Galayanee Doungchawee

Leptospirosis: current situation and trends of specific laboratory tests

Leptospirosis is re-emerging as a worldwide zoonosis and is caused by bacteria of the genus Leptospira. Human leptospirosis is associated with high temperature and humidity. Laboratory tests are indispensible for the early diagnosis and proper disease management. The demand for suitable leptospirosis point-of-care diagnostic tests grows with the awareness and number of incidences. Confirmation is achieved by the microscopic agglutination test, bacterial cultivation, PCR or histopathologic methods. However, high costs, poor standardization and/or elaborate sample preparation prevent routine use at the point of care. Cost-efficient, but insensitive serological methods dominate the diagnostic landscape and, likewise, urgently need improvement toward greater compliance with some of the point-of-care criteria. Combined application of antigen and antibody detection methods increases accuracy, but also new development or transfer of diagnostic technologies should be considered useful. Nano- and microparticle technology may play a key role in improving future antigen detection methods.

Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing.

ABSTRACT There is an urgent need for the development of serodiagnostic approaches with improved sensitivity for patients with acute leptospirosis. Immunoblots were performed on 188 sera collected from 74 patients with laboratory-confirmed early leptospiral infection to detect immunoglobulin M (IgM) antibodies to antigens pooled from 10 leptospiral strains prevalent in Thailand. Sera from patients with other febrile diseases served as controls. IgM reactivity to seven distinct antigens, with apparent molecular masses of 14 to 18, 19 to 23, 24 to 30, 32, 35/36, 37, and 41/42 kDa, was observed. The low-molecular-mass 14- to 18-kDa band was the most frequently detected antigen, being recognized in sera from 82.4% of patients during the first 3 days after the onset of symptoms. We evaluated the accuracy of the IgM immunoblot (IgM-IB) test by using reactivity to the 14- to 18-kDa band and/or at least two bands among the 19- to 23-, 24- to 30-, 32-, 35/36-, 37-, and 41/42-kDa antigens as the diagnostic criterion. The sensitivities of the IgM-IB test and the microscopic agglutination test (MAT) were 88.2% and 2.0%, respectively, with sera from patients 1 to 3 days after the onset of symptoms. In contrast, the IgM-IB test was positive with only 2/48 (4.2%) sera from patients with other febrile illnesses. The high sensitivity and specificity of the IgM-IB test for acute leptospirosis would provide greatly improved diagnostic accuracy for identification of patients who would benefit from early antibiotic intervention. In addition, the antigens identified by the IgM-IB test may serve as components of a rapid, accurate, point-of-care diagnostic test for early leptospirosis.

Development of a magnetic bead fluorescence microscopy immunoassay to detect and quantify Leptospira in environmental water samples

Climate change, world population growth, and poverty have led to an increase in the incidence of leptospirosis. Leptospirosis is caused by pathogenic spirochaete bacteria that belong to the genus Leptospira. The bacteria are maintained in the renal tubules of the reservoir hosts (typically a rodent), then shed into the environment via the urine. Water is key for environmental survival and transmission, as leptospires can survive for several weeks in a moist environment. Therefore, environmental epidemiological studies are needed to study the contamination of environmental water sources. However, few such studies have been performed using cultivation of the isolates and PCR assays. But, leptospira cultivation can be easily contaminated by other organisms and takes usually several weeks. Moreover, PCR is a complex and costly analysis for the underdeveloped countries that have the highest incidence of leptospirosis. In this study, we describe two modifications of a fluorescence microscopy assay based on immuno-magnetic separation (IMS) to detect leptospires in environmental water samples that mainly differ in fluorescent dye staining. The first type uses acridine orange fluorescent dye staining combined with multiplexed IMS for sample screening. The detection limit ranged from 10(2) to 10(3) organisms per mL and largely depended on the capture efficiency (CE) of the immuno-magnetic particles. The second type uses serogroup-specific immuno-particles and direct fluorescence antibody staining (DFA) to detect leptospires; the detection limit of this second assay was approximately 10(1) cells per mL. Both assay types were applied to natural and experimentally infected water samples, which were also analysed with DFM and real-time PCR. Our data show that the fluorescent microscopy immunoassay successfully identified experimental leptospire contamination and was as sensitive as PCR. This modified immune-fluorescence assay may therefore enable epidemiological studies of leptospirosis.

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non‐leptospirosis patients with a negative MAT were pooled and used as the negative control. 2‐D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI‐TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti‐human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.

Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Leptospirosis caused by Leptospira interrogans is the most widespread zoonosis and a major public health problem worldwide. Based on light-scattering and absorption, quantification of leptospires using UV-VIS spectroscopy was used as an indirect counting technique by measuring the optical density and comparing this to automated direct counting using a counting chamber in combination with imaging and analyzing software. Two serovars, Bangkok and Copenhagenii, from log-phase growth were used for the establishment of standard curves. They were found to be linear and slightly different in gradient for each serovar. The ease, rapidity, and reliability of these two adapted and optimized counting techniques may provide a useful alternative enumeration technique for leptospirosis research.

Effects of static magnetic field on growth of leptospire, Leptospira interrogans serovar canicola: immunoreactivity and cell division.

The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet=140+/-5 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using dark-field microscopy together with visual imaging. The data showed that the exposed Leptospira cells have lower densities and lower agglutination immunoreactivity than the unexposed control group. Interestingly, some of the exposed Leptospira cells showed abnormal morphologies such as large lengths. We discussed some of the possible reasons for these observations.

Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis

Leptospirosis is a common life-threatening disease worldwide. However, its diagnosis is frequently ineffective because the gold standard bacterial culture and microscopic agglutination test (MAT) are usually positive 1–2 weeks after the disease onset. We thus developed an immunochromatographic assay (LEPkit) to detect serum anti-leptospiral lipopolysaccharide (LPS) IgM for rapid diagnosis of acute leptospirosis. Using referenced sera of 77 leptospirosis and 91 non-leptospirosis cases, LEPkit yielded 97.4% sensitivity, 94.5% specificity, 93.8 positive predictive value (PPV), 97.7% negative predictive value (NPV), and 95.8% accuracy. The stability of this kit stored for up to 18 months and its reproducibility were confirmed. Testing in 74 new cases using samples at admission-phase and subsequent paired samples (total n = 135), overall sensitivity was 98.5%, whereas that of culture and single MAT (≥1:400) was 15.6% and 35.6%, respectively. When only the samples at admission-phase were used (n = 74), the sensitivity remained at 98.7%, whereas that of culture and single MAT (≥1:400) was 28.4% and 13.5%, respectively. In summary, our LEPkit was far more effective than any conventional methods for the diagnosis of acute leptospirosis, especially within the first few days after the disease onset. The ease of use, stability and reproducibility further enhance its feasibility for clinical use on-site.

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

Leptospirosis constitutes a major health problem in tropical and subtropical countries and is caused by pathogenic Leptospira. Immuno-magnetic separation (IMS) is considered to be an effective pre-enrichment method to isolate Leptospira from liquid specimen. We applied an inexpensive and simple IMS protocol using zero-length cross-linkers to immobilize polyclonal anti-leptospiral antibodies onto magnetic particles. The IMS-system has been optimized and evaluated by the assessment of the capture efficiency (CE). Main parameters that influence the conjugation procedure were optimized, including the amount of protein per milligram of magnetic particles, the pH and ionic strength of the conjugation buffer. The bead-bound leptospiral fraction was identified by using acridine orange fluorescence dye. The highest value for CE occurred when using high molar phosphate saline buffer at a pH around the isoelectric point of the antibodies. Finally, up to 3×108 leptospiral cells per mL could have been captured with approximately 50 μg of antibody-labelled particles. Strong particle agglutination could be observed during incubation for leptospiral concentrations in the range of 107–108 cells per mL. Despite covalent binding, we show that the physical adsorption parameters pH and ionic strength of the conjugation buffer greatly affect the entire immobilization process with regard to the CE, thus being able to increase the reactivity of the particles. We therefore conclude that a well-adjusted conjugation buffer for the used chemistry could possibly replace expensive and more complicated antibody immobilization methods.

Association between Opisthorchis viverrini and Leptospira spp. infection in endemic Northeast Thailand.

Opisthorchiasis caused by Opisthorchis viverrini is an important foodborne trematodiasis in Thailand, Laos and Cambodia. Interestingly, the opisthorchiasis endemic region overlaps with an area of leptospirosis emergence. Here we report an association between opisthorchiasis and leptospirosis in Thailand. Of 280 sera collected from villagers living around the Lawa wetland complex in Khon Kaen province, 199 (71%) were seropositive for leptospirosis by immunochromatography. Individuals with O. viverrini infection had a significantly higher rate of leptospirosis than those without (P=0.001). Significant higher leptospirosis prevalence was found in males than females (P=0.002). However, females but not males with O. viverrini infection showed a significantly higher seroprevalence of leptospirosis. Twenty-one of 35 environmental samples from the lake (water, mud and fish skin mucus) were positive for Leptospira spp. DNA sequencing, sequence alignment, and phylogenetic analysis of some positive nested PCR products revealed both pathogenic and intermediate pathogenic strains of Leptospira in the samples. Strikingly, O. viverrini metacercariae from the fish were positive for L. interrogans. These results suggest a close association between opisthorchiasis and leptospirosis. Contact with water, mud or eating raw fish harboring liver fluke metacercariae may be risk factors for Leptospira infection.

Immunodominance of LipL3293–272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies

BACKGROUND/PURPOSE Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.