Early Diagnosis of Human Leptospirosis by Detection of Antibodies to Leptospira-Secreted Virulence-Modifying Protein Exotoxins.

The aim of this study was to compare the sensitivity and specificity of polymerase chain reaction (PCR) using two primer pairs and combined with blood culture, immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA), microscopic agglutination test (MAT) and slide agglutination test (SAT) in the diagnosis of human leptospirosis. We analysed 124 serum samples: 60 from patients with confirmed leptospirosis, 20 from patients with other diseases and 44 from healthy individuals. Analysing the first serum sample collected during the first 3-8 days of disease, the sensitivities of the four tests MAT, IgM ELISA, SAT and PCR were, respectively, 69.0%, 79.3%, 72.4% and 62%. In subsequent samples, those same sensitivities were, respectively, 95.4%, 100%, 100% and 72.7% in samples collected from days 9 to 14 and 88.9%, 88.9%, 77.8% and 44.4% in those collected from days 15 to 42. The most specific method (at 100%) was PCR and the least specific (at 89.1%) was IgM ELISA. Although we found PCR to be less sensitive than the serological tests over the course of the disease, our data indicate that PCR was the most sensitive in those initial serum samples presenting no specific antibodies detectable by any of the serological methods tested. We also recommend that PCR can be used in combination with serological tests as we found that this improves the sensitivity of the diagnosis of leptospirosis in the first phase of the disease (93.1-96.5%).

Comparison of Positive Two-Step PF4-Polyvinylsulfonate Antigen Assay Results with Thrombotic Risk and Clinical Outcome among 182 Patients with Heparin-Induced Thrombocytopenia (HIT).

Does the Platelet Factor 4 (PF4) ELISA Correlate with the Clinical Diagnosis of Type II Heparin-Induced Thrombocytopenia?.

BackgroundHeparin-induced thrombocytopenia (HIT) is an adverse drug reaction caused by antibodies to the heparin/platelet factor 4 (PF4) complex, resulting in thrombocytopenia and prothrombotic state. HIT diagnosis is challenging and depends on clinical presentation and laboratory tests. We investigated the usefulness of clinical scores and heparin/PF4 ELISA optical density (OD) as a diagnostic marker and thrombosis predictor in HIT.MethodsWe analyzed 92 patients with suspected HIT. The heparin/PF4 antibody was measured using a commercial ELISA kit (GTI, USA). For each patient, the 4 T's score and Chong's score were calculated.ResultsOf the 92 patients, 28 were anti-heparin/PF4-seropositive. The 4 T's score and Chong's score showed good correlation (r=0.874). The 4 T's score and OD values showed good performance for diagnosis of the definite and unlikely HIT groups; however, OD levels showed better sensitivity (93.8%) than the 4 T's score used alone (62.5%). Of the 92 patients, 26 developed thrombosis. The OD values were significantly higher in patients with thrombosis than in those without thrombosis (0.52 vs. 0.22, P<0.001). Patients with high OD values (OD>0.4) had an increased risk of thrombosis (adjusted odds ratio 9.44 [3.35-26.6], P<0.001) and a shorter 250-day thrombosis-free survival (32.1% vs. 54.7%, P=0.012).ConclusionsELISA OD values in combination with clinical scoring can improve the diagnosis of and thrombosis prediction in HIT. More attention should be paid to the use of clinical scores and OD values as thrombosis predictors in HIT.

Serological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay. In total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets. Sera with low ELISA optical density (OD) values (0.4-1) activated on average 5.6, sera with intermediate ELISA OD values (>1-2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets. Sera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.

The diagnosis of leptospirosis remains a challenge due to its non-specific symptoms and the biphasic nature of the illness. A comprehensive diagnosis that includes both molecular (polymerase chain reaction (PCR)) and serology is vital for early detection of leptospirosis and to avoid misdiagnosis. However, not all samples could be subjected to both tests (serology and molecular) due to budget limitation, infrastructure, and technical expertise at least in resource-limited countries. We evaluated the usefulness of testing the clinically suspected leptospirosis cases with both techniques on all samples collected from the patients on the day of admission. Among the 165 patient’s blood/serum samples tested (from three hospitals in Central Malaysia), 43 (26%) showed positivity by microscopic agglutination test (MAT), 63 (38%) by PCR, while 14 (8%) were positive by both MAT and PCR. For PCR, we tested two molecular targets (lipL32 by qPCR and 16S rDNA or rrs by nested PCR) and detected lipL32 in 47 (29%) and rrs gene in 63 (38%) patients. The use of more than one target gene for PCR increased the detection rates. Hence, a highly sensitive multiplex PCR targeting more than one diagnostic marker is recommended for the early detection of Leptospira in suspected patients. When the frequencies for positivity detected either by MAT or PCR combined, leptospirosis was diagnosed in a total of 92 (56%) patients, a higher frequency compared to when samples were only tested by a single method (MAT or PCR). The results from this study suggest the inclusion of both serology and molecular methods for every first sample irrespective of the days post-onset of symptoms (DPO) collected from patients for early diagnosis of leptospirosis.

ObjectiveTo evaluate PCR assay sensitivity and specificity compared to that of microscopic agglutination test (MAT) for diagnosis of canine leptospirosis. Electronic records search was performed to identify dogs with results for both PCR and MAT testing for leptospirosis.MethodsAll dogs were clinically ill. Diagnosis of leptospirosis was defined as an unvaccinated dog with a positive MAT titer of ≥1:800 or a vaccinated dog or dog with an unknown vaccination status with a positive MAT titer of ≥1:1,600. Diagnosis of leptospirosis was excluded based on MAT titer <1:800 on both the initial and convalescent samples or an initial MAT titer <1:800 and an alternative definitive diagnosis.ResultsForty-nine samples (urine, n = 39; blood, n = 10) were evaluated. Leptospirosis was diagnosed in 17 dogs and excluded in 26 dogs. Urine PCR assay demonstrated sensitivity of 69.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 86.6%. Blood PCR assay demonstrated sensitivity of 25%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 25%. Overall PCR sensitivity was 52.4%, specificity was 100%, positive predictive value was 100%, and negative predictive value was 73.7%.ConclusionsPCR assay performed on urine or blood has high specificity and positive predictive value when compared to MAT for diagnosis of clinical canine leptospirosis. Sensitivity and negative predictive value are moderate to low, so PCR testing should be performed in conjunction with paired MAT testing for canine leptospirosis. Prior antibiotic therapy does not preclude the use of the PCR test.

Rapid confirmation of the diagnosis leptospirosis is important in order to apply appropriate treatment; in addition, infected dogs are a zoonotic risk. Culture and isolation of living leptospires from blood, urine, or tissues is considered the reference standard of diagnosis. However, sensitivity are low, and leptospires require weeks to months to grow. Therefore, diagnosis of leptospirosis is most commonly based on antibody testing or the detection of Leptospira spp. DNA by PCR. Microscopic agglutination test (MAT) is currently still the recommended confirmatory test for canine leptospirosis, despite its numerous limitations (e. g., negative results in early infection, positive results due to vaccine-associated antibodies). ELISA can differentiate between IgG and IgM antibodies and thus, discriminate current infections from previous vaccination or exposure. Point-of-care tests and in-house-PCR tests have recently been developed for use in private practice allowing easy and immediate diagnosis. However, only few data on sensitivity and specificity of these tests exist so far. A reliable diagnosis can only be established in case of a positive PCR result or a fourfold titre increase in MAT.

A commercially available slide agglutination test (SAT) for the diagnosis of human leptospirosis was evaluated by comparing it to an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) and to the microscopic agglutination test (MAT). For all 108 patients, leptospirosis was diagnosed on the basis of a fourfold or greater increase in titer by MAT (seroconversion), and all but 1 of 245 controls were MAT negative (titers, <1:100). Both SAT and the IgM ELISA failed to detect one case of infection (sensitivity, 99%). Only 3 of 145 blood donors and none of the 100 patients with other illnesses were SAT positive (specificity, 99%). The overall results were similar for the three tests; however, SAT and ELISA were statistically more sensitive as initial screening tests. For 22% of the patients, the diagnosis of leptospirosis was made earlier by SAT than by MAT. SAT detected 27 (44%) of 62 MAT-negative patients with the first serum sample. ELISA and SAT had very similar results. Follow-up of patients for 1 year after the onset of symptoms showed a decreasing rate of positivity by SAT from the third month on. The rate of positivity by ELISA decreased more slowly, to about 67% by the end of the study. By MAT all patients were persistently reactive. SAT and ELISA seem to be convenient methods for the rapid and early screening for leptospirosis and could replace the less sensitive MAT. ELISA gives less subjective results than SAT and provides information on IgM kinetics, but it can be performed only by the more sophisticated laboratories. SAT is inexpensive, can be performed more quickly and more easily than ELISA, and could be used by the less well equipped laboratories.

Usefulness of dark field microscopy (DFM), IGM ELISA and microscopic agglutination test (MAT) for the early diagnosis of acute leptospirosis - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/j.ijmr.2019.036, Indian Journal of Microbiology Research-Indian J Microbiol Res

An evaluation of dark field microscopy, culture and commercial serological kits in the diagnosis of leptospirosis

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.

Diagnosis of Canine Leptospirosis

BackgroundDiagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively.MethodologyDuring 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1st submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness.ResultsThe overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1st submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB).ConclusionsAll three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.

Leptospirosis is a common zoonosis of worldwide distribution. Diagnosis of leptospirosis is usually accomplished by serology, but the microscopic agglutination test (MAT) generally requires paired sera for detection of seroconversion and is considered too complex for routine use. A number of rapid assays have been developed in recent years. In the present study, 2 immunoglobulin (Ig) M enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the early diagnosis of acute leptospirosis in Barbados. A total of 103 patients admitted to the Queen Elizabeth Hospital for diagnosis of suspected leptospirosis were investigated. A case of leptospirosis was confirmed by a 4-fold rise in titer between 2 sera tested by MAT, an initial titer of > or = 800 in the MAT, or by isolation of leptospires from blood or urine. A total of 48 cases of leptospirosis were confirmed. In 33 cases, both commercial assays were positive in the first sample, taken at admission, a mean of 6.7 days after onset of symptoms, whereas seroconversion was detected in a further 9 cases. Both assays were negative in 5 cases, and the remaining case gave discordant results in the 2 assays. False-positive IgM results were detected in 4 patients without leptospirosis. The sensitivity of the 2 assays was 89.6 and 97.5%, respectively, and specificities were 92.7 and 96.4%, respectively. The positive predictive values were 87.8 and 95.5%, and the negative predictive values were 90.7 and 89.5%, respectively. Either of these assays can be used for early diagnosis of leptospirosis, particularly in laboratories that cannot perform more specialized leptospiral serology.