Role of underappreciated vectors in malaria transmission in an endemic region of Bangladesh-India border

Background Despite the efforts of the National Malaria Control Programme, malaria remains as an important public health problem in Bangladesh, particularly in the south-eastern region bordering India. Successful malaria control strategies rely on a detailed understanding of the underlying causes of malaria transmission. Here, an entomological survey was conducted in a malaria endemic area of Bangladesh bordering India to investigate the Anopheles mosquito community and assess their Plasmodium infection status. Methods Monthly entomological collections were undertaken from October 2010 to September 2011 in five villages in the Matiranga sub-district, Khagrachari district in Bangladesh, bordering the Indian State of Tripura. CDC miniature light traps were placed inside houses to collect adult Anopheles mosquitoes. Following morphological and molecular identification of the female Anopheles mosquitoes collected, they were screened for circumsporozoite proteins (CSP) of Plasmodium falciparum (Pf), Plasmodium vivax-210 (Pv-210) and Plasmodium vivax-247 (Pv-247), by ELISA to determine natural infection rates. Variation in Anopheles species composition, relative abundance and Plasmodium infection rates were analysed between sampled villages. Results A total of 2,027 female Anopheles were collected, belonging to 20 species. Anopheles nivipes was the most abundant species in our test villages during the peak malaria transmission season, and was observed sympatrically with An. philippinensis in the studied area. However, in the dry off-peak season, An. jeyporiensis was the most abundant species. Shannon’s diversity index was highest in October (2.12) and evenness was highest in May (0.91). The CSP ELISA positive rate overall was 0.44%. Anopheles karwari (n = 2), An. barbirostris s.l. (n = 1) and An. vagus (n = 1) were recorded positive for Pf. Anopheles kochi (n = 1) was positive for Pv-210 while An. umbrosus (n = 1), An. nivipes (n = 1) and An. kochi (n = 1) were positive for Pv-247. A mixed infection of Pf and Pv-247 was detected in An. barbirostris s.l.. Conclusion High diversity of Anopheles species was observed in areas close to the international border where species that were underestimated for malaria transmission significantly outnumbered principal vector species and these may play a significantly heightened role in malaria transmission

Clinical Validation of a Commercial LAMP Test for Ruling out Malaria in Returning Travelers: A Prospective Diagnostic Trial.

The mainstay of malaria diagnosis relies on rapid diagnostic tests (RDTs) and microscopy, both of which lack analytical sensitivity. This leads to repeat testing to rule out malaria. A prospective diagnostic trial of the Meridian illumigene Malaria assay (loop-mediated isothermal amplification [LAMP]) was conducted comparing it with reference microscopy and RDTs (BinaxNOW Malaria) in returning travelers between June 2017 and January 2018. Returning travelers with signs and symptoms of malaria were enrolled in the study. RDTs, microscopy, and LAMP assays were performed simultaneously. A total of 298 patients (50.7% male; mean age, 32.5 years) were enrolled, most visiting friends and relatives (43.3%), presenting with fever (88.9%), not taking prophylaxis (82.9%), and treated as outpatients (84.1%). In the prospective arm (n = 348), LAMP had a sensitivity of 98.1% (95% confidence interval [CI], 90.0%-100%) and a specificity of 97.6% (95% CI, 95.2%-99.1%) vs microscopy. After discrepant resolution with real-time polymerase chain reaction, LAMP had a sensitivity of 100% (95% CI, 93.7%-100%) and a specificity of 100% (95% CI, 98.7%-100%) vs microscopy. After discrepant resolution, RDTs had a sensitivity of 83.3% (95% CI, 58.6%-96.4%) and a specificity of 96.2% (95% CI, 93.2%-98.1%) vs microscopy. When including retrospective specimens (n = 377), LAMP had a sensitivity of 98.8% (95% CI, 93.2%-100%) and a specificity of 97.6% (95% CI, 95.2%-99.1%) vs microscopy, and after discrepant resolution of this set, LAMP had a sensitivity of 100% (95% CI, 95.8%-100%) and a specificity of 100% (95% CI, 98.7%-100%). A cost-benefit analysis of reagents and labor suggests savings of up to USD$13 per specimen using a novel algorithm with LAMP screening

Real-time PCR assay and rapid diagnostic tests for the diagnosis of clinically suspected malaria patients in Bangladesh

<p>Abstract</p> <p>Background</p> <p>More than 95% of total malaria cases in Bangladesh are reported from the 13 high endemic districts. <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>are the two most abundant malaria parasites in the country. To improve the detection and management of malaria patients, the National Malaria Control Programme (NMCP) has been using rapid diagnostic test (RDT) in the endemic areas. A study was conducted to establish a SYBR Green-based modified real-time PCR assay as a gold standard to evaluate the performance of four commercially-available malaria RDTs, along with the classical gold standard- microscopy.</p> <p>Methods</p> <p>Blood samples were collected from 338 febrile patients referred for the diagnosis of malaria by the attending physician at Matiranga</p> <p>Upazila Health Complex (UHC) from May 2009 to August 2010. Paracheck RDT and microscopy were performed at the UHC. The blood samples were preserved in EDTA tubes. A SYBR Green-based real-time PCR assay was performed and evaluated. The performances of the remaining three RDTs (Falcivax, Onsite Pf and Onsite Pf/Pv) were also evaluated against microscopy and real-time PCR using the stored blood samples.</p> <p>Result</p> <p>In total, 338 febrile patients were enrolled in the study. Malaria parasites were detected in 189 (55.9%) and 188 (55.6%) patients by microscopy and real-time PCR respectively. Among the RDTs, the highest sensitivity for the detection of <it>P. falciparum </it>(including mixed infection) was obtained by Paracheck [98.8%, 95% confidence interval (CI) 95.8-99.9] and Falcivax (97.6%, 95% CI 94.1-99.4) compared to microscopy and real-time PCR respectively. Paracheck and Onsite Pf/Pv gave the highest specificity (98.8%, 95% CI 95.7-99.9) compared to microscopy and Onsite Pf/Pv (98.8, 95% CI 95.8-99.9) compared to real-time PCR respectively for the detection of <it>P. falciparum</it>. On the other hand Falcivax and Onsite Pf/Pv had equal sensitivity (90.5%, 95% CI 69.6-98.8) and almost 100% specificity compared to microscopy for the detection of <it>P. vivax</it>. However, compared to real-time PCR assay RDTs and microscopy gave low sensitivity (76.9%, 95% CI 56.4-91) in detecting of <it>P. vivax </it>although a very high specificity was obtained (99- 100%).</p> <p>Conclusion</p> <p>The results of this study suggest that the SYBR Green-based real-time PCR assay could be used as an alternative gold standard method in a reference setting. Commercially-available RDTs used in the study are quite sensitive and specific in detecting <it>P. falciparum</it>, although their sensitivity in detecting <it>P. vivax </it>was not satisfactory compared to the real-time PCR assay.</p

Development and application of ultra-sensitive tools for the detection of malaria

The goal to eliminate malaria has been challenged by the lack of accurate diagnostic tools to identify symptomatic, asymptomatic, and drug-resistant malaria carriers. In this dissertation, we have shown the potential of the Loop-mediated Isothermal Amplification (LAMP)-based diagnostic approaches to be a powerful tool available for malaria elimination. We have validated the combination of the Non-instrumented Nucleic Acid (NINA) platform heater (PATH, Seattle) with a commercial LAMP kit (LoopAmp malaria Pan/Pf detection kit), with a view to deploying it in extremely resource-limited settings in the future. An ultrasensitive (US)-LAMP assay was also developed and validated to identify asymptomatic malaria reservoirs. Moreover, a novel strategy for detecting single nucleotide polymorphisms (SNPs) by the LAMP method was designed and deployed for spotting artemisinin resistance in P. falciparum. We conclude that the NINA-LAMP assay can be a convenient test for detecting symptomatic malaria cases with a sensitivity of 100% and specificity of 98.6% compared to the gold standard nested PCR. Additionally, the US-LAMP assay was able to achieve a limit of detection (LOD) between 25 to 100 parasites/mL from dried blood spots. We have also found that the overall prevalence of asymptomatic malaria was 22.1% in the Gambella region of Ethiopia, detected by the US-LAMP assay. The sensitivity and the specificity of the US-LAMP assay were 92.6% and 97.1%, respectively compared to an ultrasensitive quantitative reverse transcriptase PCR. Additionally, the SNP-LAMP assay was 100% sensitive and 97.3% specific to identify the C580Y mutation in the kelch 13 propeller gene, which is known as the major genetic determinant of artemisinin resistance in Southeast Asia. Furthermore, we conclude that artemisinin resistance-linked kelch 13 propeller mutations are absent in the Bangladeshi P. falciparum isolates. However, two cases of the A578S SNP in the kelch 13 propeller gene were found in those P. falciparum isolates, although this SNP was not associated with artemisinin resistance. In conclusion, as the LAMP-based diagnostic approaches are simple, low-cost, and accurate compared to currently available nucleic acid tests, they can be used at different aspects to diagnose malaria and expedite elimination

Performance of loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria among malaria suspected pregnant women in Northwest Ethiopia

Abstract Background Malaria is a major public health problem and an important cause of maternal and infant morbidity in sub-Saharan Africa, including Ethiopia. Early and accurate diagnosis of malaria with effective treatment is the best strategy for prevention and control of complications during pregnancy and infant morbidity and mortality. However, laboratory diagnosis has relied on the identification of malaria parasites and parasite antigens in peripheral blood using Giemsa-stained microscopy or rapid diagnostic tests (RDTs) which lack analytical and clinical sensitivity. The aim of this study was to evaluate the performance of loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria among malaria suspected pregnant women in Northwest Ethiopia. Methods A cross sectional study was conducted from January to April 2016. Pregnant women (n = 87) suspected of having malaria at six health centres were enrolled. A venous blood sample was collected from each study subject, and analysed for Plasmodium parasites by microscopy, RDT, and LAMP. Diagnostic accuracy outcome measures (sensitivity, specificity, predictive values, and Kappa scores) of microscopy, RDT and LAMP were compared to nested polymerase chain reaction (nPCR) as the gold standard. Specimen processing and reporting times were documented. Results Using nPCR as the gold standard technique, the sensitivity of microscopy and RDT was 90 and 70%, and the specificity was 98.7 and 97.4%, respectively. LAMP assay was 100% sensitive and 93.5% specific compared to nPCR. Conclusions This study showed higher sensitivity of LAMP compared to microscopy and RDT for the detection of malaria in pregnancy. Increased sensitivity and ease of use with LAMP in point-of-care testing for malaria in pregnancy was noted. LAMP warrants further evaluation in intermittent screening and treatment programmes in pregnancy

A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

International audienceBackground: Artemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing.Methods: We developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy.Results: The SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9-98.3) and 91.9% specific (95% CI, 82.6-96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9-100) and 97.3% specific (95% CI, 89.7-99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries.Conclusions: Single-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient

Ultrasensitive loop mediated isothermal amplification (US-LAMP) to detect malaria for elimination

Abstract Background Malaria elimination requires diagnostic methods able to detect parasite levels well below what is currently possible with microscopy and rapid diagnostic tests. This is particularly true in surveillance of malaria at the population level that includes so-called “asymptomatic” individuals. Methods The development of the first ultrasensitive loop mediated amplification method capable of detecting malaria from both whole blood and dried blood spots (DBS) is described. The 18S rRNA and corresponding genes that remain stable on DBS for up to 5 months are targeted. Results In the case of Plasmodium falciparum, lower limits of detection of 25 parasite/mL and 50–100 parasite/mL from whole blood and DBS were obtained, respectively. A sensitivity of 97.0% (95% CI 82.5–99.8) and specificity of 99.1% (95% CI 97.6–99.7) was obtained for the detection of all species in asymptomatic individuals from Africa and Asia (n = 494). Conclusion This tool is ideally suited for low middle-income countries where malaria is endemic and ultrasensitive surveillance of malaria is highly desirable for elimination

Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013)

Abstract Background Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. Methods Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox’s Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). Results Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. Conclusion The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation