Outdoor residual spraying for malaria vector-control in Kayin (Karen) state, Myanmar: A cluster randomized controlled trial.

Outdoor and early biting by mosquitoes challenge the efficacy of bed nets and indoor residual spraying against malaria in the Greater Mekong Subregion. The objective of this study was to assess the efficacy of outdoor residual spraying (ORS) for malaria vector-control in this region. A cluster randomized controlled trial was conducted between July 2018 and April 2019 in twelve villages in Karen (Kayin) state, Myanmar. Villages were randomly assigned to receive either a single round of ORS with a capsule suspension of lambda-cyhalothrin for two days in October or no intervention (six villages per group). The primary endpoint was the biting rate of malaria mosquitoes assessed with human-landing catch and cow-baited trap collection methods, and was analyzed with a Bayesian multi-level model. In the intervention villages, the proportion of households located within the sprayed area ranged between 42 and 100% and the application rate ranged between 63 and 559 g of active ingredient per hectare. At baseline, the median of Anopheles biting rate estimates in the twelve villages was 2 bites per person per night (inter-quartile range [IQR] 0-5, range 0-48) indoors, 6 bites per person per night (IQR 2-16, range 0-342) outdoors and 206 bites per cow per night (IQR 83-380, range 19-1149) in the cow-baited trap. In intention-to-treat analysis, it was estimated that ORS reduced biting rate by 72% (95% confidence interval [CI] 63-79) from Month 0 to Month 3 and by 79% (95% CI 62-88) from Month 4 to Month 6, considering control villages as the reference. In conclusion, ORS rapidly reduces the biting rates of malaria mosquitoes in a Southeast Asian setting where the vectors bite mostly outdoors and at a time when people are not protected by mosquito bed nets.

Assessment of Plasmodium antigens and CRP in dried blood spots with multiplex malaria array.

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12639-020-01325-2) contains supplementary material, which is available to authorized users.

A randomized controlled trial of dihydroartemisinin-piperaquine, artesunate-mefloquine and extended artemether-lumefantrine treatments for malaria in pregnancy on the Thailand-Myanmar border.

BACKGROUND: Artemisinin and artemisinin-based combination therapy (ACT) partner drug resistance in Plasmodium falciparum have spread across the Greater Mekong Subregion compromising antimalarial treatment. The current 3-day artemether-lumefantrine regimen has been associated with high treatment failure rates in pregnant women. Although ACTs are recommended for treating Plasmodium vivax malaria, no clinical trials in pregnancy have been reported. METHODS: Pregnant women with uncomplicated malaria on the Thailand-Myanmar border participated in an open-label randomized controlled trial comparing dihydroartemisinin-piperaquine (DP), artesunate-mefloquine (ASMQ) and a 4-day artemether-lumefantrine regimen (AL+). The primary endpoint for P. falciparum infections was the PCR-corrected cure rate and for P. vivax infections was recurrent parasitaemia, before delivery or day 63, whichever was longer, assessed by Kaplan-Meier estimate. RESULTS: Between February 2010 and August 2016, 511 pregnant women with malaria (353 P. vivax, 142 P. falciparum, 15 co-infections, 1 Plasmodium malariae) were randomized to either DP (n=170), ASMQ (n=169) or AL+ (n=172) treatments. Successful malaria elimination efforts in the region resulted in premature termination of the trial. The majority of women had recurrent malaria (mainly P. vivax relapses, which are not prevented by these treatments). Recurrence-free proportions (95% confidence interval [95% CI]) for vivax malaria were 20.6% (5.1-43.4) for DP (n=125), 46.0% (30.9-60.0) for ASMQ (n=117) and 28.7% (10.0-50.8) for AL+ (n=126). DP and ASMQ provided longer recurrence-free intervals. PCR-corrected cure rates (95% CI) for falciparum malaria were 93.7% (81.6-97.9) for DP (n=49), 79.6% (66.1-88.1) for AMSQ (n=55) and 87.5% (74.3-94.2) for AL+ (n=50). Overall 65% (85/130) of P. falciparum infections had Pfkelch13 propeller mutations which increased over time and recrudescence occurred almost exclusively in them; risk ratio 9.42 (95% CI 1.30-68.29). Among the women with falciparum malaria, 24.0% (95% CI 16.8-33.6) had P. vivax parasitaemia within 4 months. Nausea, vomiting, dizziness and sleep disturbance were more frequent with ASMQ. Miscarriage, small-for-gestational-age and preterm birth did not differ significantly among the treatment groups, including first trimester exposures (n=46). CONCLUSIONS: DP was well tolerated and safe, and was the only drug providing satisfactory efficacy for P. falciparum-infected pregnant woman in this area of widespread artemisinin resistance. Vivax malaria recurrences are very common and warrant chloroquine prophylaxis after antimalarial treatment in this area. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01054248 , registered on 22 January 2010.

Multiplex Human Malaria Array: Quantifying Antigens for Malaria Rapid Diagnostics.

Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT), which quantifies the antigens commonly used in RDTs-Plasmodium falciparum-specific histidine-rich protein 2 (HRP2), P. falciparum-specific lactate dehydrogenase (Pf LDH), Plasmodium vivax -specific LDH (Pv LDH), and Pan malaria lactate dehydrogenase (Pan LDH), and human C-reactive protein (CRP), a biomarker of severity in malaria. At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.

Simultaneous Quantification of Plasmodium Antigens and Host Factor C-Reactive Protein in Asymptomatic Individuals with Confirmed Malaria by Use of a Novel Multiplex Immunoassay.

Malaria rapid diagnostic tests (RDTs) primarily detect Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, P. vivax LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria. The assay also measures C-reactive protein (CRP) levels as an indicator of inflammation. Validation was conducted with clinical specimens from 397 asymptomatic donors from Myanmar and Uganda, confirmed by PCR for infection, and from participants in induced blood-stage malaria challenge studies. The assay lower limits of detection for HRP2, pan LDH, P. vivax LDH, and CRP were 0.2 pg/ml, 9.3 pg/ml, 1.5 pg/ml, and 26.6 ng/ml, respectively. At thresholds for HRP2, pan LDH, and P. vivax LDH of 2.3 pg/ml, 47.8 pg/ml, and 75.1 pg/ml, respectively, and a specificity ≥98.5%, the sensitivities for ultrasensitive PCR-confirmed infections were 93.4%, 84.9%, and 48.9%, respectively. Plasmodium LDH (pLDH) concentration, in contrast to that of HRP2, correlated closely with parasite density. CRP levels were moderately higher in P. falciparum infections with confirmed antigenemia versus those in clinical specimens with no antigen. The 4-plex array is a sensitive tool for quantifying diagnostic antigens in malaria infections and supporting the evaluation of new ultrasensitive RDTs.

Contribution of Asymptomatic Plasmodium Infections to the Transmission of Malaria in Kayin State, Myanmar.

BACKGROUND: The objective of mass antimalarial drug administration (MDA) is to eliminate malaria rapidly by eliminating the asymptomatic malaria parasite reservoirs and interrupting transmission. In the Greater Mekong Subregion, where artemisinin-resistant Plasmodium falciparum is now widespread, MDA has been proposed as an elimination accelerator, but the contribution of asymptomatic infections to malaria transmission has been questioned. The impact of MDA on entomological indices has not been characterized previously. METHODS: MDA was conducted in 4 villages in Kayin State (Myanmar). Malaria mosquito vectors were captured 3 months before, during, and 3 months after MDA, and their Plasmodium infections were detected by polymerase chain reaction (PCR) analysis. The relationship between the entomological inoculation rate, the malaria prevalence in humans determined by ultrasensitive PCR, and MDA was characterized by generalized estimating equation regression. RESULTS: Asymptomatic P. falciparum and Plasmodium vivax infections were cleared by MDA. The P. vivax entomological inoculation rate was reduced by 12.5-fold (95% confidence interval [CI], 1.6-100-fold), but the reservoir of asymptomatic P. vivax infections was reconstituted within 3 months, presumably because of relapses. This was coincident with a 5.3-fold (95% CI, 4.8-6.0-fold) increase in the vector infection rate. CONCLUSION: Asymptomatic infections are a major source of malaria transmission in Southeast Asia.

Operational Performance of a Plasmodium falciparum Ultrasensitive Rapid Diagnostic Test for Detection of Asymptomatic Infections in Eastern Myanmar.

In the Greater Mekong Subregion in Southeast Asia, malaria elimination strategies need to target all Plasmodium falciparum parasites, including those carried asymptomatically. More than 70% of asymptomatic carriers are not detected by current rapid diagnostic tests (RDTs) or microscopy. An HRP2-based ultrasensitive RDT (uRDT) developed to improve the detection of low-density infections was evaluated during prevalence surveys within a malaria elimination program in a low-transmission area of eastern Myanmar. Surveys were conducted to identify high-prevalence villages. Two-milliliter venous blood samples were collected from asymptomatic adult volunteers and transported to the laboratory. Plasmodium parasites were detected by RDT, uRDT, microscopy, ultrasensitive qPCR (uPCR), and multiplex enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, and predictive positive and negative values of RDT and uRDT were calculated compared to uPCR and ELISA. Parasite and antigen concentrations detected by each test were defined using uPCR and ELISA, respectively. A total of 1,509 samples, including 208 P. falciparum-positive samples were analyzed with all tests. The sensitivity of the uRDT was twofold higher than that of RDT, 51.4% versus 25.2%, with minor specificity loss, 99.5% versus 99.9%, against the combined reference (uPCR plus ELISA). The geometric mean parasitemia detected by uRDT in P. falciparum monospecific infections was 3,019 parasites per ml (95% confidence interval [95% CI], 1,790 to 5,094; n = 79) compared to 11,352 parasites per ml (95% CI, 5,643 to 22,837; n = 38) by RDT. The sensitivities of uRDT and RDT dropped to 34.6% and 15.1%, respectively, for the matched tests performed in the field. The uRDT performed consistently better than RDT and microscopy at low parasitemias. It shows promising characteristics for the identification of high-prevalence communities and warrants further evaluation in mass screening and treatment interventions.

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections.

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.