scholarly journals Community-based surveys for Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in selected regions of mainland Tanzania

2020 ◽  
Author(s):  
Catherine Bakari ◽  
Sophie Jones ◽  
Gireesh Subramaniam ◽  
Celine Isaack Mandara ◽  
Mercy G Chiduo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
False Negative ◽  
Malaria Diagnosis ◽  
Positive Control ◽  
Gene Deletions ◽  
Negative Results ◽  
False Negative Results ◽  
Malaria Rapid Diagnostic Tests ◽  
The Status ◽  
Relationship Of

Background: Despite recent reports of false negative results among histidine-rich protein 2 (HRP2) based-malaria rapid diagnostic tests (mRDTs) caused by pfhrp2/3 gene deletions in different countries, there is paucity of data in Tanzania. Methods: This study assessed the status of pfhrp2/3 deletions in 7,543 blood sample using laboratory multiplex antigen detection (Plasmodium lactate dehydrogenase - pLDH, aldolase, and HRP2). Samples showing mRDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped for pfhrp2/3 genes. Results: Of all samples, 2,417 (32.0%) were positive for any Plasmodium antigens while 5,126 (68.0%) were negative. About 99.8% (n=2,411) of antigen positive samples had HRP2, but 6 (0.2%) had only pLDH and/or pAldolase. Thirteen samples had atypical relationships between pan-Plasmodium antigens and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 mRDTs but positive by microscopy were also chosen; all giving 35 samples genotyped for pfhrp2/3. Of 35 samples, 4 (11.4%) failed to consistently amplify positive control genes (pfmsp1 and pfmsp2), and pfhrp2 and pfhrp3 genes were successfully amplified in 31 (88.6%) samples. Conclusions: Lack of pfhrp2 and/or pfhrp3 genes deletions in Plasmodium falciparum parasites supports continued use of HRP2-based mRDTs for routine malaria diagnosis in Tanzania.

scholarly journals Modelling the epidemiology of malaria and spread of HRP2-negative Plasmodium falciparum following the replacement of HRP2-detecting rapid diagnostic tests

2022 ◽  
Vol 2 (1) ◽  
pp. e0000106
Author(s):  
Alisha Chaudhry ◽  
Jane Cunningham ◽  
Qin Cheng ◽  
Michelle L. Gatton
Keyword(s):  
Plasmodium Falciparum ◽  
Diagnostic Tests ◽  
False Negative ◽  
Parasite Prevalence ◽  
Rapid Diagnostic Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Malaria Rapid Diagnostic Tests ◽  
The Impact

Malaria rapid diagnostic tests (RDTs) are dominated by products which use histidine-rich protein 2 (HRP2) to detect Plasmodium falciparum. The emergence of parasites lacking the pfhrp2 gene can lead to high rates of false-negative results amongst these RDTs. One solution to restore the ability to correctly diagnose falciparum malaria is to switch to an RDT which is not solely reliant on HRP2. This study used an agent-based stochastic simulation model to investigate the impact on prevalence and transmission caused by switching the type of RDT used once false-negative rates reached pre-defined thresholds within the treatment-seeking symptomatic population. The results show that low transmission settings were the first to reach the false-negative switch threshold, and that lower thresholds were typically associated with better long-term outcomes. Changing the diagnostic RDT away from a HRP2-only RDT is predicted to restore the ability to correctly diagnose symptomatic malaria infections, but often did not lead to the extinction of HRP2-negative parasites from the population which continued to circulate in low density infections, or return to the parasite prevalence and transmission levels seen prior to the introduction of the HRP2-negative parasite. In contrast, failure to move away from HRP2-only RDTs leads to near fixation of these parasites in the population, and the inability to correctly diagnose symptomatic cases. Overall, these results suggest pfhrp2-deleted parasites are likely to become a significant component of P. falciparum parasite populations, and that long-term strategies are needed for diagnosis and surveillance which do not rely solely on HRP2.

scholarly journals High prevalence and extended deletions in Plasmodium falciparum hrp2/3 genomic loci in Ethiopia

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241807
Author(s):  
Lemu Golassa ◽  
Alebachew Messele ◽  
Alfred Amambua-Ngwa ◽  
Gote Swedberg
Keyword(s):  
Plasmodium Falciparum ◽  
Target Genes ◽  
False Negative ◽  
Malaria Diagnosis ◽  
Gene Deletions ◽  
Malaria Rapid Diagnostic Tests ◽  
High Prevalence ◽  
Filter Papers ◽  
Genomic Regions ◽  
Flanking Regions

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.

scholarly journals Community-based surveys for Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in selected regions of mainland Tanzania

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Catherine Bakari ◽  
Sophie Jones ◽  
Gireesh Subramaniam ◽  
Celine I. Mandara ◽  
Mercy G. Chiduo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Single Gene ◽  
False Negative ◽  
Malaria Diagnosis ◽  
Positive Control ◽  
Cross Sectional Survey ◽  
Community Based ◽  
Cross Sectional ◽  
Gene Deletions ◽  
Antigen Assay

Abstract Background Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania. Methods A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes. Results Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes. Conclusions This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.

scholarly journals No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Mandella King ◽  
Alexander E. George ◽  
Pau Cisteró ◽  
Christine K. Tarr-Attia ◽  
Beatriz Arregui ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
False Negative ◽  
Malaria Diagnosis ◽  
Rapid Diagnostic Tests ◽  
Molecular Testing ◽  
False Negatives ◽  
Gene Deletions ◽  
History Of ◽  
Catholic Hospital ◽  
Or History

Abstract Background Malaria diagnosis in many malaria-endemic countries relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia. Methods PfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n = 951) and pregnant women at first antenatal care (ANC) visit (n = 87) enrolled in the Saint Joseph’s Catholic Hospital (Monrovia) from March to July 2019 were used to assess the frequency of false-negative RDT results. True–false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy results. Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3. Results One-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and decreased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n = 5) and 8% (n = 7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the 16 RDT-negative but microscopy-positive samples which were available for molecular testing. Among the 14 samples that were positive by qPCR but negative by RDT and microscopy, 3 only amplified pfldh, and among these 3 all were positive for pfhrp2 and pfhrp3. Conclusion There is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates that these deletions are not expected to affect the performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

scholarly journals Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control

2017 ◽  
Vol 29 (3) ◽  
pp. 351-356
Author(s):  
Amaresh Das ◽  
Ming Y. Deng ◽  
Shawn Babiuk ◽  
Michael T. McIntosh
Keyword(s):  
Real Time ◽  
False Negative ◽  
Positive Control ◽  
Lumpy Skin Disease ◽  
Outbreak Management ◽  
Negative Results ◽  
Beta Actin ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Analytical Sensitivities

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

scholarly journals PO 8271 PFHRP2 GENE DELETIONS IN PLASMODIUM FALCIPARUM AND SCHISTOSOMA MANSONI CO-INFECTIONS: AN EMERGING CHALLENGE FOR MALARIA RAPID DIAGNOSTIC TESTS

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A25.2-A25
Author(s):  
Hilda Echelibe ◽  
Masumbe Netongo Palmer ◽  
Nji Akindeh ◽  
Wilfred Mbacham
Keyword(s):  
Plasmodium Falciparum ◽  
Schistosoma Mansoni ◽  
Diagnostic Tests ◽  
False Negative ◽  
Rapid Diagnostic Tests ◽  
Sub Saharan Africa ◽  
P Value ◽  
Gene Deletions ◽  
Malaria Rdts ◽  
Malaria Rapid Diagnostic Tests

BackgroundMalaria and schistosomiasis are infections that have a great impact in sub-Saharan Africa based on their high morbidity and mortality rates. We suggest the possibility that the microenvironment created from interactions between the parasites involved generates a pressure on the malaria parasite which could in turn favour the parasite’s adaptation or escape through Pfhrp2 gene deletions. Thus, this study aimed at determining the association between the co-infection with both parasites and false-negative PfHRP2-based malaria rapid diagnostic tests which occur because of these deletions.MethodsThis pilot study was conducted in a total of 149 children aged 7–17 years living in Yorro, located in the Mbam-Inoubou division of the Center region of Cameroon. We collected fresh stool samples from each participant to identify Schistosoma mansoni (Sm) eggs by Kato Katz method and blood samples to identify the ring stages of Plasmodium falciparum (Pf) by thick smear. Malaria rapid diagnostic test and Pfhrp2 gene polymerase chain reaction were performed. The association between the co-infection with Sm/Pf and the false-negative malaria RDTs was determined by the Fisher’s exact test. A p value<0.05 was considered statistically significant.ResultsOur results showed that samples were singly infected with Sm, Pf, co-infected (Sm/Pf) and negative for both infections at frequencies of 12%, 43%, 30.2% and 14.8% respectively. False-negative PfHRP2-based RDTs were observed in 4.7% of the participants. A higher frequency (5/7) of the cases with false-negative malaria RDTs were co-infected with Sm/Pf. A p value of 0.027 showed statistical significance in the association of Sm/Pf co-infection and false-negative PfHRP2-based RDTs.ConclusionA significant association of Plasmodium falciparum and Schistosoma mansoni co-infection with false-negative PfHRP2-based RDTs supports the case for a plausible implication of Pfhrp2 gene deletions, with consequences for malaria rapid diagnostic testing.

scholarly journals Prozone-like phenomenon in travellers with fatal malaria: report of two cases

2015 ◽  
Vol 9 (03) ◽  
pp. 321-324 ◽  
Author(s):  
Lurdes Santos ◽  
Nuno Rocha Pereira ◽  
Paulo Andrade ◽  
Paulo Figueiredo Dias ◽  
Carlos Lima Alves ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
False Negative ◽  
Specific Antigen ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Rapid Diagnostic Tests ◽  
Microscopy Observation ◽  
Negative Results ◽  
False Negative Results ◽  
Potential Benefits

Malaria diagnosis remains a concern in non-endemic countries, with rapid diagnosis being crucial to improve patients’ outcome. Rapid diagnostic tests have high sensitivity but they also have flaws and false-negative results that might jeopardize malaria diagnosis. Some false-negative results might relate to a prozone-like effect. The authors describe two patients with false-negative rapid diagnostic tests in which a prozone-like effect might have been involved. The authors highlight that these tests should not be used without accompanying light microscopy observation of blood films and discuss potential benefits of using rapid diagnostic tests with more than one specific antigen for Plasmodium falciparum.

scholarly journals Secondary analysis of malaria rapid diagnostic tests from rounds 5–8 of WHO product testing with a focus on false-negative results

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Biao Xu ◽  
Bo Tu ◽  
Fang Chu ◽  
Mohamed Jalloh ◽  
Jin-Song Mu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
False Negative ◽  
Secondary Analysis ◽  
World Health ◽  
Product Testing ◽  
Negative Results ◽  
Malaria Rdts ◽  
False Negative Results ◽  
Malaria Rapid Diagnostic Tests ◽  
Health Organization

AbstractDespite the widespread use of malaria rapid diagnostic test (RDT) in clinical practice, there are a lot of challenges. We conducted a secondary analysis of 129 malaria RDT data from rounds 5–8 of the World Health Organization (WHO) product testing summary and discuss the causes of false-negative (FN) results with a focus on low parasite density, improper RDT storage, operation and interpretation, and plasmodium falciparum with a pfhrp2/3 gene deletion. The results demonstrated that the malaria RDTs currently commercially available might cause FN results in practice.

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

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