Zoonotic neglected tropical diseases (NTDs) remain a substantial but under-recognised source of human morbidity and economic concern in the Greater Mekong Subregion, particularly in settings characterised by close human-animal interaction. This article represents a narrative synthesis of zoonotic disease research conducted in Laos and Cambodia between 2000 and 2025. The review integrates published literature with findings from long-term surveillance programmes conducted by the authors and collaborating institutions in Laos, with comparative insights from Cambodia, to examine the presence, distribution, diversity, and drivers of zoonotic pathogens at the human-animal interface. Evidence demonstrates the endemic presence of a wide range of parasitic, bacterial, and viral zoonoses, including Taenia solium, Trichinella spp., Streptococcus suis, rickettsial infections, melioidosis, hepatitis E virus, and Japanese encephalitis virus. Some of these pathogens are sustained within smallholder livestock systems, informal slaughter, farming practises, food networks, and wet market environments, where limited diagnostic capacity and fragmented surveillance obscure true disease presence. Surveillance innovations, including abattoir-based sampling, cross-sectoral serological studies, environmental surveillance approaches, and molecular diagnostic tools, have improved pathogen detection but have also highlighted persistent structural and behavioural barriers to control. Socio-cultural practices, occupational exposure, wildlife trade, and economic dependencies reinforce transmission dynamics, indicating that biomedical interventions alone are insufficient. Instead, zoonotic disease persistence reflects the interaction of livestock production systems, environmental conditions, diagnostic limitations, and entrenched human behaviours. This review emphasises the need for integrated One Health approaches that combine strengthened surveillance, improved diagnostics, behavioural interventions, and regional collaboration. Addressing zoonotic NTDs in Laos and Cambodia requires coordinated strategies that account for both biological complexity and socio-economic context to achieve sustainable disease control and improved public health outcomes.

BACKGROUND:Murine typhus is a flea-borne disease caused by Rickettsia typhi that typically presents as an acute febrile illness. The diagnosis is often missed, leading to delays in appropriate treatment. A qPCR targeting a single gene (ompB) for R. typhi is widely used for diagnosis; however, it has low sensitivity for detecting bacterial DNA in patients' blood. We aimed to increase sensitivity of detection of R. typhi using qPCR by targeting a gene containing repetitive sequences, sca2 (surface cell antigen 2). METHODOLOGY:We compared diagnostic accuracy with the standard assay targeting single sequence ompB (outer membrane protein B). Specificity, sensitivity, and bacterial load measurement of both assays were compared using stored EDTA-anticoagulated buffy coat samples from 88 patients with febrile illness at Mahosot Hospital or provincial hospitals in Laos. Among these, 55 cases were confirmed as positive or negative by first culturing Rickettsia spp. from patients' EDTA blood, followed by assessment with IFA and ompB PCR, and buffy coat from 6 additional cases was confirmed by ompB PCR. Ten further positive cases were confirmed by IFA using paired sera, and 17 cases classified as negative for scrub typhus and murine typhus based on Rapid Diagnostic Test (RDT) results were included in the evaluation. RESULTS:The sca2 qPCR assay showed 59.09% sensitivity (95% CI, 38.73-76.74%) and 100% specificity (93.98-100%) for detection of R. typhi. In comparison, ompB assay demonstrated 36.36% sensitivity (95% CI, 19.73-57.05%) and 100% specificity (95% CI 93.98-100%). DNA copy number determined using the sca2 gene was approximately 2.933 log unit higher than that determined using ompB gene (median, 16,500 copies/μL; IQR, 13,045-40,000 versus median, 19.25 copies/μL; IQR, 11.11-56.62, P < 0.0001). CONCLUSION:This study suggests qPCR targeting sca2 increases frequency of detection of R. typhi in patients with low bacterial DNA concentrations.

BackgroundMurine typhus, caused by Rickettsia typhi, is a treatable febrile illness in Laos, where azithromycin treatment failure has been reported. Antibiotic susceptibility testing for Rickettsia spp. is challenging due to absence of resistant strains. We aimed to induce an azithromycin-resistant in R. typhi and investigate its genetic basis.MethodologyR. typhi Wilmington was cultured in azithromycin-containing media (R. typhiAZM), starting at a concentration of 0.0019 mg/L and gradually increased to 0.0625 mg/L. Resistant populations were selected up to 0.125 mg/L. MICs were determined using plaque assay and qPCR, and DNA sequencing was performed for rplD (L4), rplV (L22), and 23S rRNA domain V. Protein modeling of azithromycin-binding sites was conducted, and strain stability was assessed over 24 passages without azithromycin (R. typhi AZM (-)).ResultsMICs for wild type (R. typhiWT) and R. typhiAZM were 2 mg/L versus >16 mg/L (plaque assay) and 0.25 mg/L versus 8 mg/L (qPCR). A 15-nucleotides insertion (5'-AAAGGAAGAGCAACT-3') was found in the rplV of R. typhiAZM, but not other isolates. Protein modeling suggested the insertion extends the L22 loop, potentially affecting azithromycin binding site within the ribosomal exit tunnel. R. typhiAZM reverted to wild type MIC and genotype by 24 passages without azithromycin. R. typhiAZM exhibited an 8 -fold higher MIC than R. typhiWT.ConclusionRepetitive insertion in rplV was associated with azithromycin resistance and may interfere with drug binding. R. typhiAZM was unstable without selective pressure. This approach may help generate resistant strains for assay validation. The role of rplV mutations in azithromycin susceptibility warrants further investigation.

IntroductionPhlebotomine sandflies are the principal vectors of leishmaniasis. Laos is a landlocked country bordering Thailand, where autochthonous cases of leishmaniasis have been reported. However, the status of leishmaniases in Laos is unknown due to a lack of testing. In the past decades, very few studies on sandflies and sandfly-borne pathogens have been conducted in Laos. Therefore, the knowledge of sandfly diversity, distribution, and their related pathogens is lacking. We hypothesized that both known and putative sandfly-borne pathogens may silently circulate in Laos but remain undetected. Herein, we aimed to report species diversity data and Leishmania detection among sandflies collected from seven provinces of Laos.MethodsSandflies were collected using CDC light traps from various habitats, including caves and peri-caves in karstic areas, domestic animal sheds, forests, and plantations, across seven provinces in northern Laos. Sandfly collections were conducted between 2014 and 2024 from different seasons. Sandflies were identified by morphological and molecular techniques. PCR targeting the ITS1 region was used to screen for Leishmania in the sandfly samples. Though a specific Trypanosoma PCR was not used, this PCR is also able to detect the ITS1 region in some Trypanosoma spp.ResultsA total of 3,857 sandflies from 25 species belonging to five genera were collected and examined: Chinius eunicegalatiae, Idiophlebotomus longiforceps, Phlebotomus barguesae, Ph. breyi, Ph. (Adlerius) sp., Ph. kiangsuensis, Ph. mascomai, Ph. seowpohi, Ph. shadenae, Ph. sinxayarami, Ph. stantoni, Sergentomyia anodontis, Se. bailyi, Se. barraudi group, Se. brevicaulis, Se. dvoraki, Se. hivernus, Se. khawi, Se. perturbans, Se. phasukae, Se. sutherlandi n. sp., Se. sylvatica, Se. tambori, Se. gemmea-like (Se. sp 1) and Grassomyia indica. The highest diversity of sandflies was found in karstic areas where sandflies were collected from cave and peri-cave areas. One new sandfly species, Se. sutherlandi n. sp. is described. We also report for the first time in Southeast Asia a Phlebotomus female belonging to the subgenus Adlerius and we also discuss about the taxonomy of Sergentomyia brevicaulis. Although no Leishmania DNA was detected from screened sandflies, unknown Trypanosoma species were detected from 24 individual sandflies: Chinius eunicegalatiae (n = 22) and Idiophlebotomus longiforceps (n = 2) using a PCR assay that was primarily meant to screen for Leishmania.ConclusionThis study reveals a notable diversity of sandfly species across seven provinces in Laos, with the highest species richness observed in karstic cave environments. While no Leishmania DNA was detected, the unexpected identification of unknown Trypanosoma species in Chinius eunicegalatiae and Idiophlebotomus longiforceps suggests the potential presence of unrecognized trypanosomatid in the region. These findings underscore the need for broader geographic surveillance and increased sampling efforts to improve our understanding of sandfly ecology and the pathogens they may carry in Laos.

Abstract We used 2492 whole genome sequences from Laos to investigate the molecular epidemiology of SARS-CoV-2 from 2021 through 2024, covering the major waves of COVID-19 disease in Laos including time periods of travel restrictions and after relaxation of travel across international borders. We identify successive waves of COVID-19 caused by shifts in the dominant lineage, beginning with the Alpha variant in April 2021 and continuing through the Delta and Omicron variants. We quantify a shift from a small number of viral introductions responsible for widespread transmission in early waves to a larger number of introductions for each variant after travel restrictions were lifted, and identify potential routes of introduction into the country. Our study underscores the importance of genomic surveillance to public health responses to characterize viral transmission dynamics during pandemics.

Background: Serological evidence of spotted fever group rickettsioses (SFGRs) and interactions between pets and their owners in Laos have rarely been reported. We conducted a cross-sectional study to describe SFGR exposure risk and seroprevalence among companion animals and owners. Methods: Blood samples from 295 pet owners and their pets (270 dogs and 25 cats) were screened for IgG against SFGRs. Risk exposure to pathogens was assessed with clinical record forms and pet–owner interaction questionnaires. Results: The SFGR seropositivity rate among pet owners was 1.4%. Low-level SFGR antibody reactivity was detected in three cats (12%), whereas no dogs tested positive by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence assay (IFA) revealed low levels of immuno globulin G (IgG), with titers ranging from 1:4 to 1:16 in pet owners and as high as 1:64 in pets. No significant correlation was observed between seropositivity in pets and pet owners (p>0.05) or between participants with versus without ectoparasites on their pets (odds ratio 1.00, 95% CI 0.15–6.55, p=0.99). Conclusions: This study identified low-level serological reactivity to SFGR antigens in a small number of cats and pet owners. These findings might suggest possible previous exposure but should be interpreted cautiously, because the antibody titers were low, and the serological assay has not been validated for canine and feline sera.

INTRODUCTION:While pathogen genomics using next-generation sequencing (NGS) has been recommended by the WHO as an essential tool for national communicable disease surveillance programmes, procurement and supply chain management (PSM) systems for this new technology are still evolving. To assess the status of PSM systems for pathogen genomics, we examined perspectives from end-users and manufacturers across South and Southeast Asia. METHODS:Between 2022 and 2023, a cross-sectional survey was conducted among institutional partners supporting pathogen genomics among primarily low- and middle-income countries in South and Southeast Asia. This was complemented by qualitative interviews with the major regional NGS manufacturers. A PSM framework was employed to assess sales, procurement, production, distribution and post-sales support. Analyses are expressed as proportions and means or medians for continuous variables. RESULTS:A total of 42 partners across 13 countries, 3 genomics manufacturers and 22 laboratory personnel contributed data to this assessment. PSM challenges were reported by all countries and for all sequencing platforms. High costs of equipment and consumables were identified by 85% of respondents. Long equipment purchasing lead times and reagent re-supply times were reported by 69% and 77% of countries, respectively, with reagent resupply times averaging 8 weeks (IQR 6.2-9.0). Additional barriers included customs clearance, variability of import procedures, taxes and duties. Manufacturers reported a range of strategies to respond to PSM bottlenecks, including establishing regional hubs, distributor networks and financing schemes. CONCLUSION:Coordinated national and regional efforts are required to improve PSM systems for pathogen genomic sequencing to enhance timely early disease detection and response capacity in South and Southeast Asia.

BackgroundRickettsial diseases, including scrub typhus and murine typhus, are major yet persistently under-recognised causes of acute febrile illness in Southeast Asia. Limited diagnostic capacity, ecological complexity, and non-specific clinical presentation have historically contributed to the underestimation of their burden.MethodsWe synthesised 25 years (2001-2025) of integrated epidemiological, clinical, diagnostic, molecular, ecological, and treatment research conducted across Southeast Asia. Evidence from prospective surveillance, hospital-based cohorts, seroepidemiology, molecular characterisation, in vitro isolation, genomic analyses, and randomised clinical trials was reviewed to identify convergent findings and policy-relevant lessons.FindingsRickettsial infections account for 10%-25% of hospitalised acute febrile illness cases in many endemic settings and are important causes of central nervous system infection, severe disease, and adverse pregnancy outcomes. Diagnostic advances include calibrating IFA and ELISA cut-offs, evaluating rapid diagnostic tests and LAMP assays, developing highly sensitive real-time PCR platforms, and genomic analyses revealing extensive strain diversity. Whole-genome sequencing and multilocus typing demonstrate high recombination and weak geographic structuring of the core genome despite antigenic heterogeneity. Randomised trials confirm doxycycline as first-line therapy for scrub typhus, while azithromycin shows inferior efficacy for murine typhus. Integrated One Health investigations have clarified ecological drivers and vector-host dynamics, and community engagement initiatives have improved awareness in high-risk populations.InterpretationSustained regional investment has transformed rickettsial research from fragmented studies into an integrated surveillance, diagnostic, and translational research framework. This experience provides a transferable model for addressing neglected vector-borne diseases and strengthening febrile illness management in endemic settings. Continued support for laboratory capacity, genomic surveillance, and clinical research is essential to maintain progress and improve regional health system resilience.

ABSTRACT Murine typhus, caused by Rickettsia typhi, is a globally distributed flea-borne disease. Antimicrobial susceptibility data are largely based on reference strains, with little evaluation of variability across clinical isolates. The plaque assay, the gold standard for antibiotic susceptibility testing, requires up to 14 days to complete. Quantitative real-time PCR (qPCR) offers a rapid alternative for determining minimum inhibitory concentrations (MICs). Although qPCR-based susceptibility testing has been described, formal statistical assessment of agreement between methods remains limited. We estimated the MICs for azithromycin, doxycycline, and amoxicillin using qPCR and plaque assay on 24 R. typhi isolates (8 laboratory strains and 16 Lao clinical isolates) cultured in Vero cells. Heat-inactivated R. typhi (56°C, 30 min) served as a control. MIC was defined as the lowest concentration preventing plaque formation (plaque assay) or producing a Ct value greater than or equal to that of the heat-inactivated sample (qPCR). The mean MIC from three independent experiments was taken to represent each isolate. Agreement between methods was assessed using Bland-Altman analysis. Median qPCR MICs (MIC50) of all isolates were 0.130 mg/L (IQR, 0.104–0.240 mg/L) for azithromycin, 0.130 mg/L (IQR, 0.065–0.224 mg/L) for doxycycline, and 256 mg/L (IQR, 234.7–341.3 mg/L) for amoxicillin. Corresponding plaque assay MICs50 were 0.383 mg/L (IQR, 0.194–0.807 mg/L), 0.037 mg/L (IQR, 0.026–0.073 mg/L), and 170.7 mg/L (IQR, 112–256 mg/L), respectively. Bias was −0.3452 (−1.146, 0.4558) for azithromycin, 0.0969 (−0.2297, 0.4235) for doxycycline, and 85.33 (−187.3, 358) for amoxicillin. These findings demonstrate inter-isolate variability and quantitative agreement between methods.IMPORTANCEMurine typhus is a treatable febrile illness caused by Rickettsia typhi, transmitted to humans via fleas. The plaque assay, which detects bacterial-induced host cell death in the presence or absence of antibiotics, is the reference method for testing drug susceptibility. However, the method requires approximately 2 weeks to obtain results. Detection of bacterial nucleic acid provides an alternative laboratory approach that reduces the assay duration to approximately 1 week, thereby improving research efficiency and throughput when testing multiple isolates. Compared with plaque assays, quantitative real-time PCR allows downstream analysis following sample inactivation, reducing prolonged high-containment handling. In this study, we compared the antibiotic susceptibility of 24 R. typhi isolates using both methods. We tested antibiotics commonly used to treat R. typhi infection (doxycycline and azithromycin), along with amoxicillin as a negative control. Both showed comparable results for all antibiotics tested, further supporting nucleic acid detection as a reliable and faster alternative.

BACKGROUND:Scrub typhus and murine typhus, caused by Orientia tsutsugamushi and Rickettsia typhi, respectively, are important causes of febrile illness in Laos. Although several studies have assessed rickettsial infection in selected provinces, the nationwide distribution remains unclear. This study aimed to estimate exposure to scrub typhus group (STG) and typhus group (TG) across twelve provinces of Laos and identify potential hotspots. METHODOLOGY:We screened 1,200 serum samples from blood donors (100 per province) for STG and TG IgG antibodies using enzyme-linked immunosorbent assays (ELISA). Samples with optical density ≥ 0.5 were confirmed by immunofluorescence assays (IFA). RESULTS:Overall seroprevalence was 7.26% (95%CI:5.93-8.87) for STG and 4.09% (95% CI:3.11-5.37) for TG. STG seroprevalence was highest in Huaphan (27%), Oudomxay (19%) and Xiangkhuang (17%), all in northern Laos. TG seroprevalence was 10% in both Oudomxay (north) and Attapue (south). Compared with Vientiane Capital, these provinces had significantly higher seropositivity. No significant association was observed with age group. STG seropositivity was higher in males, whereas TG seropositivity was higher in females. CONCLUSION:Rickettsial infections are widespread in Laos, with STG seroprevalence concentrated in the north and TG seroprevalence present in both the north and south, guiding future research priorities and informing targeted public health interventions.

Melioidosis is a life-threatening infectious disease caused by Burkholderia pseudomallei. Early and accurate diagnosis is critical for timely treatment and improved outcomes. Although highly endemic in tropical regions, it remains underdiagnosed due to limitations of current diagnostic methods. Bacterial culture, the diagnostic gold standard, is time-consuming, prone to misidentified species, requires specialized laboratory facilities, and has low sensitivity. The indirect hemagglutination assay is also unreliable due to poor sensitivity and specificity. We developed a second-generation immunochromatography test (Hcp1-ICT) to detect anti-Hcp1 IgG antibodies against hemolysin co-regulated protein 1 of B. pseudomallei and evaluated its diagnostic performance in an international multi-center study. A total of 1,838 stored serum samples from 601 culture proven melioidosis patients, 598 healthy individuals and 639 patients with non-melioidosis infections in Thailand, Lao PDR, Vietnam, Malaysia, Sri Lanka, Cambodia, and Australia were analyzed for diagnosis of melioidosis. The sensitivities in Thailand, Lao PDR, Vietnam, Malaysia, Sri Lanka, Cambodia, and Australia were 92%, 77%, 76%, 80%, 74%, 90%, and 48%, respectively. The specificities with healthy donor samples were 96%, 79%, 98%, 100%, 100%, 87%, and 100%, while the specificities for cases with samples from other infections other than melioidosis were 97%, 87%, 98%, 98%, 100%, 90%, and 94%, respectively. These findings indicate that the Hcp1-ICT is a promising point-of-care tool for serodiagnosis of melioidosis. However, its variable performance across regions underscores the need for prospective validation locally in various regions to optimize its diagnostic utility and facilitate implementation in both referral centers and resource-limited settings.

OBJECTIVES:Rickettsioses are frequent causes of treatable febrile illness in Southeast Asia, including Myanmar. Accurate estimates of the incidence of rickettsioses are needed to inform investments in disease prevention and control. We sought to estimate the incidence of rickettsioses among adults and adolescents by combining sentinel hospital surveillance with a healthcare utilisation survey in Yangon, Myanmar. METHODS:We conducted a household-based healthcare utilisation survey in the Yangon Region from 12 March through 5 April 2018. Multipliers derived from this survey were then applied to scrub typhus, murine typhus, and spotted fever group rickettsioses infections identified from a study of adolescent and adult community-onset febrile illness done from 5 October 2015 through 4 October 2016 at Yangon General Hospital to estimate disease incidence. Acute serum was collected at enrolment and convalescent serum 14-30 days after enrolment. Confirmed acute scrub typhus, murine typhus, and spotted fever group infections were diagnosed by a ≥ 4-fold rise between acute and convalescent immunofluorescent antibody test titre to Orientia tsutsugamushi pooled Karp, Kato, and Gilliam antigens; Rickettsia typhi Wilmington strain; and Rickettsia honei and Rickettsia conorii antigens, respectively. RESULTS:After applying multipliers, we estimated the overall annual incidence of acute scrub typhus among adolescents and adults in the Yangon Region at 211 cases per 100,000 persons, and the overall estimate of acute murine typhus among adults and adolescents was 44 cases per 100,000 persons per year for 2015-2016. There were no confirmed spotted fever group infections. CONCLUSIONS:We provide the first estimates of scrub typhus and murine typhus community incidence in Myanmar. Similar research in children and from other parts of Myanmar, as well as studies of illness duration, complications, and deaths, is needed to estimate the disease burden.

OBJECTIVES:To inform patient management and disease prevention, we sought to estimate the prevalence of, and identify risk factors for, scrub typhus, murine typhus, and spotted fever group rickettsioses (SFGR) among febrile patients presenting to hospital in Myanmar. METHODS:We recruited patients ≥12 years old with fever ≥38°C among those seeking care at Yangon General Hospital from 5 October 2015 through 4 October 2016. Standardised clinical and risk factor assessments were conducted. Confirmed scrub typhus, murine typhus, and SFGR infections were defined as a positive polymerase chain reaction or ≥4-fold rise in immunofluorescence assay antibody titre to Orientia tsutsugamushi, Rickettsia typhi or Rickettsia honei or Rickettsia conorii, respectively. Probable infection was defined as IgM titre ≥1:400 to O. tsutsugamushi, an IgM titre of ≥1:800 or IgG ≥1:1600 to R. typhi or an IgG titre of ≥1:200 to R. honeii or R. conorii. Univariate and multivariable logistic regression was used to identify associations. RESULTS:Among 944 participants, the median (range) age was 37 (12-94) years, 444 (47.0%) were female, and 704 (74.6%) resided in rural areas. Among participants, 63 (6.7%) had confirmed or probable scrub typhus and 15 (1.6%) had confirmed or probable murine typhus. No SFGR infections were identified. The odds of confirmed or probable scrub typhus were lower among females than males (adjusted odds ratio [aOR] 0.5, p = 0.014), lower among those earning >300,000 Kyat per month compared with those earning less than 100,000 Kyat per month (aOR 0.28, p = 0.039), and higher among agricultural workers compared with others (aOR 2.9, p = 0.004). CONCLUSION:Scrub typhus was common among patients presenting with fever in Yangon, murine typhus was uncommon, and SFGR was not found. Empiric treatment of severe febrile illness should include an antimicrobial with activity against rickettsial diseases. Public health campaigns targeting agricultural workers are recommended.

OBJECTIVES:Globally, the circulation of influenza and other seasonal respiratory viruses changed dramatically during the COVID-19 pandemic. This study aims to determine the trends of acute respiratory infections (ARIs) caused by SARS-CoV-2, influenza A, influenza B and respiratory syncytial viruses (RSVs) in patients presenting to hospitals in the Lao People's Democratic Republic (PDR) (Laos). DESIGN:Prospective surveillance study. SETTING:Four provincial hospitals across Laos between March 2021 and July 2023. PARTICIPANTS:Participants of all ages who met our case definition for an ARI (axillary temperature ≥37.5°C or history of fever AND cough or other respiratory symptoms/signs OR loss of smell and/or taste) presenting to the hospital less than 10 days after symptom onset were eligible to be enrolled in the study. Combined nasopharyngeal and throat swabs were tested for SARS-CoV-2, influenza A, influenza B and human RSV (hRSV) using probe-based real-time RT (Reverse transcription)-PCR assays. PRIMARY OUTCOME MEASURE:The proportion of patients in whom SARS-CoV-2, influenza A, influenza B and hRSV was detected. RESULTS:There were 4203 patients recruited, of whom 898 (21%) were children aged under 5 years. SARS-CoV-2 was detected in 16.9% of patients, followed by influenza A, influenza B and hRSV (8.4%, 7.2% and 4.7%, respectively). 98 patients (2.3%) were diagnosed with probable co-infection, with at least two viruses detected. After May 2022, the number of cases of influenza A, influenza B and hRSV increased rapidly. Six per cent of patients (263) had a quick Sequential Organ Failure Assessment score of ≥2, and 34 (0.8%) patients died, of whom 11 tested positive for a respiratory virus. CONCLUSIONS:During the COVID-19 pandemic in Laos, few respiratory viruses were detected by passive surveillance until the relaxation of non-pharmaceutical interventions implemented for infection control. After restrictions were lifted, influenza A, influenza B and hRSV emerged rapidly, showing the importance of continuous surveillance.

Ticks and fleas are vectors of medically important infectious diseases globally, such as Rickettsiae. These pathogens are frequently reported in Southeast Asia, including Laos; however, there are very few comprehensive reports on their prevalence and vector diversity in urban areas. This study collected ectoparasites from companion animals to assess pathogen prevalence and exposure risk. In five veterinary clinics across Vientiane capital, ectoparasites were collected from dogs and cats and identified to the species level using both morphological and molecular methods. Ectoparasite DNA samples were screened for bacteria (17-kDa and 16S rRNA gene). Ticks were submitted to evaluate the potential of MALDI-TOF mass spectrometry for species identification. A total of 3,771 arthropod vectors (3,658 ticks, 105 fleas, 8 lice) were removed from dogs and cats. Ticks were morphologically identified as Rhipicephalus sanguineus sensu lato (s.l.) tropical lineage (currently recognised as Rhipicephalus linnaei), whilst fleas were classified as either Ctenocephalides felis felis (57.1%) or C. f. orientis (42.9%) and lice were Heterodoxus spiniger. The MALDI-TOF spectra in this study revealed similar mass-to-charge (m/z) peak profiles to those reported in previous studies for Rhipicephalus sanguineus. Rickettsia spp. (Rickettsia asembonensis and Rickettsia felis) were detected in 44.4% of pooled flea samples collected from 12 dogs and 4 cats, as well as 3.5% of tick pools collected from 142 dogs and 50% of lice pools collected from 2 dogs. In addition, Anaplasmataceae (Ehrlichia canis and Anaplasma platys) were detected in 22.5% of ticks collected from dogs. This study highlights the diversity of ectoparasite species collected from dogs and cats and provide preliminary insights into the use of MALDI-TOF MS for tick species identification. While promising, further research is needed to enhance the reliability and efficacy of this approach. The findings also reveal a high prevalence of pathogens in ectoparasites, emphasizing the need for increased awareness among pet owners, veterinarians, and addressing public health concerns.

Murine typhus, caused by infection with Rickettsia typhi , is a neglected disease contributing to infectious disease burden in south- and southeast Asia. Despite its importance, we have minimal knowledge of the genomics of R. typhi , with only four complete genomes being sequenced prior to this work. We sequenced a further 25 genomes including historical strains collected before 1976 from both human and rat hosts, and recent genomes isolated from patients at a single hospital in Laos. Whole genome SNP analysis reveals extremely low levels of genetic diversity across the 29 genomes, with overall nucleotide diversity (π) of 1.27e -05 and evidence of purifying selection, and a minimal pan-genome. Phylogenetic analysis shows clustering of the genome by historic or modern origin, with the exception of one modern strain which is most closely related to historic strains from Thailand, and no clustering by host origin. The highly conserved genome of R. typhi suggests strong constraints on genome evolution in this obligate intracellular parasite, and has implications for the design of future murine typhus diagnostic tools and vaccines.

Background Visceral leishmaniasis (VL) is a vector-borne protozoan disease with a global distribution, with higher rates of infection associated with HIV. Zoonotic species of Leishmania have also been reported infecting domestic animals. Reports of VL are increasing in Southeast Asia, with over 200 cases reported in Thailand since the first autochthonous case in 1999, and recently the first patients have been reported from Vietnam and Cambodia. However, no cases of VL have been reported from Lao PDR (Laos) and clinical awareness of the disease is limited. This study aimed to investigate whether Leishmania is circulating in Laos by screening people living with HIV, stored samples from unselected patients with fever, and ruminants taken to abattoirs. Methods People living with HIV from two specialist units in Vientiane Capital had EDTA blood taken and DNA extracted and tested for Leishmania by nested-PCR. Stored serum samples from patients presenting to Mahosot Hospital with fever and without known HIV infection, as well as serum from goats, cows and buffalo taken to abattoirs in four provinces in Laos were tested for Leishmania using the InBios Kalazar Detect Rapid Test. Results There were 1015 people living with HIV tested between 2021 and 2024 for Leishmania by nested-PCR, all of whom were negative. Of 511 human serum samples collected between 2005 and 2023, two (0.4%) tested positive by rapid test. These samples were identified as coming from the same patient, with samples taken 10 months apart. There were 5/159 (3.1%) ruminant serum samples positive by rapid test with 3/45 (6.7%) buffalo positive, 2/47 (4.3%) goat positive and 0/67 cows positive. Conclusions This study suggests Leishmania may be circulating in Laos with undetected cases. Further investigation is needed to confirm the findings, determine at-risk populations and increase clinical awareness of the disease. This study expands on the current regional knowledge on leishmaniasis and shows the need for further epidemiological studies.

The global priorities in the field of infectious diseases are constantly changing. While emerging viral infections have regularly dominated public health attention, which has only intensified after the COVID-19 pandemic, numerous bacterial diseases have previously caused, and continue to cause, significant morbidity and mortality-deserving equal attention. Three potentially life-threatening endemic bacterial diseases (leptospirosis, melioidosis, and rickettsioses) are a huge public health concern especially in low- and middle-income countries. Despite their continued threat, these diseases do not receive proportionate attention from global health organizations and are not even included on the WHO list of neglected tropical diseases (NTDs). This, in turn, has led to a vicious circle of neglect with continued, yet conceivably preventable, hospitalizations and deaths each year especially in the vulnerable population. This is a call from a group of multi-institutional experts on the urgent need to directly address the circle of neglect and raise support in terms of funding, research, surveillance, diagnostics, and therapeutics to alleviate the burden of these 3 diseases.

Asia remains vulnerable to new and emerging infectious diseases. Understanding how to improve next generation sequencing (NGS) use in pathogen surveillance is an urgent priority for regional health security. Here we developed a pathogen genomic surveillance assessment framework to assess capacity in low-resource settings in South and Southeast Asia. Data collected between June 2022 and March 2023 from 42 institutions in 13 countries showed pathogen genomics capacity exists, but use is limited and under-resourced. All countries had NGS capacity and seven countries had strategic plans integrating pathogen genomics into wider surveillance efforts. Several pathogens were prioritized for human surveillance, but NGS application to environmental and human-animal interface surveillance was limited. Barriers to NGS implementation include reliance on external funding, supply chain challenges, trained personnel shortages and limited quality assurance mechanisms. Coordinated efforts are required to support national planning, address capacity gaps, enhance quality assurance and facilitate data sharing for decision making. Surveying next generation sequencing capabilities in 13 Asian countries identifies challenges to be met for improved implementation, pathogen surveillance and integration into public health decision-making.