A STANDARD SCREENING TEST FOR THE EARLY AND RAPID DIAGNOSIS OF LEPTOSPIROSIS

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1 Indian Journal of Medical Microbiology, (2004) 22 (1):23-27 Original Article A STANDARD SCREENING TEST FOR THE EARLY AND RAPID DIAGNOSIS OF LEPTOSPIROSIS *S Chandrasekaran, S Gomathi Abstract Purpose : To perform dark field microscopy (DFM) for detection of Leptospira and to validate the results using Leptospira IgM antibody SERION ELISA test. Methods : After differential centrifugation of Ruys, DFM was done to demonstrate Leptospira in the blood and SERION ELISA was done for Leptospira IgM antibody in single or paired serum samples. One hundred and eleven cases (39 adults and 72 children) of suspected leptospirosis were included in the study. Results : Anicteric cases accounted for 66.7% (26/39) of adults and complications involving brain, liver, kidney and eyes were seen in 33.3% (13/39). In children, 90.3% (65/72) were anicteric and involvement of brain and liver was seen in 9.7% (7/72) cases. On testing 60 single samples of blood from 23 adults and 37 children, DFM exhibited greater sensitivity of 93.3% (56/60) than that of SERION ELISA for Leptospira IgM antibody (33.3%, 20/60). It was observed that the positivity of DFM decreased from 100% (15/15) to 90.9% (10/11) with increase in the duration of infection for more than one week. ELISA for Leptospira IgM, done on 51 paired blood samples, was positive in 64.7% (33/51) cases when both (first and second) samples were tested while in 45.1% (23/51) cases was positive with first sample alone. 58.8%(30/51) cases were positive by testing second sample. DFM results on paired blood samples showed persistence of Leptospira in 92.9% of cases. Conclusion : This study shows the validation of DFM results by SERION ELISA for Leptospira IgM antibody, based on which we recommend that DFM can serve as a standard screening test for early and rapid diagnosis of leptospirosis. Key words: Darkfield microscopy, SERION ELISA for Leptospira, IgM, Screening test There is a need for a screening test for the early and rapid diagnosis of leptospirosis. Wolff noted that dark field microscopy (DFM), after differential centrifugation of Ruys, may enhance the chances of seeing Leptospira and thereby make an early diagnosis possible. 1 But microscopic examination of tissues or body fluids is not recommended as a single diagnostic procedure since the concentration of Leptospira in the blood may be too low and artifacts such as fibrils and extrusions from cellular elements can be easily mistaken for Leptospira by the inexperienced. 2 Therefore, DFM is not used by most of the workers. However, White and Ristic found good correlation between DFM and fluorescence antibody technique for the detection of Leptospira in the urine from experimentally infected 8 months old splenectomized Holstein heifer 48 with 5 ml of Leptospira pomona culture. 3 In bovine urine, Leptospira remained actively motile if collected in buffered saline. Heparin was used to collect blood from infected heifer 48. They found structures resembling Leptospira by both DFM and fluorescence antibody technique. Somewhat similar *Corresponding author Institute of Microbiology, Madurai Medical College, Madurai , Tamilnadu, India. Received : Accepted : structures were seen in the plasma of normal animals also. These structures were considered as fibrin. Chandrasekaran et al used 1% solution of liquoid in sterile saline and phosphate buffered 1% sodium oxalate solution ph 8.0 to demonstrate Leptospira in varying concentrations in the blood of patients and police dogs. 4,5 In 77.9% (180/231) of cases 100 high power fields (HPFs) had to be searched to get positive result for Leptospira by DFM and in 22.1% (51/231) of cases the organisms could be seen in every HPF up to a maximum of 20 per HPF. Various other DNA based methods to detect Leptospira antigen like polymerase chain reaction (PCR) are not yet available for routine use. In this study, Leptospira IgM antibody detection by SERION ELISA was evaluated in comparision with DFM for early and rapid diagnosis of leptospirosis. Materials and Methods Patients Thirty-nine adult patients between 13 and 65 years of age from medical wards and 72 children between 2 months and 12 years of age from paediatric wards of Government Rajaji Hospital, Madurai, comprised the study subjects. Single sample of blood was collected

2 24 Indian Journal of Medical Microbiology Vol.22, No.1 in 60 cases and two samples of blood in 51 DFM positive cases. In 9 out of 51 cases blood was insufficient to perform DFM and hence 42 samples were retested by DFM. Preparation of phosphate buffered 1% sodium oxalate solution (ph 8.0) Phosphate buffer ph 8.0 was prepared as per the method given by Wilkinson. 6 To 100mL of this 2 grams of sodium oxalate crystals were added. The mixture was then made up to 200mL with distilled water. The solution was autoclaved and distributed in SV1O vials (Laxbro) each vial containing 500 L aliquot. The vials were kept in the refrigerator at 4 C. Collection of blood 5mL of blood was collected aseptically and distributed in two sterile SV1O vials, one containing 500 L of sodium oxalate solution ph 8.0 and another dry tube. The former was mixed well and used for DFM. From the latter, clear supernatant serum was transferred to a sterile SV2 vial and preserved at 20 C for use in ELISA test. Second sample of blood was collected in the same way after one week from 51 patients who were found to be positive for Leptospira by DFM. Differential centrifugation The freshly collected blood in sodium oxalate solution was centrifuged at about 3000 rpm for 5 minutes to sediment the cellular elements. The supernatant plasma (10L) was placed on a 1mm thick new microscopic slide. A cover slip was placed on the drop and pressed to form a thin film without air bubbles. 7 If the film did not show any Leptospira in 100 HPFs then the plasma was transferred to another sterile SV1O vial and centrifuged in high speed automatic refrigerated centrifuge (Hitachi Koko Co. Ltd. Tokyo, Japan) at rpm for 30 minutes. A wet film was prepared using the sediment after pouring off the supernatant. Darkfield microscopic examination (DFM) Using the high power objective (x400) of a dark field microscope (Carl Zeiss, Germany) one edge of the film was focused to see the Leptospira. The number of Leptospira seen was determined by simple counting and the report was given as Leptospira positive per HPF or per 100 HPFs depending upon the concentration. If no Leptospira was seen in 100 HPFs after high speed centrifugation, the report was given as Leptospira negative. SERION ELISA for Leptospira IgM antibody Standard ELISA procedure as per the manufacturer s instructions was followed. Optical density (OD) values were recorded in an ELISA reader using 405 nm filter. The mean of the OD values of standard serum was subtracted from the OD value of substrate blank. The table corresponding to the final OD value of standard serum was selected among the 18 tables provided by the manufacturer. The OD values of test sera after subtraction from the OD values of substrate blank were referred to the selected table to find out the corresponding value of Leptospira IgM antibody content in IU/mL. 15 to 20 IU/mL was taken as borderline. Less than this value was considered negative and more than this was taken as positive for Leptospira IgM antibody. Results Clinical categorization of suspected leptospirosis cases Table 1 shows the clinical manifestations of suspected leptospirosis in the subjects. Anicteric cases accounted for 66.7% (26/39) and later complications involving brain, liver, kidney and eyes were seen in 33.3% (13/39) of adults. Anicteric cases accounted for 90.3% (65/72) and complications involving brain and liver were found in 9.7% (7/72) of children. Table 1 : Clinical categorization of suspected cases of leptospirosis Clinical Category Number of cases Adults Children I Fever, vomiting, headache, body pain, stomach pain, breathlessness, haematemesis, hepatomegaly II Fever, altered sensorium, metabolic encephalopathy, convulsion 3 1 III Fever, hepatic encephalopathy 1 1 IV Fever, Jaundice, anaemia, abdominal pain, dimness of vision 6 5 V Fever, swelling of legs, body and stomach, renal disease, post infectious glomerulonephritis 3 0 Total Comparison of DFM and SERION ELISA on single samples of blood Results presented in table 2 show the greater detection of Leptospira (93.3%, 56/60) by DFM as compared to 33.3% (20/60) by Leptospira IgM antibody

3 January, 2004 Chandrasekaran & Gomathi A Standard Screening Test for Leptospirosis 25 ELISA. Their combined efficacy was 96.7% (58/60) and their correlation was 33.3% (20/60). Table 3 exhibits the results of DFM and SERION ELISA on these 60 cases categorized on the basis of duration of infection. It could be seen that the sensitivity of DFM decreased from 100% (15/15) to 90.9% (10/11) with increase in the duration of infection for more than one week. Sensitivity of ELISA was 45.5% (5/11) when the duration of infection was 8 to 14 days, 33.3% (5/15) when the duration was 1 to 7 days and 29.4% (10/34) when the duration of infection was 15 days and above. Table 2 : Results of DFM and SERION ELISA on single samples of blood (n=60) Clinical category Total no. of DFM+ DFM+ DFM DFM cases IgM IgM+ IgM+ IgM Fever, vomiting Fever, altered sensorium Fever, hepatic encephalopathy Fever, jaundice Fever, swelling of legs Total Negative + Positive Table 3 : Effect of duration of infection on the sensitivity of DFM and ELISA on single samples of blood (n=60) Duration of Total No. of DFM+ DFM+ DFM - DFM - Sensitivity Sensitivity Infection (days) cases IgM - IgM + IgM+ IgM - of DFM (%) of ELISA (%) > Negative + Positive Sensitivity of SERION ELISA in paired samples of blood from patients who were positive by DFM in their first samples The results shown in table 4 indicate that the sensitivity of Leptospira IgM antibody ELISA was 45.1% (23/51) with one sample alone, 58.8% (30/51) with two samples and 64.7% (33/51) with both samples. Hence the correlation of ELISA with DFM increased to 64.7% when paired samples from DFM positive cases were tested by ELISA. Table 5 shows the positivity of Leptospira IgM antibody ELISA in first and second samples from 51cases who were categorized based on the duration of infection. Maximum sensitivity of 100% (6/6) was observed when the duration of infection was between 8 and 14 days, 42.9% (6/14) when the duration was less than one week and 67.7% (21/31) when the duration was more than two weeks. Table 4 : Results of SERION ELISA on paired blood samples from DFM positive cases (n=51) Clinical Total no. of category cases Fever, vomiting Fever, convulsions Fever, hepatic encephalopathy Fever, jaundice Fever, swelling of legs Total Negative 1 First sample + Positive 2 Second sample

4 26 Indian Journal of Medical Microbiology Vol.22, No.1 Table 5 : Effect of duration of infection on the sensitivity of SERION ELISA on paired blood samples (n=51) Duration of Total no Sensitivity Infection (days) of cases of ELISA % (6/14) % (6/6) > % (21/31) - Negative 1 First sample + Positive 2 Second sample Persistence of Leptospira in the blood DFM results on 42 paired samples of blood shown in table 6 indicate that 7.1% (3/42) of cases gave negative result for Leptospira, 64.3% (27/42) of cases showed reduction in the Leptospira count and 28.6% (12/42) exhibited increase in the count of Leptospira. Hence, 92.9% (39/42) of cases showed persistence of Leptospira in the blood even after two weeks of illness. Table 6 : Results of DFM on paired samples of blood from medical and paediatric ward cases Patients Total no. Negative Decrease in Increase in tested by DFM Leptospira Leptospira count count Children Adults Total (7.1%) (64.3%) (28.6%) Discussion Out of 111 cases of leptospirosis complications were seen in 33.3% adults and 9.7% children. It was probably due to greater attention paid to children than to adults. In the Queensland study renal and hepatic involvement was seen in 14% of cases and high incidence of the disease was noted in the 25 to 29 years age group. 8 Our study has revealed high sensitivity of DFM done on fresh blood samples collected in phosphate buffered sodium oxalate solution. 64.7% correlation between DFM and Leptospira IgM antibody ELISA on 51 paired samples of blood suggested high specificity of DFM. Unpublished data of Narayan and Srinivasan from Chennai, India, also show that out of 37 DFM positive blood samples 34 were positive by Dipstick ELISA for Leptospira antibody and the remaining three became positive by the latter in the second sample collected after one week. We found varying concentration of Leptospira from less than one per 100 HPF to 7 per HPF in this study. One has to spend adequate time to locate live Leptospira with characteristic morphology and motility amidst numerous cellular elements. This would help early and rapid diagnosis and prevent later complications involving vital organs like brain, liver, kidney and eyes. 9 There was persistence of Leptospira in the blood of 92.9% cases on repeat testing of 42 cases. We believe that DFM positivity needs to be correlated with clinical findings and with tests like PCR and Leptospira IgM antibody ELISA. In this study, Leptospira could be seen repeatedly even after two weeks of infection and this contradicts the opinion of Turner 10 that the Leptospira are removed from the blood and tissues by nonspecific defence mechanisms helped by increasing concentrations of specific antibodies. The persistence of Leptospira in blood circulation is a matter of concern. It could be inferred from this work that testing of paired blood samples collected between 8 and 14 days of illness gave a sensitivity of 64.7% for Leptospira IgM antibody ELISA. The lower sensitivity of ELISA than DFM could be attributed to the fact that Leptospira antigens used in SERION ELISA kit may not have included the antigens of all the local serovars. Same is said to be true for microscopic agglutination test (MAT). 11 This study shows the validation of DFM results by SERION ELISA for Leptospira IgM antibody, based on which we recommend that DFM can serve as a standard screening test for early and rapid diagnosis of leptospirosis. Acknowledgement We thank the Dean, Government Rajaji Hospital and Head of the Departments of Medicine and Paediatrics for allowing us to collect the blood samples. We also thank the Deputy Director of Health Services, Madurai, for providing us with SERION ELISA kits for Leptospira IgM antibody.

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