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Community case management by community health workers has substantially reduced malaria across the Greater Mekong Subregion and Central America. To sustain current and achieve further reductions in malaria, surveillance and delivery platforms must be redesigned to ensure their continued use by key populations., Key Findings Community health workers (CHWs) detect nearly 30% of all malaria cases in remaining hotspots in Lao PDR and more than 50% of all reported malaria cases in select regions of Honduras. Despite having dedicated funding, both countries face challenges supervising, equipping, and supporting CHWs. As malaria test positivity declines, expanding CHWs' responsibilities may encourage individuals to seek care and enable CHWs to more promptly detect and treat remaining cases. Key Implications National malaria programs should assess vertical CHW network design, management, and financing to identify opportunities to promote care seeking for febrile illness. As likelihood of malaria as the primary cause of fever decreases, providing an integrated package of services will become more critical in ensuring that patients' needs are met. CHWs are a major source of malaria testing, treatment, and disease intelligence. Information systems capable of tracking their performance and progress to guide remedial action and attribute impact are essential., Background: Across the Greater Mekong Subregion (GMS) and Central America, governments commonly employ community health workers (CHWs) to improve access to and uptake of malaria services. Many of these networks are vertical in design, organized to extend malaria-only services to those remaining communities in which malaria persists. Methods: Between 2019 and 2020, national ministries of health (MOH) and Clinton Health Access Initiative conducted mixed-methods CHW program evaluations across the GMS and Central America. Routine surveillance and programmatic data were analyzed to quantify CHW contributions to malaria elimination objectives and identify gaps and challenges. Semistructured interviews were conducted with governmental and nongovernmental stakeholders from central to community level. This article draws comparisons between the Lao People's Democratic Republic (PDR) and Honduras CHW program evaluation results to distill broader hypotheses about how vertical CHW programs might evolve as their primary mission nears its end. Results: CHWs contribute substantially to malaria case detection and surveillance, diagnosing and treating 27% of malaria cases in Lao PDR and 55% in the department of Gracias a Dios, Honduras in 2019. In the same year, malaria test positivity neared less than 1% in both countries. In 2019, 80% of CHWs in Lao PDR and 74% in Gracias a Dios, Honduras did not report a single malaria case. From inception, both programs were organized as vertical (malaria-only) CHW programs reliant upon Global Fund financing for malaria commodities, training, supervision and, where applicable, remuneration. Conclusions: Although community case management by CHWs has been highly impactful in reducing malaria cases to near zero, new challenges of acceptability and effectiveness of malaria-only service delivery, feasibility of continued vertical program management, and sustainable financing have emerged. To achieve and sustain reductions in malaria, surveillance and delivery platforms must be redesigned to encourage (and reward) care seeking based on experience of symptoms and not on a patient or caregiver's presumptive diagnosis of disease. By expanding the roles and responsibilities of currently vertical malaria CHWs, malarial interventions can be optimized and sustained. Such a shift will also position existing community-based platforms to be resilient and responsive as epidemiology of disease and community need shift.

Author(s): Jacob, Christopher G; Thuy-Nhien, Nguyen; Mayxay, Mayfong; Maude, Richard J; Quang, Huynh Hong; Hongvanthong, Bouasy; Vanisaveth, Viengxay; Ngo Duc, Thang; Rekol, Huy; van der Pluijm, Rob; von Seidlein, Lorenz; Fairhurst, Rick; Nosten, Francois; Hossain, Md Amir; Park, Naomi; Goodwin, Scott; Ringwald, Pascal; Chindavongsa, Keobouphaphone; Newton, Paul; Ashley, Elizabeth; Phalivong, Sonexay; Maude, Rapeephan; Leang, Rithea; Huch, Cheah; Dong, Le Thanh; Nguyen, Kim-Tuyen; Nhat, Tran Minh; Hien, Tran Tinh; Nguyen, Hoa; Zdrojewski, Nicole; Canavati, Sara; Sayeed, Abdullah Abu; Uddin, Didar; Buckee, Caroline; Fanello, Caterina I; Onyamboko, Marie; Peto, Thomas; Tripura, Rupam; Amaratunga, Chanaki; Myint Thu, Aung; Delmas, Gilles; Landier, Jordi; Parker, Daniel M; Chau, Nguyen Hoang; Lek, Dysoley; Suon, Seila; Callery, James; Jittamala, Podjanee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Phyo, Aung Pyae; Smithuis, Frank; Lin, Khin; Thant, Myo; Hlaing, Tin Maung; Satpathi, Parthasarathi; Satpathi, Sanghamitra; Behera, Prativa K; Tripura, Amar; Baidya, Subrata; Valecha, Neena; Anvikar, Anupkumar R; Ul Islam, Akhter; Faiz, Abul; Kunasol, Chanon; Drury, Eleanor; Kekre, Mihir; Ali, Mozam; Love, Katie; Rajatileka, Shavanthi; Jeffreys, Anna E; Rowlands, Kate; Hubbart, Christina S; Dhorda, Mehul; Vongpromek, Ranitha; Kotanan, Namfon; Wongnak, Phrutsamon; Almagro Garcia, Jacob; Pearson, Richard D; Ariani, Cristina V; Chookajorn, Thanat; Malangone, Cinzia; Nguyen, T; Stalker, Jim; Jeffery, Ben | Abstract: BackgroundNational Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures.MethodsSamples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs.ResultsGenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs.ConclusionsGenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community.FundingThe GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002–2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

Although expansions in gamma delta T cell populations are known to occur in the peripheral blood of patients infected with Plasmodium falciparum, the role of these cells in people with naturally acquired immunity against P. falciparum who live in malaria-endemic areas is poorly understood. We used a cross-sectional survey to investigate the role of peripheral blood gamma delta T cells in people living in Lao People's Democratic Republic, a malaria-endemic area. We found that the proportion of non-V gamma 9 gamma delta T cells was higher in non-hospitalized uncomplicated falciparum malaria patients (UMPs) from this region. Notably, we found that the non-V gamma 9 gamma delta T cells in the peripheral blood of UMPs and negative controls from this region had the potential to expand and produce IL-10 and interferon-gamma when cultured in the presence of IL-2 and/or crude P. falciparum antigens for 10 days. Furthermore, these cells were associated with plasma interleukin 10 (IL-10), which was elevated in UMPs. This is the first report demonstrating that, in UMPs living in a malaria-endemic area, a gamma delta T cell subset, the non-V gamma 9 gamma delta T cells, expands and produces IL-10. These results contribute to understanding of the mechanisms of naturally acquired immunity against P. falciparum., 論文

The efficacy of the six-dose regimen of artemether-lumefantrine was compared with the combination of artesunate and mefloquine in a randomised, comparative trial in Luang Namtha Province, Northern Laos. Of 1033 screened patients, 201 were positive for Plasmodium falciparum; 108 patients of all age groups (2-66 years) with acute, uncomplicated P. falciparum malaria were enrolled in the study, 100 of whom were followed-up for 42 days. Fifty-three patients received artemether-lumefantrine and 55 received artesunante-mefloquine. Both drug combinations induced rapid clearance of parasites and malaria symptoms; there was no significant difference in the initial therapeutic response parameters. Both regimes were well tolerated. After 42 days, cure rates were 93.6% (95% CI = 82.5-98.7%; 44 of 47 patients) for artemether-lumefantrine and 100% (95% CI = 93.3-100.0%; 53 of 53 patients) for artesunate-mefloquine. The results show the excellent efficacy and tolerability of both artemether-lumefantrine and artesunate-mefloquine in Northern Laos.

Strongyloidiasis is a major soil-transmitted helminth (STH) disease that affects people worldwide. We present updated data on prevalence in the Lao People's Democratic Republic (Lao PDR) in 2015, arising from a community cross-sectional helminthiasis survey. Fecal samples were collected from 327 individuals across three provinces in Lao PDR (Luang Prabang in the north, Khammouane in the center, and Champasack in the south). Agar plate culture and Kato-Katz methods were used to examine duplicate stool samples from each participant to detect Strongyloides stercoralis and co-infecting helminths. Overall prevalences of S. strercoralis human hookworm, Taenia spp., Trichuris trichiura, Ascaris lumbricoides, and Enterobius vermicularis were 41.0, 28.1, 4.9, 4.0, 1.5, and 0.9 %, respectively. The prevalence of miscellaneous trematodiases (including opisthorchiasis) was 37.9 % and of Schistosoma mekongi infection was 0.3 %. Strongyloidiasis is a current major STH disease in Lao PDR. We also report the molecular-phylogenetic identification of S. stercoralis adult males collected from 40 representative human strongyliodiasis fecal samples. DNA was extracted, amplified, and sequenced from a portion of the mitochondrial cox1 gene and the nuclear 18S ribosomal DNA. Phylogenetic analyses indicated that all specimens sequenced belonged to S. stercoralis (Bavay, 1876) Stiles and Hassall, 1902. The cox1 sequences exhibited great diversity (24 haplotypes) in Lao PDR. This is the first molecular identification and report of genetic diversity of S. stercoralis in humans from Lao PDR. An effective parasite control program is needed to reduce the serious health impacts.

Levels of drug resistance of Plasmodium falciparum strains against antimalarials have increased in Laos. In several studies, chloroquine (CQ) resistance has been associated with point mutations in the Pfcrt and pfmdr genes, and sulphadoxine/pyrimethamine (S/P) resistance with point mutations in the genes of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). We combined a study of these molecular markers with an in vivo antimalarial drug sensitivity study in Attapeu province in the south of Lao PDR. We treated 100 patients with either CQ, S/P or a combination of both. In the CQ group, Pfcrt mutations showed a very high sensitivity (100%) but a low specificity (12.5%) to predict resistance. The combination of mutations in the Pfcrt and pfmdr genes was highly specific and had a positive predictive value of 100%. Mutations in the DHPS gene showed a high correlation with the development of resistance. The prevalence of mutations in the DHFR gene, especially codon 108 Asn, was predictive with high sensitivity (100%) but low specificity. Isolates derived from patients treated with a combination of both drugs showed a high correlation between the mutation in codon 437 of DHPS gene and in vivo-resistance (odds ratio 16.00, CI). The study provides evidence for the existence of antimalarial drug resistance in the south of Lao PDR, and offers a molecular method to predict resistance.

BACKGROUND: In a recent study one third of Lao patients presenting with uncomplicated Plasmodium falciparum malaria had biochemical evidence of thiamin deficiency, which was associated with a higher incidence of adverse events. Thiamin supplementation might, therefore, reduce adverse events in this population. METHODS: An exploratory, double-blind, parallel group, placebo-controlled, superiority trial of thiamin supplementation in patients of all ages with uncomplicated and severe falciparum malaria was conducted in Xepon District, Savannakhet Province, southern Laos. Patients were randomly assigned to either oral thiamin 10 mg/day for 7 days immediately after standard anti-malarial treatment then 5 mg daily until day 42, or identical oral placebo. RESULTS: After interim analyses when 630 patients (314 in thiamin and 316 in placebo groups) had been recruited, the trial was discontinued on the grounds of futility. On admission biochemical thiamin deficiency (alpha ≥ 25%) was present in 27% of patients and 9% had severe deficiency (alpha > 31%). After 42 days of treatment, the frequency of thiamin deficiency was lower in the thiamin (2%, 1% severe) compared to the placebo (11%, 3% severe) groups (p 0.05). Clinical, haematological, and parasitological responses to treatment did not differ significantly between the two groups. CONCLUSION: Thiamin supplementation reduced biochemical thiamin deficiency among Lao malaria patients following anti-malarial drug treatment, but it did not reduce the frequency of adverse events after anti-malarial therapy or have any detected clinical or parasitological impact. TRIAL REGISTRATION: ISRCTN 85411059.

Experimental studies have indicated that low serum zinc levels affect immune responses. However, few studies have evaluated the impact of serum zinc levels on antibody responses in the field in developing countries. We investigated an association between the anti-Plasmodium falciparum (Pf) antibody (immunoglobulin G) titer and serum zinc concentration among villagers in rural areas of the Lao People's Democratic Republic. Blood samples were collected to detect Pf infection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the anti-PfIgG antibody titer. Each serum sample was assayed to measure the concentration of zinc. Pearson's correlation coefficient was applied to the association between zinc concentration and anti-PfIgG antibody titers. Multiple linear regression analysis was used to assess the association between zinc concentration and anti-PfIgG antibody titers, controlling for age and albumin level. Of 71 blood samples, 40 were Pf positive and 31 were Pf negative. The median serum zinc concentrations were 56.0 μg/dl in the Pf-positive group and 62.5 μg/dl in the Pf-negative group. The median anti-Pf titers were 833.4 in the positive group and 1237.2 in the negative group. Unexpectedly, there was a negative correlation between serum zinc and anti-Pf IgG antibody titers; the correlation coefficient were -0.453 and (p=0.003) in the positive group and -0.461 (p=0.009) in the negative group. The results of this study indicated sustained antibody responses among the villagers, who had likely been exposed to malaria periodically throughout their lives. Further studies are necessary to determine the conditions in which zinc could be effective against malaria.

Background The Lao Government changed the national policy for uncomplicated Plasmodium falciparum malaria from chloroquine to artemether-lumefantrine (AL) in 2005. Since then, no information on AL efficacy has been reported. With evidence of resistance to artemisinin derivatives in adjacent Cambodia, there has been a concern as to AL efficacy. Monitoring of AL efficacy would help the Lao Government to make decisions on appropriate malaria treatment. Methods The efficacy of a three-day, twice daily oral artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Xepon District, Savannakhet Province, southern Laos was studied over 42 days follow-up. This was part of a trial of thiamin supplementation in falciparum malaria. Results Of 630 patients with P. falciparum enrolled in the trial of thiamin treatment, 549 (87%, 357 children ≤15 years and 192 adults) were included in this study. The per protocol 42-day cure rates were 97% (524/541) [96% (337/352) for children and 99% (187/189) for adults, p = 0.042]. By conventional intention-to-treat analysis, the 42-day cure rates adjusted for re-infection, were 97% (532/549) [96% (342/357) in children and 99% (190/192) in adults, p = 0.042]. The proportion of patients who remained parasitaemic at day 1 after treatment was significantly higher in children [33% (116/356)] compared to adults [15% (28/192)] (p Conclusions The current nationally-recommended anti-malarial treatment (artemether-lumefantrine) remains highly efficacious for the treatment of uncomplicated falciparum malaria five years after introduction in Laos. Regular monitoring is required in case artemisinin-resistant P. falciparum parasites should appear. Trial registration ISRCTN85411059.

Background Overnight stays in farming huts are known to pose a risk of malaria infection. However, studies reporting the risk were conducted in the settings of poor net coverage. This study sought to assess whether an overnight stay in a farming hut is associated with an increased risk of malaria infection if insecticide-treated bed nets (ITNs) are properly used. Methods A pair of cross-sectional surveys was carried out in the Lamarm district of Sekong province, Laos, in March (dry season) and August (rainy season) in 2008. Questionnaire-based interviews and blood examinations were conducted with farmers and their household members from three randomly selected villages in March (127 households, 891 people) and August (128 households, 919 people). Logistic regression analysis, adjusted for potential confounding factors, was used to assess the association between malaria infection status and frequency of overnight stays for the two weeks prior to the study in both the seasons. Results In March, 13.7% of participants reported staying overnight in a farming hut at least once in the previous two weeks. The percentage increased to 74.6% in August. Not only adults but also young children stayed overnight as often as adults. The use of an ITN the preceding night was common both in farming huts (66.3% in March, 95.2% in August), and in main residences (85.8% in March, 92.5% in August). Logistic regression analysis showed no statistical association between malaria infection status and frequency of overnight stays in farming huts in either study period. However, people sharing one family type net with five people or more were significantly more likely to have malaria than those sharing a net with up to two people in the dry season. Conclusions This study showed that staying overnight in farming huts was not associated with an increased risk of malaria infection in the setting where ITNs were widely used in farming huts. It suggests that malaria infection during overnight stays in farming huts might be preventable if ITNs are properly used in rural Laos.

Background Counterfeit oral artesunate has been a major public health problem in mainland SE Asia, impeding malaria control. A countrywide stratified random survey was performed to determine the availability and quality of oral artesunate in pharmacies and outlets (shops selling medicines) in the Lao PDR (Laos). Methods In 2003, 'mystery' shoppers were asked to buy artesunate tablets from 180 outlets in 12 of the 18 Lao provinces. Outlets were selected using stratified random sampling by investigators not involved in sampling. Samples were analysed for packaging characteristics, by the Fast Red Dye test, high-performance liquid chromatography (HPLC), mass spectrometry (MS), X-ray diffractometry and pollen analysis. Results Of 180 outlets sampled, 25 (13.9%) sold oral artesunate. Outlets selling artesunate were more commonly found in the more malarious southern Laos. Of the 25 outlets, 22 (88%; 95%CI 68–97%) sold counterfeit artesunate, as defined by packaging and chemistry. No artesunate was detected in the counterfeits by any of the chemical analysis techniques and analysis of the packaging demonstrated seven different counterfeit types. There was complete agreement between the Fast Red dye test, HPLC and MS analysis. A wide variety of wrong active ingredients were found by MS. Of great concern, 4/27 (14.8%) fakes contained detectable amounts of artemisinin (0.26–115.7 mg/tablet). Conclusion This random survey confirms results from previous convenience surveys that counterfeit artesunate is a severe public health problem. The presence of artemisinin in counterfeits may encourage malaria resistance to artemisinin derivatives. With increasing accessibility of artemisinin-derivative combination therapy (ACT) in Laos, the removal of artesunate monotherapy from pharmacies may be an effective intervention.

OBJECTIVE: The Pfcrt-gene encodes a transmembrane protein located in the Plasmodium falciparum digestive vacuole. Chloroquine resistant (CQR) strains of African and Southeast Asian origin carry the Pfcrt-haplotype (c72-76) CVIET, whereas most South American and Papua New Guinean CQR stains carry the SVMNT haplotype. METHOD: Eighty-eight samples from an area with reported in vivo Chloroquine and in vitro Amodiaquine-resistance were screened for the K76T mutation and their Pfcrt-haplotype (c72-76) using a new SSOP-ELISA. RESULTS: Hundred percent of the analysed samples showed the K76T mutation which is highly associated with in vivo drug failure. This very high rate of a CQR-marker is alarming in an area were CQ is still used as first line drug. The distribution of the three main Pfcrt-haplotypes was as follows: 68% CVIET, 31% SVMNT, 0% CVMNT. CONCLUSIONS: These data show, for the first time, the South American/PNG -haplotype (SVMNT) on mainland Southeast Asia. SVMNT-haplotype and others might be associated with a decreased efficacy of Amodiaquine and could therefore be potential markers for of amodiaquine resistance (AQR). If there is a correlation between AQR and the SVMNT-haplotype as suggested, 31% prevalence of a potential resistance marker is cause for concern.

Summary A longitudinal study was conducted on the prevalence of Anopheles in three malaria endemic villages in Sekong province, in the southern region of Lao PDR, from August 2000 to October 2001. All night, human landing collections took place in August and October 2000 and April and October 2001, and blood smears were taken for malaria parasites during the same period. Mosquitoes were tested for sporozoite antigen using enzyme-linked immunosorbent assay. In August 2000 (wet season) and April 2001 (dry season) the ovaries of the mosquitoes were examined for parity. A total of 16 species of Anopheles were caught in the study sites of which An. dirus A, An. maculatus sl and An. jeyporiensis were positive for sporozoites. The entomological inoculation rate (EIR) ranged from 0.06 to 0.25. There was a good correlation between EIR and vectorial capacity in the wet season, especially in Pai Mai where the prevalence of malaria was also high during the wet seasons (11.8 and 10.53). An. dirus A showed ambivalence in their choice of feeding as approximately 50% attacked man indoors and an equal proportion outdoors. An dirus A was the main vector in Pai Mai. The parous rate did not significantly differ between the wet and dry season, although it was higher in the dry season. In Takaio the parasite prevalence ranged from 8.7% (dry season) to 37.1% (wet season) and An. jeyporiensis was the vector, and the risk of infection was 0.85 in the dry season while 0.99 in the wet season. In Toumgno An. maculatus sl was the vector and infection was found only in August and October 2000. However, malaria prevalence ranged from 9.69 to 20.4% and was equally high in the dry season. Cattle were also present close to the houses in all the villages and this might be a contributory factor in the prevalence of malaria.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

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