Dataset Information

Monitoring the threatened utility of malaria rapid diagnostic tests by novel high-throughput detection of Plasmodium falciparum hrp2 and hrp3 deletions: A cross-sectional, diagnostic accuracy study.

ABSTRACT: BACKGROUND:Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. METHODS:We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. FINDINGS:In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. INTERPRETATION:The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. FUNDING:BMBF (03VP00402).

SUBMITTER: Kreidenweiss A

PROVIDER: S-EPMC6921222 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Monitoring the threatened utility of malaria rapid diagnostic tests by novel high-throughput detection of Plasmodium falciparum hrp2 and hrp3 deletions: A cross-sectional, diagnostic accuracy study.

Kreidenweiss Andrea A Trauner Franziska F Rodi Miriam M Koehne Erik E Held Jana J Wyndorps Lea L Manouana Gédéon Prince GP McCall Matthew M Adegnika Ayola Akim AA Lalremruata Albert A Kremsner Peter G PG Fendel Rolf R Sandri Thaisa Lucas TL

EBioMedicine 20191121

<h4>Background</h4>Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies.<h4>Methods</h4>We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum in ...[more]

PMID: 31761619

Similar Datasets

Project description:BackgroundMalaria has a negative effect on the outcome of pregnancy. Pregnant women are at high risk of severe malaria and severe haemolytic anaemia, which contribute 60-70% of foetal and perinatal losses. Peripheral blood smear microscopy under-estimates sequestered placental infections, therefore malaria rapid diagnostic tests (RDTs) detecting histidine rich protein-2 antigen (HRP-2) in peripheral blood are a potential alternative.MethodsHRP-2 RDTs accuracy in detecting malaria in pregnancy (MIP >28 weeks gestation) and placental Plasmodium falciparum malaria (after childbirth) were conducted using Giemsa microscopy and placental histopathology respectively as the reference standard. The study was conducted in Mbale Hospital, using the midwives to perform and interpret the RDT results. Discordant results samples were spot checked using PCR techniques.ResultsAmong 433 febrile women tested, RDTs had a sensitivity of 96.8% (95% CI 92-98.8), specificity of 73.5% (95% CI 67.8-78.6), a positive predictive value (PPV) of 68.0% (95% CI 61.4-73.9), and negative predictive value (NPV) of 97.5% (95% CI 94.0-99.0) in detecting peripheral P. falciparum malaria during pregnancy. At delivery, in non-symptomatic women, RDTs had a 80.9% sensitivity (95% CI 57.4-93.7) and a 87.5% specificity (95%CI 80.9-92.1), PPV of 47.2% (95% CI 30.7-64.2) and NPV of 97.1% (95% CI 92.2-99.1) in detecting placental P. falciparum infections among 173 samples. At delivery, 41% of peripheral infections were detected by microscopy without concurrent placental infection. The combination of RDTs and microscopy improved the sensitivity to 90.5% and the specificity to 98.4% for detecting placental malaria infection (McNemar's X(2)> 3.84). RDTs were not superior to microscopy in detecting placental infection (McNemar's X(2)< 3.84). Presence of malaria in pregnancy and active placental malaria infection were 38% and 12% respectively. Placental infections were associated with poor pregnancy outcome [pre-term, still birth and low birth weight] (aOR = 37.9) and late pregnancy malaria infection (aOR = 20.9). Mosquito net use (aOR 2.1) and increasing parity (aOR 2.7) were associated with lower risk for malaria in pregnancy.ConclusionUse of HRP-2 RDTs to detect malaria in pregnancy in symptomatic women was accurate when performed by midwives. A combination of RDTs and microscopy provided the best means of detecting placental malaria. RDTs were not superior to microscopy in detecting placental infection. With a high sensitivity and specificity, RDTs could be a useful tool for assessing malaria in pregnancy, with further (cost-) effectiveness studies.

Project description:Deletions in Plasmodium falciparum histidine rich protein 2(pfhrp2) gene threaten the usefulness of the most widely used HRP2-based malaria rapid diagnostic tests (mRDTs) that cross react with its structural homologue, PfHRP3. Parasites with deleted pfhrp2/3 genes remain undetected and untreated due to 'false-negative' RDT results. As Ethiopia recently launched malaria elimination by 2030 in certain selected areas, the availability of RDTs and the scale of their use have rapidly increased in recent years. Thus, it is important to explore the presence and prevalence of deletion in the target genes, pfhrp2 and pfhrp3. From a total of 189 febrile patients visited Adama Malaria Diagnostic centre, sixty-four microscopically-and polymerase chain reaction (PCR)-confirmed P. falciparum clinical isolates were used to determine the frequency of pfhrp2/3 gene deletions. Established PCR assays were applied to DNA extracted from blood spotted onto filter papers to amplify across pfhrp2/3 exons and flanking regions. However, analysis of deletions in pfhrp2, pfhrp3 and flanking genomic regions was successful for 50 of the samples. The pfhrp2 gene deletion was fixed in the population with all 50(100%) isolates presenting a deletion variant. This deletion extended downstream towards the Pf3D7 0831900 (MAL7PI.230) gene in 11/50 (22%) cases. In contrast, only 2/50 (4%) of samples had deletions for the Pf3D7 0831700 (MALPI.228) gene, upstream of pfhrp2. Similarly, the pfhrp3 gene was deleted in all isolates (100%), while 40% of the isolates had an extension of the deletion to the downstream flanking region that codes for Pf3D7 13272400 (MAL13PI.485).The pfhrp3 deletion also extended upstream to Pf3D7 081372100 (MAL13PI.475) region in 49/50 (95%) of the isolates, exhibiting complete absence of the locus. Although all samples showed deletions of pfhrp2 exon regions, amplification of an intron region was successful in five cases. Two different repeat motifs in the intron regions were observed in the samples tested. Pfhrp2/3 gene deletions are fixed in Ethiopia and this will likely reduce the effectiveness of PfHRP2-based mRDTs. It will be important to determine the sensitivity PfHRP 2/3-based RDTs in these populations and conduct a countrywide survey to determine the extent of these deletions and its effect on routine RDT-based malaria diagnosis.

Project description:BackgroundRapid diagnostic tests (RDTs) are central to fulfilling the WHO's recommendation for parasitologic confirmation of all suspected cases of malaria. RDT performance may be compromised when exposed to the high temperature conditions typical of most malaria endemic regions. However, a systematic method to monitor RDT quality and performance in endemic countries is lacking at the present time. Current methods to monitor RDT performance in the field include comparing results from RDTs to diagnoses made by light microscopy and observing health workers perform tests. These methods are not substitutes for direct quality control. In this study, the suitability of dried Plasmodium falciparum-infected blood as quality control samples for malaria RDTs was evaluated.MethodsThree cultured strains of P. falciparum at 200 and 2,000 parasites/?l were tested on 10 brands of RDT. After baseline testing to determine initial reactivity, aliquots of parasite-infected blood were air dried, stored at 35°C, room temperature (~25°C) or 4°C for one, four and 12 weeks and were then tested on the 10 RDTs after rehydration. Extended stability testing of dried blood stored at 4°C was done using P. falciparum strain 3D7 at 1,000 and 2,000 parasites/?l.ResultsAll dried blood samples at 2,000 parasites/?l retained reactivity (100% sensitivity) at all three temperatures and time points for all nine RDT brands that detect histidine-rich protein-2 (HRP2). The dried blood samples with 200 parasites/?l were detected by six of the nine HRP2-based RDTs at all storage temperatures and time points. The sensitivity for two of the three remaining HRP2-based RDTs was 100% up to four weeks of storage at all temperatures but dropped to 87.5% at week 12. Of the four RDTs that detect plasmodium lactate dehydrogenase (pLDH) in a pan-specific manner, alone or in combination with HRP2, the detection of pLDH in samples with 2,000 parasites/?L was 100% for two RDTs and 80% for the other two RDTs. The mean level for detection of pLDH at 200 parasites/?l was low (29%), with a range of 0% to100%, which was partly attributable to weak initial baseline reactivity. Reactivity of dried 3D7 at 1,000 and 2,000 parasites/?l stored at 4°C was retained at 100% for up to 52 weeks for both HRP2 and pLDH.ConclusionsIn the absence of native or recombinant positive control antigens, well-standardized P. falciparum-infected dried blood samples can be used as positive control samples for monitoring RDT performance, particularly with HRP2-detecting tests.

Dataset Information

Monitoring the threatened utility of malaria rapid diagnostic tests by novel high-throughput detection of Plasmodium falciparum hrp2 and hrp3 deletions: A cross-sectional, diagnostic accuracy study.

Publications

Monitoring the threatened utility of malaria rapid diagnostic tests by novel high-throughput detection of Plasmodium falciparum hrp2 and hrp3 deletions: A cross-sectional, diagnostic accuracy study.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure