A prospective study to evaluate the accuracy of rapid diagnostic tests for diagnosis of human leptospirosis: Result from THAI-LEPTO AKI study.

BACKGROUND: Rapid diagnostic tests (RDTs) have become widely used in low-resource settings for leptospirosis diagnostic. This study aims to evaluate the diagnostic performance of the five commercially available RDTs to detect human IgM against Leptospira spp. in Thai population. METHODOLOGY/PRINCIPAL FINDINGS: Ninety-nine serum samples from Leptospirosis suspicious patients were tested with five RDTs, including Medical Science Public Health, Leptocheck-WB, SD bioline, TRUSTline, and J.Mitra. The case definition was based on MAT, qPCR, and culture results. Diagnostic accuracy was determined based on the first day of enrollment in an overall analysis and stratified according to days post-onset of fever. The five RDTs had overall sensitivity ranging from 1.8% to 75% and specificity ranging from 52.3% to 97.7%. Leptocheck-WB had high sensitivity of 75.0%. The sensitivity of five RDTs increased on days 4-6 post-onset of fever, while the specificity of all tests remained relatively stable at different days post-onset of fever. CONCLUSIONS/SIGNIFICANCE: The tested RDTs showed low sensitivity. Therefore, based on the present study, five commercially available RDTs might not be an appropriate test for acute leptospirosis screening in the Thai population.

Low Prevalence of Leptospira Carriage in Rodents in Leptospirosis-Endemic Northeastern Thailand.

Leptospirosis is a neglected zoonotic disease affecting mostly the world's tropical regions. The rural people of northeastern Thailand suffer from a large number of leptospirosis infections, and their abundant rice fields are optimal rodent habitats. To evaluate the contribution of diversity and carriage rate of pathogenic Leptospira in rodent reservoirs to leptospirosis incidence, we surveyed rodents, between 2011 and 2012, in four provinces in northeastern Thailand with the highest incidence rates of human leptospirosis cases. We used lipL32 real-time PCR to detect pathogenic Leptospira in rodent kidneys, partial 16S rRNA gene sequencing to classify the infecting Leptospira species, and whole 16S rDNA sequencing to classify species of isolated Leptospira. Overall prevalence of Leptospira infection was 3.6% (18/495). Among infected rodents, Bandicotaindica (14.3%), Rattusexulans (3.6%), and R. rattus (3.2%) had renal carriage. We identified two pathogenic Leptospira species: L. interrogans (n = 15) and L. borgpetersenii (n = 3). In addition, an L. wolffii (LS0914U) isolate was recovered from the urine of B. indica. Leptospira infection was more prevalent in low density rodent populations, such as B. indica. In contrast, there was a lower prevalence of Leptospira infection in high density rodent populations of R. exulans and R. rattus.

Optimization of Culture Protocols to Isolate Leptospira spp. from Environmental Water, Field Investigation, and Identification of Factors Associated with the Presence of Leptospira spp. in the Environment.

The successful culture of Leptospira spp. from the environment is challenging. Here, we optimized the isolation of Leptospira spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic Leptospira spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of Leptospira subclade P1 were culture positive within 2 weeks. For Leptospira subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 103 colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of Leptospira spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. Leptospira spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with Leptospira spp. Cultures of Leptospira spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.

Surveillance for Scrub Typhus, Rickettsial Diseases, and Leptospirosis in US and Multinational Military Training Exercise Cobra Gold Sites in Thailand.

We report findings of field surveillance for disease vectors and the prevalence of Orientia tsutsugamushi, the causative agent for scrub typhus, and other Rickettsial species that cause murine typhus and spotted fever group rickettsioses, in chigger mites and small rodents; and Leptospira in rodent kidney, urine, and environmental water samples. The study sites included various Royal Thai Army military installations and other training sites, and surrounding areas where the multinational military training exercise Cobra Gold was conducted in Thailand in 2017 and 2018. The overall prevalence of O. tsutsugamushi and Rickettsia infection in chiggers was 1.3% (20/1,594) and 7.5% (119/1,594), respectively. Serum samples of the captured rodents indicated previous exposure to O. tsutsugamushi infection with a seropositive rate of 12.2%. Leptospira species were isolated from rodent kidneys and water samples collected from catchment areas as well as tap water used for hand washing. Findings from this surveillance are important in determining the potential for scrub typhus, rickettsioses, and leptospirosis risk to military and US government personnel, as well as for informing regional and combatant commanders for prevention, correct diagnosis, prompt treatment, and timely and focused implementation of vector control and personal protective measures.

Detection of Dengue Virus in Mosquito Extracts and Human Clinical Samples Using a Field Expedient Molecular Platform.

Dengue fever occurs in localized outbreaks and can significantly erode troop strength and mission readiness. Timely identification of dengue virus (DENV) provides for rapid and appropriate patient management decisions, such as medical evacuation and supportive therapies, as well as help to promote Force Health Protection through vector control and personal protective measures. The "Ruggedized" Advanced Pathogen Identification Device is a field-friendly PCR (Polymerase Chain Reaction) platform that can be used to facilitate early identification of DENV. We developed a dry-format PCR assay on this platform. The assay demonstrated 100% analytical specificity for detecting dengue using a cross-reactivity panel. We used a panel of 102 acute, DENV isolation positive serum samples and 25 DENV negative samples; the assay demonstrated a clinical sensitivity of 97.1% (95% C.I. 91.6-99.4%) and specificity of 96.0% (95% C.I. 79.7-99.9%) in identifying patients with dengue infection. We also used the assay to test mosquito homogenates from 28 adult female Aedes aegypti. A single DENV infected mosquito was identified using the PCR assay and confirmed using immunofluorescence as a reference method. Much of the testing was performed under austere field conditions. Together, our results demonstrate the utility of this assay for detecting DENV in vector and human samples in field environments.

Effect of peritrophic matrix C-type lectin (AdPMCTL) on blood-meal size in Anopheles dirus.

The peritrophic matrix (PM) is penetrated by Plasmodium ookinete to permit transition to oocyst in the mosquito midgut, the manner by which the ookinete interacts with glycoproteins on the PM remains poorly understood. We partially characterized peritrophic matrix C-type lectin (PMCTL) from An. gambiae (CTL10) and An. dirus (AdPMCTL). AdPMCTL protein was produced specifically in blood-fed mosquitoes. The 320 amino acid AdPMCTL exhibits 72% identity with a putative secreted An. gambiae ortholog (AGAP009316, CTL10). AdPMCTL was cloned and its expression profile determined in sugar- and blood-fed midguts. RNAi was used to determine the effect of AdPMCTL on blood meal size and on mosquito survival. AdPMCTL mRNA was present in midguts of sugar-fed mosquitoes and exhibited up-regulation following a blood meal, and AdPMCTL silencing significantly influenced the blood-meal size of engorged mosquitoes, suggesting a role for AdPMCTL as a stabilizing linker molecule, which limits PM distension after blood feeding.