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PCR on formalin-fixed necropsy tissues to diagnose leptospirosis.

Sir,

A 44 yr old male soldier who was serving somewhere in the forests of north-east India during 2004-2005 was admitted to a military unit hospital in the region with complaints of low-grade intermittent fever, arthralgia, lethargy and nausea of one-week duration. Although blood smears for malarial parasite was negative, based on clinical diagnosis and endemicity of malaria in the region, he was initially administered treatment for malaria. Ultrasonography of abdomen showed mild hepatomegaly and acalculus cholecystitis. During the next 3 days he developed icterus, oliguria and malena. Investigations at the hospital showed derangement of liver and renal functions in the form of elevated levels of serum bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine (1). Blood and urine cultures, Widal test and Weil-Felix test were negative (1). Tests for human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were non reactive. In view of his uremic status, peritoneal dialysis was started. He was then managed as a case of septicaemia with acute renal failure with a differential diagnosis of malignant tertian (MT) malaria, viral haemorrhagic fever or a rickettsial disease. He was treated with a combination of broad-spectrum antibiotics and antimalarials in addition to other supportive measures.

On the eleventh day, the patient was transferred to a tertiary hospital (Army Command hospital, Eastern Command, Kolkata). By this time he was semicomatose and had developed deep icterus in addition to erythematous blanching rashes all over the body. He was afebrile but had tachycardia and tachypnoea. Systemic examination showed a hepatomegaly of 10 cm and bilateral crackles in the scapular and infrascapular areas. He was put on haemodialysis and ventilatory support. In ICU, he had a cardio respiratory arrest and died despite rigorous resuscitative measures.

Necropsy was carried out at the tertiary hospital to establish a diagnosis. Histopathological studies on hematoxylin & eosin (HE) stained kidney sections showed well-preserved architecture but with evidence of interstitial nephritis in form of mixed inflammatory infiltrate predominantly lymphomononuclear in nature. Focus of acute tubular necrosis with area of vasculitis was also seen. Liver sections also showed well-preserved architecture with areas of mild portal and periportal inflammation. There was evidence of cholestasis and foci of fatty change were seen. When no other disease could be attributed for his illness, formalin-fixed kidney and liver tissues obtained during necropsy were sent to the WHO Collaborating Centre for leptospirosis at Regional Medical Research Centre (RMRC), Port Blair, for ruling out leptospirosis.

At the RMRC, several preparative methods were employed including the conventional phenol-chloroform extraction and direct-lysis, prior to subjecting the formalin-fixed tissues to polymerase chain reaction (PCR) for establishing diagnosis. In one variant method, when 100 mg of the thinly sliced liver and kidney tissues of the case and a control (from an established case of falciparum malaria) were washed several times with water followed by 50 mM Tris-HCl buffer, pH 8.0 and incubated in the same buffer containing 10 mg/ml proteinase K at 37[degrees]C for 7 days with gentle agitation before 100 [micro]l of the extract was subjected to isolation of DNA (2) and subsequent PCR following standard techniques and published primers (3), both the liver and kidney tissues of the case showed the presence of leptospiral DNA. The control tissues were negative. PCR attempts employing the other preparative methods failed indicating that either these methods did not yield sufficient amount of intact DNA for acting as template in PCR or the impurities remained in these processes might have inhibited PCR reactions. The PCR amplified product was sequenced along with those from a few reference strains and isolates and confirmed as a 285 bp region of sec Y gene specific to leptospires (4,5). Subsequent to getting positive result several times in PCR with the necropsy tissues, the only serum sample of the patient that was available in the archives of the tertiary hospital was also brought to the RMRC, Port Blair for further tests. PCR as well as a latex agglutination test (developed in-house at RMRC, Port Blair, unpublished) showed positive reactions on this sample also thereby lending support to the diagnosis.

A post-test enquiry revealed that the place where the soldier was posted is covered by dense tropical rain forest. The soldier had frequent prolonged exposures to wet and waterlogged soil, prior to his illness. Also, the possibility of exposure to or contact with wild animals could not be ruled out. Soldiers are a risk group for leptospirosis and some of the earliest reports of the disease have come from soldiers (6). Although leptospirosis is not commonly reported from north-east India (7,8), the findings of our study should make doctors and health authorities aware of the likelihood of human beings getting infected in non endemic areas and keep the disease high in their suspicion list.

Necropsy tissues are often not suited as specimens for conventional laboratory diagnosis of leptospirosis (9,6). Since paired sera cannot be easily obtained from cadavers, serological tests, which are based on demonstrating rising antibody titres, are generally impractical in such cases. Microscopic examination of immunoperoxidase or silver stained necropsy tissue samples is often attempted but these techniques suffer from low specificity (10-12). PCR is increasingly being used on serum and urine samples (9,13,14) to establish antemortem diagnosis but its use in demonstration of leptospiral DNA in fresh necropsy tissues has been scanty (9), perhaps because extraction of intact pathogen DNA from the hardened tissue cross-links created by formalin-fixation is difficult (15) and trace of formalin remaining with the template DNA interferes with PCR. The present case underscored the usefulness of formalin-preserved archival tissues in PCR detection of leptospires by demonstrating that proper DNA preparative methods can overcome the detrimental effects of formalin fixation by extraction of pathogen DNA templates of lengths and concentrations suitable for subsequent PCR detections. As post-mortem samples are most often limited to formalin preserved tissues and these tissues do not require maintenance of cold chain during storage or shipment, these can be easily sent to referral laboratories for PCR tests without the risk of contamination (6,9). This particular case should encourage health authorities, doctors and researchers utilize formalin-fixed necropsy tissues for retrospective diagnosis and deducing epidemiological interpretations.

V. Manu, Subarna Roy *, Anita R. DuttaRoy * S. Sharma *, P. Vijayachari * (+) V.K. Kataria ** & S.C. Seghal * INHS Dhanvantari, Port Blair Andaman & Nicobar Islands

* WHO Collaborating Centre for Diagnosis Research Training & Reference in Leptospirosis Regional Medical Research Centre (ICMR) Port Blair, Andaman & Nicobar Islands & ** Command Hospital (Eastern Command) Kolkata, India

(+) For correspondence: e-mail: pblicmr@sancharnet.in

Referenees

(1.) McClatchey KD, editor. Clinical laboratory medicine, 3rd ed. New York: Lippincott Williams and Wilkins; 2002.

(2.) Boom RC, Sol JA, Salimans MMM, Jansen CL, Wertheim-Van Dillen PME, Van der Noorda J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28 : 495-503.

(3.) Gravekamp C, Van de Kemp H, Franzen M, Carrington D, Schoone GJ, Van Eys, GJJM, et al. Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol 1993; 139 : 1691-700.

(4.) Vijayachari P, Hartskeerl A, Sharma S, Natarajaseenivisan K, Roy S, Terpstra WJ, et al. A unique strain of Leptospira isolated from a patient with pulmonary haemorrhages in the Andaman Islands: a proposal of serovar portblairi of serogroup Sehgali. Epidemiol Infect 2004; 132 : 663-73.

(5.) Roy S, Biswas D, Vijayachari P, Sugunan AP, Sehgal SC. A clone of Leptospira interrogans Sensu Stricto is the major cause of leptospirosis in the archipelago of Andaman & Nicobar Islands, India. Lett Appl Microbio1 2005; 41 : 179-85.

(6.) Faine S, Adler B, Bolin C, Perolat P. Leptospira and leptospirosis. 2nd ed. Faine Melbourne: MediSci Vic, Australia; 1999.

(7.) Sehgal SC, Sugunan AP, Vijayachari P. Leptospirosis disease burden estimation and surveillance networking in India. SE Asian J Trop Med Public Health 2003; 34 : 170-7.

(8.) Vijayachari P, Sugunan AP, Sharma S, Roy S, Natarajaseenivasan K, Sehgal SC. Leptospirosis in the Andamans Islands, India. Trans R Soc Trop Med Hyg 2007; 102 : 117-22.

(9.) Brown PD, Carrington DG, Gravekamp C, van de Kemp H, Edwards CN, Jones SR, et al. Direct detection of leptospiral material in human postmortem samples. Res Microbiol 2003; 154 : 581-6.

(10.) Salkade HP, Divate S, Deshpande JR, Kawishwar V, Chaturvedi R, Kandalkar BM, et al. A study of autopsy findings in 62 cases of leptospirosis in a metropolitan city in India. J Postgrad Med 2005; 51 : 169-73.

(11.) Scanziani E, Luini M, Fabbi M, Pizzocaro E Immunoperoxidase studies on leptospiral nephritis of swine. Vet Pathol 1989; 26 : 442-4.

(12.) Chappel RJ, Prime RW, Millar BD, Mead LJ, Jones RT, Adler B. Comparison of diagnostic procedures for porcine leptospirosis. Vet Microbiol 1992; 30: 151-63.

(13.) Lever PN. Leptospirosis. Clin Microbiol Rev 2001; 14 : 296-326.

(14.) Lucchesi PMA, Arroyo GH, Etcheverria AI, Parma AE, Seijo AC. Recommendations for the detection of Leptospira in urine by PCR. Rev Soc Bras Med Trop 2004; 37 : 131-4.

(15.) Godfrey TI, Kim SH, Chavira M, Ruff DW, Warren RS, Gray JW, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn 2000; 2 : 84-91.
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Article Details
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Title Annotation:Correspondence; Polymerase chain reaction
Author:Manu, V.; Roy, Subarna; DuttaRoy, Anita R.; Sharma, S.; Vijayachari, P.; Kataria, V.K.; Seghal, S.C.
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Geographic Code:9INDI
Date:Jan 1, 2009
Words:1522
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