|Year : 2012 | Volume
| Issue : 4 | Page : 418-422
Efficiency of two commercial kits in serodiagnosis of leptospiral uveitis
A Kannan1, CG Priya1, L Prajna2, SR Rathinam3
1 Department of Immunology and Stem Cell Biology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Anna Nagar, Madurai, Tamil Nadu, India
2 Department of Microbiology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Anna Nagar, Madurai, Tamil Nadu, India
3 Aravind Medical Research Foundation, Uveitis, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Anna Nagar, Madurai, Tamil Nadu, India
|Date of Submission||14-May-2012|
|Date of Acceptance||23-Sep-2012|
|Date of Web Publication||24-Nov-2012|
S R Rathinam
Aravind Medical Research Foundation, Uveitis, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Anna Nagar, Madurai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: Uveitis is an important complication of systemic leptospirosis that can occur months to years after systemic infection. The gold standard technique Microscopic Agglutination Test (MAT) is less sensitive and more complicated. All the commercial kits currently available are for early detection of acute systemic leptospiral infection. The purpose of this study is to evaluate the efficiency of two commercial kits in serodiagnosis of leptospiral uveitis, which is a late manifestation. Materials and Methods: Serum samples from leptospiral uveitis patients 20 MAT positive, 20 MAT negative, 15 non-leptospiral uveitis patients, 20 systemic leptospiral infected patients and 21 controls were selected. These samples were tested for the presence of leptospiral IgM antibodies by (i) MAT using a panel of 20 serovars, (ii) LEPTO IgM MICROLISA (J.Mitra & Co.Pvt. Ltd, India) and (iii) Leptocheck (Zephyr Biomedicals, India). The statistical analysis was carried out using stata 11.0. Results: Total of 96 samples were tested with two commercial kits, Lepto IgM MICROLISA and Leptocheck. The sensitivity and specificity of Lepto IgM MICROLISA was 60% and 55% and Leptocheck was 80% and 59% respectively in comparison to MAT. In comparison to clinical diagnosis the sensitivity of IgM Microlisa was 55%, Leptocheck 70% and specificity of IgM MICROLISA was 58.33% and leptocheck was 69.44%. Conclusion: Commercial kits though sensitive and specific for systemic leptospirosis, have limited diagnostic capacity for leptospiral uveitis. Therefore it is essential to develop an inhouse serodiagnostic method specific for leptospiral uveitis patients using local leptospiral isolates.
Keywords: Leptospiral uveitis, Serodiagnosis of leptospiral uveitis, Lepto IgM MICROLISA, Leptocheck
|How to cite this article:|
Kannan A, Priya C G, Prajna L, Rathinam S R. Efficiency of two commercial kits in serodiagnosis of leptospiral uveitis. Indian J Med Microbiol 2012;30:418-22
|How to cite this URL:|
Kannan A, Priya C G, Prajna L, Rathinam S R. Efficiency of two commercial kits in serodiagnosis of leptospiral uveitis. Indian J Med Microbiol [serial online] 2012 [cited 2019 Jun 26];30:418-22. Available from: http://www.ijmm.org/text.asp?2012/30/4/418/103762
| ~ Introduction|| |
Leptospirosis is a zoonotic disease caused by spirochetes of genus Leptospira, which has a worldwide distribution.  Uveitis is a potentially blinding inflammatory disease of the eye, which occurs as a late complication.  The disease is often missed due to non-specific and wide spectrum of clinical symptoms in both systemic and ocular infection.  Although a history of exposure to Leptospira can help in narrowing the differential diagnosis, a rapid, simple test with high sensitivity and specificity would be useful for definitive diagnosis and for public health surveillance. 
The diagnosis of leptospiral uveitis is usually based on clinical diagnosis and demonstration of antibodies by the gold standard serological test microscopic agglutination test (MAT). ,, However, MAT is a complex test that is time consuming, requires specialized personnel, maintenance of a large panel of live cultures and has the risk of laboratory infection. We have earlier demonstrated that the current molecular methods like PCR to be highly sensitive in diagnosis of leptospiral uveitis using aqueous humor samples, since collection of aqueous humor is a highly invasive procedure and is not always possible serology by MAT is still the gold standard for diagnosis.  As an alternate to MAT, several commercial serological kits are available to diagnose systemic infection, however their sensitivity and specificity are not known in leptospiral uveitis. In this study we report the diagnostic accuracy of commercial kits, Lepto IgM MICROLISA and Leptocheck for leptospiral uveitis.
| ~ Materials and Methods|| |
Human samples were collected after getting informed consent and Institutional Review Board approval. All patients underwent a preliminary examination by a nonophthalmologist physician and a general ophthalmologist followed by a standard uveitis work-up. To identify the patients with a specific uveitis diagnosis, laboratory, and ancillary investigations were tailored for each patient, as determined by history and physical findings on presentation. Anatomic location of the inflammation was assigned based on International Uveitis Study Group criteria. The inclusion criteria for diagnosing leptospiral uveitis were: acute, anterior or pan, non-granulomatous uveitis. When these patients had hypopyon, disc oedema, vasculitis and vitreous membrane or vitreous exudates, they suggested stronger association with past leptospiral infection.  Blood samples were obtained from patients diagnosed clinically as leptospiral uveitis. Samples from 40 clinical leptospiral uveitis (20-MAT positive and20-MAT negative). Control samples (MAT negative) included 15 non-leptospiral uveitis (2- Vogt Koyanagi Harada syndrome, 2- sympathetic ophthalmia, 1- tuberculosis, 5- Behcet's uveitis, 5- Fuch's uveitis), 10 cataract and 11 population controls (Cataract controls: Patients undergoing age-related cataract surgery were selected as controls and were MAT negative. Population controls: Population controls were samples from healthy volunteers who were tested negative for MAT). Patients suspected for systemic leptospirosis (20) from hospitals in and around our area, who were seropositive by MAT were also selected. Serum samples were stored until use at -20°C.
Microscopic agglutination test
Microscopic agglutination test (MAT) was performed with a panel of 20 serovars as shown in the [Table 1]. Two fold serial dilutions of serum sample were made with 0.01M phosphate buffered saline (pH 7.2) starting from 1:25. The diluted serum samples were incubated with equal volume of live cultures for 2 h at room temperature with suspensions of live leptospires. As per standard protocol, the end point was determined as highest dilution of serum showing 50% reduction in the number of free moving leptospires.  The cut off titre was considered as 1:100, with 50% reduction in free moving leptospires as established earlier. 
The leptospira IgM ELISA kit (LEPTO IgM MICROLISA, J. Mitra and Co. Pvt. Ltd, Delhi, India), was performed as per manufacturer's protocol. The serum samples were diluted 1:100 in the diluent provided with the ELISA kit before transferring to leptospira antigen-coated (recombinant protein mixture) microwell strips. One hundred microlitres of the diluted sample was transferred to each well and incubated at 37°C for 30 min. After washing five times with the wash buffer, the strips were incubated with 100 μl of conjugate and incubated at 37°C for 30 min. The strips were washed again five times with wash buffer and 100 μl of substrate solution was added, incubated in the dark. The reaction was stopped, after 30 min with 50 μl of stop solution and the plate was read at 450 nm with a microtiter plate reader (BIORAD, Model 680). The results were calculated as IgM units as per the manufacturer's specifications, IgM units <9 interpreted as negative, IgM units from 9 to 11 interpreted as equivocal and IgM units >11 interpreted as positive. Equivocal results were considered negative.
The dipstick assay (Leptocheck, Zephyr Biomedicals, Goa, India) for detection of Leptospira IgM antibodies, is provided with the strip coated with Leptospira genus specific antigen and with different ports A/B for addition of sample/buffer respectively; T/C as test and control window to view results Serum sample of 10 μl and five drops of sample running buffer was added to the respective ports and the results were observed in the test window after an incubation time of 15 min at room temperature. Each strip in the assay was validated and if band appeared in the test window the sample was regarded as positive (as per manufacturer's protocol).
Standard diagnostic accuracy indices of sensitivity, specificity, negative predictive values (NPVs), positive predictive values (PPVs) were calculated with MAT/clinical diagnosis as reference.  Receiver Operator Curve (ROC) analysis using Stata/SE 11.0 (Stata Corp., College Station, Texas) was carried out to measure the accuracy of MICROLISA for serodiagnosis of leptospiral uveitis. The test was considered accurate if area under curve (AUC) was >0.8.
| ~ Result|| |
The diagnostic accuracy was calculated for IgM MICROLISA and Leptocheck with reference to clinical diagnosis and MAT results. We have selected MAT positive leptospiral uveitis (20) and systemic infected cases (20) for the study. Based on the results of MAT, the major infecting serogroup were Australis (% positives in leptospiral uveitis, systemic leptospirosis: 15,10), Autumnalis (10, 20), Louisiana (35, 10) and Icterohaemorrhagie (25, 15).
Our findings show that, of the 40 clinically diagnosed leptospiral uveitis samples,  22/40 tested positive for LEPTO IgM MICROLISA, 28/40 for Leptocheck, 18/40 found to be positive for both the kits [Table 2].
Of 15 non-leptospiral uveitis samples, 2 were (1- Behcet's uveitis; 1- sympathetic ophthalmia) false positives by LEPTO IgM MICROLISA. Leptocheck was positive for 4 Fuch's samples and 1 sympathetic ophthalmia which was also positive for Lepto IgM MICROLISA.Out of 21 control samples, 6 cataract patients and 7 population controls were positive for LEPTO IgM MICROLISA and 4 cataract and 2 population controls were positive by Leptocheck. Two cataract and one population control samples were positive by both. Among the 20 MAT positive systemic leptospirosis cases only 9 were positive by IgM MICROLISA, 4 by Leptocheck and 2 were positive for both. The results are summarized in [Table 2]. The sensitivity of IgM MICROLISA and Leptocheck was found to be 60% and 80% and the specificity was 55% and 59%, respectively, in comparison to the gold standard MAT [Table 3]a and b. The area under ROC curve, used to measure accuracy of the test in comparison to MAT was calculated to be 0.6580 [Figure 1]a. The sensitivity and specificity in comparison to clinical diagnosis was 55% and 58% for IgM MICROLISA and 70% and 69% for Leptocheck [Table 4]a and b. The area under ROC curve, used to measure accuracy of the test in comparison to clinical diagnosis was calculated to be 0.6576 [Figure 1]b.
|Figure 1: Receiver-operator characteristic curve of the LeptoIgM MICROLISA Vs MAT and clinical diagnosis (a) LeptoIgM MICROLISA Vs Microscopic Agglutination Test (MAT) (b) LeptoIgM MICROLISA Vs Clinical diagnosis, The area under the ROC curve, used to measure accuracy of the test in comparison with the gold standard MAT was found to be 0.6580, and in comparison with clinical diagnosis was found to be 0.6576 indicating this kit is not suitable for diagnosis of leptospiral uveitis|
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|Table 3: Sensitivity and specificity of Lepto IgM MICROLISA (a) and Leptocheck (b) in comparison to Microscopic Agglutination Test (MAT)|
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|Table 4: Sensitivity and specificity of Lepto IgM MICROLISA (a) and Leptocheck (b) in comparison to Clinical diagnosis|
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| ~ Discussion|| |
A rapid, accurate method for the diagnosis of leptospirosis is important in order to start appropriate treatment. Although leptospirosis is one of the common causes of uveitis in developing countries, it remains under diagnosed mainly because of protean manifestations and lack of proper diagnostic technique. 
MAT is considered as the gold standard for serodiagnosis of leptospirosis, but in previous studies its sensitivity was found to vary from 30% to 76%, with a specificity of 97%.  But in combination, the sensitivity of IgM ELISA and MAT was found to increase to 70% as reported earlier by Shekathkar et al.  Several recombinant proteins (rLipL32, rLipL41) have been identified to be specific to pathogenic leptospires, but with varied sensitivity and specificity in serodiagnosis. One such recombinant protein LipL32 IgG ELISA, the sensitivity and specificity was 96.2% and 90% respectively, which was comparable with Pan Bio IgM ELISA.  Senthilkumar et al. have used rLipL 41, the sensitivity and specificity was found to be 89.7% and 90.45% with reference to MAT. 
Several IgM based, commercial kits are available for the diagnosis of systemic leptospirosis using broadly reactive leptospiral antigen.  Winslow et al., 1997, have reported Panbio kit to be highly sensitive for diagnosis of systemic leptospirosis, the sensitivity and specificity was 100% and 98%.  But some kits like, IgM Microwell ELISA kit (IVD Research Inc, Carlsbad, CA92010 USA) and SERION IgM ELISA (Serion Immundiagnostica GmbH, Germany) used for diagnosis of systemic leptospirosis were found to have low sensitivity varying from 37.5% to 47.5%.  The sensitivity and specificity of Lepto IgM MICROLISA has been reported to be 96.66% and 99.79% and Leptodipstick to be 86.8% and 92.7%. ,
Our earlier studies have shown MAT to be only 58% sensitive though highly specific (98%) for leptospiral uveitis.  We have demonstrated that Lipopolysaccharide (LPS) is responsible for agglutination in MAT and is the specific antigen for serodiagnosis of systemic leptospirosis and leptospiral uveitis. Furthermore, LPS was identified as a probable cause for the development of uveitis by demonstration of infecting serovar specific LPS in aqueous humor of leptospiral uveitis patients.  Based on these results an in house IgM ELISA using LPS as the antigen (saprophytic patoc culture (Semaranga patoc Patoc I) -- known to cross react with many pathogenic serovars) was developed and found to be 48% sensitive and 90% specific.  Similar to Shekathkar et al. report on systemic infection, combination of IgM ELISA with MAT increased the sensitivity to 77%.
The use of recombinant proteins has also been analyzed in the diagnosis of leptospiral uveitis patients. With reference to the earlier reports on diagnosis of systemic infection, use of recombinant proteins like Lig A, Lig B and Lip L32 as antigens were found to have poor sensitivity and specificity in diagnosis of systemic and leptospiral uveitis patients in our geographic location (unpublished data). In addition, two leptospiral lipoproteins Lru A and B, which were known to cross react with equine lens proteins which were established to be the candidate antigen for serodiagnosis in horses were found to be less specific for leptospiral uveitis patients as antibodies to these patients were also found in Behcet's and Fuch's uveitis patients who had cataract as one of the clinical feature.  These results suggest that recombinant proteins are not useful for serodiagnosis of leptospiral uveitis. This study was carried out to evaluate the diagnostic accuracy of the two commercial kits, Lepto IgM MICROLISA, Leptocheck to Leptospiral uveitis and was found to be less sensitive and specific making it unsuitable for diagnosis.
Most of the commercial kits were designed in developed countries, for systemic leptospirosis and proved to have higher sensitivity and specificity in their geographic location. When these commercial kits were evaluated in our setting they were found to be less sensitive or specific for diagnosis indicating that the nature of antigen used plays an important role in developing a proper laboratory diagnosis for leptospiral uveitis. Therefore, it is essential to isolate the infecting serovar in humans in and around Madurai to develop a better serodiagnostic method for leptospiral uveitis and systemic leptospirosis using LPS as antigen.
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[Table 1], [Table 2], [Table 3], [Table 4]
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