Steven Weier

Validation of a microsphere immunoassay for serological leptospirosis diagnosis in human serum by comparison to the current gold standard.

A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.

Leptospirosis from water sources

Abstract Leptospirosis outbreaks have been associated with many common water events including water consumption, water sports, environmental disasters, and occupational exposure. The ability of leptospires to survive in moist environments makes them a high-risk agent for infection following contact with any contaminated water source. Water treatment processes reduce the likelihood of leptospirosis or other microbial agents causing infection provided that they do not malfunction and the distribution networks are maintained. Notably, there are many differences in water treatment systems around the world, particularly between developing and developed countries. Detection of leptospirosis in water samples is uncommonly performed by molecular methods.

The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N = 109) and negative (N = 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55–76) and 59% (95% CI: 49–68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N = 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.

Effect of age, gender and mannose-binding lectin (MBL) status on the inflammatory profile in peripheral blood plasma of Australian blood donors.

Transfusion of blood components has been associated with poor patient outcomes and, an overall increase in morbidity and mortality. Differences in the blood components arising from donor health, age and immune status may impact on outcomes of transfusion and transfusion-related immune modulation in recipients. The aim of this study was to investigate differences in inflammatory profile in donors and association with parameters including age, gender and deficiency status of pattern recognition molecule mannose-binding lectin (MBL). MBL level was determined by ELISA. Serum levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, and IFN-γ were examined by cytometric bead array (CBA). C-reactive protein (CRP) and rheumatoid factor (RF) were examined by immunoturbidimetry. This study demonstrated age was a parameter associated with the immune profile of blood donors, with significant increases in MCP-1 (p<0.05) and RF (p<0.05) and decreases in IL-1α evident in the older donors (61-76years). Significant gender-associated differences in MCP-1, IL-12 and CRP plasma levels in the blood donor cohort were also reported. There was no significant difference in the level of any inflammatory markers studied according to MBL status. This study demonstrated that age and gender are associated with inflammatory profile in donors. These differences may be a factor impacting on outcomes of transfusion.

A comparison of two molecular methods for diagnosing leptospirosis from three different sample types in patients presenting with fever in Laos

Objectives To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. Methods Serum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. Results In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%–81.8%); 99.6% (99.2%–100%)), buffy coat (58.8% (34.4%–90.9%); 99.9% (99.6%–100%)) and urine samples (45.0% (27.0%–66.7%); 99.6% (99.3%–100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%–100%) vs. 92.5% (92.3%–92.8%), p <0.001). Sensitivities of MAT (16% (95% CI 6.3%–29.4%)) and culture (25% (95% CI 13.3%–44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p <0.001). Conclusions Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease.

Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay

Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this methods usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations.

A Prospective Hospital Study to Evaluate the Diagnostic Accuracy of Rapid Diagnostic Tests for the Early Detection of Leptospirosis in Laos.

Abstract. Leptospirosis is a globally important cause of acute febrile illness, and a common cause of non-malarial fever in Asia, Africa, and Latin America. Simple rapid diagnostic tests (RDTs) are needed to enable health-care workers, particularly in low resource settings, to diagnose leptospirosis early and give timely targeted treatment. This study compared four commercially available RDTs to detect human IgM against Leptospira spp. in a head-to-head prospective evaluation in Mahosot Hospital, Lao PDR. Patients with an acute febrile illness consistent with leptospirosis (N = 695) were included in the study during the 2014 rainy season. Samples were tested with four RDTs: (“Test-it” [Life Assay, Cape Town, South Africa; N = 418]; “Leptorapide” [Linnodee, Ballyclare, Northern Ireland; N = 492]; “Dual Path Platform” [DPP] [Chembio, Medford, NY; N = 530]; and “SD-IgM” [Standard Diagnostics, Yongin, South Korea; N = 481]). Diagnostic performance characteristics were calculated and compared with a composite reference standard combining polymerase chain reaction (PCR) (rrs), microscopic agglutination tests (MATs), and culture. Of all patients investigated, 39/695 (5.6%) were positive by culture, PCR, or MAT. The sensitivity and specificity of the RDTs ranged greatly from 17.9% to 63.6% and 62.1% to 96.8%, respectively. None of the investigated RDTs reached a sensitivity or specificity of > 90% for detecting Leptospira infections on admission. In conclusion, our investigation highlights the challenges associated with Leptospira diagnostics, particularly in populations with multiple exposures. These findings emphasize the need for extensive prospective evaluations in multiple endemic settings to establish the value of rapid tools for diagnosing fevers to allow targeting of antibiotics.

Leptospirosis in Tasmanian Devils ( Sarcophilus harrisii ) in Tasmania, 2008-12.

Abstract In 2014, we performed a diagnostic study of leptospirosis in Tasmanian devil (Sarcophilus harrisii) samples collected between 2008 and 2012 from wild and captive animals. Tasmanian devil populations have been declining because of a facial tumor disease since the 1990s, with ongoing investigations examining potential causative agents. Identifying other causative pathogens that may contribute additively to their decline is important to preserve current and future populations. We tested 81 Tasmanian devil serum samples and two tissue samples using PCR, microscopic agglutination test (MAT), and microsphere immunoassay (MIA). We found evidence of leptospirosis in Tasmanian devil populations across a wide geographic range of Tasmania. Antibodies to serovars in the serogroup Javanica, which are not considered endemic to Australia, were identified in 10 Tasmanian devils using MAT. We also identified serovar Celledoni serologically using the immunoglobulin G MIA and detected Leptospira in one sample using PCR.

Leptospirosis and Leptospires—The Silent Assassins

Leptospirosis is one of the most common yet under reported zoonoses. Leptospires, the etiological agents of leptospirosis are ubiquitous pathogens, with a world-wide distribution, causing a spectrum of disease ranging from a mild influenza-like illness to Weil’s disease, which manifests itself in multi-organ failure. The following chapter reports on the epidemiology and transmission of the disease in humans and animals. The chapter will also delineate the symptoms observed in humans and animals and in concluding outline unresolved and evolving issues for microbiologists, epidemiologists and public health officials.