Search

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.

BackgroundMosquitoes are vectors of many pathogens, such as malaria, dengue virus, yellow fever virus, filaria and Japanese encephalitis virus. Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, such as cytoplasmic incompatibility. Wolbachia has been proposed as a tool to modify mosquitoes that are resistant to pathogen infection as an alternative vector control strategy. This study aimed to determine natural Wolbachia infections in different mosquito species across Hainan Province, China.MethodsAdult mosquitoes were collected using light traps, human landing catches and aspirators in five areas in Hainan Province from May 2020 to November 2021. Species were identified based on morphological characteristics, species-specific PCR and DNA barcoding of cox1 assays. Molecular classification of species and phylogenetic analyses of Wolbachia infections were conducted based on the sequences from PCR products of cox1, wsp, 16S rRNA and FtsZ gene segments.ResultsA total of 413 female adult mosquitoes representing 15 species were identified molecularly and analyzed. Four mosquito species (Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Culex gelidus) were positive for Wolbachia infection. The overall Wolbachia infection rate for all mosquitoes tested in this study was 36.1% but varied among species. Wolbachia types A, B and mixed infections of A × B were detected in Ae. albopictus mosquitoes. A total of five wsp haplotypes, six FtsZ haplotypes and six 16S rRNA haplotypes were detected from Wolbachia infections. Phylogenetic tree analysis of wsp sequences classified them into three groups (type A, B and C) of Wolbachia strains compared to two groups each for FtsZ and 16S rRNA sequences. A novel type C Wolbachia strain was detected in Cx. gelidus by both single locus wsp gene and the combination of three genes.ConclusionOur study revealed the prevalence and distribution of Wolbachia in mosquitoes from Hainan Province, China. Knowledge of the prevalence and diversity of Wolbachia strains in local mosquito populations will provide part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Hainan Province.

The protein Griffithsin (Grft) is a lectin that tightly binds to high-mannose glycosylation sites on viral surfaces. This property allows Grft to potently inhibit many viruses, including HIV-1. The major route of HIV infection is through sexual activity, so an important tool for reducing the risk of infection would be a film that could be inserted vaginally or rectally to inhibit transmission of the virus. We have previously shown that silk fibroin can encapsulate, stabilize, and release various antiviral proteins, including Grft. However, for broad utility as a prevention method, it would be useful for an insertable film to adhere to the mucosal surface so that it remains for several days or weeks to provide longer-term protection from infection. We show here that silk fibroin can be formulated with adhesive properties using the nontoxic polymer hydroxypropyl methylcellulose (HPMC) and glycerol, and that the resulting silk scaffold can both adhere to biological surfaces and release Grft over the course of at least one week. This work advances the possible use of silk fibroin as an anti-viral insertable device to prevent infection by sexually transmitted viruses, including HIV-1.

BackgroundMyanmar bears the heaviest malaria burden in the Greater Mekong Subregion (GMS). This study assessed the spatio-temporal dynamics and environmental predictors of Plasmodium falciparum and Plasmodium vivax malaria in Myanmar.MethodsMonthly reports of malaria cases at primary health centers during 2011-2017 were analyzed to describe malaria distribution across Myanmar at the township and state/region levels by spatial autocorrelation (Moran index) and spatio-temporal clustering. Negative binomial generalized additive models identified environmental predictors for falciparum and vivax malaria, respectively.ResultsFrom 2011 to 2017, there was an apparent reduction in malaria incidence in Myanmar. Malaria incidence peaked in June each year. There were significant spatial autocorrelation and clustering with extreme spatial heterogeneity in malaria cases and test positivity across the nation (P

BackgroundMass drug administration (MDA) with primaquine (PQ) is being considered for accelerating Plasmodium vivax elimination in remaining active foci. This study aimed to determine the acceptability of MDA with PQ in malaria endemic villages in a malarious setting in the South of Thailand undergoing MDA with PQ.MethodsA cross-sectional mixed-methods approach was conducted in seven malaria endemic villages where MDA with PQ was implemented. The data were collected from community villagers and health workers using structured questionnaires, in-depth interviews, and focus group discussions. Descriptive statistics and logistic regression models were used for quantitative data analysis. Thematic analysis was applied for qualitative data.ResultsAmong a total of 469 participants from the MDA villages, 293 participants were eligible for MDA with PQ and 79.86% (234) completed 14-days of PQ. The logistic regressions indicated that males (adjusted odds ratio: 2.52 [95% confidence interval: 1.33-4.81]) and those who are farmers (2.57 [1.12-5.90]) were most likely to participate in the MDA. Among 293 participants in the post-MDA study, 74.06% had originally agreed to participate in the MDA with PQ while 25.94% had originally reported not wanting to participate in the MDA. Of those who originally reported being willing to participate in the MDA, 71.23% followed through with participation in the first or second round. Conversely, 93.24% of those who originally reported not being willing to participate in the MDA did in fact participate in the MDA. Factors contributing to higher odds of agreeing to participate and following through with participation included being male (1.98 [1.06-3.69]) and correctly responding that malaria is preventable (2.32 [1.01-5.35]) with some differences by village. Five key themes emerged from the qualitative analyses: concern about side effects from taking PQ; disbelief that malaria could be eliminated in this setting; low overall concern about malaria infections; misunderstandings about malaria; and a general need to tailor public health efforts for this unique context.ConclusionWhile the reported likelihood of participating in MDA was high in this setting, actual follow-through was relatively moderate, partially because of eligibility (roughly 71% of those in the follow-up survey who originally agreed to participate actually followed through with participation). One of the largest concerns among study participants was PQ-related side effects-and these concerns likely heavily influenced participant adherence to the MDA. The results of this study can be used to tailor future MDAs, or other public health interventions, in this and potentially other similar settings.

The occurrence and spread of insecticide resistance has had a negative effect on the efficacy of insecticide-based tools and is distributed worldwide, including the Greater Mekong Subregion (GMS). This study aims to determine the insecticide susceptibility of malaria and dengue vectors in malaria and dengue hotspots on the Thai-Myanmar border. Mosquito larvae and pupae were obtained from water sources from December 2019 to April 2020 in Tha Song Yang District, Tak province, western Thailand. WHO bioassay susceptibility tests were conducted with three classes of insecticides to evaluate the knockdown and mortality rates of Anopheles and Aedes aegypti female adults. V1016G and F1534C kdr mutations in the voltage-gated sodium channel of Ae. aegypti were identified using a multiplex PCR. A total of 5764 female mosquitoes were bioassayed in this study, including Anopheles spp. (92.63%) and F1 Ae. aegypti (7.37%). After 24 h of observation, An. minimus s.l. (n = 3885) and An. maculatus s.l. (n = 1138) in Suan Oi (SO) and Tala Oka (TO) were susceptible to pyrethroids, organophosphates and carbamates (except bendiocarb) with 98-100% mortality (MR). Resistance to bendiocarb was detected with a mortality rate of 88.80%, 88.77%, and 89.92% for An. minimus s.l. (n = 125, 125) and An. maculatus s.l. (n = 66), respectively. The first generation of Ae. aegypti adult females were suspected of resistance to deltamethrin (n = 225, MR = 96.89%) and confirmed resistance to permethrin (n = 200, MR = 20.00%). V1016G and F1534C mutations were detected in three genotypes, heterozygote and homozygote forms. The correlation between the kdr alleles and deltamethrin resistance was significant. In conclusion, bendiocarb resistance was found in primary malaria vectors, An. minimus s.l. and An. maculatus s.l. F1 Ae. aegypti population was pyrethroids-resistant, associated with kdr alleles. Therefore, molecular analysis should be conducted to gain insights into the mechanism of insecticide resistance. Routine malaria vector control programmes, such as fogging implementation in hotspot villages to induce Aedes resistance available in peri-domestic sites, are questionable.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing in vivo, in vitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

The malaria landscape in the Greater Mekong Subregion has experienced drastic changes with the ramp-up of the control efforts, revealing formidable challenges that slowed down the progress toward malaria elimination. Problems such as border malaria and cross-border malaria introduction, multidrug resistance in Plasmodium falciparum, the persistence of Plasmodium vivax, the asymptomatic parasite reservoirs, and insecticide resistance in primary vectors require integrated strategies tailored for individual nations in the region. In recognition of these challenges and the need for research, the Southeast Asian International Center of Excellence for Malaria Research has established a network of researchers and stakeholders and conducted basic and translational research to identify existing and emerging problems and develop new countermeasures. The installation of a comprehensive disease and vector surveillance system at sentinel sites in border areas with the implementation of passive/active case detection and cross-sectional surveys allowed timely detection and management of malaria cases, provided updated knowledge for effective vector control measures, and facilitated the efficacy studies of antimalarials. Incorporating sensitive molecular diagnosis to expose the significance of asymptomatic parasite reservoirs for sustaining transmission helped establish the necessary evidence to guide targeted control to eliminate residual transmission. In addition, this program has developed point-of-care diagnostics to monitor the quality of artemisinin combination therapies, delivering the needed information to the drug regulatory authorities to take measures against falsified and substandard antimalarials. To accelerate malaria elimination, this program has actively engaged with stakeholders of all levels, fostered vertical and horizontal collaborations, and enabled the effective dissemination of research findings.

BackgroundIn the Greater Mekong Subregion of Southeast Asia, Plasmodium vivax malaria is endemic and causes significant morbidity. In this study, the efficacy of chloroquine for treating uncomplicated P. vivax malaria at the eastern and western borders of Myanmar was investigated.MethodsA total of 197 participants with microscopically confirmed P. vivax infection were enrolled from three townships of the southeastern (Thanbyuzayat and Kawthoung) and western (Kyauktaw) borders of Myanmar. Patients were treated with chloroquine according to the national malaria treatment guidelines and followed for 28 days.ResultsAmong the 197 enrollments, 172 completed the 28-day follow-up. Twelve recurrent P. vivax infections, all occurring in the third and fourth week, were detected, resulting in an overall cumulative rate of recurrence of 4.7% [95% confidence interval (CI) 1.5-7.8]. The incidence rate of recurrence varied among the three sites. In Thanbyuzayat township, no patients had recurrent parasitemia between days 7 and 28. In contrast, Kyauktaw township had a day 28 cumulative incidence rate of recurrence of 7.2% (95% CI 0.6-13.9%) compared to 6.9% (95% CI 0.6-13.2) in Kawthoung township.ConclusionWhile this study confirmed the relatively high clinical efficacy of chloroquine for treating P. vivax in Myanmar with modest rates of recurrent infections within 28 days of the treatment, it also revealed considerable geographical heterogeneity of chloroquine efficacy, which warrants continuous surveillance efforts.

Chemokines are small proteins that are critical for immune function, being primarily responsible for the activation and chemotaxis of leukocytes. As such, many viruses, as well as parasitic arthropods, have evolved systems to counteract chemokine function in order to maintain virulence, such as binding chemokines, mimicking chemokines, or producing analogs of transmembrane chemokine receptors that strongly bind their targets. The focus of this review is the large group of chemokine binding proteins (CBP) with an emphasis on those produced by mammalian viruses. Because many chemokines mediate inflammation, these CBP could possibly be used pharmaceutically as anti-inflammatory agents. In this review, we summarize the structural properties of a diverse set of CBP and describe in detail the chemokine binding properties of the poxvirus-encoded CBP called vCCI (viral CC Chemokine Inhibitor). Finally, we describe the current and emerging capabilities of combining computational simulation, structural analysis, and biochemical/biophysical experimentation to understand, and possibly re-engineer, protein-protein interactions.

BackgroundMyanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region.MethodsDuring October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested.ResultsA total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen.ConclusionsThis study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.

BackgroundWhile national malaria incidence has been declining in Myanmar, some subregions within the nation continue to have high burdens of malaria morbidity and mortality. This study assessed the malaria situation in one of these regions, Banmauk Township, located near the Myanmar-India border. Our goal was to provide a detailed description of the malaria epidemiology in this township and to provide some evidence-based recommendations to formulate a strategy for reaching the national malaria elimination plan. Banmauk consistently has one of the highest malaria burdens in Myanmar.MethodsWith the implementation of strengthened malaria control and surveillance activities after the endorsement of a national malaria elimination plan in 2015, detailed incidence data were obtained for 2016-2018 for Banmauk Township. The data include patient demographics, parasite species, disease severity, and disease outcome. Data were analyzed to identify characteristics, trends, distribution, and risk factors.ResultsDuring 2016-2018, 2,402 malaria cases were reported, with Plasmodium falciparum accounting for 83.4% of infections. Both P. falciparum and P. vivax were transmitted more frequently during the rainy season (May-October). Despite intensified control, the annual parasite incidence rate (API) in 2017 (11.0) almost doubled that in 2016 (6.5). In total, 2.5% (59/2042) of the cases, of which 54 P. falciparum and 5 P. vivax, were complicated cases, resulting in 5 deaths. Malaria morbidity was high in children

BackgroundDespite major reductions in malaria burden across Myanmar, clusters of the disease continue to persist in specific subregions. This study aimed to assess the predictors of test positivity among people living in Paletwa Township of Chin State, an area of persistently high malaria burden.MethodsFour villages with the highest malaria incidence from Paletwa Township were purposively selected. The characteristics of 1045 subjects seeking malaria diagnosis from the four assigned village health volunteers from January to December, 2018 were retrospectively analyzed. Their household conditions and surroundings were also recorded using a checklist. Descriptive statistics and logistic regression models were applied to investigate potential associations between individual and household characteristics and malaria diagnosis.ResultsIn 2017, the Paletwa township presented 20.9% positivity and an annual parasite index of 46.9 cases per 1000 people. Plasmodium falciparum was the predominant species and accounted for more than 80.0% of all infections. Among 1045 people presenting at a clinic with malaria symptoms, 31.1% were diagnosed with malaria. Predictors for test positivity included living in a hut [adjusted odds ratios (a OR): 2.3, 95% confidence intervals (CI): 1.2-4.6], owning farm animals (aOR: 1.7, 95% CI: 1.1-3.6), using non-septic type of toilets (aOR: 1.9, 95% CI: 1.1-8.4), presenting with fever (aOR: 1.9, 95% CI: 1.1-3.0), having a malaria episode within the last year (aOR: 2.9, 95% CI: 1.4-5.8), traveling outside the village in the previous 14 days (aOR: 4.5, 95% CI: 1.5-13.4), and not using bed nets (a OR: 3.4, 95% CI: 2.3-5.1). There were no statistically significant differences by age or gender in this present analysis.ConclusionsThe results from this study, including a high proportion of P. falciparum infections, little difference in age, sex, or occupation, suggest that malaria is a major burden for these study villages. Targeted health education campaigns should be introduced to strengthen synchronous diagnosis-seeking behaviors, tighten treatment adherence, receiving a diagnosis after traveling to endemic regions, and using bed nets properly. We suggest increased surveillance, early diagnosis, and treatment efforts to control the disease and then to consider the local elimination.

Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as "hypothetical," is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery.IMPORTANCE Cyanobacteria, environmentally prevalent photosynthetic prokaryotes, contribute ∼25% of global primary production. Cyanobacterial biofilms elicit biofouling, thus leading to substantial economic losses; however, these microbial assemblages can also be beneficial, e.g., in wastewater purification processes and for biofuel production. Mechanistic aspects of cyanobacterial biofilm development were long overlooked, and genetic and molecular information emerged only in recent years. The importance of this study is 2-fold. First, it identifies novel components of cyanobacterial biofilm regulation, thus contributing to the knowledge of these processes and paving the way for inhibiting detrimental biofilms or promoting beneficial ones. Second, the data suggest that cyanobacteria may employ the same complex for the assembly of the motility appendages, type 4 pili, and protein secretion. A shared pathway was previously shown in only a few cases of heterotrophic bacteria, whereas numerous studies demonstrated distinct systems for these functions. Thus, our study broadens the understanding of pilus assembly/secretion in diverse bacteria and furthers the aim of controlling the formation of cyanobacterial biofilms.

Imported malaria and recurrent infections are becoming an emerging issue in many malaria non-endemic countries. This study aimed to determine the molecular patterns of the imported malaria infections and recurrence. Blood samples were collected from patients with imported malaria infections during 2016-2018 in Guangxi Zhuang Autonomous Region, China. Next-generation amplicon deep-sequencing approaches were used to assess parasite genetic diversity, multiplexity of infection, relapse, recrudescence, and antimalarial drug resistance. A total of 44 imported malaria cases were examined during the study, of which 35 (79.5%) had recurrent malaria infections within 1 year. The majority (91.4%) had one recurrent malaria episode, whereas two patients had two recurrences and one patient had three recurrences. A total of 19 recurrence patterns (the species responsible for primary and successive clinical episodes) were found in patients returning from malaria epidemic countries. Four parasite species were detected with a higher than usual proportion (46.2%) of non-falciparum infections or mixed-species infections. An increasing trend of recurrence infections and reduced drug treatment efficacy were observed among the cases of imported malaria. The high recurrence rate and complex patterns of imported malaria from Africa to non-endemic countries have the potential to initiate local transmission, thereby undermining efforts to eliminate locally acquired malaria. Our findings highlight the power of amplicon deep-sequencing applications in molecular epidemiological studies of the imported malaria recurrences.

BackgroundMosquitoes are vectors of many tropical diseases. Understanding the ecology of local mosquito vectors, such as species composition, distributions, population dynamics, and species diversity is important for designing the optimal strategy to control the mosquito-borne diseases.MethodsEntomological surveillance of adult mosquitoes was conducted in five sites representing different ecological settings across Hainan Island from January to December of 2018 using BG Sentinel (BGS) traps and Centers for Disease Prevention and Control (CDC) light traps. In each site, we selected three areas representing urban, suburban and rural settings. Eighteen trap-days were sampled in each setting at each site, and CDC light traps and BGS traps were setup simultaneously. Mosquito species composition, distribution, population dynamics, and species diversity were analyzed. Mosquito densities were compared between different study sites and between different settings.ResultsNine species of mosquitoes belonging to four genera were identified. Culex quinquefasciatus (80.8%), Armigeres subalbatus (13.0%) and Anopheles sinensis (3.1%) were the top three species collected by CDC light traps; Cx. quinquefasciatus (91.9%), Ae. albopictus (5.1%), and Ar. subalbatus (2.8%) were the top three species collected by BGS traps. Predominant species varied among study sites. The population dynamics of Ae. albopictus, An. sinensis and Cx. quinquefasciatus showed clear seasonal variation regardless of study sites with a varied peak season for different species. Mosquito abundance of all species showed significant differences among different study sites and among urban, suburban and rural areas. Danzhou had the highest mosquito biodiversity, with an α, β, and Gini-Simpson biodiversity index of 8, 1.13 and 0.42, respectively. BGS traps captured Aedes mosquito at a higher efficiency than CDC light traps, whereas CDC light traps captured significantly more Anopheles and Armigeres mosquitoes than BGS traps.ConclusionsMosquitoes were abundant on Hainan Island with clear seasonality and spatial heterogeneity. Population density, species composition, distribution, and species diversity were strongly affected by the natural environment. Different tools are required for the surveillance of different mosquito species.

IntroductionThe majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention.MethodsWe formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge.ResultsEffective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota.ConclusionsWe demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection.

Anopheles sinensis is a major malaria vector in Southeast Asia. Resistance to pyrethroid insecticides in this species has impeded malaria control in the region. Previous studies found that An. sinensis populations from Yunnan Province, China were highly resistant to deltamethrin and did not carry mutations in the voltage-gated sodium channel gene that cause knockdown resistance. In this study, we tested the hypothesis that other genomic variants are associated with the resistance phenotype. Using paired-end whole genome sequencing (DNA-seq), we generated 108 Gb of DNA sequence from deltamethrin -resistant and -susceptible mosquito pools with an average coverage of 83.3× depth. Using a stringent filtering method, we identified a total of 916,926 single nucleotide variants (SNVs), including 32,240 non-synonymous mutations. A total of 958 SNVs differed significantly in allele frequency between deltamethrin -resistant and -susceptible mosquitoes. Of these, 43 SNVs were present within 37 genes that code for immunity, detoxification, cuticular, and odorant proteins. A subset of 12 SNVs were randomly selected for genotyping of individual mosquitoes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and showed consistent allele frequencies with the pooled DNA-seq derived allele frequencies. In addition, copy number variations (CNVs) were detected in 56 genes, including 33 that contained amplification alleles and 23 that contained deletion alleles in resistant mosquitoes compared to susceptible mosquitoes. The genomic variants described here provide a useful resource for future studies on the genetic mechanism of insecticide resistance in this important malaria vector species.

With almost 2 million new HIV infections worldwide each year, the prevention of HIV infection is critical for stopping the pandemic. The only approved form of pre-exposure prophylaxis is a costly daily pill, and it is recognized that several options will be needed to provide protection to the various affected communities around the world. In particular, many at-risk people would benefit from a prevention method that is simple to use and does not require medical intervention or a strict daily regimen. We show that silk fibroin protein can be formulated into insertable discs that encapsulate either an antibody (IgG) or the potent HIV inhibitor 5P12-RANTES. Several formulations were studied, including silk layering, water vapor annealing and methanol treatment to stabilize the protein cargo and impact the release kinetics over weeks. In the case of IgG, high concentrations were released over a short time using methanol treatment, with more sustained results with the use of water vapor annealing and layering during device fabrication. For 5P12-RANTES, sustained release was obtained for 31 days using water vapor annealing. Further, we show that the released inhibitor 5P12-RANTES was functional both in vitro and in ex vivo colorectal tissue. This work shows that silk fibroin discs can be developed into formidable tools to prevent HIV infection.

Although scaled-up interventions and effective control efforts have drastically reduced malaria morbidity and mortality, malaria remains a serious threat to public health worldwide. Anopheles sinensis Wiedemann 1828 is a historically important vector of Plasmodium vivax (Haemosporida: Plasmodiidae) malaria in China. Insecticide resistance has become a major obstacle to vector-borne disease control. However, little is known about the insecticide resistance of An. sinensis in Wenzhou, an important coastal port city in Zhejiang province, China. The aim of this study was to examine insecticide resistance and mechanisms in An. sinensis field mosquito populations. Evidence of multiple insecticide resistance was found in An. sinensis adult female populations. Medium to high frequencies of target site kdr together with fixed ace-1 mutations was detected in both the Ruian and Yongjia populations. Both populations showed an association between kdr L1014 mutation and resistance phenotype when tested against deltamethrin and DDT. Significantly different metabolic enzyme activities were found between the susceptible laboratory strain and field-collected mosquitoes from both Ruian and Yongjia. Both field collected An. sinensis populations exhibited significantly higher P450 enzyme activity compared with the laboratory strain, while the field-collected resistant mosquitoes exhibited various GST and COE enzyme activities. These results indicate multiple resistance mechanisms in An. sinensis field populations. Effective implementation of insecticide resistance management strategies is urgently needed. The data collected in this study will be valuable for modeling insecticide resistance spread and vector-control interventions.

Exhaled CO2 is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO2 receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO2 is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO2 receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO2-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO2-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO2-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.

Multiplicity of infection (MOI), also termed complexity of infection (COI), is defined as the number of genetically distinct parasite strains co-infecting a single host, which is an important indicator of malaria epidemiology. PCR-based genotyping often underestimates MOI. Next generation sequencing technologies provide much more accurate and genome-wide characterization of polyclonal infections. However, complete haplotype characterization of multiclonal infections remains a challenge due to PCR artifacts and sequencing errors, and requires efficient computational tools. In this review, the advantages and limitations of current molecular approaches to determine multiplicity of malaria parasite infection are discussed.

Human immunodeficiency virus (HIV) infection continues to pose a major infectious disease threat worldwide. It is characterized by the depletion of CD4+ T cells, persistent immune activation, and increased susceptibility to secondary infections. Advances in the development of antiretroviral drugs and combination antiretroviral therapy have resulted in a remarkable reduction in HIV-associated morbidity and mortality. Antiretroviral therapy (ART) leads to effective suppression of HIV replication with partial recovery of host immune system and has successfully transformed HIV infection from a fatal disease to a chronic condition. Additionally, antiretroviral drugs have shown promise for prevention in HIV pre-exposure prophylaxis and treatment as prevention. However, ART is unable to cure HIV. Other limitations include drug-drug interactions, drug resistance, cytotoxic side effects, cost, and adherence. Alternative treatment options are being investigated to overcome these challenges including discovery of new molecules with increased anti-viral activity and development of easily administrable drug formulations. In light of the difficulties associated with current HIV treatment measures, and in the continuing absence of a cure, the prevention of new infections has also arisen as a prominent goal among efforts to curtail the worldwide HIV pandemic. In this review, the authors summarize currently available anti-HIV drugs and their combinations for treatment, new molecules under clinical development and prevention methods, and discuss drug delivery formats as well as associated challenges and alternative approaches for the future.

BackgroundIn Myanmar, civil unrest and the establishment of internally displaced person (IDP) settlements along the Myanmar-China border have impacted malaria transmission.MethodsMicrosatellite markers were used to examine source-sink dynamics for Plasmodium vivax between IDP settlements and surrounding villages in the border region. Genotypic structure and diversity were compared across the 3 years following the establishment of IDP settlements, to infer demographic history. We investigated whether human migration and landscape heterogeneity contributed to P. vivax transmission.ResultsP. vivax from IDP settlements and local communities consistently exhibited high genetic diversity within populations but low polyclonality within individuals. No apparent genetic structure was observed among populations and years. P. vivax genotypes in China were similar to those in Myanmar, and parasite introduction was unidirectional. Landscape factors, including distance, elevation, and land cover, do not appear to impede parasite gene flow.ConclusionsThe admixture of P. vivax genotypes suggested that parasite gene flow via human movement contributes to the spread of malaria both locally in Myanmar and across the international border. Our genetic findings highlight the presence of large P. vivax gene reservoirs that can sustain transmission. Thus, it is important to reinforce and improve existing control efforts along border areas.

Topical microbicides have the potential to provide effective protection against sexual transmission of HIV. Challenges in developing microbicides include their application in resource-poor settings with high temperatures and a lack of refrigeration, and low user adherence to a rigorous daily regimen. Several protein-based HIV inhibitors show great promise as microbicides, being highly specific and not expected to lead to resistance that would affect the efficacy of current antiretroviral treatments. We show that four potent protein HIV inhibitors, 5P12-RANTES, 5P12-RANTES-L-C37, Grft, and Grft-L-C37 can be formulated into silk fibroin (SF) disks and remain functional for 14 months at 25, 37, and 50 °C. These HIV inhibitor-encapsulated SF disks show excellent inhibition properties in PBMC and in human colorectal and cervical tissue explants, and do not induce inflammatory cytokine secretion. Further, the SF provides a mechanically robust matrix with versatile material formats for this type of application. Finally, a formulation was developed to allow sustained release of functional Grft for 4 weeks at levels sufficient to inhibit HIV transmission. This work establishes the suitability of HIV inhibitor-encapsulated SF disks as topical HIV microbicides that can be further developed to allow easy insertion for extended protection.

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising of these for clinical use is 5P12-RANTES, a variant of the chemokine RANTES/CCL5. The N-terminal amino acid of 5P12-RANTES is glutamine (Gln; called Q0), a residue that is prone to spontaneous cyclization when at the N-terminus of a protein. It is not known how this cyclization affects the potency of the inhibitor or whether cyclization is necessary for the function of the protein, although the N-terminal region of RANTES has been shown to be critical for receptor interactions, with even small changes having a large effect. We have studied the kinetics of cyclization of 5P12-RANTES as well as N-terminal variations of the protein that either produce an identical cyclized terminus (Glu0) or that cannot similarly cyclize (Asn0, Phe0, Ile0, and Leu0). We find that the half life for N-terminal cyclization of Gln is roughly 20 h at pH 7.3 at 37 °C. However, our results show that cyclization is not necessary for the potency of this protein and that several replacement terminal amino acids produce nearly-equally potent HIV inhibitors while remaining CC chemokine receptor 5 (CCR5) antagonists. This work has ramifications for the production of active 5P12-RANTES for use in the clinic, while also opening the possibility of developing other inhibitors by varying the N-terminus of the protein.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1β/CC-Chemokine Ligand 4 (MIP-1β/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 μs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1β. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1β, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1β. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1β interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

BackgroundThe Thai-Myanmar border is a remaining hotspot for malaria transmission. Malaria transmission in this region continues year-round, with a major peak season in July-August, and a minor peak in October-November. Malaria elimination requires better knowledge of the mosquito community structure, dynamics and vectorial status to support effective vector control.MethodsAdult Anopheles mosquitoes were collected using CDC light traps and cow bait in 7 villages along the Thai-Myanmar border in January 2011 - March 2013. Mosquitoes were determined to species by morphological characters. Plasmodium-positivity was determined by circumsporozoite protein ELISA.ResultsThe 2986 Anopheles mosquitoes collected were assigned to 26 species, with Anopheles minimus sensu lato (s.l.) (40.32%), An. maculatus s.l. (21.43%), An. annularis s.l. (14.43%), An. kochi (5.39%), An. tessellatus (5.26%), and An. barbirostris s.l. (3.52%) being the top six most abundant species. Plasmodium-infected mosquitoes were found in 22 positive samples from 2906 pooled samples of abdomens and heads/thoraxes. Four mosquito species were found infected with Plasmodium: An. minimus s.l., An. maculatus s.l., An. annularis s.l. and An. barbirostris s.l. The infectivity rates of these mosquitoes were 0.76, 0.37, 0.72, and 1.74%, respectively. Consistent with a change in malaria epidemiology to the predominance of P. vivax in this area, 20 of the 22 infected mosquito samples were P. vivax-positive. The four potential vector species all displayed apparent seasonality in relative abundance. While An. minimus s.l. was collected through the entire year, its abundance peaked in the season immediately after the wet season. In comparison, An. maculatus s.l. numbers showed a major peak during the wet season. The two potential vector species, An. annularis s.l. and An. barbirostris s.l., both showed peak abundance during the transition from wet to dry season. Moreover, An. minimus s.l. was more abundant in indoor collections, whereas An. annularis s.l. and An. barbirostris s.l. were more abundant in outdoor collections, suggesting their potential role in outdoor malaria transmission.ConclusionsThis survey confirmed the major vector status of An. minimus s.l. and An. maculatus s.l. and identified An. annularis s.l. and An. barbirostris s.l. as additional vectors with potential importance in malaria transmission after the wet season.

BackgroundMany developing countries are experiencing rapid ecological changes such as deforestation and shifting agricultural practices. These environmental changes may have an important consequence on malaria due to their impact on vector survival and reproduction. Despite intensive deforestation and malaria transmission in the China-Myanmar border area, the impact of deforestation on malaria vectors in the border area is unknown.MethodsWe conducted life table studies on Anopheles minimus larvae to determine the pupation rate and development time in microcosms under deforested, banana plantation, and forested environments.ResultsThe pupation rate of An. minimus was 3.8 % in the forested environment. It was significantly increased to 12.5 % in banana plantations and to 52.5 % in the deforested area. Deforestation reduced larval-to-pupal development time by 1.9-3.3 days. Food supplementation to aquatic habitats in forested environments and banana plantations significantly increased larval survival rate to a similar level as in the deforested environment.ConclusionDeforestation enhanced the survival and development of An. minimus larvae, a major malaria vector in the China-Myanmar border area. Experimental determination of the life table parameters on mosquito larvae under a variety of environmental conditions is valuable to model malaria transmission dynamics and impact by climate and environmental changes.

BackgroundThe Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity.MethodsEpidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates.ResultsAnalysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates.ConclusionsThe four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.

BackgroundWith the premise of diminishing parasite genetic diversity following the reduction of malaria incidence, the analysis of polymorphic antigenic markers may provide important information about the impact of malaria control on local parasite populations. Here we evaluated the genetic diversity of Plasmodium vivax apical membrane antigen 1 (Pvama1) gene in a parasite population from the China-Myanmar border and compared it with global P. vivax populations.MethodsWe performed evolutionary analysis to examine the genetic diversity, natural selection, and population differentiation of 73 Pvama1 sequences acquired from the China-Myanmar border as well as 615 publically available Pvama1 sequences from seven global P. vivax populations.ResultsA total of 308 Pvama1 haplotypes were identified among the global P. vivax isolates. The overall nucleotide diversity of Pvama1 gene among the 73 China-Myanmar border parasite isolates was 0.008 with 41 haplotypes being identified (Hd = 0.958). Domain I (DI) harbored the majority (26/33) of the polymorphic sites. The McDonald Kreitman test showed a significant positive selection across the ectodomain and the DI of Pvama1. The fixation index (F ST ) estimation between the China-Myanmar border, Thailand (0.01) and Myanmar (0.10) showed only slight geographical genetic differentiation. Notably, the Sal-I haplotype was not detected in any of the analyzed global isolates, whereas the Belem strain was restricted to the Thai population. The detected mutations are mapped outside the overlapped region of the predicted B-cell epitopes and intrinsically unstructured/disordered regions.ConclusionsThis study revealed high levels of genetic diversity of Pvama1 in the P. vivax parasite population from the China-Myanmar border with DI displaying stronger diversifying selection than other domains. There were low levels of population subdivision among parasite populations from the Greater Mekong Subregion.

BackgroundAnopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis.MethodsAdults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns.ResultsThree non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection.ConclusionOur results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

BackgroundDespite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals.ResultsWithin the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers.ConclusionsDespite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.

Malaria transmission along international borders of the Greater Mekong Subregion is a big challenge for regional malaria elimination. At the Thai-Myanmar border, Plasmodium falciparum cases have dropped dramatically; however, increasing P. vivax prevalence and the emerging reports on hidden malaria burden due to asymptomatic infections demand attention. We conducted cross-sectional surveys to detect asymptomatic malaria infections in a small village located at Thai-Myanmar border and genotyped P. vivax infections in order to understand the level of genetic diversity on such a microgeographic scale. PCR/RFLP and DNA sequencing identified high levels of genetic polymorphisms at both Pvmsp3α and Pvmsp3β loci among P. vivax infections. Combining the PCR/RFLP patterns of Pvmsp3α and Pvmsp3β, a total of 10 genotypes were observed among 17 samples, while concatenated DNA sequences of Pvmsp3α and 3β generated 14 haplotypes with haplotype diversity of 0.97. These markedly diverse parasites on a microgeographic scale suggest the circulation of a considerably large parasite population at the international border.

BackgroundInternally displaced persons (IDP) represent vulnerable populations whose public health conditions merit special attention. In the China-Myanmar border area, human movement and resettlements of IDP can influence malaria transmission. Comparison of disease incidence and vector densities between IDP camps and surrounding local villages allows for better understanding of current epidemiology and to evaluate the effectiveness of interventions in the region.MethodsMalaria and vector surveillance was conducted in three IDP camps and three local villages neighbouring the camps along the China-Myanmar border in Myanmar. Clinical malaria cases were collected from seven hospitals/clinics from April 2011 to December 2014. Malaria vector population dynamics were monitored using CDC light traps. The use of malaria preventive measures and information on aid agencies and their activities was obtained through questionnaire surveys.ResultsMalaria was confirmed in 1832 patients. Of these cases, 85.4 % were Plasmodium vivax and 11.4 % were Plasmodium falciparum malaria. Annual malaria incidence rates were 38.8 and 127.0 cases/1000 person year in IDP camps and local villages, respectively. Older children of 5-14 years had the highest incidence rate in the camps regardless of gender, while male adults had significantly higher incidence rates than females in local villages and females child-bearing age had significantly lower risk to malaria in IDP camps compare to local villages. Seasonal malaria outbreaks were observed both in the IDP camps and in the local villages from May to August 2013. The proportion of P. vivax remained unchanged in local villages but increased by approximately tenfold in IDP camps from 2011 to 2014. Anopheles vector density was tenfold higher in local villages compared to IDP camps (2.0:0.2 females/trap/night). Over 99 % of households in both communities owned bed nets. While long-lasting insecticidal nets accounted for 61 % of nets used in IDPs, nearly all residents of local villages owned regular nets without insecticide-impregnation. There were more active aid agencies in the camps than in local villages.ConclusionMalaria in IDP camps was significantly lower than the surrounding villages through effective control management. The observation of P. vivax outbreaks in the study area highlights the need for increased control efforts. Expansion of malaria intervention strategies in IDP camps to local surrounding villages is critical to malaria control in the border area.

Malaria transmission is heterogeneous in the Greater Mekong Subregion with most of the cases occurring along international borders. Knowledge of transmission hotspots is essential for targeted malaria control and elimination in this region. This study aimed to determine the dynamics of malaria transmission and possible existence of transmission hotspots on a microgeographical scale along the China-Myanmar border. Microscopically confirmed clinical malaria cases were recorded in five border villages through a recently established surveillance system between January 2011 and December 2014. A total of 424 clinical cases with confirmed spatial and temporal information were analyzed, of which 330 (77.8%) were Plasmodium vivax and 88 (20.8%) were Plasmodium falciparum, respectively. The P. vivax and P. falciparum case ratio increased dramatically from 2.2 in 2011 to 4.7 in 2014, demonstrating that P. vivax malaria has become the predominant parasite species. Clinical infections showed a strong bimodal seasonality. There were significant differences in monthly average incidence rates among the study villages with rates in a village in China being 3-8 folds lower than those in nearby villages in Myanmar. Spatial analysis revealed the presence of clinical malaria hotspots in four villages. This information on malaria seasonal dynamics and transmission hotspots should be harnessed for planning targeted control.

To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs.

BackgroundRecent emergence of artemisinin-resistant P. falciparum has posed a serious hindrance to the elimination of malaria in the Greater Mekong Subregion. Parasite clearance time, a measure of change in peripheral parasitaemia in a sequence of samples taken after treatment, can be used to reflect the susceptibility of parasites or the efficiency of antimalarials. The association of genetic polymorphisms and artemisinin resistance has been documented. This study aims to examine clearance time of P. falciparum and P. vivax parasitemia as well as putative gene mutations associated with residual or recurred parasitemia in Myanmar.MethodsA total of 63 P. falciparum and 130 P. vivax samples collected from two internally-displaced populations and one surrounding village were examined for parasitemia changes. At least four samples were taken from each patient, at the first day of diagnosis up to 3 months following the initial treatment. The amount of parasite gene copy number was estimated using quantitative real-time PCR based on a species-specific region of the 18S rRNA gene. For samples that showed residual or recurred parasitemia after treatment, microsatellites were used to identify the 'post-treatment' parasite genotype and compared such with the 'pre-treatment' genotype. Mutations in genes pfcrt, pfmdr1, pfatp6, pfmrp1 and pfK13 that are potentially associated with ACT resistance were examined to identify if mutation is a factor for residual or persistent parasitemia.ResultsOver 30% of the P. falciprium infections showed delayed clearance of parasitemia after 2-3 days of treatment and 9.5% showed recurred parasitemia. Mutations in codon 876 of the pfmrp1 corroborated significance association with slow clearance time. However, no association was observed in the variation in pfmdr1 gene copy number as well as mutations of various codonsinpfatp6, pfcrt, and pfK13 with clearance time. For P. vivax, over 95% of the infections indicated cleared parasitemia at days 2-3 of treatment. Four samples were found to be re-infected with new parasite strains based on microsatellite genotypes after initial treatment.ConclusionThe appearance of P.falciparum infected samples showing delayed clearance or recurred parasitemia after treatment raises concerns on current treatment and ACT drug resistance.

Understanding naturally acquired immunity to infections caused by Plasmodia in different malaria endemicity settings is needed for better vaccine designs and for exploring antibody responses as a proxy marker of malaria transmission intensity. This study investigated the sero-epidemiology of malaria along the international border between China and Myanmar, where malaria elimination action plans are in place. This study recruited 233 P. vivax and 156 P. falciparum infected subjects with acute malaria at the malaria clinics and hospitals. In addition, 93 and 67 healthy individuals from the same endemic region or from non-endemic region, respectively, were used as controls. Acute malaria infections were identified by microscopy. Anti-recombinant PfMSP119 and PvMSP119 antibody levels were measured by ELISA. Antibody responses to respective MSP119 were detected in 50.9% and 78.2% patients with acute P. vivax and P. falciparum infections, respectively. There were cross-reacting antibodies in Plasmodium patients against these two recombinant proteins, though we could not exclude the possibility of submicroscopic mixed-species infections. IgG1, IgG3 and IgG4 were the major subclasses. Interestingly, 43.2% of the healthy endemic population also had antibodies against PfMSP119, whereas only 3.9% of this population had antibodies against PvMSP119. Higher antibody levels were correlated with age and parasite density, but not with season, gender or malaria history. Both total IgG and individual IgG subclasses underwent substantial declines during the convalescent period in three months. This study demonstrated that individuals in a hypoendemic area with coexistence of P. vivax and P. falciparum can mount rapid antibody responses against both PfMSP119 and PvMSP119. The significantly higher proportion of responders to PfMSP119 in the healthy endemic population indicates higher prevalence of P. falciparum in the recent past. Specific antibodies against PvMSP119 could serve as a marker of recent exposure to P. vivax in epidemiological studies.

In the past decade, developing countries have been experiencing rapid land use and land cover changes, including deforestation and cultivation of previously forested land. However, little is known about the impact of deforestation and land-use changes on the life history of malaria vectors and their effects on malaria transmission. This study examined the effects of deforestation and crop cultivation on the adult survivorship of major malaria mosquitoes, Anopheles sinensis and An. minimus in the China-Myanmar border region. We examined three conditions: indoor, forested, and banana plantation. Mean survival time of An. sinensis in banana plantation environment was significantly longer than those in forested environment, and mosquitoes exhibited the longest longevity in the indoor environment. This pattern held for both males and females, and also for An. minimus. To further test the effect of temperature on mosquito survival, we used two study sites with different elevation and ambient temperatures. Significantly higher survivorship of both species was found in sites with lower elevation and higher ambient temperature. Increased vector survival in the deforested area could have an important impact on malaria transmission in Southeast Asia. Understanding how deforestation impacts vector survivorship can help combat malaria transmission.

BackgroundUnderstanding the ecology of malaria vectors such as species composition and population dynamics is essential for developing cost-effective strategies to control mosquito vector populations.MethodsAdult mosquitoes (n = 79,567) were collected in five villages along the China-Myanmar border from April 2012 to September 2014 using the CDC light trap without bait method. Mosquito community structure, Anopheles species composition and diversity were analyzed.ResultsTwenty species of Anopheles mosquitoes were identified, with An. minimus s.l. accounting for 85% of the total collections. Mosquito densities varied from 0.05 females per trap per night (f/t/n) to 3.00 f/t/n, with strong seasonality in all sites and densities peaked from June to August. An. minimus s.l. was predominant (accounting for 54-91% of total captures) in four villages, An. maculatus s.l. was predominant (71%) in the high elevation village of Dao Nong, and An. culicifacies accounted for 15% of total captures in the peri-urban area of Simsa Lawk. All 20 species have been captured in the Mung Seng Yang village, 18 and 15 species in Ja Htu Kawng and Na Bang respectively, and nine species in both Simsa Lawk and Dao Nong. Species richness peaked from April to August. Species diversity, species dominance index, and species evenness fluctuated substantially from time to time with no clear seasonality, and varied greatly amongst villages.ConclusionsMosquitoes were abundant in the China-Myanmar bordering agricultural area with clear seasonality. Species composition and density were strongly affected by natural environments. The targeted intervention strategy should be developed and implemented so as to achieve cost-effectiveness for malaria control and elimination along the border areas.

BackgroundCenters for Disease Control and Prevention miniature light traps (CDC-LT) baited with CO2 are a routine tool for adult mosquito sampling used in entomological surveys, and for monitoring and surveillance of disease vectors. The present study was aimed at evaluating the performance of baited and unbaited CDC-LT for indoor and outdoor trapping of endemic mosquito species in northwestern Thailand.MethodsCDC-LT (n = 112) with and without dry ice baits were set both indoors and outdoors in 88 selected houses for stretches of 5 consecutive nights per month in 7 villages in Tha Song Yang district, Tak province between January 2011 and March 2013. Individual traps were repeatedly placed in the same location for a median of 6 (range 1-10) times. Mosquitoes were identified by morphological characteristics and classified into blood-fed, empty, male/female and gravid. Absolute mosquito numbers were converted to capture rates (i.e., mosquitoes per trap and year). Capture rates were compared using multilevel negative binomial regression to account for multiple trap placements and adjust for regional and seasonal differences.ResultsA total of 6,668 mosquitoes from 9 genera were collected from 576 individual CDC-LT placements. Culex was the predominant captured genus (46%), followed by anopheline mosquitoes (45%). Overall, CO2 baited traps captured significantly more Culex (especially Culex vishnui Theobald) and Anopheles mosquitoes per unit time (adjusted capture rate ratio (aCRR) 1.64 and 1.38, respectively). Armigeres spp. mosquitoes were trapped in outdoor traps with significantly higher frequency (aCRR: 1.50), whereas Aedes albopictus (Skuse) had a tendency to be trapped more frequently indoors (aCRR: 1.89, p = 0.07). Furthermore, capture rate ratios between CO2 baited and non-baited CDC-LT were significantly influenced by seasonality and indoor vs. outdoor trap placement.ConclusionThe present study shows that CDC-LT with CO2 baiting capture significantly more Culex and Anopheles mosquitoes, some of which (e.g., Cx. vishnui, Cx. quinquefasciatus Say, An. minimus s.l. Theobald, An. maculatus s.l. Theobald) represent important disease vectors in Thailand. This study also shows significant differences in the capture efficiency of CDC-LT when placed indoors or outdoors and in different seasons. Our study thus provides important guidelines for more targeted future vector trapping studies on the Thai-Myanmar border, which is an important cross-border malaria transmission region in Thailand.

BackgroundEndemic malaria in Thailand continues to only exist along international borders. This pattern is frequently attributed to importation of malaria from surrounding nations. A microgeographical approach was used to investigate malaria cases in a study village along the Thailand-Myanmar border.MethodsThree mass blood surveys were conducted during the study period (July and December 2011, and May 2012) and were matched to a cohort-based demographic surveillance system. Blood slides and filter papers were taken from each participant. Slides were cross-verified by an expert microscopist and filter papers were analysed using nested PCR. Cases were then mapped to households and analysed using spatial statistics. A risk factor analysis was done using mixed effects logistic regression.ResultsIn total, 55 Plasmodium vivax and 20 Plasmodium falciparum cases (out of 547 participants) were detected through PCR, compared to six and two (respectively) cases detected by field microscopy. The single largest risk factor for infection was citizenship. Many study participants were ethnic Karen people with no citizenship in either Thailand or Myanmar. This subpopulation had over eight times the odds of malaria infection when compared to Thai citizens. Cases also appeared to cluster near a major drainage system and year-round water source within the study village.ConclusionThis research indicates that many cases of malaria remain undiagnosed in the region. The spatial and demographic clustering of cases in a sub-group of the population indicates either transmission within the Thai village or shared exposure to malaria vectors outside of the village. While it is possible that malaria is imported to Thailand from Myanmar, the existence of undetected infections, coupled with an ecological setting that is conducive to malaria transmission, means that indigenous transmission could also occur on the Thai side of the border. Improved, timely, and active case detection is warranted.

BackgroundMalaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels.MethodsThree-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals.ResultsOf 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.ConclusionThese findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

Antimalarial drugs are key tools for the control and elimination of malaria. Recent decreases in the global malaria burden are likely due, in part, to the deployment of artemisinin-based combination therapies. Therefore, the emergence and potential spread of artemisinin-resistant parasites in southeast Asia and changes in sensitivities to artemisinin partner drugs have raised concerns. In recognition of this urgent threat, the International Centers of Excellence for Malaria Research (ICEMRs) are closely monitoring antimalarial drug efficacy and studying the mechanisms underlying drug resistance. At multiple sentinel sites of the global ICEMR network, research activities include clinical studies to track the efficacies of antimalarial drugs, ex vivo/in vitro assays to measure drug susceptibilities of parasite isolates, and characterization of resistance-mediating parasite polymorphisms. Taken together, these efforts offer an increasingly comprehensive assessment of the efficacies of antimalarial therapies, and enable us to predict the emergence of drug resistance and to guide local antimalarial drug policies. Here we briefly review worldwide antimalarial drug resistance concerns, summarize research activities of the ICEMRs related to drug resistance, and assess the global impacts of the ICEMR programs.

Understanding the epidemiological features and metrics of malaria in endemic populations is a key component to monitoring and quantifying the impact of current and past control efforts to inform future ones. The International Centers of Excellence for Malaria Research (ICEMR) has the opportunity to evaluate the impact of malaria control interventions across endemic regions that differ in the dominant Plasmodium species, mosquito vector species, resistance to antimalarial drugs and human genetic variants thought to confer protection from infection and clinical manifestations of plasmodia infection. ICEMR programs are conducting field studies at multiple sites with the aim of generating standardized surveillance data to improve the understanding of malaria transmission and to monitor and evaluate the impact of interventions to inform malaria control and elimination programs. In addition, these epidemiological studies provide a vast source of biological samples linked to clinical and environmental "meta-data" to support translational studies of interactions between the parasite, human host, and mosquito vector. Importantly, epidemiological studies at the ICEMR field sites are integrated with entomological studies, including the measurement of the entomological inoculation rate, human biting index, and insecticide resistance, as well as studies of parasite genetic diversity and antimalarial drug resistance.

In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar-China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border.

Chloroquine-primaquine (CQ-PQ) continues to be the frontline therapy for radical cure of Plasmodium vivax malaria. Emergence of CQ-resistant (CQR) P. vivax parasites requires a shift to artemisinin combination therapies (ACTs), which imposes a significant financial, logistical, and safety burden. Monitoring the therapeutic efficacy of CQ is thus important. Here, we evaluated the therapeutic efficacy of CQ-PQ for P. vivax malaria in northeast Myanmar. We recruited 587 patients with P. vivax monoinfection attending local malaria clinics during 2012 to 2013. These patients received three daily doses of CQ at a total dose of 24 mg of base/kg of body weight and an 8-day PQ treatment (0.375 mg/kg/day) commencing at the same time as the first CQ dose. Of the 401 patients who finished the 28-day follow-up, the cumulative incidence of recurrent parasitemia was 5.20% (95% confidence interval [CI], 3.04% to 7.36%). Among 361 (61%) patients finishing a 42-day follow-up, the cumulative incidence of recurrent blood-stage infection reached 7.98% (95% CI, 5.20% to 10.76%). The cumulative risk of gametocyte carriage at days 28 and 42 was 2.21% (95% CI, 0.78% to 3.64%) and 3.93% (95% CI, 1.94% to 5.92%), respectively. Interestingly, for all 15 patients with recurrent gametocytemia, this was associated with concurrent asexual stages. Genotyping of recurrent parasites at the merozoite surface protein 3α gene locus from 12 patients with recurrent parasitemia within 28 days revealed that 10 of these were the same genotype as at day 0, suggesting recrudescence or relapse. Similar studies in 70 patients in the same area in 2007 showed no recurrent parasitemias within 28 days. The sensitivity to chloroquine of P. vivax in northeastern Myanmar may be deteriorating.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.