spirochetes. Laboratory diagnosis of spirochetoses (syphilis, leptospirosis, relapsing fever, Lyme disease).

RICKETTSIAE, CHLAMYDIAE, AND MYCOPLASMAS.  microbiOLogic diagnosis of diseases CAUSED BY RICKETTSIAE, CHLAMYDIAE, AND MYCOPLASMAS

 

 

TREPONEMA PALLIDUM

Morphology and identification. A. Typical Organisms: Slender spirals measur­ing about 0.2 mcm in width and 5-15 mcm in length. The spiral coils are regularly spaced at a distance of 1 mcm from each other. The organisms are actively motile, rotating steadily around their central axial filaments. The long axis of the spiral is ordinarily straight but may sometimes bend, so that the organism forms a com­plete circle for moments at a time, returning then to its normal straight position.

The spirals are so thin that they are not readily seen unless darkfield illumination or immunofluores cent stain is employed. They do not stain well with aniline dyes, but they do reduce silver nitrate to metal­lic silver that is deposited on the surface, so that treponemes can be seen in tissues (Levaditi silver impregna­tion).

 

Typical organism of Treponema pallidum from tissue fluid in dark field.

 

 

Treponema pallidum

 

 

Treponemes ordinarily reproduce by transverse fission, and divided organisms may adhere to one another for some time.

B. Culture: Treponema pallidum pathogenic for humans has never been cultured with certainty on artificial media, in fertile eggs, or in tissue culture. Nonpathogenic treponemes (eg, Reiter strain) can be cultured anaerobically in vitro. They are saprophytes antigenically related to T pallidum.

C. Growth Characteristics: Because T. pal­lidum cannot be grown, no studies of its physiology have been made. The growth requirements for one cultured probably saprophytic strain (Reiter) have, however, been established. A defined medium of 11 amino acids, vitamins, salts, minerals, and serum al­bumin supports its growth.

In proper suspending fluids and in the presence of reducing substances, T pallidum may remain motile for 3-6 days at 25 °C. In whole blood or plasma stored at 4 °C, organisms remain viable for at least 24 hours, which is of potential importance in blood trans­fusions.

D. Reactions to Physical and Chemical Agents: Drying kills the spirochete rapidly, as does elevation of the temperature to 42 °C also. Treponemes are rapidly immobilized and killed by trivalent arseni-cals, mercury, and bismuth. This killing effect is ac­celerated by high temperatures and can be partially reversed and the organisms reactivated by compounds containing -SH (eg, cysteine, BAL [dimercaprol]). Penicillin is treponemicidal in minute concentrations, but the rate of killing is slow, presumably because of the metabolic inactivity and slow multiplication rate of the organism (estimated division time is 30 hours). Resistance to penicillin has not been demonstrated in syphilis.

E. Variation: A life cycle has been postulated for T pallidum, including granular stages and cystlike spherical bodies in addition to the spirochetal form. The occasional ability of T pallidum to pass through bacteriologic filters has been attributed to the filtrabihty of the granular stage.

Antigenic Structure. The antigens of T pallidum are unknown. In the human host, the spirochete stimulates the development of antibodies capable of staining T pallidwn by indi­rect immunofluorescence, of immobilizing and killing live motile T pallidum, and of fixing complement in the presence of suspensions of T. pallidum or related spirochetes. The spirochetes also cause the develop­ment of a distinct antibodylike substance, reagin, which gives positive complement fixation and flocculation tests with aqueous suspensions of lipids ex­tracted from normal mammalian tissues. Both reagin and antitreponemal antibody can be used for the serologic diagnosis of syphilis.

Pathogenesis, Pathology, and Clinical Findings.  A. Acquired Syphilis: Natural infection with T pallidum is limited to the human host. Human infec­tion is usually transmitted by sexual contact, and the infectious lesion is on the skin or mucous membranes of genitalia. In about 10% of cases, however, the primary lesion is extragenital (often oral). T palli­dum can probably penetrate intact mucous membranes, or it may enter through a break in the epider­mis.

Spirochetes multiply locally at the site of entry, and some spread to nearby lymph nodes and then reach the bloodstream. In 2-10 weeks after infection a papule develops at the site of infection and breaks down to form an ulcer with a clean, hard base (“hard chancre”). The inflammation is characterized by a predominance of lymphocytes and plasma cells. This “primary lesion” always heals spontaneously, but 2-10 weeks later the “secondary” lesions appear. These consist of a red maculopapular rash anywhere on the body and moist, pale papules (condylomas) in the anogenital region, axillas, and mouth. There may also be syphilitic meningitis, chorioretinitis, hepatitis, ne­phritis (immune complex type), or periostitis. The secondary lesions also subside spontaneously. Both primary and secondary lesions are rich in spirochetes and highly infectious. Contagious lesions may recur within 3-5 years after infection, but thereafter the individual is not infectious. Syphilitic infection may remain subclinical, and the patient may pass through the primary or secondary stage (or both) without symp­toms or signs yet develop tertiary lesions.

In about 30% of cases, early syphilitic infection progresses spontaneously to complete cure without treatment. In another 30% the untreated infection re­mains latent (principally evident by positive serologic tests). In the remainder the disease progresses to the “tertiary stage”, characterized by the development of granulomatous lesions (gummas) in skin, bones, and liver; degenerative changes in the central nervous sys­tem (paresis, tabes); or syphilitic cardiovascular le­sions, particularly aortitis (sometimes with aneurysm formation) and aortic valve insufficiency. In all ter­tiary lesions treponemes are very rare, and the exag­gerated tissue response must be attributed to some form of hypersensitivity to the organisms. However, treponemes can occasionally be found in the eye or central nervous system in late syphilis.

 

Primary syphilis

 

 

Oral syphilis             Secondary  syphilis

 

 

Secondary  syphilis

 

 

B. Congenital Syphilis: A pregnant syphilitic woman can transmit T pallidum to the fetus through the placenta beginning about the tenth week of gesta­tion. Some of the infected fetuses die and miscarriages result; others are stillbom at term. Others are bom live but develop the signs of congenital syphilis in child­hood; interstitial keratitis, Hutchinson‘s teeth, saddle nose, periostitis, and a variety of central nervous sys­tem anomalies. Adequate treatment of the mother dur­ing pregnancy prevents congenital syphilis. The reagin titer in the blood of the child rises with active infection but falls with time if antibody was passively transmit­ted from the mother. In congenital infection the child makes IgM antitreponemal antibody.

 

 

Congenital Syphilis

 

C. Experimental Disease: Rabbits can be ex­perimentally infected in the skin, testis, and eye with human T. pallidum. The animal develops a chancre rich in spirochetes, and organisms persist in lymph nodes, spleen, and bone marrow for the entire life of the animal, although there is no progressive disease.

 

Diagnostic Laboratory Tests. A. Specimens: Tissue fluid expressed from early surface lesions for demonstration of spirochetes; blood serum for serologic tests.

B. Dark field Examination: A drop of tissue fluid or exudate is placed on a slide and a coverslip pressed over it to make a thin layer. The preparation is then examined under oil immersion with dark field illumination for typical motile spirochetes.

Treponemes disappear from lesions within a few hours after the beginning of antibiotic treatment.

C. Immunofluorescence: Tissue fluid or exu­date is spread on a glass slide, air dried, and mailed to the laboratory. It is fixed, stained with a fluorescein-labeled antitreponeme serum, and examined by means of immunofluorescence microscopy for typical fluorescent spirochetes.

D. Serologic Tests for Syphilis (STS): These use either Ireponemal or nontreponemal antigens.

1.     Nontreponemal antigen tests.

The antigens employed are lipids extracted from normal mammalian tissue. The purified cardiolipin from beef heart is a diphosphatidylglycerol. It requires the addition of lecithin and cholesterol or other “sensitizers”  to react with syphilitic “reagin.” “Reagin” is a mixture of IgM and IgA antibodies directed against some antigens widely distributed in normal tissues. Reagin is found in patients’ serum after 2-3 weeks of untreated syphilitic infection and in spinal fluid after 4-8 weeks of infec­tion. Two types of tests determine the presence of reagin.

a. Flocculation tests (VDRL [Venereal Disease Research Laboratories], etc) are based on the fact that the particles of the lipid antigen (beef heart car­diolipin) remain dispersed in normal serum but com­bine with reagin to form visible aggregates within a few minutes, particularly if the solution is agitated. The rapid plasma reagin (RPR) test is a convenient modification for rapid surveys. Positive VDRL tests revert to negative 6-24 months after effective treat­ment of early syphilis.

b. Complement Fixation (CF) tests (Wassermann, Kolmer) are based on the fact that reagin-containing sera fix complement in the presence of cardiolipin “antigen. ” It is necessary to ascertain that the serum is not “anticomplementary” (i.e., that it does not destroy complement in the absence of antigen).

Both (a) and (b) can give quantitative results. An estimate of the amount of reagin present in serum can be made by performing (a) or (b) with 2-fold dilutions of serum and expressing the liter as the highest dilution that gives a positive result. Quantitative results are valuable in establishing a diagnosis and in evaluating the effect of treatment.

Nontreponemal tests are subject to false-positive results. These either are due to technical difficulties of the test or are  ‘biologic ‘ ‘ false positives attributable to the occurrence of “reagins” in a variety of human disorders. Prominent among the latter are other infec­tions (malaria, leprosy, measles, infectious mononu-cleosis, etc), vaccinations, collagen-vascular diseases (systemic lupus erythematosus, polyarteritis nodosa, rheumatic disorders), and other conditions. Non­treponemai antibody tests may become negative spon­taneously in progressive tertiary syphilis; thus, a nega­tive VDRL does not rule out such disease activity.

 

 

Complement Fixation (CF) test

 

2. Treponemal antibody tests

a. Fluorescent treponemal antibody (FTA-ABS) test-A test employing indirect immunofluores-cence (killed Tpallidum + patient’s serum + labeled antihuman gamma globulin) shows excellent spec-ificity and sensitivity for syphilis antibodies if the patient’s serum, prior to the FTA test, has been ab­sorbed with sonicated Reiter spirochetes. The FTA-ABS test is the first to become positive in early syphilis, and it usually remains positive many years after effective treatment of early syphilis. The test cannot be used to judge the efficacy of treatment. The presence of IgM FTA in the blood of newboms is good evidence of in utero infection (congenital syphilis).

b. TPI test- Demonstration of T pallidum im­mobilization (TPt) by specific antibodies in the pa­tient’s serum after (he second week of infection. Dilu­tions of serum are mixed with complement and with live, actively motile T pallidum extracted from the testicular chancre of a rabbit, and the mixture is ob­served microscopically. If specific antibodies are pres­ent, spirochetes are immobilized: iormal se­rum, active motion continues. This test requires live treponemes from infected animals and is hard to per­form.

c. T pallidum complement Fixation test- Spiro­chetes extracted from syphilomas of rabbits form spe­cific antigens for complement fixation tests that proba­bly measure the same antibody as the TPI lest, above. Such spirochetal suspensions are difficult to prepare. Antigens prepared from cultured Reiter spirochetes ‘are occasionally employed in the Reiter complement fixa­tion test.

d. T. pallidum hemagglutination (TPHA) test– Red blood cells are treated to adsorb treponemes on their surface. When mixed with serum containing antitreponemal antibodies, the cells become clumped. This test is similar to the FTA-ABS test in speciflcity and sensitivity, but it becomes positive somewhat later in the course of infection.

VDRL and FTA-ABS tests can also be performed on spinal fluid. Antibodies do not reach the cerebro-spinal fluid from the bloodstream but are probably formed in the central nervous system in response to syphilitic infection,

Immunity. A person with active syphilis or yaws appears to be resistant to superinfection with T pallidum. How­ever, if early syphilis or yaws is treated adequately and the infection is eradicated, the individual again be­comes fully susceptible.

Treatment. Penicillin in concentrations of 0.003 unit/mL has definite treponemlcidal activity, and penicillin is the treatment of choice. In early syphilis, penicillin levels are maintained for 2 weeks (eg, a single injection of benzathine penicillin G, 2.4 million units intramuscu-larly); in latent syphilis, benzathine penicillin G, 2.4 million units intramuscularly, is given 3 times at weekly intervals. Ieurosyphilis, the same therapy is acceptable, but larger amounts of penicillin (eg, aque­ous penicillin G, 20 million units intravenously daily for 2-3 weeks) are sometimes recommended. Other antibiotics can occasionally be substituted. Prolonged follow-up is essential. Ieurosyphilis, treponemes occasionally survive such treatment. A typical Jarisch-Herxheimer reaction may occur within hours after treatment is begun. It is probably due to the sudden release of endotoxin from spirochetes.

Epidemiology, Prevention, and Control. At present, the incidence of syphilis (and other sexually transmitted diseases) is rising in most parts of the world. With the exceptions of congenital syphilis and the rare occupational exposure of medical person­nel, syphilis is acquired through sexual exposure. An infected person may remain contagious for 3-5 years during “early” syphilis. “Late” syphilis, of more than 5 years’ duration, is usually not contagious. Con­sequently, control measures depend on (1) prompt and adequate treatment of all discovered cases; (2) follow-up on sources of infection and contacts so they can be treated; (3) sex hygiene; and (4) prophylaxis at the time of exposure. Both mechanical prophylaxis (condoms) and chemoprophylaxis (eg, penicillin after exposure) have .great limitations. Washing the genitalia after exposure may afford some protection to the male. Several venereal diseases can be transmitted simultaneously. Therefore, it is important to consider the possibility of syphilis when any one sexually transmitted disease has been found.

 

DISEASES RELATED TO SYPHILIS.  These diseases are all caused by treponemes in­distinguishable from T pallidum. All give biologic tme-positive serologic tests for syphilis, and some cross-immunity can be demonstrated in experimental animals and perhaps in humans. All are nonvenereal diseases and are commonly transmitted by direct con­tact. None of the causative organisms have been cul­tured on artificial media.

 

Bejel. Bejel occurs chiefly in Africa but also in the Middle East, in Southeast Asia, and elsewhere, par­ticularly among children, and produces highly infec­tious skin lesions; late visceral complications are rare. Penicillin is the dmg of choice.

 

 

Yaws (Frambesia). Yaws is endemic, particularly among children, in many humid, hot tropical countries. It is caused by Treponema pertenue. The primary lesion, an ulcerat­ing papule, occurs usually on the arms or legs. Trans­mission is by person-to-person contact in children under age 15. Transplacental, congenital infection does not occur. Scar formation of skin lesions and bone destruction are common, but visceral or nervous sys­tem complications are very rare. It has been debated whether yaws represents a variant of syphilis adapted to nonvenereal transmission in hot climates. There appears to be cross-immunity between yaws and syphilis. Diagnostic procedures and therapy are simi­lar to those for syphilis. The response to penicillin treatment is dramatic.

 

 

 

 

Pinta. Pinta is caused by Treponema carateum and oc­curs endemically in all age groups in Mexico, Central and South America, the Philippines, and some areas of the Pacific. The disease appears to be restricted to dark-skinned races. The primary lesion, a nonulcerat-ing papule, occurs on exposed areas. Some months later, flat, hyperpigmented lesions appear on the skin; depigmentation and hyperkeratosis take place years afterward. Late cardiovascular and nervous system involvement probably occurs. Transmission is nonve­nereal , either by direct contact or through the agency of a fly (Hippelates). Diagnosis and treatment are the same as for syphilis.

 

 

Rabbit Syphilis.  Rabbit syphilis (Treponema cuniculi) is a natural venereal infection of rabbits producing minor lesions of the genitalia. The causative organism is morpholog-ically indistinguishable from T pallidum and may lead to confusion in experimental work.

 

Additional material about laboratory diagnosis

Syphilis is a chronic illness which affects the skin, infernal organs, and central nervous system. Its causative agent, is Treponema pallidum.

Methods of the laboratory diagnosis of syphilis are determined by the pathogenesis of tills disease. In the primary period of the illness (less commonly in the secondary and tertiary ones) the major tool of diagnosis is bacterioscopic examination, whereas in the second­ary and tertiary periods one largely relics on serological methods.

Bacterioscopic examination. The material to be studied is tissue fluid obtained from the bottom of an ulcer (hard chancre) or aspira­tion biopsy sample of regional lymph nodes. Before taking the material, wipe the surface of a chancre with a sterile cotton tampon soaked with isotonic sodium chloride solution. Then slightly irritate the bottom of the ulcer with a platinum spatula or a scalpel, express tissue fluid with fingers protected by a rubber glove, collect it in a capillary of a Pasteur pipette, and immediately prepare a wet-mount preparation to be examined by dark-field microscopy. A positive result is recognized by the detection of motile treponemas with regular large curls. Non-pathogenic borrelias (Borrelia refringens) which may be found in the material taken from the mucosa of tlie external genitalia differ morphologically from pathogenic treponemas (by tliickness, irregularity of curls). Moreover, they are unable to move progressively. If the chancre is located on the oral mucosa, differentiation between T. pallidum and T. microdentium (usual inhabitants of the oral cavity) presents much difficulty. In this case one examines aspiration biopsy sample of regional lymph nodes. Detection of typical treponemas confirms the diagnosis of syphilis.

Preparations stained by the Romanowsky-Giemsa method are also studied for the diagnostic purpose. Following fixation with methyl alcohol or Nikiforov’s mixture, stain smears for 2-4 hrs or heat the dye three times until fumes rise, replacing the dye every minute with a new portion, the last portion of the dye being allowed to act for 5 min. The stained preparations are washed with water and dried in the air, T. pallidum acquire a light-pink colour, B. refringens stain red-violet. Saprophyte treponemas are characterized by a more intense staining.

In Burri’s negative preparations one can easily see the form of treponemas and their curls against a black background.

In the secondary and tertiary periods of the disease the material obtained from the involved foci on the skin is examined in the same manner.

Serological diagnosis. The most prevalent of serological methods employed in the diagnosis of syphilis is the Wassermann reaction. To carry it out, one utilizes the patient’s blood serum or the cerebrospinal fluid (when the nervous system is involved).

The procedure of conducting the Wassermann reaction (Table) is almost exactly the same as that of the CF test. The only difference lies in the fact that in the Wassermann reaction lipids of syphilis affected tissue and normal tissue are used as an antigen.

Table

Schematic Representation of the Main Stage of the Wassermann Reaction

Ingredient, ml	Number of the test tubes
	1	2	3	4
Patient’s serum, inactivated and diluted 1:5	0.5	0.5	0.5	0.5
Antigen 1 (specific) 0.5	0.5	–	–	–
Antigen 2 (non-specific)	–	0.5	–	–
Antigen 3 (non-specific)	–	–	0.5	–
Isotonic sodium chloride solution	–	–	–	0.5
Complement	0.5	0.5	0.5	0.5
Incubation at 37 °C for 45 min
Haemolytic system, sensitized	1,0	1,0	1,0	1,0
Incubation at 37 °C for 1 h
Results

 

Specific antigens (alcohol extracts) present potent lipids of the liver from a foetus that has died of syphilis and testicular tissue of an infected rabbit. Non-specific antigens are lipids of the normal tissue (heart muscle of ox or horse), which are less active. Antigens for the Wassermann reaction are available commercially.

Before running the test, mix the antigens with isotonic sodium chloride solution, according to the rules described in the manual. Since the commercial extracts differ in specificity, use several antigens (one specific and two non-specific ones). All the remaining ingredients of the reaction are prepared as it is done for the CF test.

Known positive (from a syphilitic patient) and negative (from a healthy subject) reacting sera are used in the reaction as a control.

When the cerebrospinal fluid (GSF) is examined by the Wasser­mann reaction, the inactivation procedure is unnecessary since the CSF contains no complement. Undiluted cerebrospinal fluid and that diluted 1 : 1 and 1 : 5 with isotonic saline are used. The cerebrospi­nal fluid possesses no anticomplement properties, so complement is added in an amount corresponding to its titre.

When making the serological diagnosis of syphilis, apart from the Wassermann reaction, one should employ at least two sedimentation reactions based on precipitation induced by the interaction of the patient’s serum with antigens (tissue lipids) whose sensitivity is enhanced by adding cholesterol. The most accurate and feasible for any laboratory are tests proposed by Kahn and Sachs-Witebsky.

During Kahns test (Table) the patient’s serum is inactivated at 56 °C for 30 min. The antigen is diluted with isotonic sodium chloride solution as prescribed on the label. Pour the antigen into one test tube and isotonic saline into another, then rapidly pour the saline into the antigen (and not in the reverse order), pour the mix­ture from one test tube into the other several times, and let it stand for 10 min. Mix the patient’s serum with the antigen, shake the tube, incubate at 37 °C for 10 min, add isotonic sodium chloride solution, and read the results of the reaction.

Table

Schematic Representation of the Kahn and Sachs-Witebsky Reactions

 

The Sachs-Witebsky test (cyt.ocholic) is a modified Kahn’s test. The employment in this reaction of a concentrated antigen leads to a faster sedimentation of grossly dispersed particles and allows a more accurate assessment of the results. The antigen is prepared in the following manner. One part of tlie lipid extract is rapidly poured into a test tube with two parts of isotonic sodium cliloride solution, the mixture being allowed to stand for 10 min. Mix the patient’s serum with the antigen, shake the tube, add isotonic saline, and read the results of the reaction.

Positive results of the Kahn and Sachs-Witebsky tests are evi­denced by the appearance, following the addition of saline, of a preci­pitate in the form of large or small Hakes which are better detected by the agglutinoscope.

The sedimentation reactions are particularly sensitive in the diagnosis of primary and latent syphilis. The Wassermann reaction yields more definite results in congenital syphilis.

The reaction of treponema immobilization is also widely utilized in the serological diagnosis of syphilis.

Procedure. Prepare a special antigen (a suspension of live T. pallidum from the teslicular tissue of a rabbit infected with a laboratory strain (Nikols’ strain No. 8 isolated by the CDVI) [Central Dermato-Venereological Institute] of the causative agent). The antigen is considered suitable if over 50 motile treponemas are detected by microscopy. Blood samples from patients are taken under sterile conditions. The reaction should not be undertaken if within the last 3 weeks the patient has received antibiotics or other antisyphilitic drugs.

To carry out the reaction, mix in a test tube 0.05 ml of the blood serum. 0.3 ml of the antigen, anil 0.2 ml of the complement. The test is attended by the control of the serum, antigen, and comple­ment. Place the test tubes into an anaerobic jar which, following the creation of vacuum, is filled witli a mixture of nitrogen and carbon dioxide and kept at 35 C. In 18 hrs, make wet-mount preparations from the contents of each test tube, count 25 treponemas. and deter­mine tlie ratio of mobile and immobile ones. The percentage of tre­ponema immobilization is calculated by tlie formula:

(A-B) x 100    ,  

A

where A is the number of motile treponemas in llie control, B is tlie number of mobile microorganisms in tlie test. When the proportion of immobile treponemas varies   from 0 to 20  per cent, the reaction is considered negative, from 21 to 50 per  cent weakly positive, and from 51 to 100 per cent, positive. If the patient has syphilis, the first preparation shows immobilization of treponemas, while in the control ones they remain mobile. This test is more specific and sensitive than llie Wassermann reaction and sedimentation reactions witli both the patient’s serum and cer.ebrospinal fluid, particularly in congenital and visceral syphilis and syphilis of the nervous system.

One can carry out tlie test of immobilization of T. pallidutn in cases where the volume of ingredients constitutes 0.35 mi (0.05 ml of the studied serum. 0.1 ml ol’ complement, 0.2 ml of antigen). If the results are doubtful, the reaction is repeated after increasing the dose of the serum tested (up to 0.1 ml).

The microreaction of immobilization of T. pallidum may be used as a presumptive test in the diagnosis of syphilis.

A blood sample from the patient’s linger is collected iiioueuf tlie capillar­ies of Panchenkov’s apparatus. Into a ceutrifiigc test tube with 0.025 ml of 5 per cent solution of sodium citrate intruduce the blood from the capillary, al­low the mixture to settle down for 1.5-2 hrs or centrifuge for 5-10 min at 1000-2000 X g. To one drop of the obtained plasma add 1 drop of antigcuic emulsion, and then rock the glass slide for 3-5 min.  A positive reaction is witnessed by the formation of large flakes, a weakly positive one. by small Gakes, a negative one, by opalescence.

Antigenic emulsion for the microreaction is prepared in tlie following man­ner: 1 ml of cardiolipid antigen is supplemented with 1 nil of isotonic sodium chloride solution, the mixture obtained is permitted to stand at room tempera­ture for 30 min, and then ceiitrifuged at 1000-2000 X g for 15 min. Decant the supernatant, add 3.5 ml of 10 per cent cholin chloride, and sliake tlie mixture. The obtained emulsion is kept at 4 °C under a cork plug. The shelf life is 7 days.

Another reaction employed in llie serological diagnosis of syphi­lis is the indirect IF test.

On a glass slide put an antigen (T. pallidum obtained from the testicular tissue of an infected rabbit) in siicli an amount that there are 50-60 microorgan­isms per microscopic field. Dry the smears in the air and fix (horn in acetone for 5 min. The patient’s serum in a 1:200 dilution is placed on the preparations and kept in a moist chamber at. 35 °C for 30 min (phase 1). Then the smears are washed for 10 min in a buffer isotonic saline and dried in the air. After that, place a drop of fluorescent seriim against human globulins onto the preparation and reincubate in a moist chamber at room temperature for another 30 min {phase II). Wash the smears in two portions of hiil’frr saline and examine them by luminescent microscopy- Tlie reaction is assessed by the degree of yellow-green fluorescence of treponemas. Using this reaction, one can determine the litre of antibodies in the patient’s blood serum by placing its different dilu­tions on a series of preparations.

The reactions of treponema immobilization and IF are consid­ered as the most specific tests in flip laboratory diagnosis of syphilis.

 

FRAMBESIA. Frambesia (synonyms: yaws. pian. boiiba, parangi, patek. dube, gatto kegeti, mombar) is chronic recurrent treponeniatosis involving the skin, mucosal membranes, bone–, and joints. The internal organs are never affected. Tlie disease occurs in the countries of Africa, South-East Asia, South America, and on the Oceania islands. The causative ag-ent of the illness is Treponema pertenue.

The material used for examination includes the secretion of cuta­neous frambesides (papules, papillomas). which is examined bacterio-scopically for the presence of Ireponemas. Morphologically, the causative organism of tills diseases is absolutely identical with Treponema pallidum. Motility of T. perienue is determined by dark-held microscopy with the help of an oil-immersion system.

Serologieal diagnosis of frambesia is based on the detection of antibodies in the patients’ sera by means of the Wassermann reaction, Kahn’sandcytocholic tests and the reaction of treponema immobilization.

 

PINTA. Pinta (synonyms: mal del pinto, carate, azul, boiissarole) presents chronic treponematosis characterized by skin lesions in the form of pigmentation disorders and hyperkeratosis. It is encountered in the countries of Central America {Mexico, etc.) and the Caribbean basin. Tlie causative agent of this illness is Treponema carateum which is similar to other treponemas.

The material to be examined includes scrapings from the damaged sites of the skin and patients’ serum.

Bacterioscopic examination consists in detecting treponemas during microscopic examination of preparations stained by the -Romanowsky-Giemsa technique.

Serologieal diagnosis. Antibodies in the patients’ sera are revealed with the help of the Wassermann, Kahn’s. and cytocholic reactions’ and the test of treponema immobilization. Because of a very close similarity of the causative organism of pinta and the causative agents of syphilis and frambesia the diagnostic value of serological reactions is low.

 

LEPTOSPIRAE

Morphology and Identification

A. Typical Organisms: Tightly coiled, thin, flexible spirochetes 5-15 JU,m long, with very fine spirals 0.1-0.2 jU-m wide. One end of the organism is often bent, forming a hook. There is active rotational motion, but no flagella have been discovered. Electron micrographs show a thin axial filament and a delicate membrane. The spirochete is so delicate that in the dark field it may appear only as a chain of minute cocci. It does not stain readily but can be impregnated with silver.

 

 

Leptospira interrogans

 

 

B. Culture: Leptospirae grow best aerobically at 30 °C in peptone broth containing 10% inactivated (rabbit) serum. On semisolid meat extract media (Fletcher) containing 10% rabbit semm, round colo­nies 1-3 mm in diameter develop in 6-10 days. Lep­tospirae also grow on chorioallantoic membranes of embryonated eggs.

C. Growth Requirements: Leptospirae derive energy from oxidation of long chain fatty acids and cannot use amino acids or carbohydrates as major energy sources- Ammonium salts are a main source of nitrogen. Leptospirae can survive for weeks in water, particularly at alkaline pH.

Antigenic Structure. The main strains of leptospirae isolated from hu­mans or animals in different parts of the world are all serologically related and exhibit marked cross-reactivity in serologic tests. This indicates con­siderable overlapping in antigenic structure, and quan­titative tests and antibody absorption studies are neces­sary for a specific serologic diagnosis. From many strains of leptospirae, a serologically reactive lipopolysaccharide has been extracted that has group reactivity.

Pathogenesis and Clinical Findings. Human infection results usually from ingestion of water or food contaminated with leptospirae. More rarely, the organisms may enter through mucous membranes or breaks in the skin. After an incubation period of 1-2 weeks, there is a variable febrile onset during which spirochetes are present in the blood­stream. They then establish themselves in the paren-chymatous organs (particularly liver and kidneys), producing hemorrhage and necrosis of tissue and re­sulting in dysfunction of those organs (jaundice, hemorrhage, nitrogen retention). The central nervous system is frequently invaded, and this results in a clinical picture of ‘ ‘aseptic meningitis.  There may be lesions in skin and muscles also. Often there is episcleral injection of the eye. The degree and distribu­tion of organ involvement vary in the different diseases produced by different leptospirae in various parts of the world. Many infections are mild or subclinical. Hepatitis is particularly frequent in pa­tients with leptospirosis. It is often associated with elevation of serum creatine phosphokinase, whereas that enzyme is present iormal concentrations in viral hepatitis.

Kidney involvement in many animal species is chronic and results in the elimination of large numbers of leptospirae in the urine; this is probably the main source of contamination and infection of humans. Human urine also may contain spirochetes in the sec­ond and third weeks of disease.

Agglutinating, complement-fixing, and lytic an­tibodies develop during the infection. Serum from convalescent patients protects experimental animals against an otherwise fatal infection. Immunity result­ing from infection in humans and animals appears to be specific for leptospirae. Dogs have been artificially immunized with killed cultures of leptospirae.

Diagnostic Laboratory Tests. Specimens consist of blood for microscopic ex­amination, culture, and inoculation of young hamsters or guinea pigs; and serum for agglutination tests.

A. Microscopic Examination: Darkfield exam­ination or thick smears stained by Giemsa’s technique occasionally show leptospirae in fresh blood from early infections. Darkfield examination of centrifuged urine may also be positive.

B. Culture: Whole fresh blood can be cultured in diluted serum or on Fletcher’s semisolid medium or Stuart Leptospira broth (each of which contains 10% rabbit serum).

C. Animal Inoculation: A sensitive technique for the isolation of leptospirae consists of the intraperi-toneal inoculation of young hamsters or guinea pigs with fresh plasma or urine. Within a few days, spirochetes become demonstrable in the peritoneal cavity; on the death of the animal (8-14 days), hemor-rhagic lesions with spirochetes are found in many organs.

D. Serology: Agglutinating antibodies attaining very high liters (1:10,000 or higher) develop slowly in leptospiral infection, reaching a peak at 5-8 weeks after infection. For agglutination tests, cultured leptospirae are used live and are observed microscopically for clumping. Cross-absorption of sera may permit identification of a species-specific antibody response. With live suspensions, agglutination may be followed by lysis. Leptospiral cultures can adsorb to red blood cells. These will clump in the presence of antibody. These hemagglutination reactions are group-specific.

Immunity. A solid species-specific immunity (directed against individual serotypes) follows leptospiral infec­tion.

Treatment. In very early infection, antibiotics (penicillin, tetracyclines) have some therapeutic effect but do not eradicate the infection.

Epidemiology, Prevention, and Control. The leptosplroses are essentially animal infec­tions; human infection is only accidental, following contact with water or other materials contaminated with the excreta of animal hosts. Rats, mice, wild rodents, dogs, swine, and cattle are the principal sources of human infection. They excrete leptospirae in urine and feces both during the active illness and during the asymptomatic carrier state. Leptospirae re­main viable in stagnant water for several weeks; drink­ing, swimming, bathing, or food contamination may lead to human infection. Persons most likely to come in contact with water contaminated by rats (eg, miners, sewer workers, farmers, fishermen) run the greatest risk of infection. Children acquire the infection from dogs more frequently than do adults. Control consists of preventing exposure to potentially contaminated water and reducing contamination by rodent control. Dogs can receive distemper-hepatitis-leptospirosis vaccinations.

 

Additional material about laboratory diagnosis

LEPTOSPIROSIS. Leptospirosis is an acute infectious zoonotic disease characterized by the primary involvement of the kidneys, liver, nervous system, and circulatory organs. The causative agent of leptospirosis is Leptospira interrogans.

Bacterioscopic, bacteriological, biological, and serological methods of investigation arc employed for the laboratory diagnosis of the disease. Tlie material to be studied is blood, urine, and cerebrospinal fluid obtained from the patient. Depending on the epidemiological peculiarities, one might additionally study water, foodsluus, and animal excreta.

Bacterioscopic examination. Within 5 days after the onset of the disease Leptospira can be detected in the blood, and later on, in the urine, cerebrospinal fluid, and parenchymatoiis organs.

Withdraw 2-3 ml of blood from the vein and mix it with 2-3 ml of 2 per cent sodium citrate. Isolate the plasma by 30-min centrifuga-Lion at 3000 X g and examine the pellet.

Samples of urine, cerebrospinal fluid, and suspension of post­mortem organs are examined immediately after tlieir collection, and their deposit is studied after 2-hour centriHigation at. 4000 X g.

The material to be investigated is placed on a thin glass slide (no thicker than 1 mm) and covered with a cover slip (no thicker than 0.2 mm). The preparation obtained is examined by dark-field micro­scopy with the aid of the dry objective. Microscopic examination reveals motile thin Leptospira with typical curls on tlie ends {second­ary curls), which are known as C- or S-shaped Leptospira; occasion­ally, small primary curls can also be visualized (Fig. 2). Micro­scopy of blood rarely yields positive results and other methods of investigation have to be employed.

 

Leptospira in a dark microscopic field

 

Bacteriological examination. Samples of blood, urine, and cerebro­spinal fluid are inoculated (in an amount of 5-20 drops) into 3-5 test tubes with a nutrient medium or distilled water.

To obtain a nutrient medium, to 3-5 ml of sterile distilled water (pH 7.2-7.4) add, under sterile conditions, 30 per cent of normal rabbit serum inactivated for 30 min at 56 °C.

A non-serum Vervoort-WoIff medium (900 ml of distilled water, 0.5 g of sodium chloride,   1 g of peptone, and 100 ml of phosphate buffer) can also be used for this purpose. Inoculated cultures are cultivated at 28-30 °C.

Leptospira propagate in a nutrient medium without affecting its appearance. The medium mimiiis transparent throughout the period of observation. To detect the growth of Leptospira. make a wet-mount preparation from each specimen 10 days after inoculation and examine it by dark-field microscopy.

From the test tube where the growth of Leptospira lias beeoted transfer 0.5 ml portions of the medium into 3 test tubes conlaining 5 ml of fresh nutrient. medium each. The cultures are incubated for 7-10 days at 28-30 °C.

To differentiate between pathogenic and saprophytic Leptospira, study their biological, cultural, and biochemical properties. The most demonstrative in this respect is tlie bicarbonate test which consists in the following: add 0.1 g of sodium hydrocarhonate to 100 ml of distilled water, sterilize tlie mixture for 30 min at 120 °C, check pH (7.2). and add 10 per cent of inactivated rabbit serum. Decant the medium in 10-ml portions, heal three times (with a 24-hour interval) on a water hath al 56 °C for 30 min, and check for sterility. 0.5-ml volumes of 7-day culture of Leptospira are introduced into 3-5 test tubes. The inoculated cultures are stored for 8 days at room temperature and then examined under tlie microscope. Pathogenic Leptospira are either absent or there may he no more then 1-2 organ­isms per microscopic field. Tlie number of saprophytes per prepara­tion is usually around 100.

Isolated Leptospira are identilied by determining their serological groups witli the lielp of a kit of agglutinating sera. To date, 18 sero-groups have been identified.

Serological diagnosis. Antibodies in the patient’s blood serum appear beginning from tlie second week of the disease. Their number grows and reaches tlie peak on the 14th-17th day of the disease. To reveal antibodies, one employs the reactions of microscopic agglutina­tion and lysis of Leptospira. Dilute the patient’ serum in multiple ratios varying from 1 : 10 to 1 : 1000. Into a series of test tubes introduce 0.2~ml amounts of diluted serum and the same quantity of a live culture of the diagnostic kit of Leptospira strains. Following one-hour incubation at 37 °C, prepare a wet-mount preparation from the contents of each tube and examine it by dark-field micro­scopy.

Serum antibodies in the first dilutions induce lysis (complete dissolvement of Leptospira), partial lysis or granular swelling of Leptospi­ra. In subsequent dilutions of the serum one can see agglutination (appearance of agglomerates). A positive reaction in a serum dilution of at least 1 : 1000 is diagnostically significant.

A positive serological result may be observed not only with Lepto­spira of a serogroup responsible for the disease but also with Lepto­spira belonging to other serological groups. To clinch the accurate serological diagnosis, one should reveal IgG which, unlike IgM, are strictly specific. For this purpose, utilize 2-merkaptoethanol and cysteine which selectively split IgM without affecting the serological activity of IgG.

Inactivation of IgM by cysteine is carried out by Chernokhvostova’s tech­nique. Cysteine (314 mg) is diluted in 10 ml of 0.2 M solution of sodium hydrox­ide. Each tested serum is diluted with buffer solution in a 1:5 ratio and divided into 2 parts; to one part add an equal volume of cysteine solution; to the other, 0.85 per cent solution of sodium chloride. The test tubes with the obtained mix­tures are closed with rubber plugs and placed in a 37 °C incubator for 18-20 hrs. The patient’s blood serum is diluted 1:10-1:1600 and used for the reaction of microscopic agglutination.

Compare the results of the reaction in cysteine-treated and native sera. If there is no difference in concentrations of antibodies in the two samples, they arc believed to beiong to class G. The presence of IgM is indicated by the complete absence of antibodies in the treated serum or by at least a four-fold decrease in their titres in the test versus control sera.

Biological examination is carried out on guinea pigs, rabbits, and 2-4-week-old puppies. Citrate blood, urine residue, and the examined culture are admin­istered to animals intraperitoneally or subcutaneously.  If the mate­rial contains Leptospira, the test animals develop fever in 5-7 days; their visible mucous membranes become yellowish. Several days after inoculation the animals die with manifestations of’hypother­mia. All post-mortem organs are yellowish in colour. Leptospira are detected in the liver, kidneys, lungs, and adrenals, less frequently in other organs and tissues.

Puppies infected with Leptospira-containing material develop a chronic process during which Leptospira can be detected in the urine for a period of 3 months.

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.

 

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

 

*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

 

 

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, toxieutralization, 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. Iucleic 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 toxieutralization 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 ionendemic 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. Ieonatal—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 ion­gonococcal urethritis and ionpregnant 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.

Ionbacterial 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 ithe 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 iormal 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 withiormal 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.

 

 

 

 

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

 

 

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