Leptospirosis, a global zoonotic disease caused by Leptospira spp., presents high morbidity and mortality risks, especially in tropical regions like Thailand. Military personnel deployed in endemic areas, such as during the Cobra Gold Joint exercise, face heightened exposure. This study assessed Leptospira’s prevalence in rodents and environmental reservoirs at military training sites from 2017 to 2022. A surveillance program was conducted at Engineering Civil Assistance Program (ENCAP) training sites using real-time PCR, dark-field microscopy, and 16S rRNA gene sequencing to detect Leptospira in rodents and environmental samples. Results showed a 1.3% infection rate in rodents (15 of 1161), while Leptospira was detected in 10.2% of water samples (42 of 413) and 23.1% of soil samples (30 of 130). Diverse Leptospira interrogans strains circulated among rodents, and three groups of naturally circulating Leptospira strains were detected in environmental reservoirs. These findings underscore Leptospira’s survival and transmission potential within exercise sites, informing Force Health Protection (FHP) decisions. By integrating pre-exercise data on primary hosts and environmental reservoirs with historical local outbreak records and research on risk factors, this study identifies key areas for public health intervention and potential mitigation strategies.

Background: Identifying effective malaria elimination strategies for remote forested regions in Southeast Asia is challenging given limited resources. In this study, two malaria elimination strategies were evaluated in partnership with the Royal Cambodian Armed Forces - monthly malaria prophylaxis (MMP) and focused screening and treatment (FSAT). Methods: Eight primarily military clusters (1,050 volunteers total) along the Cambodian-Thai border were randomized to 3 months of MMP or FSAT with monthly malaria testing by RDT, PCR, and microscopy for six months. Clusters were sub-randomized to permethrin treated (ITU) or sham water-treated clothing (sITU). Volunteers in MMP clusters were given three full monthly dihydroartemisinin-piperaquine (DP) treatment courses with 12 weekly 22.5mg primaquine. Volunteers in FSAT clusters were treated with appropriate first-line antimalarials if malaria-positive by microscopy or PCR. Results: Pf positivity in MMP clusters was reduced by 90% (10% at enrollment to 1% at 6 months; absolute risk reduction (ARR) 9%) at 6 months. However, 32% of Pf cases treated with DP as MMP at baseline recrudesced, requiring rescue treatment at 1 month with artesunate-mefloquine. Pf positivity in FSAT clusters declined 66% over 6 months (7.6% to 2.7%; ARR 4.9%). MMP reduced Pv positivity from 9% to 0% at 3 months, but Pv rebounded to 6.7% at 6 months. FSAT failed to significantly reduce Pv positivity during the study. The 22.5mg weekly primaquine MMP regimen was safe, even for the 15% of volunteers with G6PD-deficiency. Those wearing ITU had additional Pv parasitemia reductions compared to sITU in the FSAT but not MMP groups. PCR was more sensitive than microscopy and RDT for detecting both species. Conclusions: MMP was safe, and superior to FSAT to reduce Pf and Pv, suggesting greater utility to achieve malaria elimination in Cambodia. Low dose (22.5mg) weekly primaquine was a safe adjunct in this setting, even for those with G6PD-deficiency. Permethrin-treated clothing further reduced Pv parasitemia for FSAT but not MMP. MMP may be more easily scaled to eliminate malaria. The military may provide substantial support for regional elimination efforts. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT02653898 ### Funding Statement This study was funded by the Bill and Melinda Gates Foundation and the Defense Malaria Assistance Program. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IRB of the Walter Reed Army Institute of Research gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Prior to the devastating earthquake in Nepal in 2015, scrub typhus was not recognized as a highly endemic disease in the country. This contrasted with neighboring India, where scrub typhus is endemic and there have been sporadic outbreaks of severe forms. This discrepancy underscores the limitations in our comprehensive understanding of the scrub typhus epidemiological patterns in Nepal, especially before 2015. To better understand the dynamic and current status of scrub typhus, this study investigated its prevalence among patients with acute febrile illness in two hospitals located in Pokhara city, Kaski district and Bharatpur city, Chitwan district during 2009–2010. Our findings revealed that 31.5% (239 of 759 patients) of the cases were positives for scrub typhus based on serological and pathogen detection assays. These results provide crucial insights into the pre-earthquake endemicity of scrub typhus in Nepal, implying its long-standing presence in the region prior to the significant environmental transformations caused by the 2015 earthquake. This study also emphasizes the need for heightened awareness and improved diagnostic capabilities to effectively manage and control scrub typhus, which remains a significant public health concern in Nepal.

Factory-treated permethrin uniforms are the primary method used by the US Army to prevent arthropod bites and transmission of arthropod-borne diseases. In this study previously worn uniforms were collected from cadets at the United States Military Academy in West Point, NY to determine the amount of permethrin remaining after prolonged wear and subsequent effects on ticks. Six trousers were collected from cadets in the sophomore, junior, and senior classes. A new, unwashed uniform served as a positive control and an untreated maternity uniform served as a negative control. Fabric samples were removed from each trouser and used in bioassays with laboratory-reared Ixodes scapularis Say nymphs. Contact irritancy bioassays measured the nymphs' ability to remain in contact with fabric for a period of 5 min. Toxicity bioassays measured tick survival at 1 and 24 h after contacting uniform samples. liquid chromatography-mass spectrometry was used to quantify the permethrin content (mg/cm2) in each uniform after the bioassays were complete. The results showed significant amounts of permethrin were lost after extended wear and it was related to the cadet year group. The contact irritancy assays found uniforms with less permethrin did not irritate ticks and cause them to dislodge. Mortality was also affected by permethrin levels, with less ticks dying at 24 h on older uniforms. The results from this study show older uniforms lose most of their permethrin and no longer provide the same levels of protection.

INTRODUCTION:The Army uses permethrin-treated uniforms as the primary method to protect soldiers from tick-borne diseases. Permethrin binds strongly to fabric and provides long-term protection against many blood-feeding arthropods. However, protection decreases if the uniforms are not washed and cared for according to label instructions. This study was conducted among cadets at the United States Military Academy (USMA) at West Point, NY, to determine what the cadets know about permethrin and how they care for and wear their uniforms. West Point is in an area with high rates of tick-borne disease transmission. A survey was developed to determine what cadets know about the threat of tick-borne diseases and if they wear and maintain their uniforms in a manner that effectively maintains permethrin levels. MATERIALS AND METHODS:A 16-question survey was developed and submitted to the local review board for approval. The study was classified as human subjects research according to 32CFR219 and met the requirements for exempt status under 32CFR219.104(d)(2)(i). After receiving approval, a hard copy survey was distributed among cadets with efforts to sample at least 50 members from each cadet class. RESULTS:A total of 319 cadets provided responses to the survey questions, representing more than 7% of the cadet population at the USMA. The results showed most cadets knew their uniforms were treated with permethrin, but less than half knew there are specific instructions attached to their uniforms describing how the uniforms should be laundered. From the cadets who knew there were instructions, most admittedly did not follow the instructions. Sixteen percent of cadets said they had dry-cleaned their uniforms. This is a process known to remove most of the permethrin. The majority of cadets viewed the risk of tick-borne disease at West Point to be moderate or lower. CONCLUSIONS:This study provides a basic understanding of the wear patterns of permethrin-treated uniforms among cadets at the USMA. It is also one of the few studies to measure knowledge and uniform-wearing behavior among service members since the Army switched to factory-treated uniforms in 2013. The results indicate that compliance with uniform laundry and care instructions is low. This information is useful to develop training plans and educate cadets how they can wear and take care of their permethrin-treated uniforms to better protect themselves from tick-borne diseases.

There are eight Anopheles spp. present in the Republic of Korea (ROK), including five members of the Anopheles Hyrcanus Group that cannot be identified using current morphological methods. The vector competence of only Anopheles sinensis s.s., An. lesteri, and An. kleini have been investigated. As the geographical distribution of Anopheles spp. varies in the ROK, determining the relative vector competence of the Anopheles spp. provides a basis for delineating malaria risks to Korean populations and U.S. military/civilian populations deployed to the ROK. Anopheles belenrae and An. pullus, collected from a malaria high-risk area in the ROK, were evaluated for vector competence of P. vivax. A total of 1,000 each of An. dirus (Thai strain), and Korean strains of An. pullus and An. belenrae were fed on P. vivax infected blood collected from Thai patients via artificial membrane feeding. The overall oocyst infection rates for An. dirus, An. pullus, and An. belenrae dissected on days 8-9 postfeed were 64.1, 12.0, and 11.6%, respectively. The overall sporozoite infection rates for those species dissected on days 14-15 postfeed were 84.5, 3.4, and 5.1% respectively. The salivary gland sporozoite indices for positive females with +4 (>1,000 sporozoites) were observed in An. dirus (72.8%), but not observed for either An. pullus or An. belenrae. Most sporozoite-positive An. pullus (83.3%) and An. belenrae (71.4%) females were observed with only +1 (1-10 sporozoites) salivary glands. These data indicate that both An. belenrae and An. pullus are very poor vectors of P. vivax.

Tick-borne diseases are a major public health concern in Mongolia. Nomadic pastoralists, which make up ~ 26% of Mongolia’s population, are at an increased risk of both tick bite exposure and economic loss associated with clinical disease in herds. This study sought to further characterize tick-borne pathogens present in Dermacentor ticks ( n = 1,773) sampled in 2019 from 15 of Mongolia’s 21 aimags (provinces). The ticks were morphologically identified and sorted into 377 pools which were then screened using Next-Generation Sequencing paired with confirmatory PCR and DNA sequence analysis. Rickettsia spp. were detected in 88.33% of pools, while Anaplasma spp. and Bartonella spp. were detected in 3.18 and 0.79% of pools, respectively. Khentii had the highest infection rate for Rickettsia spp. (76.61%; CI: 34.65–94.79%), while Arkhangai had the highest infection rate for Anaplasma spp. (7.79%; CI:4.04–13.72%). The exclusive detection of Anaplasma spp. in tick pools collected from livestock supports previous work in this area that suggests livestock play a significant role in disease maintenance. The detection of Anaplasma, Bartonella, and Rickettsia demonstrates a heightened risk for infection throughout Mongolia, with this study, to our knowledge, documenting the first detection of Bartonella melophagi in ticks collected in Mongolia. Further research deploying NGS methods is needed to characterize tick-borne pathogens in other endemic tick species found in Mongolia, including Hyalomma asiaticum and Ixodes persulcatus .

The livestock industry in Mongolia accounts for 24% of national revenue, with one third of the population maintaining a pastoral lifestyle. This close connection between Mongolian population and livestock is a major concern for pathogen transfer, especially given the increase in vector-borne diseases globally. This study examines blood samples from livestock to assess the prevalence of tick-borne bacterial infections across three provinces in Mongolia (Dornogovi, Selenge, Töv). Whole blood samples from 243 livestock (cattle=38, camel=11, goat=85, horse=22, sheep=87) were analyzed with 16S metagenomics next-generation sequencing (NGS) to screen for bacterial pathogens. Positive-NGS samples for Anaplasma, Bartonella, Ehrlichia, Francisella, Leptospira, and Rickettsia were confirmed by conventional PCR and DNA sequencing. Prevalence rates of Anaplasma, Bartonella, and Ehrlichia were 57.6%, 12.8%, and 0.4%, respectively. A significant difference in the prevalence of Anaplasma spp. in livestock by province was observed, with a higher prevalence in Selenge (74.2%, p<0.001) and Töv (64.2% p = 0.006) compared to the semi-arid region of Dornogovi (39.8%). In contrast, no association was observed in Bartonella prevalence by provinces. All Anaplasma sequences (N = 139) were characterized as A. ovis. For Bartonella species characterization, phylogenetic analyses of gltA and rpoB genes identified three Bartonella species including B. bovis, B. melophagi and Candidatus B. ovis. Bartonella bovis was detected in all 22-positive cattle, while B. melophagi and Candidatus B. ovis were found in four and three sheep, respectively. This study identifies a high prevalence of tick-borne pathogens within the livestock population and to our knowledge, is the first time NGS methods have been used to explore tick-borne diseases in Mongolia. Further research is needed in Mongolia to better understand the clinical and economic burdens associated with tick-borne diseases in both livestock and pastoral herder populations.

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3-99.9%) than Japanese (96.9-97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

Abstract Background Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand. Methods Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection. Results A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) (87.16%, n = 5035), An. dirus s.l. (7.05%, n = 407) and An. barbirostris s.l. (2.86%, n = 165). Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), of the An. dirus (s.l.) collected, 100% were An. baimaii (n = 348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (n = 44) and 6.38% were An. sawadwongporni (n = 3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (Kruskal–Wallis H-test: χ2 = 80.89, P < 0.0001, n = 5666), with more An. barbirostris (s.l.) and An. maculatus (s.l.) caught earlier compared to An. minimus (s.l.) (P = 0.0001 and P < 0.0001, respectively) and An. dirus (s.l.) (P = 0.0082 and P < 0.001, respectively). The proportion of parous An. minimus (s.l.) captured by hour increased throughout the night (Wald Chi-square: χ2 = 17.31, P = 0.000, odds ratio = 1.0535, 95% confidence interval 1.0279–1.0796, n = 3400). Overall, An. minimus (s.l.) parity was 67.68% (2375/3509) with an intra-cluster correlation of 0.0378. A power calculation determined that an An. minimus (s.l.) parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster, at an α = 0.05, will provide 82% power to detect a significant difference following ivermectin MDA. Conclusions The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity. Graphical abstract

Background Scrub typhus is a largely ignored tropical disease and a leading cause of undifferentiated febrile illness in the areas of tsutsugamushi triangle caused by Orientia tsutsugamushi . It is frequently diagnosed in South Asian countries, although clear epidemiological information is not available from Nepal. After the 2015 earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported. The objective of this study was to investigate epidemiology of scrub typhus and its causative agents in humans, animals, and chigger mites to understand the ongoing transmission ecology. Methods Scrub typhus cases with confirmed diagnosis throughout the country were included in the analysis. Studies were concentrated in the Chitwan district, the site of a major outbreak in 2016. Additional nation-wide data from 2015 to 2017 available from the government database included to analyse the disease distribution by geographical mapping. Results From 2015 to 2017, 1239 scrub typhus cases were confirmed with the largest outbreak occurring in 2016 with 831 (67.1%) cases. The case fatality rate was 5.7% in 2015 which declined to 1.1% in 2017. A nationwide outbreak of scrub typhus was declared as the cases were detected in 52 out of the 75 districts of Nepal. Seasonal trend was observed with a peak during August and September. In addition to the human cases, the presence of O . tsutsugamushi was also confirmed in animals (rodents) and chigger mites (Leptotrombidium imphalum) from the outbreak areas of southern Nepal. Conclusion The detection of O. tsutsugamushi in humans, animals, and chigger mites from outbreak locations and wide-spread reports of scrub typhus throughout the country consecutively for 3 years confirms the ongoing transmission of O. tsutsugamushi with a firmly established ecology in Nepal. The country’s health system needs to be strengthened for systematic surveillance, early outbreak detection, and immediate actions including treatment and preventive measures.

Several light trap devices have been invented and developed to assess the abundance of sand flies. Traps available in the market have different designs and attractant combinations to catch sand fly vectors. We evaluated the efficacy of four commercial light traps and determined the effect of trap placement and carbon dioxide (CO2) on sand fly collection in northern Thailand. Trap evaluations were conducted at two natural caves located in Chiang Rai province, Thailand. In the first part of the study, the efficacies of four trap types including the Centers for Disease Control miniature light trap (CDC LT), Encephalitis Vector Survey trap (EVS), CDC Updraft Blacklight trap (CDC UB), and Laika trap (LK) were evaluated and compared using a Latin square experimental design. The second half of the study evaluated the influence of trap placement and CO2 on sand fly collection. Additionally, CDC LT were placed inside, outside, and at the entrance of caves to compare the number of sand flies collected. For the trap efficacy experiment, a total of 11,876 phlebotomine sand flies were collected over 32 trap-nights. Results demonstrated that CDC LT, CDC UB, and LK collected significantly more sand flies than EVS (P > 0.05). However, there were no significant differences between the numbers of sand flies collected by CDC LT, CDC UB, and LK. A total of 6,698 sand flies were collected from the trap placement and CO2 experiment over 72 trap-nights. Results showed that CO2 did not influence the numbers of sand flies captured (P < 0.05), whereas trap placement at the entrance of the caves resulted in collection of significantly more sand flies than traps placed inside and outside of the caves. We found the CDC LT, CDC UB, and LK without CO2 captured the greatest amount of sand flies. This was particularly observed when traps were placed at the entrance of a cave, perhaps because of the greater passage of stimuli caused by wind flow at the entrance of the cave. The light traps in this study can be used effectively to collect sand fly vectors in northern Thailand.

BACKGROUND:Scrub typhus is an important disease in the Asia-Pacific countries with increasing relevance for public health worldwide. Entomological risk assessment for scrub typhus and rickettsial disease in Phu Chi Fah village-Chiang Rai was performed to determine areas at greatest risk for disease transmission in order to increase awareness of diseases to travelers and healthcare workers.METHODS:From 2016 to 2018, rodents and chiggers were collected from 7 sites covering residential, grassland, and forest areas to determine the prevalence of pathogen of interest. The correlation between land type and vector-host-pathogen interaction system was investigated.RESULT:High prevalence of O. tsutsugamushi-infected and Rickettsia-infected chiggers was observed especially in areas with grassland and forest ecotones. Chigger and rodent species composition were negatively associated with the level of human disturbance. Increased density of rodents was responsible for a higher abundance of chigger population and increased prevalence of O. tsutsugmaushi infection in chigger in the following year.CONCLUSION:Communities in the study areas have an increased exposure risk to scrub typhus and potentially spotted fever group rickettsiosis. Travellers to this endemic area should pay more attention pathogen risks so as to avoid vector and disease exposure. Seasonal rodenticidal activity may help migitate the risk of pathogen transmission.

Entomological surveillance for arthropod-borne viruses is vital for monitoring vector-borne diseases and informing vector control programs. In this study, we conducted entomological surveillance in Zika virus endemic areas. In Thailand, it is standard protocol to perform mosquito control within 24 h of a reported dengue case. Aedes females were collected within 72 h of case reports from villages with recent Zika–human cases in Kamphaeng Phet Province, Thailand in 2017 and 2018. Mosquitoes were bisected into head-thorax and abdomen and then screened for Zika (ZIKV) and dengue (DENV) viruses using real-time RT-PCR. ZIKV RNA was detected in three samples from two female Ae. aegypti (1.4%). A partial envelope sequence analysis revealed that the ZIKV sequences were the Asian lineage identical to sequences from ZIKV-infected cases reported in Thailand during 2016 and 2017. Dengue virus-1 (DENV-1) and dengue virus-4 (DENV-4) were found in four Ae. aegypti females (2.8%), and partial capsid sequences were nearly identical with DENV-1 and DENV-4 from Thai human cases reported in 2017. Findings in the current study demonstrate the importance of entomological surveillance programs to public health mosquito-borne disease prevention measures and control.

BACKGROUND:Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens.METHODS:Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells.RESULTS:Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP.CONCLUSIONS:PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery.TRIAL REGISTRATION:ClinicalTrials.gov NCT01994525.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

Mass administration of antimalarial drugs and ivermectin are being considered as potential accelerators of malaria elimination. The safety, tolerability, pharmacokinetics, and mosquito-lethal effects of combinations of ivermectin, dihydroartemisinin-piperaquine, and primaquine were evaluated. Coadministration of ivermectin and dihydroartemisinin-piperaquine resulted in increased ivermectin concentrations with corresponding increases in mosquito-lethal effect across all subjects. Exposure to piperaquine was also increased when coadministered with ivermectin, but electrocardiograph QT-interval prolongation was not increased. One subject had transiently impaired liver function. Ivermectin mosquito-lethal effect was greater than predicted previously against the major Southeast Asian malaria vectors. Both Anopheles dirus and Anopheles minimus mosquito mortality was increased substantially (20-fold and 35-fold increase, respectively) when feeding on volunteer blood after ivermectin administration compared with in vitro ivermectin-spiked blood. This suggests the presence of ivermectin metabolites that impart mosquito-lethal effects. Further studies of this combined approach to accelerate malaria elimination are warranted.

Malaria is a serious health problem in Southeast Asia. The malaria vectors Anopheles cracens (Stenogamous) and Anopheles dirus (Eurygamous) were used to evaluate the impact of first and second blood meals on feeding, survival and hatching rates, and fecundity. Females were divided as follows: mated with oviposition, mated without oviposition, and virgin. The blood-feeding rates of An. cracens groups were significantly lower for the second blood meal than for the first. Additionally, for An. dirus mated without oviposition and virgin groups, blood-feeding rates were significantly lower for the second blood meal compared with the first blood meal. Survivorship and the number of eggs laid increased for An. cracens after the second blood meal, but the opposite was true for An. dirus. There were no differences in hatching rates. Thus, blood meals affected survivorship and fecundity in naturally mating An. cracens and induced-mating An. dirus.

Background Scrub typhus is a severely ignored tropical disease and a leading cause of undifferentiated febrile illness worldwide caused by infection of an obligate intracellular bacteria Orientia tsutsugamushi . It has been rapidly expanding in South Asian countries, although clear epidemiological information is not available from Nepal. After the 2015 earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported. The objective of this study was to investigate scrub typhus and its causative agents in human, rodent and chigger mites to better understand the ongoing transmission ecology.Methods Scrub typhus cases with confirmed diagnosis throughout the country were included in the analysis. Studies were concentrated in the Chitwan district, the site of a major outbreak in 2016. Additional country-wide data from 2015 to 2017 was made available from the government database to analyse the disease distribution using geographical mapping.Results During 2015-2017, 1,239 scrub typhus cases were confirmed with the largest outbreak occurring in 2016 with 831 (67.1%) cases. The remainder 267 cases were reported in 2017. The case fatality rate was 5.7% in 2015 and declined to 1.1% in 2017. Nationwide outbreak of scrub typhus was identified as the cases were found from 52 of the 75 districts of Nepal. A seasonal trend was observed with a peak during August and September (p = 0.01). In addition to the human cases, the presence of O. tsutsugamushi was also confirmed in rodents and chigger mites from the outbreak areas of southern Nepal.Conclusion The detection of O. tsutsugamushi in human, rodent, and chigger mites from outbreak locations and wide-spread reports of scrub typhus throughout the country over two years confirms the ongoing transmission of O. tsutsugamushi with a firmly established ecology in Nepal. The country’s health system needs to be strengthened for systematic surveillance, early outbreaks detection, and immediate response actions including treatment and preventive measures.Author Summary Scrub typhus is a disease caused by a bacteria called Orientia tsutsugamushi and transmitted to people through bites of infected chiggers (larval mites). After the 2015 Gorkha earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported with repeated outbreaks from different parts of the country. This study has documted epidemiology of scrub typhus and its causative agents in human, rodent and chigger mites confimring the local transmission O . tsutsugamushi with a firmly established ecology in Nepal. The local transmission of the diseases from most parts of the country demands strengthening for systematic surveillance, early outbreaks detection, and immediate response actions including treatment and preventive measures.