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Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

ABSTRACT: The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.

SUBMITTER: Guirou EA

PROVIDER: S-EPMC7378824 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

Guirou Etienne A EA Schindler Tobias T Hosch Salome S Donfack Olivier Tresor OT Yoboue Charlene Aya CA Krähenbühl Silvan S Deal Anna A Cosi Glenda G Gondwe Linda L Mwangoka Grace G Masuki Heavenlight H Salim Nahya N Mpina Maxmillian M Said Jongo J Abdulla Salim S Hoffman Stephen L SL Nlavo Bonifacio Manguire BM Maas Carl C Falla Carlos Cortes CC Phiri Wonder P WP Garcia Guillermo A GA Tanner Marcel M Daubenberger Claudia C

Scientific reports 20200723 1

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on ...[more]

PMID: 32703999

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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:BACKGROUND: The World Health Organization recommends parasitological confirmation of all malaria cases. Tanzania is implementing a phased rollout of malaria rapid diagnostic tests (RDTs) for routine use in all levels of care as one strategy to increase parasitological confirmation of malaria diagnosis. This study was carried out to evaluated artemisinin combination therapy (ACT) prescribing patterns in febrile patients with and without uncomplicated malaria in one pre-RDT implementation and one post-RDT implementation area. METHODS: A cross-sectional health facility surveys was conducted during high and low malaria transmission seasons in 2010 in both areas. Clinical information and a reference blood film on all patients presenting for an initial illness consultation were collected. Malaria was defined as a history of fever in the past 48 h and microscopically confirmed parasitaemia. Routine diagnostic testing was defined as RDT or microscopy ordered by the health worker and performed at the health facility as part of the health worker-patient consultation. Correct diagnostic testing was defined as febrile patient tested with RDT or microscopy. Over-testing was defined as a non-febrile patient tested with RDT or microscopy. Correct treatment was defined as patient with malaria prescribed ACT. Over-treatment was defined as patient without malaria prescribed ACT. RESULTS: A total of 1,247 febrile patients (627 from pre-implementation area and 620 from post-implementation area) were included in the analysis. In the post-RDT implementation area, 80.9% (95% CI, 68.2-89.3) of patients with malaria received recommended treatment with ACT compared to 70.3% (95% CI, 54.7-82.2) of patients in the pre-RDT implementation area. Correct treatment was significantly higher in the post-implementation area during high transmission season (85.9% (95% CI, 72.0-93.6) compared to 58.3% (95% CI, 39.4-75.1) in pre-implementation area (p?=?0.01). Over-treatment with ACT of patients without malaria was less common in the post-RDT implementation area (20.9%; 95% CI, 14.7-28.8) compared to the pre-RDT implementation area (45.8%; 95% CI, 37.2-54.6) (p?<?0.01) in high transmission. The odds of overtreatment was significantly lower in post- RDT area (adjusted Odds Ratio (OR: 95% CI) 0.57(0.36-0.89); and much higher with clinical diagnosis adjusted OR (95% CI) 2.24(1.37-3.67) CONCLUSION: Implementation of RDTs increased use of RDTs for parasitological confirmation and reduced over-treatment with ACT during high malaria transmission season in one area in Tanzania. Continued monitoring of the national RDT rollout will be needed to assess whether these changes in case management practices will be replicated in other areas and sustained over time. Additional measures (such as refresher trainings, closer supervisions, etc.) may be needed to improve ACT targeting during low transmission seasons.

Project description:BackgroundLarge-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging.MethodsBetween 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n?=?2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n?=?1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13.ResultsThe study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation.ConclusionsThis study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

Project description:BackgroundA new more highly sensitive rapid diagnostic test (HS-RDT) for Plasmodium falciparum malaria (Alere™/Abbott Malaria Ag P.f RDT [05FK140], now called NxTek™ Eliminate Malaria Ag Pf) was launched in 2017. The test has already been used in many research studies in a wide range of geographies and use cases.MethodsIn this study, we collate all published and available unpublished studies that use the HS-RDT and assess its performance in (i) prevalence surveys, (ii) clinical diagnosis, (iii) screening pregnant women, and (iv) active case detection. Two individual-level data sets from asymptomatic populations are used to fit logistic regression models to estimate the probability of HS-RDT positivity based on histidine-rich protein 2 (HRP2) concentration and parasite density. The performance of the HS-RDT in prevalence surveys is estimated by calculating the sensitivity and positive proportion in comparison to polymerase chain reaction (PCR) and conventional malaria RDTs.ResultsWe find that across 18 studies, in prevalence surveys, the mean sensitivity of the HS-RDT is estimated to be 56.1% (95% confidence interval [CI] 46.9-65.4%) compared to 44.3% (95% CI 32.6-56.0%) for a conventional RDT (co-RDT) when using nucleic acid amplification techniques as the reference standard. In studies where prevalence was estimated using both the HS-RDT and a co-RDT, we found that prevalence was on average 46% higher using a HS-RDT compared to a co-RDT. For use in clinical diagnosis and screening pregnant women, the HS-RDT was not significantly more sensitive than a co-RDT.ConclusionsOverall, the evidence presented here suggests that the HS-RDT is more sensitive in asymptomatic populations and could provide a marginal improvement in clinical diagnosis and screening pregnant women. Although the HS-RDT has limited temperature stability and shelf-life claims compared to co-RDTs, there is no evidence to suggest, given this test has the same cost as current RDTs, it would have any negative impacts in terms of malaria misdiagnosis if it were widely used in all four population groups explored here.

Dataset Information

Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

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Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

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