Dataset Information

Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification.

ABSTRACT:

Background

Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated.

Methods

Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g^®, MALBAC^TM, PicoPLEX^®, and GenomePlex^®, for which DNA quantity and quality were compared between original DNA and post-WGA products.

Results

The proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value?®, MALBAC^TM, and PicoPLEX^® increased the quantity of DNA 60 to 750-fold, whereas the ratio of parasite DNA amplification over human DNA was most favourable for MALBAC^®. Sequencing results of pfkelch13, P. falciparum chloroquine resistance transporter (pfcrt), P. falciparum dihydrofolate reductase (pfdhfr) and six microsatellite markers assessed from the post-WGA product was the same as from the original DNA.

Conclusions

Blood spots from RDTs are a good source for molecular analysis of P. falciparum, even after storage up to 32 months. WGA of RDT-derived parasite DNA reliably increase DNA quantity with sufficient quality for molecular analysis of resistance markers.

SUBMITTER: Srisutham S

PROVIDER: S-EPMC7251736 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification.

Srisutham Suttipat S Suwannasin Kanokon K Mathema Vivek Bhakta VB Sriprawat Kanlaya K Smithuis Frank M FM Nosten Francois F White Nicholas J NJ Dondorp Arjen M AM Imwong Mallika M

Malaria journal 20200527 1

<h4>Background</h4>Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated.<h4>Methods</h4>Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January ...[more]

PMID: 32460780

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Project description:BACKGROUND:The first pillar of the UNAIDS 90-90-90 goal seeks to accurately identify persons living with HIV (PLHIV), a process that is predicated on facilities having the necessary HIV tests available to perform the task. In many rural settings, the identification of PLHIV is accomplished through a two-step process involving the sequential use of 2 separate rapid diagnostic tests (RDTs). Inadequate inventory of either test has ramifications for the success of HIV-related programs. The purpose of this study was to evaluate the inventory levels of HIV RDT kits at specific healthcare facilities in Zambézia province, Mozambique. METHODS:Using facility-level pharmacy stock surveillance data from October 2015 through September 2016, we assessed the inventory levels of HIV RDTs at 75 health facilities in 8 districts within Zambézia province, Mozambique. Using programmatically established categories (good, sufficient, threatened, or stockout), defined in conjunction with the provincial health authorities, descriptive statistics were performed to determine inventory control of HIV RDTs at the district and health facility levels. Monthly proportions of adequate (good + sufficient) inventory were calculated for each district to identify inventory trends over the evaluation period. To assess whether the proportion of inadequate stocks differed between RDT, a mixed-effects logistic regression was conducted, with inadequate inventory status as the outcome of interest. RESULTS:When viewed as a whole, the inventory of each test kit was reported as being at adequate levels more than 89% of the time across the 75 facilities. However, disaggregated analysis revealed significant variability in the inventory levels of HIV RDTs at the district level. Specifically, the districts of Inhassunge, Namacurra, and Pebane reported inadequate inventory levels (threatened + stockout), of one or both test kits, for more than 10% of the study period. In addition, a disparity between inventory levels of each test kit was identified, with the odds of reporting inadequate inventory levels of the confirmatory test (Uni-Gold™) being approximately 1.8-fold greater than the initial test (Determine™) (odds ratio: 1.82, 95% CI: 1.40-2.38). CONCLUSION:As Test and Treat programs evolve, a significant emphasis should be placed on the "test" component of the strategy, beginning with assurances that health facilities have the adequate inventory of RDT necessary to meet the needs of their community. As national policy-makers rely predominantly on data from the upstream arm of the supply chain, it is unlikely the disparity between inventory levels of HIV RDTs identified at individual districts and specific health facilities would have been recognized. Moving forward, our findings point to a need for (1) renewed efforts reinforcing appropriate downstream forecasting of essential medicines and diagnostic tests in general and for Uni-Gold™ test kits specifically, and (2) simple metrics that may be routinely collected at all health facilities and which may then easily and quickly flow upstream so that policy-makers may optimally allocate resources.

Project description: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.

Project description:BackgroundThe need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.MethodsDNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance.ResultsThe P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples.ConclusionsThe results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

Project description:BackgroundWith the documentation of cases of falciparum malaria negative by rapid diagnostic tests (RDT), though at low frequency from natural isolates in a small pocket of Odisha, it became absolutely necessary to investigate the status of HRP-2 based RDT throughout the state and in different seasons of the year.MethodsSuspected individuals were screened for malaria infection by microscopy and RDT in 25/30 districts of Odisha, India. Discrepancies in results were confirmed by PCR. False negative RDT samples for Plasmodium falciparum mono-infection were evaluated for detection of HRP2 antigen in ELISA and genotyped for pfhrp2, pfhrp3 and their flanking genes. Multiplicity of infection was ascertained based on msp1 and msp2 genotyping and parasitaemia level was determined by microscopy.ResultsOf the total 1058 patients suspected for malaria, 384 were microscopically confirmed for P. falciparum mono-infection and RDT failure was observed in 58 samples at varying proportion in different regions of the state. The failure in detection was due to undetectable level of HRP-2. Although most of these samples were screened during rainy season (45/345), significantly high proportion (9/17) of RDT negative samples were obtained during the summer compared to rainy season (P = 0.0002; OR = 7.5). PCR genotyping of pfhrp2 and pfhrp3 in RDT negative samples showed 38/58 (65.5) samples to be pfhrp2 negative and 24/58 (41.4) to be pfhrp3 negative including dual negative in 17/58 (29.3). Most of the RDT negative samples (39/58) were with single genotype infection and high proportions of pfhrp2 deletion (7/9) was observed in summer. No difference in parasitaemia level was observed between RDT positive and RDT negative patients.ConclusionHigh prevalence of parasites with pfhrp2 deletion including dual deletions (pfhrp2 and pfhrp3) is a serious cause of concern, as these patients could not be given a correct diagnosis and treatment. Therefore, HRP2-based RDT for diagnosing P. falciparum infection in Odisha is non-reliable and must be performed in addition to or replaced by other appropriate diagnostic tools for clinical management of the disease.

Dataset Information

Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification.

Background

Methods

Results

Conclusions

Publications

Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification.

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