Factors contributing to a measles outbreak in a hard-to-reach rural village in Xaisomboun Province, Lao People's Democratic Republic.

Objective: An increase in measles cases was reported in the north-western of the Lao People's Democratic Republic beginning in January 2019, with outbreaks quickly spreading throughout the country. Following identification of two laboratory-confirmed cases in Xaisomboun Province, we conducted an outbreak investigation to identify factors contributing to the measles outbreak in hard-to-reach Village X. Methods: Active case-finding was undertaken at the provincial hospital and primary health care centre via a retrospective search through admission logbooks and house-to-house surveys in Village X and surrounding villages. Clinical samples were collected from suspected cases, and data were collected using a standard case investigation form. Vaccine coverage data were reviewed. Results: Of the 40 suspected measles cases with rash onset during 12 February-27 April 2019, 83% (33/40) resided in Village X and 98% (39/40) were of Hmong-Lu Mien ethnicity. Ages ranged from 22 days to 5 years, with 70% (28) aged < 24 months. Almost half of cases aged 9 to < 18 months (5/11) and 67% (8/12) of cases aged 324 months had received a measles-containing vaccine (MCV). Reported MCV coverage in Xaisomboun for children aged < 1 year in 2017-2018 was < 50%. In 55% (22/40) of cases, case notification was delayed by 36 days. The final case classification comprised 10% laboratory-confirmed, 20% clinically compatible, 60% epidemiologically linked and 10% non-cases. Discussion: This measles outbreak was likely associated with low immunization coverage, compounded by delays in reporting. Effective strategies are needed to address beliefs about and health literacy barriers to immunization and measles awareness. Such strategies may improve MCV coverage and early diagnosis, enabling timely public health interventions and reducing mortality and morbidity.

Pooling samples to increase testing capacity with Xpert Xpress SARS-CoV-2 during the Covid-19 pandemic in Lao People's Democratic Republic.

The COVID-19 pandemic created the need for large-scale testing of populations. However, most laboratories do not have sufficient testing capacity for mass screening. We evaluated pooled testing of samples, as a strategy to increase testing capacity in Lao PDR. Samples of consecutive patients were tested in pools of four using the Xpert Xpress SARS CoV-2 assay. Positive pools were confirmed by individual testing, and we describe the performance of the test and savings achieved. We also diluted selected positive samples to describe its effect on the assays CT values. 1,568 patients were tested in 392 pools of four. 361 (92.1%) pools were negative and 31 (7.9%) positive. 29/31 (93.5% (95%CI 77-99%) positive pools were confirmed by individual testing of the samples but, in 2/31 (6.5%) the four individual samples were negative, suggesting contamination. Pools with only one positive sample had higher CT values (lower RNA concentrations) than the respective individual samples, indicating a dilution effect, which suggested an increased risk of false negative results with dilutions >1:10. However, this risk may be low if the prevalence of infection is high, when pools are more likely to contain more than one positive sample. Pooling saved 67% of cartridges and substantially increased testing capacity. Pooling samples increased SARS-CoV-2 testing capacity and resulted in considerable cartridge savings. Given the need for high-volume testing, countries may consider implementation of pooling for SARS-CoV-2 screening.

Corrigendum to: Phylogenetic Analysis Revealed the Dissemination of Closely Related Epidemic Vibrio cholerae O1 Isolates in Laos, Thailand, and Vietnam.

[This corrects the article DOI: 10.1093/ofid/ofaa492.].

Low seroprevalence of COVID-19 in Lao PDR, late 2020.

BACKGROUND: In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure. METHODS: Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex. FINDINGS: In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative). INTERPRETATION: We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses. FUNDING: Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs ("PaReCIDS II").

Prevalence of pharmacogenomic variants in 100 pharmacogenes among Southeast Asian populations under the collaboration of the Southeast Asian Pharmacogenomics Research Network (SEAPharm).

Pharmacogenomics can enhance the outcome of treatment by adopting pharmacogenomic testing to maximize drug efficacy and lower the risk of serious adverse events. Next-generation sequencing (NGS) is a cost-effective technology for genotyping several pharmacogenomic loci at once, thereby increasing publicly available data. A panel of 100 pharmacogenes among Southeast Asian (SEA) populations was resequenced using the NGS platform under the collaboration of the Southeast Asian Pharmacogenomics Research Network (SEAPharm). Here, we present the frequencies of pharmacogenomic variants and the comparison of these pharmacogenomic variants among different SEA populations and other populations used as controls. We investigated the different types of pharmacogenomic variants, especially those that may have a functional impact. Our results provide substantial genetic variations at 100 pharmacogenomic loci among SEA populations that may contribute to interpopulation variability in drug response phenotypes. Correspondingly, this study provides basic information for further pharmacogenomic investigations in SEA populations.

The Impact of Preparedness in Defying COVID-19 Pandemic Expectations in the Lower Mekong Region: A Case Study.

Dire COVID-19 expectations in the Lower Mekong Region (LMR) can be understood as Cambodia, the Lao PDR, Myanmar, Thailand, and Vietnam have stared down a succession of emerging infectious disease (EID) threats from neighboring China. Predictions that the LMR would be overwhelmed by a coming COVID-19 tsunami were felt well before the spread of the COVID-19 pandemic had been declared. And yet, the LMR, excepting Myanmar, has proved surprisingly resilient in keeping COVID-19 contained to mostly sporadic cases. Cumulative case rates (per one million population) for the LMR, including or excluding Myanmar, from January 1 to October 31 2020, are 1,184 and 237, respectively. More telling are the cumulative rates of COVID-19-attributable deaths for the same period of time, 28 per million with and six without Myanmar. Graphics demonstrate a flattening of pandemic curves in the LMR, minus Myanmar, after managing temporally and spatially isolated spikes in case counts, with negligible follow-on community spread. The comparable success of the LMR in averting pandemic disaster can likely be attributed to years of preparedness investments, triggered by avian influenza A (H5N1). Capacity building initiatives applied to COVID-19 containment included virological (influenza-driven) surveillance, laboratory diagnostics, field epidemiology training, and vaccine preparation. The notable achievement of the LMR in averting COVID-19 disaster through to October 31, 2020 can likely be credited to these preparedness measures.

Secondhand Smoke Exposure in Lao People's Democratic Republic: Results From the 2015 National Adult Tobacco Survey.

Objectives: Second-hand smoke (SHS) exposure causes >600,000 deaths annually worldwide, however, information regarding SHS exposure in Lao People's Democratic Republic (Lao PRD) is limited; we report SHS exposure prevalence at home, inside workplaces, and indoor public spaces in Lao PDR. Methods: Data were from the 2015 Lao National Adult Tobacco Survey, a nationally representative sample of 7,562 participants aged ≥15 years recruited through a stratified 2-stage cluster sampling approach. Results: 88.3% (83.9% of non-smokers) reported SHS exposure at home and 63.0% (54.0% of non-smokers) at workplaces. Among non-smokers, women had greater exposure at home than men (86.6 vs. 77.0%). Lower education levels were associated with exposure at home or the workplace. 99.2% reported SHS exposure at any public place; specifically for restaurants/food stores 57.7%, government offices 56.2%, public transport 31.6%, and health care facilities 11.7%. Conclusion: SHS exposure at home and workplace in Lao PDR is among the highest in South-East Asia. Comprehensive smoke-free policies at government-owned workplaces and facilities, stricter enforcement of these smoke-free policies, and strategies to encourage smoke-free environments at homes and in public places are urgently needed.

A pilot study to investigate the risk of SARS-CoV-2 household transmission following hospital discharge of confirmed cases in Vientiane Capital, Lao PDR.

Background: Global guidelines from the World Health Organization on discharging patients diagnosed with COVID-19 changed in 2021 to a symptom-based rather than negative PCR-based approach. Studies have shown that shedding of viable virus continues for approximately eight days after symptom onset in most patients. In Vientiane, Laos, until now, patients diagnosed with asymptomatic or mild COVID-19 are hospitalised for 2 weeks and then, if they still test PCR positive for SARS-CoV-2, stay for a further week in a designated quarantine hotel before being discharged home. Objective: The aim of this pilot study was to investigate the risk of transmission of SARS-CoV-2 to household contacts of discharged patients who are still PCR-positive following 2-3 weeks quarantine in Vientiane, Lao PDR. Methods: Adult participants, who were resident in Vientiane Capital and who were about to be discharged from hospital (after 2 weeks hospitalisation), or from a quarantine hotel, following a further one-week quarantine, were screened to assess eligibility for the study. The household of each case was visited a maximum of 48 hours before or up to 24 hours after the participant was discharged and a nasopharyngeal swab was taken from all household members. Repeat nasopharyngeal swabs from cases and contacts were taken on day 7 and day 14 after discharge home of each case. Results: Between 20th May 2021 and 27th August 2021, 55 cases and 84 contacts in 27 households were enrolled in the study. The median [range] age of all 139 included participants was 26.5 years [3 months to 83 years] and 83 (60%) were female. By household, the median [range] number of cases and contacts were 1 [1-6] and 3 [1-13] respectively. At discharge home 32/48 (67%) cases tested positive for SARS-CoV-2. By day 7 11 of 47 cases (23%) still tested positive for SARS-CoV-2 by PCR and by day 14 this number was 2/24 (8%). No contacts tested positive during follow up and the numbers tested at the time of discharge of the case, 7 days later and 2 weeks later were 56, 57 and 37 respectively. Loss to follow up at day 7 and day 14 ranged from 15-50% (participants not at home at the time of visits). Conclusion: In this pilot study we found no evidence of onward transmission of SARS-CoV-2 to contacts of cases discharged home with a positive PCR result. This suggests the current discharge policy for mild to moderate COVID-19 case following 2 weeks in hospital in the Lao PDR is safe.

Laboratory evaluation of two point-of-care detection systems for early and accurate detection of influenza viruses in the Lao People's Democratic Republic.

BACKGROUND: We evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing. METHODS: The performance characteristics of 1) insulated isothermal polymerase chain reaction (PCR) on a POCKIT™ device and 2) real-time reverse transcription-PCR (rRT-PCR) on a MyGo Mini™ device were evaluated using human clinical specimens, beta-propiolactone-inactivated influenza viruses, and RNA controls. The rRT-PCR carried out on a CXF-96™ real-time detection system was used as a gold standard for comparison. RESULTS: Both systems demonstrated 100% sensitivity and specificity and test results were in 100% agreement with the gold standard. POCKIT™ only correctly identified influenza A (M gene) in clinical specimens due to the unavailability of typing and subtyping reagents for human influenza viruses, while MyGo Mini™ had either a one log higher or the same sensitivity in detecting influenza viruses in clinical specimens compared to the gold standard. For inactivated viruses and/or viral RNA, the analytic sensitivity of POCKIT™ was shown to be comparable to, or more sensitive, than the gold standard. The analytic sensitivity of MyGo Mini™ had mixed results depending on the types and subtypes of influenza viruses. CONCLUSIONS: The performance of the two systems in a laboratory is promising and supports further evaluation in field settings.

Phylogenetic Analysis Revealed the Dissemination of Closely Related Epidemic Vibrio cholerae O1 Isolates in Laos, Thailand, and Vietnam.

We performed whole-genome sequencing of Vibrio cholerae O1 isolates from Laos, Thailand, and Vietnam, where cholera outbreaks occurred, to determine their genetic lineages. Core genome phylogenetic analysis revealed that the isolates located in same lineage without regional clusters, which suggests that closely related strains circulated in Southeast Asia.

Factors Associated with Cigarette Smoking Cessation in Lao People's Democratic Republic: Results from the 2015 National Adult Tobacco Survey.

Cigarette smoking represents a major public health problem in Lao People's Democratic Republic (Lao PDR). This study aims to examine factors associated with cigarette smoking cessation attempts and intention to quit. Data were from the Lao National Adult Tobacco Survey that consisted of 7562 participants ≥15 years old. Multivariable logistic regression models were used to evaluate the associations, adjusted for sex, age groups, education level, income per day, and smoking frequency. Results show that past quit attempts were associated with visiting a healthcare provider in the past year (adjusted odds ratio [AOR]: 1.74, 95% confidence intervals [CI]: 1.28-2.35), home smoking bans (AOR: 5.52, 95% CI: 2.13-14.33), noticing media-based messages informing the dangers of smoking or encouraging quitting (AOR: 3.25, 95% CI: 2.28-4.63), noticing health warnings on cigarette packages in the past 30 days (AOR: 3.33, 95% CI: 2.21-5.03), and believing that smoking is seriously harmful to their health (AOR: 3.45, 95% CI: 1.24-9.57). The Lao PDR government should continue implementing tobacco control policies that demonstrated associations with cessation attempts or intention to quit, such as smoke-free environments and required health warnings on cigarette packages. Tobacco cessation treatment programs are pressingly needed in Lao PDR.

Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii.

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ~90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (~1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research.

Southeast Asian Pharmacogenomics Research Network (SEAPharm): Current Status and Perspectives.

Pharmacogenomics (PGx) is increasingly being recognized as a potential tool for improving the efficacy and safety of drug therapy. Therefore, several efforts have been undertaken globally to facilitate the implementation process of PGx into routine clinical practice. Part of these efforts include the formation of PGx working groups working on PGx research, synthesis, and dissemination of PGx data and creation of PGx implementation strategies. In Asia, the Southeast Asian Pharmacogenomics Research Network (SEAPharm) is established to enable and strengthen PGx research among the various PGx communities within but not limited to countries in SEA; with the ultimate goal to support PGx implementation in the region. From the perspective of SEAPharm member countries, there are several key elements essential for PGx implementation at the national level. They include pharmacovigilance database, PGx research, health economics research, dedicated laboratory to support PGx testing for both research and clinical use, structured PGx education, and supportive national health policy. The status of these essential elements is presented here to provide a broad picture of the readiness for PGx implementation among the SEAPharm member countries, and to strengthen the PGx research network and practice in this region.

Malaria among foreign migrant workers in Savannakhet Province, Lao People's Democratic Republic.

BACKGROUND: Although mobile and migrant populations are considered an important group in malaria elimination settings, there is currently a lack of understanding about foreign migrant workers in the Lao People's Democratic Republic (Lao PDR). The present study aimed to document the migration characteristics, positive rate of malaria infection, and preventive and treatment-seeking behavior for malaria among foreign migrant workers in the malaria-endemic districts of Savannakhet province, Lao PDR. METHODS: A community-based survey was undertaken in four districts of Savannakhet province between February and June, 2015. Questionnaire-based interviews and blood examinations, including rapid diagnostic tests and PCR assays, were conducted with 391 migrant workers who were registered at local police departments. RESULTS: Most of the study participants were men (75.7%) and Vietnamese (92.6%). The median age (interquartile range) was 31 (25 to 41) years old. Most common occupation was factory worker (47.6%), followed by trader/shopkeeper (21.5%) and plantation worker/farmer (16.4%). The median length of stay (interquartile range) in the districts was 405 (183 to 1207) days. The majority of the participants (85.9%) had not worked in a province other than the study province, nor had the majority (92.6%) worked in a foreign country other than the Lao PDR. Although most of the participants (62.7%) reportedly used a bed net daily, these nets were mostly conventional untreated ones. No one tested positive for malaria. However, 10.0% of the participants reported a malaria-like illness episode that had occurred in the Lao PDR. The most common measure taken for the episode was to visit a hospital/health center in the Lao PDR, followed by conducting self-medication alone. Forty-one participants reported an experience of working in the forest while living in the Lao PDR. CONCLUSIONS: Foreign migrant workers who are registered at local police departments are unlikely to play a major role in maintaining local transmissions and spreading drug-resistant malaria in the study province. However, some of them were involved in forest-related activities, suggesting that these workers are potentially at risk of malaria. The Lao National Malaria Control Program should educate foreign migrant workers about the risk of malaria when living in Lao PDR.

Tobacco use in Lao People's Democratic Republic: Results from the 2015 National Adult Tobacco Survey.

INTRODUCTION: Tobacco use is a burden for Lao People's Democratic Republic (Lao PDR). No published report has examined determinants of various tobacco uses to inform appropriate policies and prevention strategies. This paper reports tobacco uses by sociodemographic characteristics using data from the most recent Lao National Adult Tobacco Survey (NATS) in 2015. METHODS: The NATS included a nationally representative sample of 7562 people aged ≥15 years, recruited through a stratified 2-stage cluster sampling approach in 18 provinces. All analyses were weighted. Multivariable logistic regression models were used to evaluate unadjusted and adjusted associations between variables of interest. RESULTS: The NATS results showed that 32.4% of Lao people aged ≥15 years were current tobacco users (men: 51.2%, women: 15.4%). Cigarette smoking accounted for approximately 95% of all tobacco use in men, while tobacco chewing accounted for 60% of tobacco use in women. Current tobacco use was strongly associated with older ages and lower education levels (p<0.001). There were interactions between sex, education level, and income associated with tobacco use; specifically, women were more likely to have a lower education level and lower income than men, and these women were more likely to use tobacco. CONCLUSIONS: Tobacco use prevalence in Lao PDR was among the highest in the region. There were variations in types and prevalence of tobacco use across sociodemographic subpopulations. The Lao government should continue current national tobacco control efforts and implement additional proven strategies to reduce tobacco use.

Mothers' autonomy and childhood stunting: evidence from semi-urban communities in Lao PDR.

BACKGROUND: Childhood stunting (height-for-age z-scores below - 2), a form of chronic undernutrition, remains a global health burden. Although a growing literature has examined the association between mothers' autonomy and childhood stunting, these studies have been limited to countries in South Asia or Sub-Saharan Africa where women have relatively lower social status than do men. Little research has analyzed the effect of mothers' autonomy on childhood stunting in Lao PDR, where women's social status is relatively high compared to that in other countries. METHODS: We conducted a cross-sectional questionnaire and body scale measurement targeting 100 mothers and their 115 children (<5 years old) from semi-urban communities in Lao PDR, which is the country with the highest prevalence of childhood stunting in the Indochina region. As dimensions of women's autonomy, we measured self-esteem, self-efficacy, decision-making power, freedom of mobility, and control of money. We then analyzed how each dimension was associated with the likelihood of childhood stunting. RESULTS: The likelihood of childhood stunting was significantly lower if mothers had higher self-efficacy for health care (OR = 0.15, p = 0.007), self-esteem (OR = 0.11, p = 0.025), or control of money (OR = 0.11, p = 0.041). In contrast, mothers' decision-making power and freedom of mobility were not significantly associated with childhood stunting. CONCLUSIONS: We clarified which dimensions of women's autonomy were associated with childhood stunting in Lao PDR. A closer examination of mothers' autonomy will aid proper understanding of the determinants of childhood stunting.

Socioeconomic determinants of accessibility to birth registration in Lao PDR.

BACKGROUND: The global coverage rate of birth registration is only around 65% for the population of children under five although birth registration secures protection and access to health services that are fundamental rights for all babies. This study aimed to perform a basic analysis of the accessibility to birth registration to better understand how to improve the birth registration system in the Lao PDR. METHODS: For the analysis of birth registration and related socioeconomic factors, 9576 mother-child pairs were chosen from the data set of The Lao Social Indicator Survey 2011-12. After bivariate analysis with statistical tests including the chi-square test were conducted, logistic regression was performed to determine the variables that statistically influence accessibility to birth registration. RESULTS: Ethno-geographic factors and place of delivery were observed to be the factors associated with birth registration in this analysis. CONCLUSION: Many mothers in the Lao PDR deliver in their local communities. Therefore, capacity development of various human resources, such as Skilled Birth Attendant, to support the local administrative procedure of birth registration in their communities could be one option to overcoming the bottlenecks in the birth registration process in the Lao PDR.

Rapid identification of genes controlling virulence and immunity in malaria parasites.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+)-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N)/d(S) ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.

Stable Allele Frequency Distribution of the Plasmodium falciparum clag Genes Encoding Components of the High Molecular Weight Rhoptry Protein Complex.

Plasmodium falciparum Clag protein is a candidate component of the plasmodial surface anion channel located on the parasite-infected erythrocyte. This protein is encoded by 5 separated clag genes and forms a RhopH complex with the other components. Previously, a signature of positive diversifying selection was detected on the hypervariable region of clag2 and clag8 by population-based analyses using P. falciparum originating from Thailand in 1988-1989. In this study, we obtained the sequence of this region of 3 clag genes (clag2, clag8, and clag9) in 2005 and evaluated the changes over time in the frequency distribution of the polymorphism of these gene products by comparison with the sequences obtained in 1988-1989. We found no difference in the frequency distribution of 18 putatively neutral loci between the 2 groups, evidence that the background of the parasite population structure has remained stable over 14 years. Although the frequency distribution of most of the polymorphic sites in the hypervariable region of Clag2, Clag8, and Clag9 was stable over 14 years, we found that a proportion of the major Clag2 group and one amino acid position of Clag8 changed significantly. This may be a response to a certain type of pressure.