Active case detection of malaria in pregnancy using loop-mediated amplification (LAMP): a pilot outcomes study in South West Ethiopia.

BACKGROUND: 125 million women are pregnant each year in malaria endemic areas and are, therefore, at risk of Malaria in Pregnancy (MiP). MiP is the direct consequence of Plasmodium infection during pregnancy. The sequestration of Plasmodium falciparum parasites in the placenta adversely affects fetal development and impacts newborn birth weight. Importantly, women presenting with MiP commonly develop anaemia. In Ethiopia, the Ministry of Health recommends screening symptomatic women only at antenatal care visits with no formal intermittent preventive therapy. Since MiP can display low-level parasitaemia, current tests which include microscopy and RDT are challenged to detect these cases. Loop mediated isothermal Amplification (LAMP) technology is a highly sensitive technique for DNA detection and is field compatible. This study aims to evaluate the impact of active malaria case detection during pregnancy using LAMP technology in terms of birth outcomes. METHODS: A longitudinal study was conducted in two health centres of the Kafa zone, South West Ethiopia. Both symptomatic and asymptomatic pregnant women were enrolled in the first or second trimester and allocated to either Standard of Care (SOC-microscopy and RDT) or LAMP (LAMP, microscopy and RDT). Women completed at least three visits prior to delivery, and the patient was referred for treatment if Plasmodium infection was detected by any of the testing methods. The primary outcome was to measure absolute birth weight, proportion of low birth weight, and maternal/neonatal haemoglobin in each arm. Secondary outcomes were to assess the performance of microscopy and RDT versus LAMP conducted in the field. RESULTS: One hundred and ninety-nine women were included and assigned to either LAMP or SOC. Six were lost to follow up. In this cohort, 66.8% of women did not display any clinical symptoms and 70.9% were multi-parous. A reduced proportion of low birth weight newborns was observed in the LAMP group (0%) compared to standard of care (14%) (p <0.001). Improved neonatal haemoglobin was observed in the LAMP (13.1 g/dL) versus the SOC (12.8 g/dL) (p = 0.024) arm. RDT and microscopy had an analytical sensitivity of 66.7% and 55.6% compared to LAMP as a reference standard. CONCLUSIONS: These results support the use of highly sensitive tools for rapid on-site active case detection of MiP which may improve birth outcomes in the absence of IPT. However, further large-scale studies are required to confirm this finding.

Prevalence and Epidemiological Characteristics of Asymptomatic Malaria Based on Ultrasensitive Diagnostics: A Cross-sectional Study.

BACKGROUND: As the global public-health objectives for malaria evolve from malaria control towards malaria elimination, there is increasing interest in the significance of asymptomatic infections and the optimal diagnostic test to identify them. METHOD: We conducted a cross-sectional study of asymptomatic individuals (N = 562) to determine the epidemiological characteristics associated with asymptomatic malaria. Participants were tested by rapid diagnostic tests (CareStart, Standard Diagnostics [SD] Bioline, and Alere ultrasensitive RDT [uRDT]), loop-mediated isothermal amplification (LAMP), and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine malaria positivity. Hemoglobin values were recorded, and anemia was defined as a binary variable, according to World Health Organization guidelines. RESULTS: Compared to reference qRT-PCR, LAMP had the highest sensitivity (92.6%, 95% confidence interval [CI] 86.4-96.5), followed by uRDT Alere Malaria (33.9%, 95% CI 25.5-43.1), CareStart Malaria (14.1%, 95% CI 8.4-21.5), microscopy (5.0%, 95% CI 1.8-10.5), and SD Bioline (5.0%, 95% CI 1.8-10.5). For Plasmodium falciparum specimens only, the sensitivity for uRDT Alere Malaria was 50.0% (95% CI 38.8-61.3) and SD Bioline was 7.3% (95% CI 2.7-15.3). Based on multivariate regression analysis with qRT-PCR as the gold standard, for every 3.2% increase in the prevalence of asymptomatic malaria, hemoglobin decreased by 1 gram per deciliter (prevalence ratio 0.968, 95% CI 0.940-0.997; P = .032). Deletions (4.8%) in hrp2 were noted. CONCLUSIONS: While uRDT Alere Malaria has superior sensitivity to rapid diagnostic tests and microscopy in detecting asymptomatic malaria, LAMP is superior still. Ultrasensitive diagnostics provide the accurate prevalence estimates of asymptomatic malaria required for elimination.

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

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.

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

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.

In vitro and in vivo anti-malarial activity of novel harmine-analog heat shock protein 90 inhibitors: a possible partner for artemisinin.

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum strains poses a serious challenge to the control of malaria. This necessitates the development of new anti-malarial drugs. Previous studies have shown that the natural beta-carboline alkaloid harmine is a promising anti-malarial agent targeting the P. falciparum heat-shock protein 90 (PfHsp90). The aim of this study was to test the anti-malarial activity of harmine analogues. METHODS: Forty-two harmine analogues were synthesized and the binding of these analogues to P. falciparum heat shock protein 90 was investigated. The in vitro anti-malarial activity of two of the analogues, 17A and 21A, was evaluated using a 72-h growth inhibition assay. The in vivo anti-malarial activity was tested in Plasmodium berghei infection of BALB/c mice. The potential of 21A for a combination treatment with artemisinin was evaluated using in vivo combination study with dihydro-artemisinin in BALB/c mice. Cytotoxicity of the harmine analogues was tested in vitro using HepG2 and HeLa cell lines. RESULTS: 17A and 21A bound to PfHsp90 with average IC50 values of 12.2 ± 2.3 and 23.1 ± 8.8 µM, respectively. They also inhibited the P. falciparum W2 strain with average IC50 values of 4.2 ± 1.3 and 5.7 ± 1.7 µM, respectively. In vivo, three daily injections of P. berghei-infected BALB/c mice with 100 mg/kg of either 17A or 21A showed significant reduction in parasitaemia with a 51.5 and 56.1% reduction, respectively. Mice treated with 17A and 21A showed a median survival time of 11 and 14 days, respectively, while the vehicle control mice survived a median of only 8.5 days. A dose-ranging experiment with 21A showed that the compound has a dose-dependent anti-malarial effect. Furthermore, treatment of infected mice with a combination of 21A and dihydroartemisinin (DHA) showed a dramatic reduction in parasitaemia compared to treatment with DHA alone. CONCLUSION: A novel and non-toxic harmine analogue has been synthesized which binds to PfHsp90 protein, inhibits P. falciparum in vitro at micromolar concentration, reduces parasitaemia and prolongs survival of P. berghei-infected mice with an additive anti-malarial effect when combined with DHA.

Genetic diversity of Plasmodium vivax in clinical isolates from Bangladesh.

BACKGROUND: Plasmodium vivax is the second most prevalent human malaria parasite in Bangladesh; however, there are no data of its genetic diversity. Several molecular markers are available where Pvcsp, Pvmsp 1 and Pvmsp 3α are most commonly used for P. vivax genotyping studies. The aim of the study was to investigate the population structure of P. vivax in Bangladesh. METHODS: A total of 102 P. vivax-positive blood samples were collected from different malaria-endemic areas in Bangladesh and subsequently analysed for those three genotyping markers. Nested PCR was performed for diagnosis and genotyping analysis followed by PCR-RFLP to detect genetic diversity using Pvcsp, Pvmsp 1 and Pvmsp 3α markers. RESULTS: Analysis of Pvcsp showed that the VK210 repeat type was highly prevalent (64.7%, 66/102) compared to VK247 (35.3%, 36/102), although the prevalence of VK247 was higher than other Southeast Asian countries. Analysis of these three genes revealed a diverse, circulating population of P. vivax where a total of ten, 56 and 35 distinct genotypes were detected for Pvcsp, Pvmsp 1 and Pvmsp 3α, respectively. CONCLUSION: This genotyping observation of P. vivax is the first report from Bangladesh and will provide valuable information for establishing the genotyping methods and circulating genetic variants of these three markers available in Bangladesh.

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

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.

Performance of a HRP-2/pLDH based rapid diagnostic test at the Bangladesh-India-Myanmar border areas for diagnosis of clinical malaria.

BACKGROUND: The rapid diagnostic test (RDT) has been adopted in contemporary malaria control and management programmes around the world as it represents a fast and apt alternative for malaria diagnosis in a resource-limited setting. This study assessed the performance of a HRP-2/pLDH based RDT (Parascreen® Pan/Pf) in a laboratory setting utilizing clinical samples obtained from the field. METHODS: Whole blood samples were obtained from febrile patients referred for malaria diagnosis by clinicians from two different Upazila Health Complexes (UHCs) located near the Bangladesh-India and Bangladesh-Myanmar border where malaria is endemic. RDT was performed on archived samples and sensitivity and specificity evaluated with expert microscopy (EM) and quantitative PCR (qPCR). RESULTS: A total of 327 clinical samples were made available for the study, of which 153 were Plasmodium falciparum-positive and 54 were Plasmodium vivax-positive. In comparison with EM, for P. falciparum malaria, the RDT had sensitivity: 96.0% (95% CI, 91.2-98.3) and specificity: 98.2% (95% CI, 94.6-99.5) and for P. vivax, sensitivity: 90.7% (95% CI, 78.9-96.5) and specificity: 98.9% (95% CI, 96.5-99.7). Comparison with qPCR showed, for P. falciparum malaria, sensitivity: 95.4% (95% CI, 90.5-98.0) and specificity: 98.8% (95% CI, 95.4-99.7) and for P. vivax malaria, sensitivity: 89.0% (95% CI,77.0-95.4) and specificity: 98.8% (95% CI, 96.5-99.7). Sensitivity varied according to different parasitaemia for falciparum and vivax malaria diagnosis. CONCLUSION: Parascreen® Pan/Pf Rapid test for malaria showed acceptable sensitivity and specificity in border belt endemic areas of Bangladesh when compared with EM and qPCR.

Ultrasensitive Reverse Transcriptase Loop-Mediated Isothermal Amplification (US-LAMP)-Based Detection of Malaria Infection from Dried Blood Spots.

Submicroscopic malaria diagnosis requires highly sensitive tools instead of the conventional microscopy and rapid diagnostic tests (RDTs). While polymerase chain reaction (PCR) is more sensitive than RDTs and microscopy, the required capital cost and technical expertise hinder implementation of PCR in low- and middle-income countries. This chapter describes an ultrasensitive reverse transcriptase loop-mediated isothermal amplification (US-LAMP) test for malaria with a high sensitivity and specificity, while also being practical to implement in low-complexity laboratory settings. The workflow combines a silica spin column-based total nucleic extraction from dried blood spots (DBS) with US-LAMP amplifying the Plasmodium (Pan-LAMP) target and subsequent identification Plasmodium falciparum (Pf-LAMP).

Evaluation of the OnSite (Pf/Pan) rapid diagnostic test for diagnosis of clinical malaria.

BACKGROUND: Accurate diagnosis of malaria is an essential prerequisite for proper treatment and drug resistance monitoring. Microscopy is considered the gold standard for malaria diagnosis but has limitations. ELISA, PCR, and Real Time PCR are also used to diagnose malaria in reference laboratories, although their application at the field level is currently not feasible. Rapid diagnostic tests (RDTs) however, have been brought into field operation and widely adopted in recent days. This study evaluates OnSite (Pf/Pan) antigen test, a new RDT introduced by CTK Biotech Inc, USA for malaria diagnosis in a reference setting. METHODS: Blood samples were collected from febrile patients referred for malaria diagnosis by clinicians. Subjects were included in this study from two different Upazila Health Complexes (UHCs) situated in two malaria endemic districts of Bangladesh. Microscopy and nested PCR were considered the gold standard in this study. OnSite (Pf/Pan) RDT was performed on preserved whole blood samples. RESULTS: In total, 372 febrile subjects were included in this study. Of these subjects, 229 (61.6%) tested positive for Plasmodium infection detected by microscopy and nested PCR. OnSite (Pf/Pan) RDT was 94.2% sensitive (95% CI, 89.3-97.3) and 99.5% specific (95% CI, 97.4-00.0) for Plasmodium falciparum diagnosis and 97.3% sensitive (95% CI, 90.5-99.7) and 98.7% specific (95% CI, 96.6-99.6) for Plasmodium vivax diagnosis. Sensitivity varied with differential parasite count for both P. falciparum and P. vivax. The highest sensitivity was observed in febrile patients with parasitaemia that ranged from 501-1,000 parasites/µL regardless of the Plasmodium species. CONCLUSION: The new OnSite (Pf/Pan) RDT is both sensitive and specific for symptomatic malaria diagnosis in standard laboratory conditions.

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

BACKGROUND: More than 95% of total malaria cases in Bangladesh are reported from the 13 high endemic districts. Plasmodium falciparum and Plasmodium vivax 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. METHODS: Blood samples were collected from 338 febrile patients referred for the diagnosis of malaria by the attending physician at MatirangaUpazila 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. RESULT: 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 P. falciparum (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 P. falciparum. 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 P. vivax. However, compared to real-time PCR assay RDTs and microscopy gave low sensitivity (76.9%, 95% CI 56.4-91) in detecting of P. vivax although a very high specificity was obtained (99-100%). CONCLUSION: 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 P. falciparum, although their sensitivity in detecting P. vivax was not satisfactory compared to the real-time PCR assay.

A metagenomics workflow for SARS-CoV-2 identification, co-pathogen detection, and overall diversity.

An unbiased metagenomics approach to virus identification can be essential in the initial phase of a pandemic. Better molecular surveillance strategies are needed for the detection of SARS-CoV-2 variants of concern and potential co-pathogens triggering respiratory symptoms. Here, a metagenomics workflow was developed to identify the metagenome diversity by SARS-CoV-2 diagnosis (npositive = 65; nnegative = 60), symptomatology status (nsymptomatic = 71; nasymptomatic = 54) and anatomical swabbing site (nnasopharyngeal = 96; nthroat = 29) in 125 individuals. Furthermore, the workflow was able to identify putative respiratory co-pathogens, and the SARS-CoV-2 lineage across 29 samples. The diversity analysis showed a significant shift in the DNA-metagenome by symptomatology status and anatomical swabbing site. Additionally, metagenomic diversity differed between SARS-CoV-2 infected and uninfected asymptomatic individuals. While 31 co-pathogens were identified in SARS-CoV-2 infected patients, no significant increase in pathogen or associated reads were noted when compared to SARS-CoV-2 negative patients. The Alpha SARS-CoV-2 VOC and 2 variants of interest (Zeta) were successfully identified for the first time using a clinical metagenomics approach. The metagenomics pipeline showed a sensitivity of 86% and a specificity of 72% for the detection of SARS-CoV-2. Clinical metagenomics can be employed to identify SARS-CoV-2 variants and respiratory co-pathogens potentially contributing to COVID-19 symptoms. The overall diversity analysis suggests a complex set of microorganisms with different genomic abundance profiles in SARS-CoV-2 infected patients compared to healthy controls. More studies are needed to correlate severity of COVID-19 disease in relation to potential disbyosis in the upper respiratory tract. A metagenomics approach is particularly useful when novel pandemic pathogens emerge.

A rapid near-patient detection system for SARS-CoV-2 using saliva.

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

Persistence of Markers of Chloroquine Resistance in Plasmodium falciparum in Bangladesh.

The control of malaria, in terms of drug resistance, remains a significant global challenge, with Bangladesh, a malaria-endemic country, being no exception. The aim of this study was to explore antimalarial resistance in Bangladesh by molecular analysis of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance transporter 1 (pfmdr1) genetic markers of P. falciparum. Samples were obtained from uncomplicated malaria patients between 2009 and 2014 from six malaria-endemic districts. Based on parasite transmission intensity, the endemic districts were divided into high-transmission (Chittagong Hill Tracts [CHT]) and low-transmission (non-CHT) regions. Falciparum malaria-positive isolates were genotyped for K76T of the pfcrt gene, and N86Y and Y184F of the pfmdr1 gene: in total, 262 P. falciparum clinical isolates were analyzed. In CHT areas, the prevalence of polymorphisms was 70.6% for 76T, 14.4% for 86Y, and 7.8% for 184F. In non-CHT areas, 76T and 86Y mutations were found in 78.0% and 19.5% of the samples, respectively, whereas no 184F mutations were observed. We compared our data with previous similar molecular observations, which shows a significant decrease in pfcrt 76T mutation prevalence. No pfmdr1 amplification was observed in any of the samples suggesting an unaltered susceptibility to amino alcohol drugs such as mefloquine and lumefantrine. This study provides an updated assessment of the current status of pfcrt and pfmdr1 gene mutations in Bangladesh, and suggests there is persistent high prevalence of markers of resistance to aminoquinoline drugs.

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2.

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.

Host Selection of Field-Collected Anopheles jeyporiensis and Anopheles nivipes in Bangladesh.

Anopheles jeyporiensis and Anopheles nivipes appear to play an important role in contemporary malaria transmission in Bangladesh. However, very little is known about the natural host selection of these vectors. Therefore, host selection of these two species was investigated in Bandarban, the most malarious region of Bangladesh. A total of 480 engorged mosquitoes were analyzed. The human blood index (HBI) of An. jeyporiensis varied from 4.17% in outdoor to 19.17% in indoor collections. Similarly, HBI of An. nivipes ranged between 0.83% and 22.50% from outdoor to indoor. For both species, cow blood indices were significantly higher than HBI in both indoor and outdoor collections. These data demonstrate the opportunistic and zoophilic nature of An. jeyporiensis and An. nivipes, which suggests that approaches such as zooprophylasis may prove beneficial as a control strategy.

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

BACKGROUND: 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.

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.

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh.

Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial to case management, because severity and treatment varies between species. Diagnoses can be made using rapid diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South Asia.