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Laboratory Diagnosis of Spirochetes Infection

Laboratory Diagnosis


In its primary and secondary stages, syphilis can often be diagnosed by dark-field examination of fresh exudate obtained from lesions. Because the exudates from non-syphilitic lesions may also contain spiral organisms, the result must be interpreted with care. An experienced observer can often differentiate T. pallidum from other spirochetes.  T. pallidum may also be identified by immunfluorescent staining. Drolark field microscopy is particularly important in suspected early primary syphilis before serological tests become reactive. Specimens from genital ulcers or other lesions consistent with a chancre and from skin lesions of secondary syphilis, especially condylomata lata should be examined by dark field microscopy. Specimens for DFM should be collected in a room adjacent to a laboratory and examined immediately.

       Flocculation tests for Wasserman Abs are of great value in the presumptive diagnosis of syphilis. The Wasserman antigen is a phospholipid known as cardiolipin which is a normal constituent of host tissue. The standard is the VDRL test. The RPR (Rapid  Plasma Reagin) test is another widely used test. However, a wide variety of other diseases e.g. malaria, SLE, leprosy, and other infections may give a false-positive result. Wasserman antibody is usually detected 1 to 3 weeks after the primary lesion appears and reaches a maximum during the second stage of infection. Subsequently, this antibody may remain at an elevated level. However, it is only positive in 75% of patients with tertiary syphilis and thus a negative VDRL test does not exclude late syphilis.

       Tests for treponemal antibody, such as the TPI (T. pallidum immobilisation) test, are more specific but are not generally available. The more widely performed fluorescent antibody (FTA) test is positive in 80% of patients with primary syphilis and in almost all patients with secondary or tertiary syphilis. It should therefore not be used as a screening test. The TPHA test is similar to the FTA test in its sensitivity and specificity. Antitreponemal antibodies decline more slowly than Wasserman antibody after treatment, and positive reactions may persist for years.



Treatment and prevention


Penicillin is the drug of choice in the treatment of syphilis. It is less effective in the late stages of disease since irreparable damage had already been sustained. Many patients with early syphilis are unaware they have the disease since the early lesions may be symptomless. Hence control of the spread of syphilis is important. All known sexual contacts of an infected individual should be examined. Most authorities recommend treatment of all contacts recently exposed to primary or secondary syphilis.





T. pertenue is the causative agent of the nonvenereal tropical disease known as yaws. It is virtually indistinguishable from T. pallidum except for the character of the lesions it produces. Both organisms induce the formation of Wasserman Abs and serologically identical treponemal Abs. There is also a high degree of cross-resistance. Yaws occur in the tropics. Children often become infected at an early age. In areas of high endemicity, 75% of the population may acquire the disease before the age of 20.

       The primary lesion appears 3 to 4 weeks after exposure and is known as the mother yaw. It begins as a painless red papule surrounded by a zone of erythema often referred to as a frambiose. Generalised secondary lesions of a similar nature appear 6 weeks to 3 months afterwards and commonly occur in successive crops. They may last a period of months or even years. On the soles of the feet, tender hyperkeratotic lesions known as crab yaws often appear. The late tertiary lesions are generally restricted to the skin and bones; gummatous nodules and deep chronic ulcerations may disfigure the nose and face and are often disabling. However, the disease is not as grave as syphilis as it rarely involves the viscera. Like syphilis, yaws respond dramatically to treatment with penicillin, often only requiring a single injection of a long-acting drug.




T. carateum is the causative agent of Pinta, which also occurs primarily in the tropics. The organism is morphologically identical to T. pallidum but differs in being difficult to propagate in laboratory animals. The disease may be acquired at any age through contact. The primary lesion is non-ulcerating and is followed within 5 to 18 months by successive crops of flat, erythematous skin lesions. Tertiary lesions are extremely rare. Penicillin is effective against Pinta.





Relapsing fever


Spirochetes of the genus Borrelia are usually longer than the treponemes, their spirals are more loosely wound and more flexible. In man, spirochetes of this genus cause relapsing fever in an epidemic or endemic form. The epidemic form is caused by B. recurrentis and is transmissible from person to person through the louse. There is no animal reservoir and the lice do not transmit the disease transovarially. The endemic form is caused by various species of Borrelia and is spread from various animal reservoirs (often rodents) to man by ticks.

       After an incubation period of 3 to 10 days, there is a sudden onset of fever, which usually lasts for 4 days. The organism may be recovered from the blood, urine, and the CSF. The fever then subsides and the organisms disappear from the blood. As they decrease in numbers, they become less and less motile, assume pleomorphic forms, and agglutinate, often in rossettes. During the afebrile period, the blood is not infectious, but after 3 to 10 days, it again teems with organisms and the fever returns. The recurrent attacks usually number from 3 to 10 and becomes progressively less severe until they subside altogether. Although the case fatality rate is less than 5% for the endemic disease, it may exceed 50% in severe louse-borne epidemics. Miliary necrotic lesions containing large number of organisms may be found in the spleen and liver.

       The most remarkable feature of this disease is its tendency to relapse at regular intervals. The pathogenesis of the sequential relapses is unique: the organisms in each successive attack show antigenic differences, and circulating antibodies specific for the organisms of each onset appear in the blood. These antibodies are responsible for the agglutination and disappearance of the spirochetes. DNA rearrangement affecting the variable major protein is responsible for these defined shifts.

       The diagnosis is usually made by microscopic examination of blood samples obtained during a febrile attack. The organisms can be identified by dark-field microscopy or in stained smears. Because of the numerous antigenic variants encountered, serological tests are of little diagnostic value. Animal inoculation may help when direct examinations are negative: infected blood injected into young rats will usually result in readily demonstrable bacteraemia within 24 to 72 hours. Relapsing fever responds well to penicillin. Tetracylcines and chloramphenicol are also effective. Tick and louse-control measures are the most effective means of prevention.


Lyme disease


B. burgdorferi is the agent of Lyme’s disease. It is harboured by ticks of the Ixodes species and transmitted to man during the spring and summer. The disorder is named after a unique cluster of manifestations observed in patients around Lyme, Connecticut. The syndrome consists of a characteristic skin rash (erythema chronicum migrans), flu-like symptoms, and sequelae such as arthritis, carditis, and neuritis. The unusual clinical and epidemiological features of Lyme disease, highlighted by a history of tick bites, travel in endemic areas, and development of a migratory ring-like skin lesion are important diagnostic indicators. Serological tests are useful in making a diagnosis. Direct observation of spirochetes in clinical specimens and culture are also diagnostic but are infrequently successful even in definite cases.





Leptospires are characterised by extremely fine tightly wound spirals. They are obligate aerobes and can be grown readily in a variety of artificial media supplemented with 10% heat-inactivated rabbit serum. Saprobic leptospires can be differentiated from the pathogenic types by several cultural differences. All leptospires possess a common somatic Ag. It can be divided into 19 serogroups and 200 serotypes. On the basis of G + C content of DNA, the leptospires have also been divided into 4 distinct genetic groups which bear little relation to groupings based on antigenic structure. Leptospirosis is primarily a zoonosis, with rodents and domestic animals providing the principal reservoirs.

       Different seroptypes cause a similar pattern of illness after an incubation period of 8 to 12 days. The onset is abrupt, often with chills followed by high fever. Headache, photophobia, and severe muscular pains, particularly in the back and legs, are prominent symptoms, as is conjunctivitis. Jaundice occurs in less than 10% of clinically recognisable cases. Lymphocytic meningitis is often present. The serotype Leptospira interrogans icterohaemorrhagiae is responsible for Weil’s disease, which consists of jaundice, haemorrhagic tendencies, and involvement of the kidneys.

       A diagnosis based on direct microscopic examination of the blood should be made only by experienced observers. Leptospiraemia may be detected by culturing the blood, preferably at 30oC, in broth or agar media enriched with 10% serum or by inoculating it intraperitoneally into young guinea pigs or hamsters. Penicillin and tetracyclines are the drugs of choice. However, treatment of leptospirosis is unsatisfactory. Man usually acquires leptospirosis from infected animals through contact with their urine or tissues. Workers in rat-infested slaughterhouses, fish-cleaning establishments, miners, farmers, sewage workers, and swimmers in stagnant pools and canals run the greatest risks. Preventive measures should be directed at diminishing the chances of contact with contaminated water.


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