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During infections with the malaria parasites Plasmodium vivax , patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreira et al. , Science 368 , 746-753 (2020); Smith et al ., Science 368 , 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

Background: Estimating malaria risk associated with work locations and travel across a region provides local health officials with information useful to mitigate possible transmission paths of malaria as well as understand the risk of exposure for local populations. This study investigates malaria exposure risk by analysing the spatial pattern of malaria cases (primarily Plasmodium vivax) in Ubon Ratchathani and Sisaket provinces of Thailand, using an ecological niche model and machine learning to estimate the species distribution of P. vivax malaria and compare the resulting niche areas with occupation type, work locations, and work-related travel routes., Methods: A maximum entropy model was trained to estimate the distribution of P. vivax malaria for a period between January 2019 and April 2020, capturing estimated malaria occurrence for these provinces. A random simulation workflow was developed to make region-based case data usable for the machine learning approach. This workflow was used to generate a probability surface for the ecological niche regions. The resulting niche regions were analysed by occupation type, home and work locations, and work-related travel routes to determine the relationship between these variables and malaria occurrence. A one-way analysis of variance (ANOVA) test was used to understand the relationship between predicted malaria occurrence and occupation type., Results: The MaxEnt (full name) model indicated a higher occurrence of P. vivax malaria in forested areas especially along the Thailand-Cambodia border. The ANOVA results showed a statistically significant difference between average malaria risk values predicted from the ecological niche model for rubber plantation workers and farmers, the two main occupation groups in the study. The rubber plantation workers were found to be at higher risk of exposure to malaria than farmers in Ubon Ratchathani and Sisaket provinces of Thailand., Conclusion: The results from this study point to occupation-related factors such as work location and the routes travelled to work, being risk factors in malaria occurrence and possible contributors to transmission among local populations., (© 2023. The Author(s).)

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). Multiple copies of pfpm2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC 90 values of piperaquine. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.

Background: Primaquine is an approved radical cure treatment for Plasmodium vivax malaria but treatment can result in life-threatening hemolysis if given to a glucose-6-phosphate dehydrogenase deficient (G6PDd) patient. There is a need for reliable point-of-care G6PD diagnostic tests., Objectives: To evaluate the performance of the CareStart™ rapid diagnostic test (RDT) in the hands of healthcare workers (HCWs) and village malaria workers (VMWs) in field settings, and to better understand user perceptions about the risks and benefits of PQ treatment guided by RDT results., Methods: This study enrolled 105 HCWs and VMWs, herein referred to as trainees, who tested 1,543 healthy adult male volunteers from 84 villages in Cambodia. The trainees were instructed on G6PD screening, primaquine case management, and completed pre and post-training questionnaires. Each trainee tested up to 16 volunteers in the field under observation by the study staff., Results: Out of 1,542 evaluable G6PD volunteers, 251 (16.28%) had quantitative enzymatic activity less than 30% of an adjusted male median (8.30 U/g Hb). There was no significant difference in test sensitivity in detecting G6PDd between trainees (97.21%), expert study staff in the field (98.01%), and in a laboratory setting (95.62%) (p = 0.229); however, test specificity was different for trainees (96.62%), expert study staff in the field (98.14%), and experts in the laboratory (98.99%) (p < 0.001). Negative predictive values were not statistically different for trainees, expert staff, and laboratory testing: 99.44%, 99.61%, and 99.15%, respectively. Knowledge scores increased significantly post-training, with 98.7% willing to prescribe primaquine for P.vivax malaria, an improvement from 40.6% pre-training (p < 0.001)., Conclusion: This study demonstrated ability of medical staff with different background to accurately use CareStart™ RDT to identify G6PDd in male patients, which may enable safer prescribing of primaquine; however, pharmacovigilance is required to address possible G6PDd misclassifications., Competing Interests: The authors have declared that no competing interests exist.

Background: The recent dramatic decline in dihydroartemisinin-piperaquine (DHA-PPQ) efficacy in northwestern Cambodia has raised concerns about the rapid spread of piperaquine resistance just as DHA-PPQ is being introduced as first-line therapy in neighbouring countries., Methods: Ex vivo parasite susceptibilities were tracked to determine the rate of progression of DHA, PPQ and mefloquine (MQ) resistance from sentinel sites on the Thai-Cambodian and Thai-Myanmar borders from 2010 to 2015. Immediate ex vivo (IEV) histidine-rich protein 2 (HRP-2) assays were used on fresh patient Plasmodium falciparum isolates to determine drug susceptibility profiles., Results: IEV HRP-2 assays detected the precipitous emergence of PPQ resistance in Cambodia beginning in 2013 when 40 % of isolates had an IC 90 greater than the upper limit of prior years, and this rate doubled to 80 % by 2015. In contrast, Thai-Myanmar isolates from 2013 to 14 remained PPQ-sensitive, while northeastern Thai isolates appeared to have an intermediate resistance profile. The opposite trend was observed for MQ where Cambodian isolates appeared to have a modest increase in overall sensitivity during the same period, with IC 50 declining to median levels comparable to those found in Thailand. A significant association between increased PPQ IC 50 and IC 90 among Cambodian isolates with DHA-PPQ treatment failure was observed. Nearly all Cambodian and Thai isolates were deemed artemisinin resistant with a >1 % survival rate for DHA in the ring-stage assay (RSA), though there was no correlation among isolates to indicate cross-resistance between PPQ and artemisinins., Conclusions: Clinical DHA-PPQ failures appear to be associated with declines in the long-acting partner drug PPQ, though sensitivity appears to remain largely intact for now in western Thailand. Rapid progression of PPQ resistance associated with DHA-PPQ treatment failures in northern Cambodia limits drugs of choice in this region, and urgently requires alternative therapy. The temporary re-introduction of artesunate AS-MQ is the current response to PPQ resistance in this area, due to inverse MQ and PPQ resistance patterns. This will require careful monitoring for re-emergence of MQ resistance, and possible simultaneous resistance to all three drugs (AS, MQ and PPQ).

Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

Introduction: Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses., Methods: In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose., Results: Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated., Conclusion: Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed., Trial Registration: ClinicalTrials.gov NCT01044095.

A hospital-based study was conducted along the Thai-Myanmar border to provide greater knowledge of the causes of febrile illness and to determine what zoonotic and vector-borne emerging infectious diseases might be present. A total of 613 adults were enrolled from June 1999 to March 2002. Cases were classified based on clinical findings and laboratory results. An etiologic diagnosis was made for 48% of subjects. Malaria was the most common diagnosis, accounting for 25% of subjects, with two-thirds Plasmodium falciparum. Serologic evidence for leptospirosis was found in 17% of subjects. Other etiologic diagnoses included rickettsial infections, dengue fever, and typhoid. The most frequent clinical diagnoses were nonspecific febrile illness, respiratory infections, and gastroenteritis. Clinical associations were generally not predictive of etiologic diagnosis. Apparent dual diagnoses were common, particularly for malaria and leptospirosis. Findings have been used to modify treatment of unspecified febrile illness in the area.

We assessed the prophylactic efficacy of azithromycin (250 mg/day) against malaria in 276 adults in western Thailand in a randomized, double-blind, placebo-controlled trial. After antimalarial suppressive treatment, volunteers were randomized in a 2:1 ratio to either the azithromycin or placebo, respectively. Study medication was given for an average of 74 days. The azithromycin group (n = 179) had five endpoint parasitemias (1 Plasmodium vivax and 4 P. falciparum), and the placebo group (n = 97) had 28 endpoint parasitemias (21 P. vivax, 5 P. falciparum, and 2 mixed infections). Adverse events and compliance and withdrawal rates were similar in both groups. The protective efficacy (PE) of azithromycin was 98% for P. vivax (95% confidence interval [CI] = 88-100%). There were too few cases to reliably estimate the efficacy of azithromycin for P. falciparum (PE =71%, 95% C =-14-94%). We conclude that daily azithromycin was safe, well-tolerated, and had a high efficacy for the prevention of P. vivax malaria.

Molecular markers have been proposed as a method of monitoring malaria drug resistance and could potentially be used to prolong the life span of antimalarial drugs. Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum gene pfmdr1 and increased gene copy number have been associated with in vitro drug resistance but have not been well studied in vivo. In a prospective cohort study of malaria patients receiving mefloquine treatment on the Thai-Myanmar border, there was no significant association between either pfmdr1 SNPs or in vitro drug sensitivity and mefloquine resistance in vivo. Increased pfmdr1 gene copy number was significantly associated with recrudescence (relative risk 2.30, 95% CI 1.27-4.15). pfmdr1 gene copy number may be a useful surveillance tool for mefloquine-resistant falciparum malaria in Thailand.

We have performed a case-control analysis to determine the significance of clinical, laboratory and epidemiological features as predictive factors of rickettsioses among patients in Sangkhla Buri, Thailand (Thai-Myanmar border). Fifteen serologically-confirmed rickettsiosis patients including Spotted Fever Group (SFG) rickettsioses, scrub typhus, and murine typhus were classified as 'cases'; one hundred and sixty-three acutely febrile patients presenting to the same hospital during the same time period, who had no serological evidence of acute rickettsiosis, were classified as 'controls'. Patients' report of rash/arthropod bite [Odds ratio (OR) 22.90, 95% CI (confidence interval) 6.23, 84.13] and history of jungle trips (OR 5.30, 95% CI 1.69-16.62) were significant risk factors. Elevated ALT (OR 3.04, 95% CI 1.04, 8.88) and depressed platelet count (OR 3.38, 95% CI 1.13, 10.10) were also useful differentiating markers of rickettsioses in this population. Definitive diagnosis of rickettsioses is difficult without specialized diagnostic capabilities that are rarely available in remote areas such as Sangkhla Buri, where other acute febrile illnesses with similar presentation are commonly found. The relative importance of predictive factors presented here may provide clinicians with some useful guidance in distinguishing rickettsioses from other acute febrile illnesses. Timely administration of empiric treatment in highly suspicious cases can deter potential morbidity from these arthropod-borne infections.