Microbiological diagnosis of diseases, which caused by pathogenic Rickettsia, Chlamydia and Mycoplasma.

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Rickettsiae

Rickettsiae are small bacteria that are obligate intracellular parasites and – except for Q fever – are transmitted to humans by arthropods. At least 4 rickettsiae (Rickettsia rickettsii, Rickettsia conorii, Rickettsia tsuisugamushi. Rickettsia akari) – and perhaps others – are transmitted transovarially in the ar-thropod, which serves as both vector and reservoir. Rickettsial diseases (except Q fever) typically exhibit fever, rashes, and vasculitis. They are grouped on the basis of clinical features, epidemiologic aspects, and immunologic characteristics (see Table).

Table.

Rickettsial diseases

Disease	Rickettsia	Geographic Area of Prevalence	Insect Vector	Mammalian Reservoir	Weil-Felix Agglutination
					0X19	0X2	OXK
Typhus group Epidemic typhus	Rickettsia prowazekii	South America, Africa, Asia	Louse	Humans	++	+	-
Murine typhus	Rickettsia typhi	Worldwide; small	Flea	Rodents	++	-	-
Scrub typhus	Rickettsia tsutsugamushi	Southeast Asia, Japan	Mite*	Rodents	-	-	++
Spotted fever group Rocky Mountain spotted fever (RMSF)	Rickettsia rickettsii	Western hemisphere	Tick*	Rodents, dogs	+	+	-
Fievre boutonneuse Kenya tick Typhus South African tick fever Indian tick typhus	Rickettsia conorii	Africa, India, Mediterranean	Tick*	Rodents, dogs	+	+	–
Queensland tick typhus	Rickettsia australis	Australia	Tick*	Rodents, marsupials	+	+	–
North Asian tick typhus	Rickettsia sibirica	Siberia, Mongolia	Tick*	Rodents	4	+	-
Rickettsial pox	Rickettsia akari	USA. Korea, Russia	Mite*	Mice	-	-	-
RMSF-like	Rickettsia Canada	North America	Tick*	Rodents	?	?	-
Other Q fever	Coxiella burnetii	Worldwide	None**	Cattle, sheep, goats	–	–	-–
Trench fever	Rochalimaea quintana	Rare	Louse	Humans

*Also serve as arthropod reservoir, by maintaining the rickettsiae through transovarian transmission.

**Human infection results from inhalation of dust.

Properties of Rickettsiae. Rickettsiae are pleomorphic, appearing either as short rods, 600 x 300 nm in size, or as cocci, and they occur singly, in pairs, in short chains, or in filaments. When stained, they are readily visible under the optical microscope. With Giemsa's stain they stain blue; with Macchiavello's stain they stain red and contrast with the blue-staining cytoplasm in which they appear.

A wide range of animals are susceptible to infection with rickettsial organisms- Rickettsiae grow readily in the yolk sac of the embryonated egg (yolk sac suspensions contain up to 10⁹ rickettsial particles per milliliter). Pure preparations of rickettsiae can be obtained by differential centrifugation of yolk sac suspensions. Many rickettsial strains also grow in cell culture.

Purified rickettsiae contain both RNA and DNA in a ratio of 3.5:1 (similar to the ratio in bacteria). Rickettsiae have cell walls made up of peptidoglycans containing muramic acid, resembling cell walls of gram-negative bacteria, and they divide like bacteria. In cell culture, the generation time is 8-10 hours at 34 °C.

Purified rickettsiae contain various enzymes concerned with metabolism. Thus they oxidize intermediate metabolites like pyruvic, succinic, and glutamic acids and can convert glutamic acid into aspartic acid. Rickettsiae lose their biologic activities when they are stored at 0 °C; this is due to the progressive loss of nicotinamide adenine dinucleotide (NAD). All of these properties can be restored by subsequent incubation with NAD. They may also lose their biologic activity if they are starved by incubation for several hours at 36 °C. This loss can be prevented by the addition of glulamate, pyruvate, or adenosine triphosphate (ATP). Subsequent incubation of the starved organism with glutamate at 30 °C leads to recovery of activity.

Rickettsiae may grow in different parts of the celt. Those of the typhus group are usually found in the cytoplasm; those of the spotted fever group, in the nucleus. Thus far, one of the rickettsiae, Rochalimaea quintana, has been grown on cell-free media. It has been suggested that rickettsiae grow best when the metabolism of the host cells is low. Thus, their growth is enhanced when the temperature of infected chick embryos is lowered to 32 °C. If the embryos are held at 40 °C, rickettsial multiplication is poor. Conditions that influence the metabolism of the host can alter its susceptibility to rickettsial infection.

Rickettsial growth is enhanced in the presence of sulfonamides, and rickettsial diseases are made more severe by these drugs. Para-aminobenzoic acid (PABA), the structural analog of the sulfonamides, inhibits the growth of rickettsial organisms. Tetracy-clines or chloramphenicol inhibits the growth of rickettsiae and can be therapeutically effective.

In general, rickettsiae are quickly destroyed by heat, drying, and bactericidal chemicals. Although rickettsiae are usually killed by storage at room temperature, dried feces of infected lice may remain infective for months at room temperature.

The organism of Q fever is the rickettsial agent most resistant to drying. This organism may survive pasteurization at 60 °C for 30 minutes and can survive for months in dried feces or milk. This may be due to the formation of endospores by Coxiella burneiii.

Rickettsial Antigens and Antibodies. A variety of rickettsial antibodies are known; all of them participate in the reactions discussed below. The antibodies that develop in humans after vaccination generally are more type-specific than the antibodies developing after natural infection.

A. Agglutination of Proteus vulgaris (Weil-Felix Reaction): The Weil-Felix reaction is commonly used in diagnostic work. Rickettsiae and Proteus organisms appear to share certain antigens. Thus, during the course of rickettsial infections, patients develop antibodies that agglutinate certain strains ofP vulgaris. For example, the Proteus strain 0X19 is agglutinated strongly by sera from persons infected with epidemic or endemic typhus; weakly by sera from those infected with Rocky Mountain spotted fever; and not at all by those infected with Q fever. Convalescent sera from scrub typhus patients react most strongly with the Proteus strain OXK (Table 1).

B. Agglutination of Rickettsiae: Rickettsiae are agglutinated by specific antibodies. This reaction is very sensitive and can he diagnostically useful when heavy rickettsial suspensions are available for mi-croagglutination tests.

C. Complement Fixation With Rickettsial Antigens: Complement-fixing antibodies are commonly used in diagnostic laboratories. A 4-fold or greater antibody titer rise is usually required as laboratory support for the diagnosis of acute rickettsial infection. Convalescent liters often exceed 1:64. Group-reactive soluble antigens are available for the typhus group, the spotted fever group, and Q fever. They originate in the cell wall. Some insoluble antigens may give species-specific reactions.

D. Immunonuorescence Test With Rickettsial Antigens: Suspensions of rickettsiae can be partially purified from infected yolk sac material and used as antigens in indirect immunofluorescence tests (see p 168) with patient's serum and a fluore see in-labeled antihuman globulin. The results indicate the presence of partly species-specific antibodies, but some cross-reactions are observed. Antibodies after vaccination are IgG; early after infection, IgM.

E. Passive Hemagglutination Test: Treated red blood cells adsorb soluble antigens and can then be agglutinated by antibody.

F. Neutralization of Rickettsial Toxins: Rickettsiae contain toxins that produce death in animals within a few hours after injection. Toxin-neutralizing antibodies appear during infection, and these are specific for the toxins of the typhus group, the spotted fever group, and scrub typhus rickettsiae. Toxins exist only in viable rickettsiae—inactivated rickettsiae are nontoxic.

Pathology

Rickettsiae multiply in endothelial cells of small blood vessels and produce vasculitis. The ceils become swollen and necrotic; there is thrombosis of the vessel, leading to rupture and necrosis. Vascular lesions are prominent in the skin, but vasculitis occurs in many organs and appears to be the basis of hemostatic disturbances. In the brain, aggregations of lym-phocytes, polymorphonuclear leukocytes, and mac-rophages are associated with the blood vessels of the gray matter; these are called typhus nodules. The heart shows similar lesions of the small bloodvessels. Other organs may also be involved.

Immunity. In cell cultures of macrophages, rickettsiae are phagocytosed and replicate intracellularly even in the presence of antibody. The addition of lymphocytes from immune animals stops this multiplication in vitro. Infection in humans is followed by partial immunity to reinfection from external sources, but relapses occur.

Clinical Findings. Except for Q fever, in which there is no skin lesion, rickettsial infections are characterized by fever, headache, malaise, prostration, skin rash. and enlargement of the spleen and liver.

A. Typhus Group:

1. Epidemic typhus – In epidemic typhus, systemic infection and prostration are severe, and fever lasts for about 2 weeks. The disease is more severe and is more often fatal in patients over 40 years of age. During epidemics, the case mortality rate has been 6-30%.

2. Endemic typhus-The clinical picture of endemic typhus has many features in common with that of epidemic typhus, but the disease is milder and is rarely fatal except in elderly patients.

B. Spotted Fever Group: The spotted fever group resembles typhus clinically; however, unlike the rash in other rickettsial diseases, the rash of the spotted fever group usually appears first on the extremities, moves centripetally, and involves the palms and soles. Some, like Brazilian spotted fever, may produce severe infections; others, like Mediterranean fever, are mild. The case mortality rate varies greatly. In untreated Rocky Mountain spotted fever, it is usually much greater in older age groups (up to 60%) than in younger people.

Rickettsialpox is a mild disease with a rash resembling that of varicella. About a week before onset of fever, a firm red papule appears at the site of the mite bite and develops into a deep-seated vesicle that in turn forms a black eschar (see below).

C. Scrub Typhus: This disease resembles epidemic typhus clinically. One feature is the eschar, the punched-out ulcer covered with a blackened scab that indicates the location of the mite bite. Generalized lymphadenopathy and lymphocytosis are common. Localized eschars may also be present in the spotted fever group.

D. Q Fever: This disease resembles influenza, nonbacterial pneumonia, hepatitis, or encephalopathy rather than typhus. There is no rash or local lesion. The Weil-Felix test is negative. Transmission results from inhalation of dust contaminated with rickettsiae from dried feces, urine, or milk.

E. Trench Fever: The disease is characterized by the headache, exhaustion, pain, sweating, coldness of the extremities, and fever associated with aroseolar rash. Relapses occur. Trench fever has been known only among armies during wars in central Europe.

Laboratory Findings. Isolation of rickettsiae is technically quite difficult and so is of only limited usefulness in diagnosis. Whole blood (or emulsified blood clot) is inoculated into guinea pigs, mice, or eggs. Rickettsiae are recovered most frequently from blood drawn soon after onset, but they have been found as late as the 12th day of the disease.

If the guinea pigs fail to show disease .(fever, scrotal swellings, hemorrhagic necrosis, death), serum is collected for antibody tests to determine if the animal has had an inapparent infection.

Some rickettsiae can infect mice, and rickettsiae are seen in smears of peritoneal exudate. In Rocky Mountain spotted fever, skin biopsies taken from patients between the fourth and eighth days of illness reveal rickettsiae by immunofluorescence stain.

The most sensitive and specific serologic tests are microimmunofluorescence, microagglutination, and complement fixation. An antibody rise should be demonstrated during the course of the illness.

Treatment. Tetracyclines and chloramphenicol are effective provided treatment is started early. Tetracycline, 2-3 g, or chloramphenicol, 1.5-2 g, is given daily orally and continued for 3-4 days after defervescence. In severely ill patients, the initial doses can be given intravenously. Sulfonamides enhance the disease and are con-traindicated. The antibiotics do not free the body of rickettsiae, but they do suppress their growth. Recovery depends in part upon the immune mechanisms of the patient.

Epidemiology. A variety of arthropods, especially ticks and mites, harbor Rickettsia organisms in the cells that line the alimentary tract. Many such organisms are not evidently pathogenic for humans.

The life cycles of different rickettsiae vary:

(1) Ricketlsia prowazekii has a life cycle limited to humans and to the human louse (Pediculus corporis and Pediculus capitis). The louse obtains the organism by biting infected human beings and transmits the agent by fecal excretion on the surface of the skin of another person. Whenever a louse bites, it defecates at the same time. The scratching of the area of the bite allows the rickettsiae excreted in the feces to penetrate the skin. As a result of the infection the louse dies, but the organisms remain viable for some time in the dried feces of the louse. Rickettsiae are not transmitted from one generation of lice to another. Typhus epidemics have been controlled by delousing large proportions of the population with insecticides.

Brill's disease is a recrudescence of an old typhus infection. The rickettsiae can persist for many years in the lymph nodes of an individual without any symptoms being manifest. The rickettsiae isolated from such cases behave like classic R prowaz.ekii; this suggests that humans themselves are the reservoir of the rickettsiae of epidemic typhus. Flying squirrels in the USA may provide an extrahiiman reservoir, and human cases have occurred after bites by ectopara-sites. Epidemic typhus epidemics have been associated with war and the lowering of standards of personal hygiene, which in turn have increased the opportunities for human lice to flourish. If this occurs at the time of recrudescence of an old typhus infection, an epidemic may be set off. Brill's disease occurs in local populations of typhus areas as well as in persons who migrate from such areas to places where the disease does not exist. Serologic characteristics readily distinguish Brill's disease from primary epidemic typhus. Antibodies arise earlier and are IgG rather than the IgM detected after primary infection. They reach a maximum by the tenth day of disease. The Weil-Felix reaction is usually negative. This early IgG antibody response and the mild course of the disease suggest that partial immunity is still present from the primary infection.

(2) Rickettsia typhi has its reservoir in the rat, in which the infection is inapparent and long-lasting. Rat fleas carry the rickettsiae from rat to rat and sometimes from rat to humans, who develop endemic typhus. Cat fleas can serve as vectors. In endemic typhus, the flea cannot transmit the rickettsiae transovarially.

(3) Rickettsia tsuisugumushi has its true reservoir in the mites that infest rodents. Rickettsiae can persist in rats for over a year after infection. Mites transmit the infection transovariaily. Occasionally, infected mites or rat fleas bite humans, and scrub typhus results. The rickettsiae persist in the mite-rat-mite cycle in the scrub or secondary jungle vegetation that has replaced virgin Jungle in areas of partial cultivation. Such areas may become infested with rats and trombiculid mites.

(4) Rickettsia rickettsii may be found in healthy wood ticks (Dermacentor andersoni) and is passed transovarially. Vertebrates such as rodents, deer, and humans are occasionally bitten by infected ticks in the western USA. In order to be infectious, the tick carrying the rickettsiae must be engorged with blood, for this increases the number of rickettsiae in the tick. Thus, there is a delay of 45-90 minutes between the time of the attachment of the tick and its becoming infective. In the eastern USA, Rocky Mountain spotted fever is transmitted by the dog tick Dermacentor variahiiis. Dogs are hosts to dog ticks but rarely, if ever. serve as a continuing source of tick infection. Most Rocky Mountain spotted fever in the USA now occurs in the eastern and the southeastern regions.

(5) Rickettsia akari has its vector in blood-sucking mites of the species Allodermanyssus san-guineus. These mites may be found on the mice (Mus musculus) trapped in apartment houses in the USA where rickettsialpox has occurred. Transovarial transmission of the rickettsiae occurs in the mite. Thus the mite may act as a true reservoir as well as a vector. R akari has also been isolated in Korea.

(6) Rochalimaea quintana is the causative agent of trench fever; it is found in lice and in humans, and its life cycle is like that of R prowazekii. The disease has been limited to fighting armies. This organism can be grown on blood agar in 10% COa.

(7) Coxiella burnetii is found in ticks, which transmit the agent to sheep, goats, and cattle. Workers in slaughterhouses and in plants that process wool and cattle hides have contracted the disease as a result of handling infected animal tissues. C burnetii is transmitted by the respiratory pathway rather than through the skin. There may be a chronic infection of the udder of the cow. In such cases the rickettsiae are excreted in the milk and occasionally may be transmitted to humans by ingestion or inhalation.

Infected sheep may excrete C burnetii in the feces and urine. The placentas of infected cows and sheep contain the rickettsiae, and parturition creates infectious aerosols, The soil may be heavily contaminated from one of the above sources, and the inhalation of infected dust leads to infection of humans and livestock. It has been proposed that endospores formed by C burnetii contribute to its persistence and dissemina-tion. Coxiella infection is now widespread among sheep and cattle in the USA. Coxiella can cause endocarditis in humans in addition to pneumonitis and hepatitis.

Geographic Occurrence. A. Epidemic Typhus: Potentially worldwide, it has disappeared from the USA, Britain, and Scandinavia. It is still present in the Balkans, Asia, Africa, Mexico, and the Andes. In view of its long duration in humans as a latent infection (Brill's disease), it can flourish quickly under proper environmental conditions, as it did in Europe during World War II as a result of the deterioration of community sanitation.

B. Endemic, Murine Typhus: Worldwide, especially in areas of high rat infestation. It may exist in the same areas as—and may be confused with— epidemic typhus or scrub typhus.

C. Scrub Typhus: Far East, especially Burma, India, Ceylon, New Guinea, Japan, and Taiwan. Trombicula pallida, the chigger most often found in Korea, maintains the infection among wild rodents of Korea (Apodemus agrarius), but only infrequently does it transfer scrub typhus to humans.

D. Spotted Fever Group: These infections occur around the globe, exhibiting as a rule some epidemiologic and immunologic differences in different areas. Transmission by a tick of the Ixodidae family is common to the group. The diseases that are grouped together include Rocky Mountain spotted fever (western and eastern RMSF), Colombian, Brazilian, and Mexican spotted fevers; Mediterranean (boutonneuse), South African tick, and Kenya fevers;

North Queensland tick typhus; and North Asian tick-borne rickettsiosis.

E. Rickettsialpox: The human disease has been found among inhabitants of apartment houses in the northern USA. However, the infection also occurs in Russia, Africa, and Korea.

F. Q Fever: The disease is recognized around the world and occurs mainly in persons associated with goats, sheep, or dairy cattle. It has attracted attention because of outbreaks in veterinary and medical centers where large numbers of people were exposed to animals shedding Coxiella.

Seasonal Occurrence. Epidemic typhus is more common in cool climates, reaching its peak in winter and waning in the spring. This is probably a reflection of crowding, lack of fuel, and low standards of personal hygiene, which favor louse infestation.

Rickettsial infections that must be transmitted to the human host by vector reach their peak incidence at the time the vector is most prevalent—the summer and fall months.

Control. Control is achieved by breaking the infection chain or by immunizing and treating with antibiotics.

A. Prevention of Transmission by Breaking the Chain of Infection:

1. Epidemic typhus-Delousing with insecticide.

2. Murine typhus-Rat-proofing buildings and using rat poisons.

3. Scrub typhus-Clearing from campsites the secondary jungle vegetation in which rats and mites live.

4. Spotted fever-Similar measures for the spotted fevers may be used; clearing of infested land;

personal prophylaxis in the form of protective clothing such as high boots, socks worn over trousers; tick repellents; and frequent removal of attached ticks.

5. Rickettsial pox-Elimination of rodents and their parasites from human domiciles.

B. Prevention of Transmission of Q Fever by Adequate Pasteurization of Milk: The presently recommended conditions of "high-temperature, short-time "pasteurization at 71.5 °C (161 °F) for 15 seconds are adequate to destroy viable Coxiella.

C. Prevention by Vaccination: Active immunization may be carried out using formalinized antigens prepared from the yolk sacs of infected chick embryos or from cell cultures. Such vaccines have been prepared for epidemic typhus (R prowazekii), Rocky Mountain spotted fever (R ricketlsii), and Q fever (C burnelii). However, commercially produced vaccines are not available in the USA in 1982. Cell culture-grown , inactivated suspensions of rickettsiae are under study as vaccines. A live vaccine (strain E) for epidemic typhus is effective and used experimentally but produces a self-limited disease.

D. Chemoprophylaxis: Chloramphenicol has been used as a chemoprophylactic agent against scrub typhus in endemic areas. Oral administration of 3-g doses at weekly intervals controls infection so that no disease occurs even though rickettsiae appear in the blood. The antibiotic must be continued for a month after the initiation of infection to keep the person well. Tetracyclines may be equally effective.

Diagnosis of RICKETTSIAL INFECTIONS in details

Rickettsioses present a group of infectious diseases wliicli are induced by Rickettsia and affect both humans and animals.

The laboratory diagnosis of rickettsioses largely relies on the use of serological methods of investigation, including such reactions as A, CF, IHA, IF, and some others. Some peculiarities of conducting-these reactions in individual rickettsioses are described in the corresponding sections. Rickettsia are isolated from the patient’s blood and other material only for scientific purposes. Isolation is performed in special laboratories by infecting experimental animals, chicken embryos, or insects.

Epidemic Typhus Fever. The causal organism of epidemic typhus and Brill-Symmers disease (Rickettsia prowazekii) induces in man an acute illness characterized by a sudden onset, liigli pyrexia, and roseolopetechial rash. For the laboratory diagnosis of these diseases one usually employs such tests as CF, IHA, A with R. prowazeku, indirect haemolysis, and IF.

Serological diagnosis. The CF reaction allows to diagnose both an active pathological process and a history of the illness (retrospective diagnosis). The reaction is performed according to the conventional technique employed for isolating antibodies in the blood serum of patients and individuals with a history of the disease. Blood samples are withdrawn from the patient on the 5th-7lh day of the illness. The serum is diluted in ratios ranging from 1 : 10 to 1 : 320. Pickettsial suspension (a corpuscular antigen), well purified and vacuum-dried, is usually used as an antigen.

Complement fixation (phase 1 of the reaction) is carried out at + 4 °C for 18-20 hrs or at 37 C for 1 h. The reaction is considered positive if inhibition of haemolysis is recorded in a serum dilution of 1:160 and in dilution 1:10 in retrospective diagnosis.

The agglutination reaction with R. prowazekii is positive in patients with epidemic typhus fever in almost all cases. The reaction is diagnostically significant in serum dilutions of 1:40-1:80 but increasing litres of antibodies in paired sera are a more reliable diagnostic indicator (tables).

Table

Agglutination reaction with Rickettsia prowazekii antigen or Rickettsia typhi antigen

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8	9	10
Isotonic sodium chloride solution	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Patient's serum diluted 1:5	0,2	®	®	®	®	®	®	¯	0,2	–
Dilution	1:10	1:20	1:40	1:80	1:160	1:320	1:640	1:1280	–	–
Rickettsia prowazekii antigen or Rickettsia typhi antigen	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2	–	0,2
	Temperature 37 °C, 18-20 h
Results

The IHA reaction is considered positive if haemagglutination is observed in a 1:250 serum dilution (tabl.).

Table

Indirect hemagglutination test

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8	9	10	11
Isotonic sodium chloride solution	0,4	0,4	0,4	0,4	0,4	0,4	0,4	0,4	–	0,4	0,4
Patient's serum diluted 1:62,5	0,4	®	®	®	®	®	®	¯	–	–	–
Dilution	1:125	1:250	1:500	1:1000	1:2000	1:4000	1:8000	1:16000	Control of serum	Control of antigen	Control of erythrocytess
Erythrocytic diagnosticum with Rickettsial antigens	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1	–	0,1	–
Sheep erythrocytes 1 %	–	–	–	–	–	–	–	–	0,1	–	0,1
	Temperature 30 °C 18 h
Results

The reaction of indirect haemolysis is distinguished by high sensitivity and speciticity. Moreover, it allows the detection of antibodies at early stages and can be used as a rapid method of diagnosis since one can read the presumptive results already 30 min after its performance.

A typhus antigen required for the reaction is adsorbed on sheep erythrocytes (to 4 ml of the antigen add 0.15 ml of washed off sheep red blood cells). The mixture is incubated for 1 h and then centrifuged for 8-10 min at 2500 X s. The residue of erythrocytes is suspended in 10 ml of isotonic sodium chloride solution. The serum tested is inactivated at 56 °C for 30 min and diluted with isotonic saline. To 0.4 ml of each serum dilution add 0.1 ml of complement and 0.1 ml of the antigen. Incubate the mixture at 37 °C. Read the results of the reaction in 30 min and then in 1 h. The minimal diagnostic titre of the reaction is 1:100-1:200.

A drop modification of the reaction of indirect haemolysis. The patient's serum preliminarily diluted 1:50 is introduced into an agglutination tube containing 4 drops of isotonic sodium chloride solution to obtain a 1:100 dilution. From this test tube transfer 4 drops into the next test tube with 4 drops of saline solution (dilution 1:200) and so on until a 1:3200 dilution is achieved. Then, to each test tube add 1 drop of the typhus antigen and 1 drop of complement diluted 1:10. Shake the tubes and incubate them for 1 h at 37 °C. A positive reaction is witnessed by erythrocyte haemolysis, there being no haemolysis in test tubes with the control of erythrocytes, antigen, and serum.

The IF reaction is conducted in the following manner: on fixed Rickettsia smears place sequential dilutions of the tested serum and incubate them in a moist chamber at 37 "C for 15-20 min. Then, wash the smears for 1 min with a light stream of water and place for 10 min into a cuvette with phosphate buffer solution, wash with distilled water, and dry in the air. After that, apply on the smears antiglobulin luminescent serum taken in the working dilution indicated on the ampoule label, incubate them for 20 min in a moist chamber, wash with water, dry, and study by luminescent microscopy.

Endemic Typhus Fever. The causal organism of endemic (murine) typhus fever (Rickettsia typhi) induces a disease whose clinical manifestations are similar to those of epidemic typhus fever.

Serological diagnosis. To differentiate between endemic and epidemic typhus fever, one sets up at the same time agglutination reactions with antigens from R. prowazekii and R. mooseri, as well as theCF and IHA tests. In case of endemic typhus fever the diagnostic titre of the serum with R. mooseri is 3-4 times as high as that of the serum with R. prowazekii.

Biological examination. Male guinea pigs are infected intra-peritoneally with patients' blood. The appearance of a scrotal reaction, fever, and also detection of Rickettsia in scrapings from the testi-cular membranes verifies the diagnosis of endemic typhus fever.

Q Fever. The causative agent of Q fever (Coxiella burnetii) induces an acute infectious disease characterized by a polymorphic clinical picture, sometimes by a subacute and chronic course. In the laboratory diagnosis of this type of rickettsiosis one utilizes serological reactions (A, CF, IHA), allergy, and biological tests.

Serological diagnosis. The agglutination reaction is made at the second week of the disease (on the 10th-12th day). The patient's blood serum, because of its low levels of agglutinins, is diluted from 1:4 to 1:64 and decanted into the test tubes in 0.25-ml portions. Then, 0.25 ml of the antigen is added to each test tube. The results of the reaction are read after 18-20-hour incubation of the test tubes. The presence of the reaction in dilutions 1:4 and over is considered positive. The agglutination reaction should be repeated to establish a growth in the antibody titre (the principle of paired sera).

The CF test may be positive at the end of the first week of the disease. Complement-fixating antibodies reach their highest concentration in the serum during the third week of the disease. The procedure of the reaction is the same as in typhus fever. The IHA test is more sensitive than complement fixation.

The allergy cutaneous test is carried out according to the standard procedure. Corpuscular antigen from Rickettsia burnetii is used as an allergen. The results of the test are read 24-48 hrs after the administration of 0.1 ml of the allergen and assessed by the size of the infiltrate and hyperaemia which is occasionally attended by oedema. The reaction is specific but can be used only for retrospective diagnosis. In individuals with a history of Q fever the test remains positive for 9-10 years.

Biological examination. Inoculation of guinea pigs makes it possible to isolate the causal organism of Q fever. Take 3-5 ml of blood from patients in the febrile period and administer to a guinea pig by either intraperitoneal or intratesticular route (0.3-0.5 ml of the infective blood deep into the testicles). With material from the infected guinea pig inoculate chicken embryos by the Cox procedure. In repeated passages Rickettsia transform from filtrable to visible forms and are detected by the Romanowsky-Giemsa and Zdrodovsky techniques.

Tsuteugamushi Disease (Scrub Typhus). Scrub typhus is an acute anthropozoonotic rickettsiosis characterized by multiple vasculites, fever, and involvement of the nervous system and circulatory organs.

The causal organism of this infection is Rickettsia tsutsugamushi. For the diagnostic purpose, one uses blood and serum obtained from febrile patients.

Scrological diagnosis is carried out beginning from the second week of the disease and involves the agglutination and complement-fixation tests. Proteus OX-K is utilized as an antigen in the agglutination test.

R. tsutsugamushi may be found by infecting with the material examined the subculture of L cells and primary trypsin-treated fibroblasts of the chicken embryo.

Biological examination consists of the intraperitoneal inoculation of white mice with the patient's blood. The animal dies 6-14 days after the inoculation. Prepare impression-smears of the internal organs of the animal and stain them by the Romanowsky-Giemsa method. As a result, the cytoplasm of Rickettsia stains blue, while the nuclei are red. Following Zdrodovsky's staining, Rickettsia are ruby-red.

Rickettsia may also be revealed with the help of the IF test.

Rickettsioses of the Spotted Fever Group. This group of rickettsioses includes the following diseases: Marseilles (Boutonneuse) fever caused by Rickettsia conorii; East-African tick-borne fever caused by Rickettsia pi]perii\ Rickettsialpox induced by Rickettsia akari; Rocky Mountain spotted fever induced by Rickettsia rickettsii; and Australian (Queensland) tick-borne fever caused by Rickettsia australis.

The laboratory diagnosis of these rickettsioses is based on the isolation of the causative agent from patients' blood with the aid of a biological test, its cultivation in the yolk sac of the chicken embryo, and also on the determination of specific antibodies in the patient's blood.

Biological examination. Withdraw 3-5 ml of blood from the patient's vein and inject it intraperitoneally to male guinea pigs. Take some vectors (ticks), treat them with alcohol, wash, grind in a mortar, and prepare a suspension in isotonic sodium chloride solution, which is administered intraperitoneally to male guinea pigs. In 6-14 days after the inoculation the animals develop periorchitis. The causal organisms of Rocky Mountain spotted fever induce a scro-tal phenomenon characterized by scrotal necrosis.

Intraperitoneal administration to white mice of blood from a patient with rickettsialpox (vesicular rickettsiosis) induces (in 8-10 days) rickettsial peritonitis with a dramatic spleen enlargement.

The causal organisms of many rickettsioses can be relatively easily isolated by inoculating chicken embryos according to Cox's technique.

Serological diagnosis. Sufficient amounts of antibodies in patients' blood sera are accumulated on the second week of the disease. The CF and IffA reactions are typically employed for their detection. Determination of an increase in the antibody titres makes the diagnosis unquestionable.

Specific antigens from Rickettsia inducing a given disease are used for serological reactions.

Chlamydiae

Chlamydiae are a large group of obligate intracellular parasites closely related to gram-negative bacteria. They are divided into 2 species, Chlamydia psittaci and Chlamydia trachomatix, on the basis of antigenic composition, intracellular inclusions, sulfonamide susceptibility, and disease production (see below). All chlamydiae exhibit similar morphologic features, share a common group antigen, and multiply in the cytoplasm of their host cells by a distinctive developmental cycle.

Because of their obligate intracellular parasitism, chlamydiae were once considered viruses. Chlamydiae differ from viruses in the following important characteristics:

(l) Like bacteria, they possess both RNA and DNA.

(2) They multiply by binary fission; viruses never do.

(3) They possess bacterial type cell walls with peptidoglycans probably containing muramic acid.

(4) They possess ribosomes; viruses never do.

(5) They have a variety of metabolically active enzymes, eg, they can liberate C0₂ from glucose. Some can synthesize folates.

(6) Their growth can be inhibited by many antimicrobial drugs,

Chlamydiae can be viewed as gram-negative bacteria that lack some important mechanisms for the production of metabolic energy- This defect restricts them to an intracellular existence, where the host cell furnishes energy-rich intermediates.

Developmental Cycle. All chlamydiae share a general sequence of events in their reproduction. The infectious particle is a small cell ("elementary body") with an electron-dense nucleoid. It is taken into the host cell by phagocytosis. A vacuole derived from the host cell surface membranes, forms around the small particle. This small particle is reorganized into a large one ("initial body") measuring about 0.5-1 Jiim and devoid of an electron-dense nucleoid. Within the membrane-bound vacuole, the large particle grows in size and divides repeatedly by binary fission. Eventually the entire vacuole becomes filled with small particles derived by binary fission from large bodies to form an "inclusion" in the host cell cytoplasm. The newly formed small particles may be liberated from the host cell to infect new cells. The developmental cycle takes 24-48 hours.

Structure and Chemical Composition. Examination of highly purified suspensions of chlamydiae, washed free of host cell materials, indicates the following: the outer cell wall resembles the cell wall of gram-negative bacteria. It has a relatively high Upid content, and the peptidoglycan perhaps contains muramic acid. Cell wall formation is inhibited by penicillins and cycloserine, substances that inhibit peptidoglycan synthesis in bacteria. Both DNA and RNA are present in both small and large particles, In small particles, most DNA is concentrated in the electron-dense central nucleoid. In large particles, the DNA is distributed irregularly throughout the cytoplasm. Most RNA probably exists in ribosomes, in the cytoplasm. The large particles contain about 4 times as much RNA as DNA, whereas the small, infective particles contain about equal amounts of RNA and DNA.

The circular genome of chlamydiae (MW 7 x 10⁸) is similar to bacterial chromosomes. Chlamydiae contain large amounts of lipids, especially phos-pholipids, which are well characterized. A toxic principle is intimately associated with infectious chlamydiae. It kills mice after the intravenous administration of more than 10⁸ particles. Toxicity is destroyed by heat but not by ultraviolet light.

Staining Properties. Chlamydiae have distinctive staining properties (similar to those ofrickettsiae) that differ somewhat at different stages of development. Single mature particles (elementary bodies) stain purple with Giemsa's stain and red with Macchiavello's stain, in contrast to the blue of host cell cytoplasm. The larger, noninfec-live bodies (initial bodies) stain blue with Giemsa's stain. The Gram reaction of chlamydiae is negative or variable, and Gram's stain is not useful in the identification of the agents.

Fully formed, mature intracellular inclusions are compact masses near the nucleus which are dark purple when stained with Giemsa's stain because of the densely packed mature particles. If stained with dilute Lugol's iodine solution, the inclusions formed by some chlamydiae (mouse pneumonitis, lym-phogranuloma venereum [LGV], trachoma, inclusion conjunctivitis) appear brown because of the glycogen-like matrix that surrounds the particles.

Antigens. Chlamydiae possess 2 types of antigens. Both are probably located in the cell wall. Group antigens are shared by all chlamydiae. These are heat-stable lipoprotein-carbohydrate complexes, with 2-keto-3-deoxy-octonic acid as an immunodominant component. Antibody to these group antigens can be detected by complement fixation and immunofluorescence. Specific antigens (species-specific or immunotype-specific) remain attached to cell walls after group antigens have been largely removed by treatment with fluorocarbon or deoxycholate. Some specific antigens are membrane proteins that have been purified by immunoadsorption. Specific antigens can best be detected by immunofluorescence. Specific antigens are shared by only a limited number of chlamydiae, but a given organism may contain several specific antigens. Fifteen immunotypes of C trachomatis have been identified (A, B, Ba, C-K, L1-L3), and the last 3 are LGV immunotypes. The toxic effects of chlamydiae are associated with antigens. Specific neutralization of these toxic effects by antiserum permits similar an-tigenic grouping of organisms.

A very unstable hemagglutinin capable of clumping some chicken and mouse erythrocytes is present in chlamydiae. This he maggluti nation is blocked by group antibody.

Growth and Metabolism. Chlamydiae require an intracellular habitat, presumably because they lack some essential feature of energy metabolism. All types of chlamydiae proliferate in embryonated eggs, particularly in the yolk sac. Some also grow in cell cultures and in various animal tissues. Cells have attachment sites for chlamydiae. Removal of these sites prevents easy uptake of chlamydiae.

Chlamydiae appear to have an endogenous metabolism similar to that of some bacteria but participate only to a limited extent in potentially energy-yielding processes. They can liberate CO; from glucose, pyru-vate, and glutamate; they also contain dehydrogen-ases. Nevertheless, they require energy-rich intermediates from the host cell to carry out their biosynthetic activities.

Reactions to Physical and Chemical Agents. Chlamydiae are rapidly inactivated by heat. They lose infectivity completely after 10 minutes at 60 °C. They maintain infectivity for years at -50 °C to -70 °C. During the process of freeze-drying, much of the infectivity is lost. Some air-dried chlamydiae may remain infective for long periods. Chlamydiae are rapidly inactivated by ether (in 30 minutes) or by phenol (0.5% for 24 hours).

The replication of chlamydiae can be inhibited by many antibacterial drugs. Cell wall inhibitors such as penicillins and cycloserine result in the production of morphologically" defective forms but are not effective in clinical diseases. Inhibitors of protein synthesis (tetracyc lines, erythromycins) are effective in laboratory models and at times in clinical infections. Some chlamydiae synthesize folates and are susceptible to inhibition by sulfonamides. Aminoglycosides have little inhibitory activity for chlamydiae.

Characteristics of Host-Parasite Relationship. The outstanding biologic feature of infection by chlamydiae is the balance that is often reached between host and parasite, resulting in prolonged, often lifetime persistence. Spread from one species (eg, birds) to another (eg, humans) more frequently leads to disease. Antibodies to several antigens of chlamydiae are regularly produced by the infected host. These antibodies have little protective effect. Commonly the infectious agent persists in the presence of high antibody titers. Treatment with effective antimicrobial drugs (eg, tetracyclines) for prolonged periods may eliminate the chlamydiae from the infected host. Very early, intensive treatment may suppress antibody formation. Late treatment with antimicrobial dmgs in moderate doses may suppress disease but permit persistence of the infecting agent in tissues.

The immunization of susceptible animals with various inactivated or living vaccines tends to induce protection against death from the toxic effect of living challenge organisms. However, such immunization in animals or humans has been singularly unsuccessful in protecting against infection. At best, immunization or prior infection has induced some resistance and resulted in milder disease after challenge or reinfec-tion.

Classification. Historically, chlamydiae were arranged according to their pathogenic potential and their host range. Antigenic differences were defined by antigen-antibody reactions studied by immunofluorescence, toxin neutralization, and other methods. The 2 presently accepted species and their characteristics are as follows:

(1) C psillaci: This species produces diffuse intracytoplasmic inclusions that lack glycogen; it is usually resistant to sulfonamides. It includes agents of psittacosis in humans, omithosis in birds, meningo-pneumomtis, feline pneumonitis, and many other animal pathogens.

(2) C trachnmatis: This species produces compact intracytoplasmic inclusions that contain glycogen; it is usually inhibited by sulfonamides, It includes agents of mouse pneumonitis and several human disorders such as trachoma, inclusion conjunctivitis, nongonococcal urethritis, salpingitis, cervicitis, pneumonitis of infants, and lymphogranuloma venereum. In nucleic acid hybridization experiments, the 2 species appear not to be closely related.

PSITTACOSIS (Ornithosis). Psittacosis is a disease of birds that may be transferred to humans. In humans, the agent, C psillaci, produces a spectrum of clinical manifestations ranging from severe pneumonia and sepsis with a high mortality rafe to a mild inapparent infection.

Properties of the Agent. A. Size and Staining Properties: Similar to other members of the group (see above).

B. Animal Susceptibility and Growth of Agent: Psittacosis agent can be propagated in em-bryonated eggs, in mice and other animals, and in some cell cultures. In all these host systems, growth can be inhibited by tetracyclines and, to a limited extent, by penicillins. In intact animals and in humans, tetracyclines can suppress illness but may not be able to eliminate the infectious agent or end the carrier state.

C. Antigenic Properties: The heat-stable group-reactive complement-fixing antigen resists pro-teolytic enzymes but is destroyed by potassium perio-date. It is probably a lipopolysaccharide.

Infected tissue contains a toxic principle, intimately associated with the agent, that rapidly kills mice upon intravenous or intraperitoneal infection. This toxic principle is active only in particles that are infective.

Specific serotypes characteristic for certain mammalian and avian species may be demonstrated by cross-neutralization tests of toxic effect. Neutralization of infectivity of the agent by specific antibody or cross-protection of immunized animals can also be used for serotyping and parallels immunofluorescence.

D. Cell Wall Antigens: Walls of the infecting agent have been prepared by treatment with deoxycho-late followed by trypsin. The deoxycholate extracts contained group-reactive complement-fixing antigens, while the cell walls retained the species-specific antigens. The cell wall antigens were also associated with toxin neutralization and infectivity neutralization.

Pathogenesis and Pathology. The agent enters through the respiratory tract, is found in the blood during the first 2 weeks of the disease, and may be found in the sputum at the time (he lung is involved.

Psittacosis causes a patchy inflammation of the lungs in which consolidated areas are sharply demarcated. The exudate is predominantly mononuclear. Only minor changes occur in the large bronchioles and bronchi. The lesions are similar to those found in pneumonitis caused by some viruses and mycoplas-mas. Liver, spleen, heart, and kidney are often enlarged and congested.

Clinical Findings. A sudden onset of illness taking the form of influenza or nonbacterial pneumonia in a person exposed to birds is suggestive of psittacosis. The incubation period averages 10 days. The onset is usually sudden, with malaise, fever, anorexia, sore throat, photophobia, and severe headache. The disease may progress no further and the patient may improve in a few days. In severe cases the signs and symptoms of bronchial pneumonia appear at the end of the first week of the disease. The clinical picture often resembles that of influenza, nonbacterial pneumonia, or typhoid fever. The fatality rate may be as high as 20% in untreated cases, especially in the elderly.

Laboratory Diagnosis. A. Recovery of Agent: Laboratory diagnosis is dependent upon the recovery of psittacosis agent from blood and sputum or, in fatal cases, from lung tissues. Specimens are inoculated intra-abdominally into mice, into the yolk sacs of embryonated eggs, and into cell cultures. Infection in the test systems is confirmed by the serial transmission of the infectious agent, its microscopic demonstration, and serologic identification of the recovered agent.

B. Serology: A variety of antibodies may develop in the course of infection. In humans, complement fixation with group antigen is the most widely used diagnostic test. Acute and later phase sera should be run in the same test in order to establish an antibody rise. In birds, the indirect CF test may provide additional diagnostic information. Although antibodies usually develop within 10 days, the use of antibiotics may delay their development for 20-40 days or suppress it altogether.

Sera of patients with other chlamydiat infections may fix complement in high liter with psittacosis antigen. In patients with psittacosis, the high liter persists for months and, in carriers, even for years. Infection of live birds is suggested by a positive CF test and by the presence of an enlarged spleen or liver. This can be confirmed by demonstration of particles in smears or sections of organs and by passage of the agent in mice and eggs.

Immunity. Immunity in animals and humans is incomplete. A carrier state in humans can persist for 10 years after recovery. During this period the agent may continue to be excreted in the sputum.

Skin tests with group-reactive antigen are positive soon after infection with any member of the group. Specific dermal reactions may be obtained by the use of some skin-testing antigens prepared by extracting suspensions of agent with dilute hydrochloric acid or with detergent. Live or inactivated vaccines induce only partial resistance in animals.

Treatment. Tetracyclines are the drugs of choice. Psittacosis agents are not sensitive to aminoglycosides, and most strains are not susceptible to sulfonamides. Although antibiotic treatment may control the clinical evidence of disease, it may not free the patient from the agent, ie, the patient may become a carrier. Intensive antibiotic treatment may also delay the normal course of antibody development. Strains may become drug-resistant.

With the introduction of antibiotic therapy, the fatality rate has dropped from 20% to 2%. Death occurs most frequently in patients 40-60 years of age.

Epidemiology. The term psittacosis is applied to the human disease acquired from contact with birds and also the infection ofpsittacine birds (parrots, parakeets, cockatoos, etc). The term ornithosis is applied to infection with similar agents in all types of domestic birds (pigeons, chickens, ducks, geese, turkeys, etc) and free-living birds (gulls, egrets, petrels, etc). Outbreaks of human disease can occur whenever there is close and continued contact between humans and infected birds that excrete or shed large amounts of infectious agent. Birds often acquire infection as fledglings in the nest; may develop diarrheal illness or no illness; and often carry the infectious agent for their normal life span. When subjected to stress (eg, malnutrition, shipping), birds may become sick and die. The agent is present in tissues (eg, spleen) and is often excreted in feces by healthy birds. The inhalation of infected dried bird feces is a common method of human infection. Another source of infection is the handling of infected tissues (eg, in poultry rendering plants) and inhalation of an infected aerosol.

Birds kept as pets have been an important source of human infection. Foremost among these were the many psittacine birds imported from South America, Australia, and the Far East and kept in aviaries in the USA. Latent infections often flared up in these birds during transport and crowding, and sick birds excreted exceedingly large quantities of infectious agent. Control of bird shipment, quarantine, testing of imported birds for psittacosis infection, and prophylactic tetra-cyclines in bird feed help to control this source. Pigeons kept for racing or as pets or raised for squab meat have been important sources of infection. Pigeons populating civic buildings and thoroughfares in many cities are not infrequently infected but shed relatively small quantities of agent.

Among the personnel of poultry farms involved in the dressing, packing, and shipping of ducks, geese, turkeys, and chickens, subclinical or clinical infection is relatively frequent. Outbreaks of disease among birds have at times resulted in heavy economic losses and have been followed by outbreaks in humans.

Persons who develop psittacosis may become infectious for other persons if the evolving pneumonia results in expectoration of large quantities of infectious sputum. This has been an occupational risk to hospital personnel.

Control. Shipments of psittacine birds should be held in quarantine to ensure that there are no obviously sick birds in the lot. A proportion of each shipment should be tested for antibodies and examined for agent. An intradermal test has been recommended for detecting ornithosis in turkey flocks. The incorporation of tetra-cyclines into bird feed has been used to reduce the number of carriers. The source of human infection should be traced, if possible, and infected birds should be killed.

OCULAR, GENITAL, AND RESPIRATORY INFECTIONS DUE TO CHLAMYDIA TRACHOMATIS. TRACHOMA. Trachoma is an ancient eye disease, well described in the Ebers Papyrus, which was written in Egypt 3800 years ago. It is a chronic keratoconjunctivitis that begins with acute inflammatory changes in the conjunctiva and cornea and progresses to scarring and blindness.

Properties of C trachomatis.

A. Size and Staining Properties: See chlamydiae

B. Animal Susceptibility and Growth: Humans are the natural host for C trachomatis. Monkeys and chimpanzees can be infected in the eye and genital tract. Alt chlamydiae multiply in the yolk sacs of embryonated hens' eggs and cause death of the embryo when the number of particles becomes sufficiently high. C trachomatis also replicates in various cell lines, particularly when cells are treated with cycloheximide, cytochalasin B, or idoxuridine. C trachomulis of different immunotypes replicates differently . Isolates from trachoma do not grow as well as those from LGV or genital infections. Intracytoplasmic replication results in a developmental cycle that leads to formation of compact inclusions with a glycogen matrix in which particles are embedded.

A toxic factor is associated with C trachomatis provided the particles are viable. Neutralization of this toxic factor by immunotype-specific antisera permits typing of isolates that gives results analogous to those achieved by typing by immunofluorescence. The immunotypes specifically associated with endemic trachoma are A, B, Ba, and C.

Clinical Findings. In experimental infections, the incubation period is 3-10 days. In endemic areas, initial infection occurs in early childhood and the onset is insidious. Chlamyd-ial infection is often mixed with bacterial conjunctivitis in endemic areas, and the 2 together produce the clinical picture. The earliest symptoms of trachoma arelacrimation, mucopurulent discharge, conjunctiva! hyperemia, and follicular hypertrophy. Biomicro-scopic examination of the cornea reveals epithelial keratitis, subepithelial infiltrates, and extension of limba! vessels into the cornea (pannus).

As the pannus extends downward across the cornea, there is scarring of conjunctiva, lid deformities (entropion, trichiasis), and added insult caused by eyelashes sweeping across the cornea. With secondary bacterial infection, loss of vision and blindness occur over a period of years. There are, however, no systemic symptoms or signs of infection.

Laboratory Diagnosis. A. Recovery of C trachomatis: Typical cyto-plasmic inclusions are found in epithelial cells of conjunctiva! scrapings stained with fluorescent antibody or by Giemsa's method. These occur most frequently in the early stages of the disease and on the upper tarsal conjunctiva.

Inoculation of conjunctival scrapings into embryonated eggs or cycloheximide-treated cell cultures permits growth of C trachomatis if the number of viable infectious particles is sufficiently large. Cen-trifugation of the inoculum into treated cells increases the sensitivity of the method. The diagnosis can sometimes be made in the first passage by looking for inclusions after 2-3 days of incubalion by immunofluorescence, iodine staining, or staining by Giemsa's method.

B. Serology: Infected individuals often develop both group-reactive and immunotype-specific antibodies in serum and in eye secretions. Immunofluorescence is the most sensitive method for their detection. Neither ocular nor serum antibodies confer significant resistance to reinfection.

Treatment. In endemic areas, sulfonamides, erythromycins, and tetracyclines have been used to suppress chlamydiae and bacteria that cause eye infections. Periodic topical application of these drugs to the conjunctivas of all members of the community is sometimes supplemented with oral doses; the dosage and frequency of administration vary with the geographic area and the severity of endemic trachoma. Drug-resistant C Irachomatis has not been definitely identified except in laboratory experiments. Even a single monthly dose of 300 mg of doxycycline can result in significant clinical improvement, reducing the danger of blindness. Topical application of corticosteroids is not indicated and may reactivate latent trachoma. Chlamydiae can persist during and after drug treatment. and recurrence of activity is common.

Epidemiology and Control. It is believed that over 400 million people throughout the world are infected with trachoma and that 20 million are blinded by it. The disease is most prevalent in Africa, Asia, and the Mediterranean Basin, where hygienic conditions are poor and water is scarce. In such hyperendemic areas, childhood infection may be universal, and severe, blinding disease (resulting from frequent bacterial superinfections) is common. In the USA, trachoma occurs sporadically in some areas, and endemic foci persist on Indian reservations.

Control of trachoma depends mainly upon improvement of hygienic standards and drug treatment.

When socioeconomic levels rise in an area, trachoma becomes milder and eventually may disappear. Experimental trachoma vaccines have not given encouraging results, Surgical correction of lid deformities may be necessary in advanced cases.

GENITAL CHLAMYDIAL INFECTIONS and INCLUSION CONJUNCTIVITIS. C trachomatis, immunotypes D-K, is a common cause of sexually transmitted diseases that may also produce infection of the eye (inclusion conjunctivitis). In sexually active adults, particularly in the USA and western Europe—and especially in higher socioeconomic groups—C trachomatis is a prominent cause of nongonococcat urethritis and, rarely, epididymitis in males. In females, C trachomatis causes urethritis, cervicitis, salpingitis, and pelvic inflammatory disease. Any of these anatomic sites of infection may give rise to symptoms and signs, or the infection may remain asymptomatic but communicable to sex partners. Up to 50% of nongonococcal or postgonococcal urethritis or the urethra! syndrome is attributed to chlamydiae and produces dysuria, non-purulent discharge, and frequency of urination.

This enormous reservoir of infectious chlamydiae in adults can be manifested by symptomatic genital tract illness in adults or by an ocular infection that closely resembles trachoma. In adults, this inclusion conjunctivitis results from self-inoculation of genital secretions and was formerly thought to be "swimming pool conjunctivitis".

The neonate acquires the infection during passage through an infected birth canal. Inclusion conjunctivitis of the newborn begins as a mucopurulent conjunctivitis 7-12 days after delivery. It tends to subside with erythromycin or tetracycline treatment, or spontaneously after weeks or months. Occasionally, inclusion conjunctivitis persists as a chronic chlamyd-ial infection with a clinical picture indistinguishable from subacute or chronic childhood trachoma in nonendemic areas and usually not associated with bacterial conjunctivitis.

Laboratory Diagnosis. A. Recovery of C trachomatis: Scrapings of epithelial cells from urethra, cervix, vagina, or conjunctiva and biopsy specimens from salpinx or epididymis can be inoculated into chemically treated cell cultures for growth of C irachomatis (see above). Isolates can be typed by microimmunofluorescence wilh specific sera. In neonatal—and sometimes adult—inclusion conjunctivitis, the cytoplasmic inclusions in epithelial cells are so dense that they are readily detected in conjunclival exudate and scrapings examined by immunofluorescence or stained by Giem-sa's method.

B. Serologic Tests: Because of the relatively great antigenic mass of chlamydiae in genital tract infections, serum antibodies occur much more commonly than in trachoma and are of higher titer. A liter rise occurs during and after acute chlamydial infection. In genital secretions (eg, cervical), antibody can be detected during active infection and is directed against the infecting immunotype.

Treatment. It is essential that chlamydial infections be treated simultaneously in both sex partners and in offspring to prevent reinfection.

Tetracyclines (eg, doxycycline, 100 mg/d by mouth for 10-20 days) are commonly used in nongonococcal urethritis and in nonpregnant infected females. Erythromycin, 250 mg 4-6 times daily for 2 weeks, is given to pregnant women. Topical tetracycline or erythromycin is used for inclusion conjunctivitis, sometimes in combination with a systemic drug.

Epidemiology and Control. Genital chlamydial infection and inclusion conjunctivitis are sexually transmitted diseases that are spread by indiscriminate contact with multiple sex partners. Neonatal inclusion conjunctivitis originates in the mother's infected genital tract. Prevention of neonatal eye disease depends upon diagnosis and treatment of the pregnant woman and her sex partner. As in all sexually transmitted diseases, the presence of multiple etiologic agents (gonococci, treponemcs, Trichomonas, herpes, mycoplasmas, etc) must be considered. Instillation of 1% silver nitrate into the newbom 's eyes does not prevent development of chlamydial conjunctivitis. The ultimate control of this—and all—sexually transmitted disease depends on reduc-tion in promiscuity, use of condoms, and early diagnosis and treatment of the infected reservoir.

RESPIRATORY TRACT INVOLVEMENT WITH C. TRACHOMATIS

Adults with inclusion conjunctivitis often manifest upper respiratory tract symptoms (eg, otalgia, otitis, nasal obstruction, pharyngitis), presumably resulting from drainage of infectious chlamydiae through the nasolacrimal duct. Pneumonitis is rare in adults.

Neonates infected by the mother may develop respiratory tract involvement 2-12 weeks after birth, culminating in pneumonia. There is striking tachypnea, paroxysmal cough, absence of fever, and eosinophilia. Consolidation of lungs and hyperinflation can be seen by x-ray. Diagnosis can be established by isolation of C tracbomutis from respiratory secretions and can be suspected if pneumonitis develops in a neonate who has inclusion conjunctivitis. Systemic erythromycin (40 mg/kg/d) is effective treatment in severe cases.

LYMPHOGRANULOMA VENEREUM (LGV). LGV is a sexually transmitted disease, characterized by suppurative inguinal adenitis, that is common in tropical and temperate zones. The agent is C irachomatis of immunotypes L1-L3.

Properties of the Agent. A. Size and Staining Properties: Similar to other chlamydiae.

B. Animal Susceptibility and Growth of Agent: The agent can be transmitted to monkeys and mice and can be propagated in tissue cultures or in chick embryos. Most strains grow in cell cultures; their infectivity for cells is not enhanced by pretreatment with DEAE-dextran.

C. Antigenic Properties: The particles contain complement-fixing heat-stable chlamydial group antigens that are shared with all other chlamydiae. They also contain one of 3 specific antigens (L1-L3), which can be defined by immunofluorescence. Infective particles contain a toxic principle.

Clinical Findings. Several days to several weeks after exposure, a small, evanescent papule or vesicle develops on any part of the external genitalia, anus, rectum, or elsewhere. The lesion may ulcerate, but usually— especially in women—it remains unnoticed and heals in a few days. Soon thereafter, the regional lymph nodes enlarge and tend to become matted and often painful. In males, inguinal nodes are most commonly involved both above and below Poupart's ligament, and the overlying skin often turns purplish as the nodes suppurate and eventually discharge pus through multiple sinus tracts. In females and in homosexual males, the perirectal nodes are prominently involved, with proctitis and a bloody mucopurulent anal discharge. Lymphadenitis may be most marked in the cervical chains.

During the stage of active lymphadenitis, there are often marked systemic symptoms including fever, headaches, meningismus, conjunctivitis, skin rashes, nausea and vomiting, and arthralgias. Meningitis, arthritis, and pericarditis occur rarely. Unless effective antimicrobial drug treatment is given at that stage, the chronic inflammatory process progresses to fibrosis, lymphatic obstruction, and rectal strictures. The lymphatic obstruction may lead to elephantiasis of the penis, scrotum, or vulva. The chronic proctitis of women or homosexual males may lead to progressive rectal strictures, rectosigmoid obstruction, and fistula formation.

Laboratory Diagnosis. A. Smears: Pus, buboes, or biopsy material may be stained, but particles are rarely recognized.

B. Isolation of Agent: Suspected material is inoculated into the yolk sacs of embryonated eggs, into cell cultures, or into the brains of mice. Streptomycin (but not penicillin or ether) may be incorporated into the inoculum to lessen bacterial contamination. The agent is identified by morphology and serologic tests.

C. Serologic Tests: The CF reaction is the simplest serologic test for the presence of antibodies. Antigen is prepared from infected yolk sac. The test becomes positive 2-4 weeks after onset of illness, at which time skin hypersensitivity can sometimes also be demonstrated. In a clinically compatible case, a rising antibody level or a single liter of more than 1:64 is good evidence of active infection. If treatment has eradicated the LGV infection, the complement fixation riter falls. Serotogic diagnosis of LGV can employ immunofluorescence, but the antibody is broadly reactive with many chlamydial antigens. A more specific antibody can be demonstrated by counterimmunoelec-trophoresis with a chlamydial protein extracted from LGV.

D. Frei Test: Intradermal injection of heat-inactivated egg-grown LGV (0.1 mL) is compared to control material prepared from noninfected yolk sac. The skin test is read in 48-72 hours- An inflammatory nodule more than 6 mm in diameter at the test (but not the control) site constitutes a positive reaction. This can occur with different chlamydiae that share the group-reactive lipopolysaccharide. Thus, the Frei test lacks diagnostic specificity. The preparations of antigen available commercially have given unreliable results and have not been licensed in the USA since 1979.

Immunity. Untreated infections tend to be chronic, with persistence of the agent for many years. Little is known about active immunity. The coexistence of latent infection, antibodies, and cell-mediated reactions is typical of many chlamydial infections.

Treatment. The sulfonamides and tetracyclines have been used with good results, especially in the early stages. In some drug-treated persons there is a marked decline in complement-fixing antibodies, which may indicate that the infective agent has been eliminated from the body. Late stages require surgery.

Epidemiology. The disease is most often spread by sexual contact, but not exclusively so. The portal of entry may sometimes be the eye (conjunctivitis with an oculo-glandular syndrome). The genital tracts and rectums of chronically infected (but at times asymptomatic) persons serve as reservoirs of infection.

Although the highest incidence of LGV has been reported from subtropical and tropical areas, the infection occurs all over the world.

Control. The measures used for the control of other sexually transmitted diseases apply also to the control of LGV. Case-finding and early treatment and control of infected persons are essential.

DIAGNOSIS CHLAMYDIAL INFECTIONS IN DETAILS

CHLAMYDIAL INFECTIONS. In human beings Chlamydia cause ornithosis, trachoma, lymplio-granuloma venereum, neonatal inclusion blennorrhoea, adult inclusion conjunctivitis, urogenital infections, atypical pneumonia, and other illnesses which may run in the form of acute or chronic infection, or an asymptomatic carrier state.

The following types of examination are employed for the laboratory diagnosis of chlamydial infections.

Bacteriological examination involves the isolation of the causative agent by infecting experimental animals, chicken. embryos (see p. 182) or cell cultures (see p. 16ti). The causal organism is identified by the presence of elementary particle accumulation in impression smears from the internal organs of animals, allantoic fluid, cells of tissue cultures stained with the Romanowsky-Giemsa dye or acridine orange.

Serological methods of investigation include such tests as CF, HAI, and IF, which are carried out according to the ordinary scheme with chlamydial antigens.

The allergy intracutaneous test provides the earliest and most accessible method of diagnosis.

Since the laboratory diagnosis of all chlamydial infections is analogous, we w^ll consider in detail the diagnosis of only trachoma and ornithosis.

Trachoma. Trachoma is a contagious keratoconjunctivitis characterized by a chronic course. The causal organism of this disease is Chlamydia trachomatis.

To obtain biological material for the laboratory diagnosis, first anaesthetize the eye, then remove pus and mucus from the conjunctiva with the help of a cotton swab, and scrape off tha conjunctival epithelium with a blunt scalpel.

Bacterioscopic and bacteriological examination. Place a scraping onto glass slides, fix the preparations, and stain for 3-4 hrs, using the Romanowsky-Giemsa technique or acridine orange. In positive cases one finds coccal inclusions in epithelial cells (Plate VIII, 4), which measure up to 10 u,m (Prowazek-Halberstaedter bodies). To detect antigens by the IF reaction, preparations of scrapings are fixed in cold acetone for 10-15 min and treated with fluorescent antibodies. Upon luminescent microscopy C. trachomatis appear as fluorescent inclusions in the cytoplasm of conjunctival cells. Cytoplasmic .inclusions are also formed in cells of cultures of the human thyroid tissue infected with material obtained from patients.

It is recommended that the causative agent of trachoma be grown in the yolk sac of the chicken embryo. The material is treated with antibiotics for several hours at -4- 4: °C and introduced in 0.3-ml portions into the yolk sac of chicken embryos. Microscopic examination of impression smears prepared from the yolk sac of dead embryos reveals large numbers of Chlamydia.

To detect antibodies in trachoma patients' sera, an indirect IF reaction may be used.

Ornithosis. Ornithosis, which is a communicable disease caused by Chlamydia psittaci, is characterized by general intoxication and lung involvement.

To isolate the causal organism, take 5-10 ml of blood from the patient within the first two weeks of the illness. To perform serologi-cal tests, blood samples should be obtained within the first days and then 30-45 days after the onset of the disease. Washings from the nasal portion of the throat are made in the same manner which is utilized in examination for influenza (see p. 190). Sputum and vomitus are collected in sterile vessels. Post-mortem samples include damaged sites of the lung, liver, and spleen. The material to be examined is brought to the laboratory in the frozen form, checked for the absence of other bacteria, and used to prepare suspensions.

Biological examination. To isolate the causative agents of ornitho-sis, use white mice and chicken embryos. Inject 0.5 ml of a suspension of the tested material into the brain of mice. Make three sequential passages. In positive cases prepare histological sections and impression smears from the brain and stain them by the Romanowsky-Giemsa dye or acridine orange. C. psittaci are usually seen as accumulations of elementary particles in the cell cytoplasm.

Upon the intranasal inoculation of white mice the causative agent of ornithosis induces the disease and death of the animals from pneumonia in 3-4 days. Examination of impression smears of the lung tissue reveals cytoplasmic inclusions and elementary particles in the epithelial cells of alveoles and bronchioles, and also in phagocytes. Rabbits, young guinea pigs, rats, and Syrian hamsters are also sensitive to C. psittaci.

Bacteriological examination. Inoculate cliicken embryos via the yolk sac, allantoic cavity, and chorio-allantoic membrane, and then incubate them at 35-36 °C for 3-4 days. After 3-5 passages, the yolk culture of the causative agent of ornithosis achieves a higli degree of toxicity. Examination of impression smears of the allantoic membr.iiie and allantoic fluid discloses accumulations of the causative agent in the form of elementary bodies. On the surface of the chorio-allantoic membrane one can see patches similar to smallpox ones. Elementary bodies are also detected in the impression smears of yolk sacs stained with Romanowsky-Giemsa stain or with acridine orange.

Using the examined material, inoculate continuous cultures of cells, for example, L, IIela, Hep-2, etc.

The efficacy of cell culture inoculation increases when forced adsorption is utilized. For this purpose, after streaking of the tested material onto a monolayer of cells the cultures are centrifuged at 1000-1500 Xg for 20-30 min. Furthermore, it is recommended that one should use cell cultures which have been either irradiated or treated withcytostatics. After 24-48-hour incubation, monolayers of . cells on glass slides are stained with the Romanowsky-Giemsa stain or acridine orange. Microscopic examination of the resultant preparations reveals cytoplasmic inclusions of the causal organism in the form of roundish formations.

Serological diagnosis. The CF reaction is made with patients' paired sera, according to the ordinary technique and using the standard ornithosis antigen (ornithin) which is commercially available. A two-fold or greater increase in the antibody titre in the second serum is diagnostically important.

The indirect IF test is also employed for recovering antibodies in the patient's serum.

The intracutaneous allergy test with ornithin is assessed in 24 and 48 hrs. The test is positive in the acute period of the disease (from the 3rd day to approximately the 3rd-4th week of the disease).

OTHER AGENTS OF THE GROUP. Many mammals are subject to chlamydial infections, mainly with C. pstiaci. Common animal disease entities are pneumonitis, arthritis, enteritis, and abortion, but infection is often latent. Some of these agents may also be transmitted to humans and cause disease in them.

Chlamydiae have been isolated from Reiter's disease in humans, both from the involved joints and from the urethra. The causative role of these agents remains uncertain.

In nonbacterial regional lymphadenitis (cat-scratch fever), a skin test with heat-inactivated pus gives a delayed positive reaction. Chlamydiae have been proposed as a possible cause but without proof. The usefulness of tetracyclines in this syndrome is dubious

MYCOPLASMAS (PPLO) and WALL-DEFECTIVE MICROBIAL VARIANTS.

Mycoplasmas (previously called pleuropneumonia-like organisms or PPLO) are a group of organisms with the following characteristics: (1) The smallest reproductive units have a size of 125-250 nm. (2) They are highly pleomorphic because they lack a rigid cell wall and instead are bounded by a triple-layered "unit membrane." (3) They are completely resistant to penicillin but inhibited by tetracycllne or erythromycln. (4) They can reproduce in cell-free media; on agar the center of the whole colony is characteristically embedded beneath the surface (“fried egg” appearance). (5) Growth is inhibited by specific antibody. (6) Mycoplasmas do not revert to, or originate from, bacterial parental forms. (7) Mycoplasmas have an affinity for cell membranes.

L phase variants are wall-defective microbial forms (WDMF) that can replicate serially as nonrigid cells and produce colonies on solid media. Some L phase variants are stable; others are unstable and revert to bacterial parent forms. Wall-defective microbial forms are not genetically related to mycoplasmas. WDMF can result from spontaneous mutation or from the effects of chemicals. Treatment of eubacteria with cell wall-inhibiting drugs or lysozyme can produce WDMF. Protoplasts are WDMF derived from gram-positive organisms; they are osmotically fragile, with external surfaces free of cell wall constituents. Spheroplasts are WDMF derived from gram-negative bacteria; they retain some outer membrane material.

Morphology and Identification. A. Typical Organisms: Mycoplasmas cannot be studied by the usual bacteriologic methods because of the small size of their colonies, the plasticity and delicacy of their individual cells (due to the lack of a rigid cell wall), and their poor staining with aniline dyes. The morphology appears different according to the method of examination (eg, darkfield, immunoflu-orescence, Giemsa-stained films from solid or liquid media, agar fixation).

Mycoplasma pneumoniae

Mycoplasma hominis

Ureaplasma urealyticum

Growth in fluid media gives rise to many different forms, including rings, bacillary and spiral bodies, filaments, and granules. Growth on solid media consists principally of plastic protoplasmic masses of indefinite shape that are easily distorted. These structures vary greatly in size, ranging from 50 to 300 nm in diameter.

B. Culture: Many strains of mycoplasmas grow in heart infusion peptone broth with 2% agar (pH 7.8) to which about 30% human ascitic fluid or animal serum (horse, rabbit) has been added. Following incubation at 37 °C for 48-96 hours, there may be no turbidity; but Giemsa stains of the centrifuged sediment show the characteristic pleomorphic structures, and subculture on solid media yields minute colonies.

After 2-6 days on special agar medium incubated in a Petri dish that has been sealed to prevent evaporation, isolated colonies measuring 20-500 /n.m can be detected with a hand tens. These colonies are round, with a granular surface and a dark center nipple typically buried in the agar. They can be subcultured by cutting out a small square of agar containing one or more colonies and streaking this material on a fresh plate or dropping it into liquid medium. The organisms can be stained for microscopic study by placing a similar square on a slide and covering the colony with a coverglass onto which an alcoholic solution of methylene blue and azure has been poured and then evaporated (agar fixation). Such slides can also be stained with specific fluorescent antibody.

Mycoplasma colonies

C. Growth Characteristics: Mycoplasmas are unique in microbiology because of (1) their extremely small size and (2) their growth on complex but cell-free media.

Mycoplasmas pass through filters with 450-nm pore size and thus are comparable to chlamydiae or large vimses. However, parasitic mycoplasmas grow on cell-free media that contain lipoprotein and sterol. The sterol requirement for growth and membrane synthesis is unique. Mycoplasmas are resistant to thallium acetate in a concentration of 1:10,000, which can be used to inhibit bacteria. Many mycoplasmas use glucose as a source of energy; ureaplasmas require urea.

Many human mycoplasmas produce peroxides and hemolyze red blood cells. In cell cultures and in vivo, mycoplasmas develop predominantly at cell surfaces. Many established cell line cultures carry mycoplasmas as contaminants.

D. Variation: The extreme pleomorphism of mycoplasmas is one of their principal characteristics. There is no genetic relationship between mycoplasmas and WDMF or their parent bacteria. The characteristics of WDMF are similar to those of mycoplasmas, but, by definition, mycoplasmas do not revert to parent bacteria or originate from them. WDMF continue to synthesize some antigens that are normally located in the cell wall of the parent bacteria (eg, streptococcal L forms produce M protein and capsular polysaccharide.

Reversion of L forms to the parent bacteria is enhanced by growth in the presence of 15-30% gelatin or 2.5% agar, whereas reversion is inhibited by inhibitors of protein synthesis.

Antigenic structure. Many antigenicaily distinct species of mycoplasmas have been isolated from animals (eg, mice, chickens, turkeys).

In humans, at least 11 species can be identified, including Mycoplasma hominis, Mycoplasma salivarium, Mycoplasma orale, Mycoplasma fermentans, Mycoplasma pneumoniae, Ureaplasma urealyticum, and others.

The last 2 species are of pathogenic significance.

The species are classified by biochemical and serologic features. The complement-fixing antigens of mycoplasmas are glycolipids. Some species have more than one serotype.

It is doubtful that WDMF cause tissue reactions resulting in disease. They may be important for the persistence of microorganisms in tissues and recurrence of infection after antimicrobial treatment.

The parasitic mycoplasmas appear to be strictly host-specific, being communicable and potentially pathogenic only within a single host species. In animals , mycoplasmas appear to be intracellular parasites with a predilection for mesothelial cells (pleura, peritoneum, synovia of joints). Several extracellular products can be elaborated, eg, hemolysins.

A. Diseases of Animals: Bovine pleuropneumonia is a contagious disease of cattle producing pulmonary consolidation and pleural effusion, with occasional deaths. The disease probably has an airborne spread. Mycoplasmas are found in inflammatory exudates.

Agalactia of sheep and goats i n the Mediterranean area is a generalized infection with local lesions in the skin, eyes, joints, udder, and scrotum; it leads to atrophy of lactating glands in females. Mycoplasmas are present in blood early; in milk and exudates later.

In poultry, several economically important respiratory diseases are caused by mycoplasmas. The organisms can be transmitted from hen to egg and chick. Swine, dogs, rats, mice, and other species harbor mycoplasmas that can produce infection involving particularly the pleura, peritoneum, joints, respiratory tract, and eye. In mice, a Mycoplasma of spiral shape (Spiroplasma) can induce cataracts.

B. Diseases of Humans: Mycoplasmas have been cultivated from human mucous membranes and tissues, particularly from the genital, urinary, and respiratory tracts and from the mouth. Some mycoplasmas are inhabitants of the normal genitourinary tract, particularly in females. In pregnant women, carriage of mycoplasmas on the cervix has been associated with chorioamnionitis and low birth weight of infants. U urealyticum (formerly called T strains of mycoplasmas), requiring 10% urea for growth, is found in some cases of urethritis and prostatitis in men who suffer from "nongonococcal urethritis. " This organism and M. hominis have also been associated infrequently with salpingitis and pelvic inflammatory disease. U. urealyticum may play a causative role and is suppressed by tetracycline or spectinomycin. However, a majority of cases of “nongonococcal urethritis” are caused by Chlamydia trachomatis.

Infrequently, mycoplasmas have been isolated from brain abscesses and pleural or joint effusions. Mycoplasmas are part of the normal flora of the mouth and can be grown from normal saliva, oral mucous membranes, sputum, or tonsillar tissue.

M. hominis and M. salivarium can be recovered from the oral cavity of many healthy adults, but an association with clinical disease is uncertain. Over half of normal adults have specific antibodies to M. hominis.

M. pneumoniae is one of the causative agents of nonbacterial pneumonia (see p 276). In humans, the effects of infection with M. pneumoniae range from inapparent infection to mild or severe upper respiratory disease, ear involvement (myringitis), and bronchial pneumonia (see p 276).

C. Diseases of Plants: Aster yellows, corn stunt, and other plant diseases appear to be caused by mycoplasmas. They are transmitted by insects and can be suppressed by tetracyclines.

Specimens consist of throat swab, sputum, inflammatory exudates, and respiratory, urethral, or genital secretions.

A. Microscopic Examination: Direct examination of a specimen is useless. Cultures are examined as described above.

B. Culture: The material is inoculated onto special solid media and incubated for 3-10 days at 37 °C (often under anaerobic conditions), or into special broth (see above) incubated aerobically. One or 2 transfers of media may be necessary before growth appears that is suitable for microscopic examination by staining or immunofluorescence. Colonies may have a "fried egg" appearance on agar.

C. Serology: Antibodies develop in humans infected with mycoplasmas and can be demonstrated by several methods. CF tests can be performed with glycolipid antigens extracted with chloroform-methanol from cultured mycoplasmas. HI tests can be applied to tanned red cells with adsorbed Mycoplasma antigens. Indirect immunofluorescence may be used. The test that measures growth inhibition by antibody is quite specific. When counterimmunoelectrophoresis is used, antigens and antibody migrate toward each other, and precipitin lines appear in 1 hour. With all these serologic techniques, there is adequate specificity for different human Mycoplasma species, but a rising antibody liter is required for diagnostic significance because of the high incidence of positive serologic tests in normal individuals.

Treatment. Many strains of mycoplasmas are inhibited by a variety of antimicrobial drugs, but most strains are resistant to penicillins, cephalosporins, and vancomy-cin. Tetracyclines and erythromycins are effective both in vitro and in vivo and are, at present, the drugs of choice in mycoplasmal pneumonia.

Epidemiology, Prevention, and Control. Isolation of infected livestock will control the highly contagious pleuropneumonia and agalactia in limited areas. No vaccines are available. Mycoplasmal pneumonia behaves like a communicable viral respiratory disease (see next section).

Acute nonbacterial pneumonitis may be due to many different infectious agents, including adeno-viruses, influenza viruses, respiratory syncytia! virus, parainfluenza type 3 virus, chlamydiae, and Coxiella burnetii, the etiologic agent of Q fever. However, the single most prominent causative agent, especially for those between ages 5 and 15, is M pneumoniae. Mycoplasmal pneumonia appears to be much more common in military recruit populations than in college populations of comparable age.

The first step in M pneumoniae infection is the attachment of the tip of the organism to a receptor on the surface of respiratory epithelial cells. The clinical spectrum of M pneumoniae ranges from asymptomatic infection to serious pneumonitis, with occasional neu-rologic and hematologic (ie, hemolytic anemia) involvement and a variety of possible skin lesions. Bul-lous myringitis occurs in spontaneous cases and in experimentally inoculated volunteers. Typical cases during epidemic periods might show the following:

The incubation period varies from I to 3 weeks. The onset is usually insidious, with lassitude, fever, headache, sore throat, and cough. Initially, the cough is nonproductive, but it is occasionally paroxysmal. Later there may be blood-streaked sputum and chest pain. Early in the course, the patient appears only moderately ill, and physical signs of pulmonary consolidation are often negligible compared to the striking consolidation seen on x-rays. Later, when the infiltration is at a peak, the illness may be severe. Resolution of pulmonary infiltration and clinical improvement occur slowly for 1—4 weeks. Although the course of the illness is exceedingly variable, death is very rare and is usually attributable to cardiac failure. Complications are uncommon, but hemolytic anemia may occur. The most common pathologic findings are interstitial and peribronchial pneumonitis and necrotizing bronchiolitis.

The following laboratory findings apply to M pneumoniae pneumonia: The white and differential counts are within normal limits. The causative Myco-plasma can be recovered by culture early in the disease from the pharynx and from sputum. Immunofluores-cent stains of mononuclear cells from the throat may reveal the agent. There is a rise in specific antibodies to M pneumoniae that is demonstrable by complement fixation, immunofluorescence, passive hemagglutina-tion, and growth inhibition.

A variety of nonspecific reactions can be observed . Cold hemagglutinins for group O human eryth-rocytes appear in about 50% of untreated patients, in rising liter, with the maximum reached in the third or fourth week after onset. A titer of 1:32 or more supports the diagnosis of M pneumonias infection.

Tetracyclines or erythromycins in full systemic doses (2 g daily for adults) can result in clinical improvement but do not eradicate the mycoplasmas.

M pneumoniae infections are endemic all over the world. In populations of children and young adults where close contact prevails, and in families, the infection rate may be high (50-90%), but the incidence of pneumonitis is variable (3-30%). For every case of frank pneumonitis, there exist several cases of milder respiratory illness. M pneumoniae is apparently transmitted mainly by direct contact involving respiratory secretions. Second attacks are infrequent. The presence of antibodies to M pneumoniae has been associated with resistance to infection but may not be responsible for it. Cell-mediated immune reactions occur. The pneumonic process may be in part attributed to an immunologic response rather than only to infection by mycoplasmas. Experimental vaccines have been prepared from agar-grown M pneumoniae. Several such killed vaccines have aggravated subsequent disease; a degree of protection has been claimed with the use of other vaccines.

On rare occasions, central nervous system involvement has accompanied or followed mycoplasmal pneumonia.

Borrelia

BORRELIA RECURRENTIS

Morphology and Identification

A. Typical Organisms: Borrelia recurrentis is an irregular spiral 10-30 mcm long and 0.3 mcm wide. The distance between turns varies from 2 to 4 mcm (fig.1). The organisms are highly flexible and move both by rotation and by twisting. B recurrentis stains readily with bacteriologic dyes as well as with blood stains such as Giemsa's or Wright's stain.

Borrelia recurrentis in blood smear.

B. Culture: The organism can be cultured in fluid media containing blood, serum, or tissue; but it rapidly loses its pathogenicity for animals when transferred repeatedly in vitro. Multiplication is rapid in chick embryos when blood from patients is inoculated into the chorioallantoic membrane.

C. Growth Characteristics: Virtually nothing is known of the metabolic requirements or activity of borreliae. At 4 °C, the organisms survive for several months in infected blood or in culture. In some ticks (but not in lice), spirochetes are passed from generation to generation.

D. Variation: The only significant variation of Borrelia is with respect to its antigenic structure.

Antigenic Structure. Isolates of Borrelia from different parts of the world, from different hosts, and from different vectors (ticks or lice) either have been given different species names or have been designated strains of B recurrenris. Biologic differences between these strains or species do not appear to be stable.

Agglutinins, complement-fixing antibodies, and lytic antibodies develop in high titer after infection with borreliae. Apparently the antigenic structure of the organisms changes in the course of a single infection. The antibodies produced initially may act as a selective factor that permits the survival only of anti genically distinct variants. The relapsing course of the disease appears to be due to the multiplication of such antigenic variants, against which the host must then develop new antibodies. Ultimate recovery (after 3-10 relapses) is associated with the presence of antibodies against several antigenic variants.

Pathology. Fatal cases show spirochetes in great numbers in the spleen and liver, necrotic foci in other parenchyma-tous organs, and hemorrhagic lesions in the kidneys and the gastrointestinal tract. Spirochetes have been occasionally demonstrated in the spinal fluid and brains of persons who have had meningitis. In experimental animals (guinea pigs, rats), the brain may serve as a reservoir of borreliae after they have disappeared from the blood.

Pathogenesis and Clinical Findings. The incubation period is 3-10 days. The onset is sudden, with chills and an abrupt rise of temperature. During this time spirochetes abound in the blood. The fever persists for 3-5 days and then declines, leaving the patient weak but not ill. The afebrile period lasts 4-10 days and is followed by a second attack of chills, fever, intense headache, and malaise. There are from 3 to 10 such recurrences, generally of diminishing severity. During the febrile stages (especially when the temperature is rising), organisms are present in the blood; during the afebrile periods they are absent. Organisms appear less frequently in the urine.

Antibodies against the spirochetes appear during the febrile stage, and it is possible that the attack is terminated by their agglutinating and lytic effects. These antibodies may select out antigenically distinct variants that multiply and cause a relapse. Several distinct antigenic varieties of borreliae may be isolated from a single patient's several relapses, even following experimental inoculation with a single organism.

Diagnostic Laboratory Tests

A. Specimens: Blood obtained during the rise in fever, for smears and animal inoculation.

B. Stained Smears: Thin or thick blood smears stained with Wright's or Giemsa's stain reveal large, loosely coiled spirochetes among the red cells.

C. Animal Inoculation: White mice or young rats are inoculated intraperituneally with blood-Stained films of tail blood are examined for spirochetes 2-4 days later.

D. Serology: Spirochetes grown in culture can serve as antigens for CF tests, but the preparation of satisfactory antigens is difficult. Patients suffering from epidemic (louse-borne) relapsing fever may develop agglutinins for Proteus OXK and also a positive VDRL.

Immunity. Immunity following infection is usually of short duration.

Treatment. The great variability of the spontaneous remissions of relapsing fever makes evaluation of chemotherapeutic effectiveness difficult. Tetracy-clines, erythromycin, and penicillin are all believed to be effective. Treatment for a single day may be sufficient to terminate an individual attack.

Epidemiology, Prevention, and Control. Relapsing fever is endemic in many parts of the world. Its main reservoir is the rodent population, which serves as a source of infection for ticks of the genus Ornithodorus. The distribution of endemic foci and the seasonal incidence of the disease are largely determined by the ecology of the ticks in different areas. In the USA, infected ticks are found throughout the West, especially in mountainous areas, but clinical cases are rare. In the tick, Borrelia may be transmitted transovarially from generation to generation.

Spirochetes are present in all tissues of the tick and may be transmitted by the bite or by crushing the tick. The tick-home disease is not epidemic. However, when an infected individual harbors lice, the lice become infected by sucking blood; 4-5 days later, they may serve as a source of infection for other individuals . The infection of lice is not transmitted to the next generation, and the disease is the result of rubbing crushed lice into bite wounds. Severe epidemics may occur in louse-infested populations, and transmission is favored by crowding, malnutrition, and cold climate.

In endemic areas human infection may occasionally result from contact with the blood and tissues of infected rodents. The mortality rate of the endemic disease is low, but in epidemics it may reach 30%.

Prevention is based on avoidance of exposure to ticks and lice and on delousing (cleanliness, insecticides). No vaccines are available.

Lyme disease. Lyme disease was named for the small Connecticut community in which the disease was first recognized in 1975 It is an inflammatory disorder caused by the spirochete Borrelia burgdorferi.

FILM: Lyme bacteria Cyst formation with detail http://www.youtube.com/watch?v=lVmCa70bAxE

The disease is transmitted to humans by Ixodes ticks The whitetailed deer and white-tailed mouse are the reservoirs of B burgdorfen. The distribution of the disease is restricted to regions with these animal reservoirs and Ixodes ticks. Increases in deer populations and human activities such as clearance of woodland and hiking and camping in wooded areas have increased the likelihood of being bitten by a deer tick. This has resulted in an increased number of cases of Lyme disease Although the territory over which this disease has occurred is increasing, the majority of the approximately 7,000 reported cases of Lymc disease have occurred in the Northeast, upper Midwest, and California

Lyme disease usually begins with a distinctive skin lesion. The circularly expanding annular skin lesions are hardened (indurated) with wide borders and central clearing Although these lesions may reach diameters of 12 inches or more, they are painless Accompanying symptoms may resemble a mild flu, with some patients experiencing symptoms resembling mild meningitis or encephalitis, hepatitis, musculoskeletal pam, enlarged spleen, and cough Weeks to months later, the patient often shows signs of the second stage of the disease, developing arthritic joint pain, and sometimes neurologic or cardiac abnormalities. The neurological complications may include visual, emotional, and memory disturbances, temporary paralysis of a facial nerve, and movement difficulties The third stage, which may appear months to years after infection, is characterised by crippling arthritic symptoms in one or more joints, especially in the knees, and severe neurological symptoms that mimic multiple sclerosis. These symptoms are believed to be caused by the body's immune defense system's attempts to fight the infective agent, rather than by the organism itself The antibody complexes produced in response to the infective agent cause the joints to become inflamed. Treatment with trivalent antibodies neutralizes the toxins produced by B. burgdorferi.

Lyme disease

Recent epidcmiologk and laboratory investigations indicate that Lyme disease may be a different manifestation of erythema chronicum miggrans, a syndrome long recognized in Europe. Both diseases arc caused by the spirochete, Borrelia burgdorferi, transmitted to humans by the bites of Ixodes ticks Similar spirochetes have been recovered from the blood of Lvme disease patients and cultured from ixodes ticks, and patients with Lyme disease have antibodies to the cultured spirochetes. When this disease is diagnosed, penicillin is an effective treatment. There is also a new vaccine that appears to be effective for preventing Lyme disease

Epidemiology of Lyme disease. In October 1975 the Connucticut Department received independent calls reporting multtpk eases of what speared to be arthritis m chiMren in lyme and Old Lyme, rural towns in that state. Despite being assured by their physicians that arthritis was not infectious, the callers were not satisfied An epidemic investigation ensued in which the extent, characteristicis, mode of transmission, and etiology of the cluster of cases were studied. As characteristic of many epidemidiogical investigations, public health officiate began by trying to locate all individuals who had sudden onset of swelling and pain in the knee or other large joints lasting a weelk to several months. An old, large skin rash, repeated attacks at intervals of a few months, fever, and fatigue were the reported symptoms. State epidemiologists questioned parents and physidans – asking were the cases related, were there other similar cases, wasthis an infectious form of arthritis, and what forms of arthritis are infectious? Next the epidemiologists determined the time, place, and personal characteristics of these cases. The incidence of onset of disease seemed to cluster in late spring and summer and lasted from a week to a few months The cases were concentrated in three adjacent towns on the eastern side of the Connecticut River and most patients lived in wooded areas near lakes and streams. Of the 51 cases, 39 were children about evenly split between boys and girls There were no familial patterns. Epidemiologists created an epidemic curve, listing the cases by the time of onset, and began calling the disease "Lyme arthritis".

The clustering of cases, the fact that most began in late spring or summer and that they were most frequently located in wooded areas along lakes or streams suggested a disease transmitted by an arthropod A study was undertaken to determine if this was a communicable disease. Cases of the disease were matched with a similar group of control or unaffected persons for age, sex, and other relevant factors. It was found that affected people were more likely to have a household pet than those who were unaffected. Pet owners are more likely to come in contact with ticks that their dogs and cats might pick up m me woods. The importance of this finding was emphasized when combined with the fact that one fourth of the patients reported that their arthritic symptoms were preceded by an unusual skin rash that started as a red spot that spread to a 6-inch ring. A dermatology consultant recalled a similar skin outbreak reported in Switzerland in 1910 that was attributed to tick bites.

This was only suggestive evidence that a tick bite might initiate an infectious disease. The connection between the rash and the disease had to be strengthed.

Now public health authorities had to ask if patients with such a rash always progress to develop Lyme arthritis. A prospective study lookcd for new patients with a rash. Of 32 new cases of the characteiastic skin rash, 19 progressed to show signs and symptoms of Lyme disease. The tick connection was strengthened after an entomological study found that adult ticks were 16 times more abundant on the east side of the Connecticut River than the west This corresponded to the proportion of incidence of the disease on each side of the river. Also, more tick bites were reported by the arthritis sufferers then by their unaffected neighbors A surveillance network was set up in. Connecticut and surrounding stales to gather information about other cases. These investigations showed more adult victims than children and also more serious manifestations, including neurological and heart diseases.

The Rocky Mountain Public Health Laboratory in Montana was asked to assist in the investigation because of its expertise in the area of tickborne disease. They found unusual spirochetes in the guts of many of the ticks sent from Connecticute. Spirochetes, which are bacteria with curved cells wound around a central filament, are often difficult to culture and so it would be difficult to prove that these were the causative organisms of Lyme disease Therefore they first tried to infect laboratoiy animals with the infected ticks The rabbits developed rashes resembling those seen in humans. A spirochete was isolated ftom the ticks and when pure cultures were inoculated into rabbite, the rabbits developed the characteristic rash. The infected rabbits contained antispirochetal antibodies in their serum. The identification was complete when the spirochete was isolated from human cases. The spirochete was classified as a member of the Borrelia genus and named Borrelia burgdorferi after the entomologist who discovered the organisms in the ticks.

Lyme disease accounted for more than 90 % of the vector-home infectious diseases in the United States in 1992. The distribution of the disease was highly correlated with the distribution, of the principal tick vectors. The nearly twerrtyfold increase in cases since the early 1980s may be a consequense of increased surveillance and improved diagnostic methods, or a real increase in disease prevalence due to increases in deer and tick populations and closer human contact with these animals.

Summary. Lyme disease is characterized by the development of arthritis and neurological symptoms that result when the body's immune defenses react to infections with the spirochete Borrelid burgdorferi.

The occurrence of Lyme disease has been concentrated in the Northeast and other areas where Ixodes ticks carry Borrelia burgdorferi.

Additionala material abour laboratory diagnosis

RECURRENT FEVER (EPIDEMIC). The causative agent of epidemic (louse-borne) relapsing fever (Borrelia recurrentis), of endemic (tick-borne) typhus (Borrelia persica) and some other microorganisms induce clinically similar acute infectious diseases whose course is characterized by fever attacks, general intoxication, and liver and spleen enlargement.

The main method of investigation in recurrent types of typhus is bacterioscopic examination, that is visualization of the causal organism in the patient's blood.

Bacterioscopic examination. During or before an attack of relapsing fever take a sample of blood from the patient and make a thick-drop preparation and a smear. First, stain a thick-drop preparation, since its examination provides better possibilities to obtain a positive result. On a dried unfixed preparation of a blood drop pour the Romanowsky-Giemsa dye and allow it to act for 35-45 min. Haemo-lysis occurs in a drop (the dye is diluted with distilled water), so 5 min later replace the dye with a fresh portion which is kept in place for 30-40 min. Then, the preparation is carefully washed with water and dried. Under the microscope Borrelia look like thin blue-violet threads with several large coils (secondary coils). The length of Borrelia varies from 10 to 30 u-m or more. Sometimes their size is several times larger than the diameter of leucocytes (the preparation contains only leucocytes because erythrocytes are lysed). When Borrelia are present in large numbers, which is characteristic of relapsing fever, their aggregates may pose difliculty with regard to their differentiation from fibrin threads. In this case examine micro-scopically a smear which is dried, fixed with alcohol or Nikiforov's mixture, and stained with the Romanowsky-Giemsa dye or Pfeiffer's fuchsine. Borrelia are readily visualized by microscopy. They are at least twice as long as the diameter of red blood cells. If no Borrelia are found in a thick blood film, smears are not subjected to examination.

When the patient is afebrile, Borrelia cannot he detected by the ordinary methods of investigation. Hence, enrichment methods are recommended to be used in such cases. Withdraw from the vein 8-10 ml of blood, wait until it has coagulated, separate the serum, and centrifuge it at 3000 X g for 45-60 min. Then, quickly pour off the fluid by inverting the test tube, make wet-mount preparations from the sediment (which are examined by dark-field microscopy), and thick smears (which are fixed, stained, and studied in the manner used with blood smears).

Biological examination. To confirm the diagnosis of tick-borne recurrent typhus, inject, pubciitaneously 0.5-1 ml of citrate blood to guinea pigs. Five-six day? later one can observe large numbers of Borrelia in the blood of the infected animals. This method permits the differentiation of the causative agents of epidemic versus tick-borne recurrent typhus fever. The latter induce the disease in guinea pigs, whereas B. recurrentis is non-pathogenic for them

Borrelia

BORRELIA RECURRENTIS

Morphology and Identification

A. Typical Organisms: Borrelia recurrentis is an irregular spiral 10-30 mcm long and 0.3 mcm wide. The distance between turns varies from 2 to 4 mcm (fig.1). The organisms are highly flexible and move both by rotation and by twisting. B recurrentis stains readily with bacteriologic dyes as well as with blood stains such as Giemsa's or Wright's stain.

Borrelia recurrentis in blood smear.

B. Culture: The organism can be cultured in fluid media containing blood, serum, or tissue; but it rapidly loses its pathogenicity for animals when transferred repeatedly in vitro. Multiplication is rapid in chick embryos when blood from patients is inoculated into the chorioallantoic membrane.

C. Growth Characteristics: Virtually nothing is known of the metabolic requirements or activity of borreliae. At 4 °C, the organisms survive for several months in infected blood or in culture. In some ticks (but not in lice), spirochetes are passed from generation to generation.

D. Variation: The only significant variation of Borrelia is with respect to its antigenic structure.

Antigenic Structure. Isolates of Borrelia from different parts of the world, from different hosts, and from different vectors (ticks or lice) either have been given different species names or have been designated strains of B recurrenris. Biologic differences between these strains or species do not appear to be stable.

Agglutinins, complement-fixing antibodies, and lytic antibodies develop in high titer after infection with borreliae. Apparently the antigenic structure of the organisms changes in the course of a single infection. The antibodies produced initially may act as a selective factor that permits the survival only of anti genically distinct variants. The relapsing course of the disease appears to be due to the multiplication of such antigenic variants, against which the host must then develop new antibodies. Ultimate recovery (after 3-10 relapses) is associated with the presence of antibodies against several antigenic variants.

Pathology. Fatal cases show spirochetes in great numbers in the spleen and liver, necrotic foci in other parenchyma-tous organs, and hemorrhagic lesions in the kidneys and the gastrointestinal tract. Spirochetes have been occasionally demonstrated in the spinal fluid and brains of persons who have had meningitis. In experimental animals (guinea pigs, rats), the brain may serve as a reservoir of borreliae after they have disappeared from the blood.

Pathogenesis and Clinical Findings. The incubation period is 3-10 days. The onset is sudden, with chills and an abrupt rise of temperature. During this time spirochetes abound in the blood. The fever persists for 3-5 days and then declines, leaving the patient weak but not ill. The afebrile period lasts 4-10 days and is followed by a second attack of chills, fever, intense headache, and malaise. There are from 3 to 10 such recurrences, generally of diminishing severity. During the febrile stages (especially when the temperature is rising), organisms are present in the blood; during the afebrile periods they are absent. Organisms appear less frequently in the urine.

Antibodies against the spirochetes appear during the febrile stage, and it is possible that the attack is terminated by their agglutinating and lytic effects. These antibodies may select out antigenically distinct variants that multiply and cause a relapse. Several distinct antigenic varieties of borreliae may be isolated from a single patient's several relapses, even following experimental inoculation with a single organism.

Diagnostic Laboratory Tests

A. Specimens: Blood obtained during the rise in fever, for smears and animal inoculation.

B. Stained Smears: Thin or thick blood smears stained with Wright's or Giemsa's stain reveal large, loosely coiled spirochetes among the red cells.

C. Animal Inoculation: White mice or young rats are inoculated intraperituneally with blood-Stained films of tail blood are examined for spirochetes 2-4 days later.

D. Serology: Spirochetes grown in culture can serve as antigens for CF tests, but the preparation of satisfactory antigens is difficult. Patients suffering from epidemic (louse-borne) relapsing fever may develop agglutinins for Proteus OXK and also a positive VDRL.

Immunity. Immunity following infection is usually of short duration.

Treatment. The great variability of the spontaneous remissions of relapsing fever makes evaluation of chemotherapeutic effectiveness difficult. Tetracy-clines, erythromycin, and penicillin are all believed to be effective. Treatment for a single day may be sufficient to terminate an individual attack.

Epidemiology, Prevention, and Control. Relapsing fever is endemic in many parts of the world. Its main reservoir is the rodent population, which serves as a source of infection for ticks of the genus Ornithodorus. The distribution of endemic foci and the seasonal incidence of the disease are largely determined by the ecology of the ticks in different areas. In the USA, infected ticks are found throughout the West, especially in mountainous areas, but clinical cases are rare. In the tick, Borrelia may be transmitted transovarially from generation to generation.

Spirochetes are present in all tissues of the tick and may be transmitted by the bite or by crushing the tick. The tick-home disease is not epidemic. However, when an infected individual harbors lice, the lice become infected by sucking blood; 4-5 days later, they may serve as a source of infection for other individuals . The infection of lice is not transmitted to the next generation, and the disease is the result of rubbing crushed lice into bite wounds. Severe epidemics may occur in louse-infested populations, and transmission is favored by crowding, malnutrition, and cold climate.

In endemic areas human infection may occasionally result from contact with the blood and tissues of infected rodents. The mortality rate of the endemic disease is low, but in epidemics it may reach 30%.

Prevention is based on avoidance of exposure to ticks and lice and on delousing (cleanliness, insecticides). No vaccines are available.

Lyme disease. Lyme disease was named for the small Connecticut community in which the disease was first recognized in 1975 It is an inflammatory disorder caused by the spirochete Borrelia burgdorferi.

FILM: Lyme bacteria Cyst formation with detail http://www.youtube.com/watch?v=lVmCa70bAxE

The disease is transmitted to humans by Ixodes ticks The whitetailed deer and white-tailed mouse are the reservoirs of B burgdorfen. The distribution of the disease is restricted to regions with these animal reservoirs and Ixodes ticks. Increases in deer populations and human activities such as clearance of woodland and hiking and camping in wooded areas have increased the likelihood of being bitten by a deer tick. This has resulted in an increased number of cases of Lyme disease Although the territory over which this disease has occurred is increasing, the majority of the approximately 7,000 reported cases of Lymc disease have occurred in the Northeast, upper Midwest, and California

Lyme disease usually begins with a distinctive skin lesion. The circularly expanding annular skin lesions are hardened (indurated) with wide borders and central clearing Although these lesions may reach diameters of 12 inches or more, they are painless Accompanying symptoms may resemble a mild flu, with some patients experiencing symptoms resembling mild meningitis or encephalitis, hepatitis, musculoskeletal pam, enlarged spleen, and cough Weeks to months later, the patient often shows signs of the second stage of the disease, developing arthritic joint pain, and sometimes neurologic or cardiac abnormalities. The neurological complications may include visual, emotional, and memory disturbances, temporary paralysis of a facial nerve, and movement difficulties The third stage, which may appear months to years after infection, is characterised by crippling arthritic symptoms in one or more joints, especially in the knees, and severe neurological symptoms that mimic multiple sclerosis. These symptoms are believed to be caused by the body's immune defense system's attempts to fight the infective agent, rather than by the organism itself The antibody complexes produced in response to the infective agent cause the joints to become inflamed. Treatment with trivalent antibodies neutralizes the toxins produced by B. burgdorferi.

Lyme disease

Recent epidcmiologk and laboratory investigations indicate that Lyme disease may be a different manifestation of erythema chronicum miggrans, a syndrome long recognized in Europe. Both diseases arc caused by the spirochete, Borrelia burgdorferi, transmitted to humans by the bites of Ixodes ticks Similar spirochetes have been recovered from the blood of Lvme disease patients and cultured from ixodes ticks, and patients with Lyme disease have antibodies to the cultured spirochetes. When this disease is diagnosed, penicillin is an effective treatment. There is also a new vaccine that appears to be effective for preventing Lyme disease

Epidemiology of Lyme disease. In October 1975 the Connucticut Department received independent calls reporting multtpk eases of what speared to be arthritis m chiMren in lyme and Old Lyme, rural towns in that state. Despite being assured by their physicians that arthritis was not infectious, the callers were not satisfied An epidemic investigation ensued in which the extent, characteristicis, mode of transmission, and etiology of the cluster of cases were studied. As characteristic of many epidemidiogical investigations, public health officiate began by trying to locate all individuals who had sudden onset of swelling and pain in the knee or other large joints lasting a weelk to several months. An old, large skin rash, repeated attacks at intervals of a few months, fever, and fatigue were the reported symptoms. State epidemiologists questioned parents and physidans – asking were the cases related, were there other similar cases, wasthis an infectious form of arthritis, and what forms of arthritis are infectious? Next the epidemiologists determined the time, place, and personal characteristics of these cases. The incidence of onset of disease seemed to cluster in late spring and summer and lasted from a week to a few months The cases were concentrated in three adjacent towns on the eastern side of the Connecticut River and most patients lived in wooded areas near lakes and streams. Of the 51 cases, 39 were children about evenly split between boys and girls There were no familial patterns. Epidemiologists created an epidemic curve, listing the cases by the time of onset, and began calling the disease "Lyme arthritis".

The clustering of cases, the fact that most began in late spring or summer and that they were most frequently located in wooded areas along lakes or streams suggested a disease transmitted by an arthropod A study was undertaken to determine if this was a communicable disease. Cases of the disease were matched with a similar group of control or unaffected persons for age, sex, and other relevant factors. It was found that affected people were more likely to have a household pet than those who were unaffected. Pet owners are more likely to come in contact with ticks that their dogs and cats might pick up m me woods. The importance of this finding was emphasized when combined with the fact that one fourth of the patients reported that their arthritic symptoms were preceded by an unusual skin rash that started as a red spot that spread to a 6-inch ring. A dermatology consultant recalled a similar skin outbreak reported in Switzerland in 1910 that was attributed to tick bites.

This was only suggestive evidence that a tick bite might initiate an infectious disease. The connection between the rash and the disease had to be strengthed.

Now public health authorities had to ask if patients with such a rash always progress to develop Lyme arthritis. A prospective study lookcd for new patients with a rash. Of 32 new cases of the characteiastic skin rash, 19 progressed to show signs and symptoms of Lyme disease. The tick connection was strengthened after an entomological study found that adult ticks were 16 times more abundant on the east side of the Connecticut River than the west This corresponded to the proportion of incidence of the disease on each side of the river. Also, more tick bites were reported by the arthritis sufferers then by their unaffected neighbors A surveillance network was set up in. Connecticut and surrounding stales to gather information about other cases. These investigations showed more adult victims than children and also more serious manifestations, including neurological and heart diseases.

The Rocky Mountain Public Health Laboratory in Montana was asked to assist in the investigation because of its expertise in the area of tickborne disease. They found unusual spirochetes in the guts of many of the ticks sent from Connecticute. Spirochetes, which are bacteria with curved cells wound around a central filament, are often difficult to culture and so it would be difficult to prove that these were the causative organisms of Lyme disease Therefore they first tried to infect laboratoiy animals with the infected ticks The rabbits developed rashes resembling those seen in humans. A spirochete was isolated ftom the ticks and when pure cultures were inoculated into rabbite, the rabbits developed the characteristic rash. The infected rabbits contained antispirochetal antibodies in their serum. The identification was complete when the spirochete was isolated from human cases. The spirochete was classified as a member of the Borrelia genus and named Borrelia burgdorferi after the entomologist who discovered the organisms in the ticks.

Lyme disease accounted for more than 90 % of the vector-home infectious diseases in the United States in 1992. The distribution of the disease was highly correlated with the distribution, of the principal tick vectors. The nearly twerrtyfold increase in cases since the early 1980s may be a consequense of increased surveillance and improved diagnostic methods, or a real increase in disease prevalence due to increases in deer and tick populations and closer human contact with these animals.

Summary. Lyme disease is characterized by the development of arthritis and neurological symptoms that result when the body's immune defenses react to infections with the spirochete Borrelid burgdorferi.

The occurrence of Lyme disease has been concentrated in the Northeast and other areas where Ixodes ticks carry Borrelia burgdorferi.

Additionala material abour laboratory diagnosis

RECURRENT FEVER (EPIDEMIC). The causative agent of epidemic (louse-borne) relapsing fever (Borrelia recurrentis), of endemic (tick-borne) typhus (Borrelia persica) and some other microorganisms induce clinically similar acute infectious diseases whose course is characterized by fever attacks, general intoxication, and liver and spleen enlargement.

The main method of investigation in recurrent types of typhus is bacterioscopic examination, that is visualization of the causal organism in the patient's blood.

Bacterioscopic examination. During or before an attack of relapsing fever take a sample of blood from the patient and make a thick-drop preparation and a smear. First, stain a thick-drop preparation, since its examination provides better possibilities to obtain a positive result. On a dried unfixed preparation of a blood drop pour the Romanowsky-Giemsa dye and allow it to act for 35-45 min. Haemo-lysis occurs in a drop (the dye is diluted with distilled water), so 5 min later replace the dye with a fresh portion which is kept in place for 30-40 min. Then, the preparation is carefully washed with water and dried. Under the microscope Borrelia look like thin blue-violet threads with several large coils (secondary coils). The length of Borrelia varies from 10 to 30 u-m or more. Sometimes their size is several times larger than the diameter of leucocytes (the preparation contains only leucocytes because erythrocytes are lysed). When Borrelia are present in large numbers, which is characteristic of relapsing fever, their aggregates may pose difliculty with regard to their differentiation from fibrin threads. In this case examine micro-scopically a smear which is dried, fixed with alcohol or Nikiforov's mixture, and stained with the Romanowsky-Giemsa dye or Pfeiffer's fuchsine. Borrelia are readily visualized by microscopy. They are at least twice as long as the diameter of red blood cells. If no Borrelia are found in a thick blood film, smears are not subjected to examination.

When the patient is afebrile, Borrelia cannot he detected by the ordinary methods of investigation. Hence, enrichment methods are recommended to be used in such cases. Withdraw from the vein 8-10 ml of blood, wait until it has coagulated, separate the serum, and centrifuge it at 3000 X g for 45-60 min. Then, quickly pour off the fluid by inverting the test tube, make wet-mount preparations from the sediment (which are examined by dark-field microscopy), and thick smears (which are fixed, stained, and studied in the manner used with blood smears).

Biological examination. To confirm the diagnosis of tick-borne recurrent typhus, inject, pubciitaneously 0.5-1 ml of citrate blood to guinea pigs. Five-six day? later one can observe large numbers of Borrelia in the blood of the infected animals. This method permits the differentiation of the causative agents of epidemic versus tick-borne recurrent typhus fever. The latter induce the disease in guinea pigs, whereas B. recurrentis is non-pathogenic for them

References:

1.     Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 149-165.

2.     Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 151-164.

3.     Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P. 285-314.

4.     Essential of medical microbiology /W. A. Volk.. et al.– 5 ed., 1995

SUPPLEMENT

http://en.wikipedia.org/wiki/Treponema_pallidum

http://www.cehs.siu.edu/fix/medmicro/trepo.htm

http://microbewiki.kenyon.edu/index.php/Treponema

http://www.gsbs.utmb.edu/microbook/ch036.htm

http://www.bacteriamuseum.org/species/Tpallidum.shtml

http://dermatlas.med.jhmi.edu/derm/result.cfm?Diagnosis=1596362672 !!!!! Atlas

http://en.wikipedia.org/wiki/Bejel

http://en.wikipedia.org/wiki/Pinta_(disease)

http://microbewiki.kenyon.edu/index.php/Leptospira

http://en.wikipedia.org/wiki/Leptospira

http://www.gsbs.utmb.edu/microbook/ch035.htm

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.chapter.1929

http://microbewiki.kenyon.edu/index.php/Borrelia

http://en.wikipedia.org/wiki/Borrelia_burgdorferi

http://textbookofbacteriology.net/Lyme.html

http://www.wadsworth.org/databank/borreli.htm

http://www.kcom.edu/faculty/chamberlain/Website/Lects/RICKETT.HTM !!!

www.Healthline.com

http://en.wikipedia.org/wiki/Coxiella_burnetii

http://www.cdc.gov/ncidod/dvrd/qfever/

http://microbewiki.kenyon.edu/index.php/Coxiella

http://menshealth.about.com/b/a/190382.htm

http://en.wikipedia.org/wiki/Chlamydia

http://www.netdoctor.co.uk/diseases/facts/chlamydia.htm

www.gsbs.utmb.edu/microbook/ch039.htm

http://pathmicro.med.sc.edu/mayer/chlamyd.htm

http://en.wikipedia.org/wiki/Mycoplasma

www.emedicine.com/EMERG/topic467.htm

www.health.state.ny.us/diseases/communicable/mycoplasma/fact_sheet.htm

http://www.gsbs.utmb.edu/microbook/ch037.htm