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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 2  |  Page : 209-211
Early diagnosis of leptospirosis by conventional methods: One-year prospective study

Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

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Several techniques are available for the diagnosis of leptospirosis and microscopic agglutination test is considered as gold standard, but these require sophisticated techniques and instruments. This study was conducted to know the sensitivity and specificity of dark field microscopy (DFM) as compared to IgM enzyme-linked immunosorbent assay (ELISA) in correlation with clinical diagnosis. It is a prospective study of 297 samples received from clinically suspected cases of leptospirosis for DFM and IgM ELISA. We tried to evaluate the easily available technique, DFM. We got promising results with DFM. Specificity and sensitivity were observed to be 61% and 60%, respectively, alone and 95.8% efficacy when combined with IgM ELISA. Thus, DFM should be tried for where other diagnostic tools are not easily available. It is an easy and rapid technique, which can help in the early diagnosis and management of patients

Keywords: Dark field microscopy, leptospirosis, microscopic agglutination test, Weil′s disease

How to cite this article:
Sharma KK, Kalawat U. Early diagnosis of leptospirosis by conventional methods: One-year prospective study. Indian J Pathol Microbiol 2008;51:209-11

How to cite this URL:
Sharma KK, Kalawat U. Early diagnosis of leptospirosis by conventional methods: One-year prospective study. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Feb 28];51:209-11. Available from: http://www.ijpmonline.org/text.asp?2008/51/2/209/41687

   Introduction Top

Leptospirosis is an emerging zoonotic disease prevalent worldwide. It commonly occurs in tropical, subtropical and temperate zones. Most commonly, the infection is acquired by direct or indirect exposure to urine of reservoir animals through contaminated soil, mud and water entering via small abrasions or breaches in the skin during occupational, recreational, habitational or vocational activities. Infection may also be acquired by drinking or inhalation of contaminated water. The infection is commonly associated with field workers, with increased incidence in rural than urban population. [1]

The causative organism is a spirochete, which is 10-20 µm and 0.1-0.15 µm, spirally coiled bacteria with hooked ends and motile by flagella. They are not visible under ordinary microscope, but can be visualized under dark field microscope and phase contrast microscope. Leptospirosis presents with protean clinical manifestations ranging from mild flu-like symptoms to very severe conditions such as meningitis and hemorrhages. The signs and symptoms resemble a wide range of bacterial and viral diseases and sometimes can present as food poisoning, chemical poisoning and snake bite also, due to which the diagnosis is often missed. The most severe form is called Weil's syndrome, which consists of jaundice, bleeding and oliguria or anuria. In India, leptospires were isolated for the first time in Andaman Islands. [2] Several epidemics have been reported from the southern and western parts of India in the last century. [3],[4] The incidence of leptospirosis from the northern parts of the country was not known due to lack of awareness and the diagnostic techniques, but recently the situation has changed and several reports have been published from these areas also. [3] Several techniques have been devised for early and accurate diagnosis, but all have their own limitations. Most of the techniques available are costly, labour intensive and require sophisticated and costly equipment available only in a few centres. Dark field microscopy (DFM) is a cost-effective and rapid technique, but is not considered to be sensitive as it requires at least 10 leptospires / ml to be seen by DFM. [5]

   Materials And Methods Top

It is a single-sample prospective study of 297 samples, from clinically suspected cases of Leptospirosis with various signs and symptoms such as bleeding tendencies, fever, headache, muscle aches, renal failure etc., received in the department of Microbiology for IgM ELISA and DFM.

Processing for DFM

For DFM, 5-ml blood samples were collected in EDTA vial. The sample was transferred to a sterile test tube and was centrifuged at 1000 rpm for 15 min. After this, 10 µl of Buffy coat or plasma was transferred to a new, clean slide and a cover slip was placed over it and the preparation was examined under dark field microscope (Zeiss). Then the plasma was spun at 3000-4000 rpm for 20 min, at the end of which the supernatant was discarded and wet preparation was done with a drop of sediment, which was examined under a dark field microscope. [6] The sample was reported negative if no spirochete was observed after screening of approximately 100 fields in each of the preparations.

Processing for ELISA

ELISA was done with Lepto-M Leptospira Microwell Serum ELISA kit. The test procedure was performed according to the protocol provided along with the kit. The results were interpreted according to the manufacturer's instructions. Negative and positive controls were kept with each test run. Cut-off was calculated and reporting of results was done as positive, negative and equivocal as per the manufacturer's guidelines provided along with the kit. After 2-3 weeks, a second blood sample was collected from equivocal cases and once again tested by ELISA for IgM antibodies.

   Results Top

This is a hospital-based single sample prospective study conducted from January 2005 to December 2005. Two hundred and ninety-seven blood samples were received for IgM ELISA and DFM. Fever with chills and rigour was the most common complaint followed by fever with vomiting. The least common manifestation was haematemesis and haemoptysis. Jaundice was seen in 43 (14.5%) patients. Complications involving the central nervous system were observed in 34 (11.4%) patients. Twenty-six (8%) patients presented with renal failure. Headache was the least common manifestation. The oldest patient was a 91-year-old male and the youngest patient was a 2-month-old infant.

Comparison of DFM and IgM ELISA was done. Results of DFM and IgM ELISA are presented in [Table 1]. Alternative diagnosis was made in 54 samples, which were negative for leptospirosis by DFM and IgM ELISA, as shown in [Table 2]. The combined efficacy of DFM and IgM ELISA was 96% (233/243), sensitivity of ELISA was 79% (192/243), sensitivity of DFM was 60.5% (157/243) and the correlation between ELISA and DFM was 54% (160/297). Second samples from equivocal cases tested positive for IgM ELISA, indicating seroconversion.

All the patients recovered well after treatment with parenteral penicillins. One patient expired due to unknown reasons with rapid deterioration of vital functions. This patient was positive for leptospira by IgM ELISA as well as DFM.

   Discussion Top

Leptospirosis is an emerging infectious disease with a rising trend in India as well. A large number of tests have been described, but in practice, availability of appropriate laboratory test still remains a problem. Positive diagnosis of leptospirosis in humans is tedious and time-consuming. Conventionally, either culture positivity, or recent seroconversion, or a four-fold rise in microagglutination titres (MAT) should be demonstrated for confirmation. Performance of the gold standard MAT for routine clinical testing is expensive and requires technical expertise. At present, there is a need for development of an economic, rapid and sensitive test for diagnosis. DFM is considered to be insensitive as artefacts like lysed RBC's fibrils and tissue debris can be confused with leptospires.

The leptospires change their morphology when grown in artificial conditions and so the appearance in vitro is entirely different from in vivo . [7] Various forms of leptospires can be visualized under dark field microscope, such as hooked, straight, coccal and other forms, which pose problems in identification by a new and untrained person. [8] In this study, we tried to evaluate DFM, which is a very rapid and economic test. We found that the sensitivity (60.5%) was slightly lower than ELISA (79%) and other rapid techniques available, which are comparatively costly and labour intensive techniques and require trained personnel and costly equipments. [9] There was 53.87% correlation between DFM and ELISA, which can be even better with paired blood samples as reported by Chandrasekaran [10] Combined efficacy of ELISA and DFM was 96%. Therefore DFM can serve as a very good alternative to the unaffordable tests at a peripheral centre and also as an excellent supplementary test to IgM ELISA. So, we recommend that more and more laboratory personnel should be trained in the screening of samples by DFM as it can be a very useful screening test for leptospires in peripheral centres where facilities for more sophisticated tests such as ELISA and MAT are lacking.

   Conclusions Top

Diagnosis of the early stage of leptospirosis is difficult, since it has varied clinical presentations. There are several techniques available, but none of them is considered as gold standard except MAT, which is difficult and have to maintain several reference strains, but it has little role in the early diagnosis of leptospirosis. One should develop expertise in DFM to reduce the morbidity and mortality. DFM may be considered for rapid and early diagnosis of leptospirosis.

   References Top

1.Rao SR, Gupta N, Bhalla P, Agarwal SK. Leptospira in India and rest of the world. Braz J Infect Dis 2003; 7:178-93.  Back to cited text no. 1    
2.Taylor J, Goyle A N. Leptospirosis in the Andaman. Indian Med Res Memoirs 1931; 20:55-6.  Back to cited text no. 2    
3.Chaudhary R, Premlatha MM, Mohanty S, Dhawan B, Singh KK, Dey AB. Emerging leptospirosis, North India. Emerg Infect Dis 2002;7:8.  Back to cited text no. 3    
4.Sharma KK, Gururajkumar A, Mohan A, Siva Kumar V, Kalawat U. Preliminary study on the prevalence of leptospirospira serovars among suspected cases of Leptospirosis in Tirupati, Andhra Pradesh. Indian J Med Microbiol 2006;24:302.  Back to cited text no. 4    
5.Levett PN. Leptospirosis. Clin Microbiol Rev 2001;14:296-326.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Coghlan JD. Leptospira, Borrelia, Sprillum. In Mackie and McCartney. Practical Medical Microbiology 1996.14 th Edition (Vol. 2):559- 62.  Back to cited text no. 6    
7.Czekalowki JW, Eaves G. Formation of granular structures by Leptospirae as revealed by the electron microscope. J Bacteriol 1954;67:619-27.  Back to cited text no. 7    
8.Simpson CF, White FH. Ultrastructural variations between hooked and non hooked leptospires. J Infect Dis 1964;114:69-74.  Back to cited text no. 8  [PUBMED]  
9.Vijayachari P, Sehgal SC. Recent advances in the laboratory diagnosis of Leptospirosis and characterization of leptospires. Indian J Med Microbiol 2006;24:320-2.  Back to cited text no. 9    
10.Chandrasekaran SG. A standard test for the early and rapid diagnosis of leptospirosis. Indian J Med Microbiol 2004;22:23-7  Back to cited text no. 10    

Correspondence Address:
Krishna Kanchan Sharma
Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati - 517 507, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.41687

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