusion syndrome”). The incubation period is about 30-40 days. There is cytomegaloviruria and a rise of cytomegalovirus antibody. Cytomegalovirus has been isolated from the peripheral blood leukocytes of such patients. Perhaps the postperfusion syndrome is caused by cytomegalovirus harboured in the leukocytes of the blood donors.
Patients with malignancies or immunologic defects or those undergoing immunosuppressive therapy for organ transplantation may develop cytomegalo-virus pneumonitis or hepatitis and occasionally generalized disease. In such patients a latent infection may be reactivated when host susceptibility to infection is increased by immunosuppression. In seronegative patients without evidence of previous cytomegalovirus infection, the virus may be transmitted exogenously. Eighty-three percent of seronegative patients who received kidneys from seropositive transplant donors developed infection. Thus, the kidneys seemed to be the source of virus.
Laboratory Diagnosis. Recovery of Virus: The virus can be recovered from mouth swabs, urine, liver, adenoids, kidneys, and peripheral blood leukocytes by inoculation of human fibroblastic cell cultures. In cultures, 1-2 weeks are usually needed for cytotogic changes consisting of small foci of swollen, rounded, translucent cells with large intranuclear inclusions. Cell degeneration progresses slowly, and the virus concentration is much higher within the cell than in the fluid. Prolonged serial propagation is needed before the virus reaches high titters.
Rapid diagnosis of cytomegalovirus infection in infants is possible by detection of inclusion-bearing “owl cells” in the urine. These are desquamated cells from infected kidney tubules.
Serology. Antibodies may be detected by neutralization, complement fixation, or immunofluorescence tests. Such tests may be useful in detecting congenitally infected infants with no clinical manifestations of disease.
Immunity. Complement-fixing and neutralizing antibodies occur in most human sera. In young children possessing CF antibodies, virus may be detected in the mouth and in the urine for many months.
Virus may occur in the urine of children even though serum-neutralizing antibody is present. This suggests that the virus propagates in the urinary tract rather than being filtered from the bloodstream. Virus is not found in young children who lack antibody.
Intrauterine infection may produce a serious disease in the newborn. Infants infected during fetal life may be born with antibody that continues to rise after birth in the presence of persistent virus excretion. (This is similar to the situation in congenital rubella infection.)
Most infants infected with cytomegalovirus in the perinatal period are asymptomatic, and infection continues in the presence of high antibody titters.
Treatment. There is no specific treatment. Neither immune gamma globulior DNA virus-inhibitory drugs have any effect.
Epidemiology. The mechanism of virus transmission in the population remains unknown except in congenital infections and those acquired by organ transplantation, blood transfusion, and reactivation of latent virus. Infection with cytomegaloviruses is widespread. Antibody is found in 80% of individuals over 35 years of age. The prolonged shedding of virus in urine and saliva suggests a urine-hand-oral route of infection. Cytomegalovirus can also be transmitted by sexual contact.
Control. Specific control measures are not available. Isolation of newborns with generalized cytomegalic inclusion disease from other neonates is advisable.
Screening of transplant donors and recipients for cytomegalovirus antibody may prevent some transmissions of primary cytomegalovirus. The cytomegalovirus-seronegative transplant recipient population represents a high-risk group for cytomegalovirus infections as well as other lethal superinfections and would be a target population for a vaccine.
A live cytomegalovirus “vaccine” has been developed and has had some preliminary clinical trials, Since cytomegalovirus, like other herpesviruses, causes latent persistent infection, there is doubt that such a “vaccine” would be useful for the population at large. The possible benefits and dangers of a vaccine program for prevention of cytomegalovirus congenital infections require further study.
EB HERPESVIRUS (Human Herpesvirus 4).
(Infectious Mononucleosis, Burkitt’s Lymphoma, Nasopharyngeal Carcinoma).
EB (Epstein-Barr) virus is the causative agent of infectious mononucleosis and has been associated with Burkitt’s lymphoma and nasopharyngeal carcinoma. The virus is an antigenically distinct herpesvirus.
Properties of the Virus. Morphology: EB virus is indistinguishable in size and structure from other herpesviruses.
Antigenic Properties: EB virus is distinct from all other human herpesviruses. Many different EB virus antigens can be detected by CF, immunodiffusion, or immunofluorescence tests. A lymphocyte-detected membrane antigen (LYDMA) is the earliest-detected virus-determined antigen. EBNA is a complement-fixing nuclear antigen. Early antigen (EA) is formed in the presence of DNA inhibitors and membrane antigen (MA), the neutralizing antigen, is a cell surface antigen. The virus capsid antigen (VCA) is a late antigen representing virions and structural antigen.
C. Virus Growth: Human blood B lymphocytes infected in vitro with EB virus have resulted in the establishment of continuous cell lines, suggesting that these cells have been transformed by the virus.
This transformation by EB virus enables B lymphocytes to multiply continuously, and all cells contain many EB virus genomes and express EBNA. Some EB virus cell lines express certain antigens but produce no virus particles or VCA; others produce virus particles. EB virus is carried in lymphoid cell lines derived from patients with African Burkitt’s lymphoma, nasopharyngeal carcinoma, or infectious mononucleosis. Non-virus-producing B lymphocyte cell lines can be established in vitro from the blood of patients with infectious mononucleosis. Such lines represent a latent state of the virus; the cells contain EB virus genomes but express only the earliest antigen (LYDMA) and possibly EBNA.
Owl monkeys and marmosets inoculated with cell-free EB virus can develop fatal malignant lymphomas. Lymphoblastoid cells from such monkeys cultured as continuous cell lines give positive reactions with EB virus antisera by immunofluorescence.
Immunity. The most widely used and most sensitive serologic procedure for detection of EB virus infection is the indirect immunofluorescence test with acetone-fixed smears of cultured Burkitt’s lymphoma cells. The cells containing the EB virus exhibit fluorescence after treatment with fluorescent antibody. Detectable levels of antibody persist for many years.
Early in acute disease, a transient rise in IgM antibodies to VCA occurs, replaced within 2 weeks by IgG antibodies to VCA, which persist for life. Slightly later, antibodies to MA and to EBNA arise and persist throughout life.
Epidemiology. Seroepidemiologic studies using the immunofluorescence technique and CF reaction indicate that infection with EB virus is common in different parts of the world and that it occurs early in life. In some areas, including urban parts of the USA, about 50% of children 1 year old. 80-90% of children over age 4, and 90% of adults have antibody to EB virus.
In groups at a low socioeconomic level, EB virus infection occurs in early childhood without any recognizable disease. These inapparent infections result in permanent seroconversion and total immunity to infectious mononucleosis. In groups living in comfortable social circumstances, infection is often postponed until adolescence and young adulthood. Again, the majority of these adult infections are asymptomatic, but In almost half of cases the infection is manifested by heterophil-positive infectious mononucleosis.
Antibody to EB virus is also present ionhuman primates.
EB Virus & Human Disease. Most EB virus infections are clinically inapparent. The virus causes infectious mononucleosis and is strongly associated with Burkitt’s lymphoma and nasopharyngeal carcinoma.
Infectious mononucleosis (glandular fever) is a disease of children and young adults characterized by fever and enlarged lymph nodes and spleen. The total white blood count may range from 10,000/mcL to 80,000/mc, with a predominance of lymphocytes. Many of these are-large “atypical” cells with vacuo-lated cytoplasm and nucleus. These atypical lymphocytes, probably T cells, are diagnostically important. During mononucleosis, there often are signs of hepatitis.
During the course of infection, the majority of patients develop heterophil antibodies, detected by sheep cell agglutination or the mononucleosis spot test.
Although the pathogenesis of infectious mononucleosis is still not understood, infectious EB virus can be recovered from throat washings and saliva of patients (“kissing disease”). Infectious virus is produced by B lymphocytes in the oropharynx and perhaps in special epithelial cells of this region. Virus cannot be recovered from blood, but EB virus genome-containing B lymphocytes are present in up to 0.05% of me circulating mononuclear leukocytes as demonstrated by me establishment of cell lines. These EB virus genome-containing cells express the earliest antigen, LYDMA, which is specifically recognized by killer T cells.
These T cells reach large numbers and can lyse EB virus genome-positive but not EB virus genome-negative target cells. Part of the infectious mononucleosis syndrome may reflect a rejection reaction against virally converted lymphocytes.
Patients with infectious mononucleosis develop antibodies against EB virus, as measured by immunofluorescence with virus-bearing cells. Antibodies appear early in the acute disease, rise to peak levels within a few weeks, and remain high during convalescence. Unlike the short-lived heterophil antibodies, those against EB virus persist for years.
The role that EB virus may play in Burkitt’s lymphoma (a tumour of the jaw in African children and young adults) and nasopharyngeal carcinoma (common in males of Chinese origin) is less well established. The association with EB virus is based primarily on the finding that the prevalence of antibody is greater and the antibody titters are higher among patients with Burkitt’s lymphoma and nasopharyngealcarcinoma than in healthy matched controls or individuals with other types of malignancies. The significance of these associations is uncertain at present. All cells from Burkitt’s lymphoma of African origin and from nasopharyngeal carcinoma carry multiple copies of the EB virus genome and express the antigen EBNA.
Students Practical activities
1. To inoculate the pig embryo kidneys cell culture by blood of the patient with suspicion on a tick-borne encephalitis.
There is pig embryo kidneys cell culture the sterile bottle. It is on the side of bottle opposite to vertical line. In sterile conditions it is necessary to pour out the medium and to fill in the bottle 1,5 ml of the defibrinated patient’s blood. To close the bottle and to put it on a horizontal surface by the line upwards for 1 h at 37 °C for adsorption of the viruses on the cells surface. After that sterilely to add in the bottle 10,0 ml of medium 199.
In 72-96 hours material is inoculating into the brain of newborn white mice for the identification of viruses.
2. To carry out neutralization test with type specific sera in the pig embryo kidneys cell culture.
The scheme of the neutralization test for viruses identification
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Ingredients
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Tubes |
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1
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2
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Patient’s defibrinated blood or serum (virus-containing specimen)
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0,5 ml
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0,5 ml
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Tick-borne encephalitis viruses antiserum
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0,5 ml
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–
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Japanese encephalitis viruses antiserum
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–
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0,5 ml
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Incubation 1 h, temperature 18-20 °C
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Pig embryo kidneys cell culture
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5,0 ml
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5,0 ml
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Incubation 4-7 days, temperature 37 °C
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Results
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3. To carry out Complement fixation test with paired sera for serological diagnosis tick-borne encephalitis.
The scheme of the Complement fixation test
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Number of the test tubes
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1
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2
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3
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4
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5
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6
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7
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8
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Isotonic sodium chloride solution
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0,2
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0,2
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0,2
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0,2
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0,2
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0,2
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0,2
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0,2
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Patient’s serum diluted 1:5
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I
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0,2
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–
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0,2
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II
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0,2
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–
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0,2
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Serum dilution
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1:10
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1:20
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1:40
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1:80
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1:160
|
1:320
|
–
|
–
|
|||||||
|
Viral diagnosticum (tick-borne encephalitis viruses)
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
–
|
|||||||
|
Complement
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
|||||||
|
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
|
|||||||||||||||
|
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension )
|
0,4
|
0,4
|
0,4
|
0,4
|
0,4
|
0,4
|
0,4
|
0,4
|
|||||||
|
Incubation for 30-60 min, temperature 37 °C
|
|||||||||||||||
|
Results
|
|
|
|
|
|
|
|
|
|||||||
|
Patient’s serum
|
|
|
|
|
|
|
|
|
|||||||
|
I
|
|
|
|
|
|
|
|
|
|||||||
|
II
|
|
|
|
|
|
|
|
|
|||||||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
4. To carry out put Complement fixation test with specific serum against Crimean-Congo hemorrhagic fever viruses.
The scheme of Complement fixation test for laboratory diagnosis of
Crimean-Congo hemorrhagic fever
|
Ingredient, ml
|
Number of the test tubes
|
|||||||
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
Dilution of antigen
|
1:4
|
1:8
|
1:16
|
1:32
|
1:4
|
1:4
|
1:8
|
1:8
|
|
Investigated antigen
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
|
Specific serum
|
0,1
|
0,1
|
0,1
|
0,1
|
–
|
–
|
–
|
–
|
|
Non-specific serum
|
–
|
–
|
–
|
–
|
0,1
|
–
|
0,1
|
–
|
|
Isotonic sodium chloride solution
|
–
|
–
|
–
|
–
|
–
|
0,1
|
–
|
0,1
|
|
Complement (2 U)
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
|
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
|
||||||||
|
Hemolytic system
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
|
Incubation for 30-60 min, temperature 37 °C
|
||||||||
|
Results
|
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
5. To carry out Hemagglutination inhibition test with paired sera for diagnosis of rubella.
The scheme of Hemagglutination inhibition test for serological diagnosis of rubella
|
Ingredient, ml
|
Number of the test tubes
|
|||||||
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
Dextrose-gelatin-veronal buffer with 0,4 % of bovine albumin
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
|
Patient’s serum diluted 1:5
|
|
|
|
|
|
|
|
|
|
I
|
0,1
|
® |
® |
® |
® |
¯ |
0,1
|
–
|
|
II
|
0,1
|
® |
® |
® |
® |
¯ |
0,1
|
–
|
|
Dilution
|
1:10
|
1:20
|
1:40
|
1:80
|
1:160
|
1:320
|
–
|
–
|
|
Viral diagnosticum (Rubella virus, 4 HAU/ml)
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
–
|
0,1
|
|
Incubation for 30 min, temperature 18-
|
||||||||
|
1 % suspension of chicken erythrocytes
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
|
Incubation for 45 min, temperature 18-20 °C
|
||||||||
|
Result: Serum I
|
|
|
|
|
|
|
|
|
|
II
|
|
|
|
|
|
|
|
|
Test results are assessed after complete erythrocyte sedimentation in control (7 well). In the experimental well a markedly localized erythrocytes sediment (“rouleaus”), and in the control well (8) the rapid erythrocytes agglutination with star-like, marginally festooned sediment (“umbrella”) on the bottom are observed. The titer of serum is its biggest dilution, which inhibits hemagglutination. The growth of patient’s antiviral antibodies titers at least in 4 times testifies about disease.
6. To do Complement fixation test for serological diagnosis of rotavirus infection.
The scheme of the Complement fixation test
|
Ingredient, ml
|
Number of the test tubes
|
|||||||
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
Isotonic sodium chloride solution
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
|
Patient’s serum diluted 1:5 I
|
0,1
|
® |
® |
® |
® |
¯ |
–
|
0,1
|
|
II
|
0,1
|
® |
® |
® |
® |
¯ |
–
|
0,1
|
|
Serum dilution
|
1:10
|
1:20
|
1:40
|
1:80
|
1:160
|
1:320
|
–
|
–
|
|
Viral diagnosticum (rotavirus)
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
–
|
|
Complement
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
0,1
|
|
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
|
||||||||
|
Hemolytic system
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
0,2
|
|
Incubation for 30-60 min, temperature 37 °C
|
||||||||
|
Result: Serum I
|
|
|
|
|
|
|
|
|
|
II
|
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood). The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
7. To carry out complement fixation test with patient’s paired sera for serological diagnosis of Herpes simplex.
The scheme of Complement fixation test
|
Ingredient, ml |
Number of the test tubes
|
|||||||
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
Isotonic sodium chloride solution
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
|
Patient’s serum diluted 1:5
|
|
|
|
|
|
|
|
|
|
I
|
0,5
|
® |
® |
® |
® |
¯ |
–
|
0,5
|
|
II
|
0,5
|
® |
® |
® |
® |
¯ |
–
|
0,5
|
|
Serum dilution
|
1:10
|
1:20
|
1:40
|
1:80
|
1:160
|
1:320
|
–
|
–
|
|
Viral diagnosticum
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
0,5
|
–
|
|
Incubation for 45 min, temperature 37 °C
|
||||||||
|
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension )
|
1,0
|
1,0
|
1,0
|
1,0
|
1,0
|
1,0
|
1,0
|
1,0
|
|
Incubation for 30-60 min, temperature 37 °C
|
||||||||
|
Results
|
|
|
|
|
|
|
|
|
|
Patient’s serum
|
|
|
|
|
|
|
|
|
|
I
|
|
|
|
|
|
|
|
|
|
II
|
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++ , ++), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
The titer of antibody in the second serum must increase in 4 times as compared with the first one.
References
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