scholarly journals Deletions of pfhrp2 and pfhrp3 genes in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

2020 ◽  
Author(s):  
Sam L Nsobya ◽  
Andrew Walakira ◽  
Elizabeth Namirembe ◽  
Moses Kiggundu ◽  
Joaniter I Nankabirwa ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
False Negative ◽  
Plasmodium Species ◽  
Pcr Amplification ◽  
Rapid Diagnostic Tests ◽  
False Negatives ◽  
Cross Sectional ◽  
True Prevalence ◽  
Blood Smears

Abstract Background: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the homologous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on protein expression, and our understanding of the true prevalence of deletions is incomplete. Methods: We investigated pfhrp2 / pfhrp3 deletions in blood samples from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. We evaluated samples with positive Giemsa-stained thick blood smears and negative PfHRP2-based RDTs by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression ofPfHRP2. Results: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum was identified in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum -positive samples there was a failure to amplify pfhrp2 or pfhrp3 :in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum -positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion: False negative RDTs were uncommon, and deletions in pfhrp2 and pfhrp3 explained some of these findings, although most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

scholarly journals Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sam L. Nsobya ◽  
Andrew Walakira ◽  
Elizabeth Namirembe ◽  
Moses Kiggundu ◽  
Joaniter I. Nankabirwa ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
Diagnostic Tests ◽  
False Negative ◽  
Pcr Amplification ◽  
Rapid Diagnostic Tests ◽  
False Negatives ◽  
Cross Sectional ◽  
True Prevalence ◽  
Blood Smears

Abstract Background Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. Methods Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. Results Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

scholarly journals Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

2020 ◽  
Author(s):  
Sam L Nsobya ◽  
Andrew Walakira ◽  
Elizabeth Namirembe ◽  
Moses Kiggundu ◽  
Joaniter I Nankabirwa ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
Diagnostic Tests ◽  
False Negative ◽  
Pcr Amplification ◽  
Rapid Diagnostic Tests ◽  
False Negatives ◽  
Cross Sectional ◽  
True Prevalence ◽  
Blood Smears

Abstract BackgroundRapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete.MethodsDeletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2.ResultsOf 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay.ConclusionFalse negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

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 Genetic Variations of Plasmodium Falciparum Histidine-rich Protein 2 and 3 in Assosa Zone, Ethiopia: Its Impact on the Performance of Malaria Rapid Diagnostic Tests

2021 ◽  
Author(s):  
Gezahegn Solomon Alemayehu ◽  
Alebachew Messele ◽  
Karen Lopez ◽  
Eugenia Lo ◽  
Daniel Janies ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Diagnostic Tests ◽  
False Negative ◽  
Dried Blood Spots ◽  
Genetic Variations ◽  
Rapid Diagnostic Tests ◽  
Repeat Type ◽  
Amino Acid Repeat ◽  
Cross Sectional

Abstract Background: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of Plasmodium falciparum malaria. However, false negative results of RDT caused by genetic variations of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. Methods: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger prick samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. Results: The PfHRP2 repeat type isolates were less conserved compared to the PfHRP3 repeat type. A total of eleven and eight different PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 isolates. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3 % in PfHRP3 sequence isolate at parasitemia levels >250 parasites/µl. Using the Baker model, 100 % of the isolates in group A and 73.7% of the isolates in group B were predicted to be detected by PfHRP2 RDT at parasitemia level> 250 parasite/μl. Conclusion: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

scholarly journals Genetic variation of Plasmodium falciparum histidine-rich protein 2 and 3 in Assosa zone, Ethiopia: its impact on the performance of malaria rapid diagnostic tests

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Gezahegn Solomon Alemayehu ◽  
Alebachew Messele ◽  
Kayla Blackburn ◽  
Karen Lopez ◽  
Eugenia Lo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Variation ◽  
Amino Acid ◽  
Diagnostic Tests ◽  
False Negative ◽  
Dried Blood Spots ◽  
Rapid Diagnostic Tests ◽  
Repeat Type ◽  
Amino Acid Repeat ◽  
Cross Sectional

Abstract Background Rapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. Methods A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. Results The PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50–99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/μl. Conclusion The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

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 Submicroscopic Infections with Plasmodium falciparum during Pregnancy and Their Association with Circulating Cytokine, Chemokine, and Cellular Profiles

2014 ◽  
Vol 21 (6) ◽  
pp. 859-866 ◽  
Author(s):  
Samad A. Ibitokou ◽  
Stéphanie Boström ◽  
Laurent Brutus ◽  
Nicaise Tuikue Ndam ◽  
Bertin Vianou ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Multivariate Analyses ◽  
Cell Types ◽  
Interleukin 10 ◽  
Peripheral Blood Mononuclear ◽  
Cross Sectional ◽  
Content Type ◽  
Blood Smears ◽  
Inducible Protein ◽  
A Prospective Study

ABSTRACTThe immunological consequences of pregnancy-associated malaria (PAM) due toPlasmodium falciparumhave been extensively investigated in cross-sectional studies conducted at delivery, but there have been very few longitudinal studies of changes due to PAM during pregnancy. We conducted a prospective study in Benin to investigate the changes associated with PAM in groups of 131 and 111 women at inclusion in the second trimester and at delivery, respectively. Infected women were identified by standard microscopic examinations of blood smears and by quantitative PCR (qPCR) assays and were matched to uninfected control women by age, gestational age, and gravidity. We quantified plasma levels of a panel of soluble immunological mediators and other mediators, as well as the frequencies of peripheral blood mononuclear cell types. Comparisons of these variables in infected and uninfected women used multivariate analyses, and we also assessed the predictive value of variables measured at inclusion for pregnancy outcomes at delivery. In multivariate analyses, peripheral plasma interleukin 10 (IL-10) and gamma interferon-inducible protein 10 (IP-10) levels were associated with PAM at inclusion and at delivery, while higher IL-10 levels distinguished qPCR-detectable submicroscopic infections at inclusion but not at delivery. Maternal anemia at delivery was associated with markers of proinflammatory (increased frequency of monocytes) and anti-inflammatory (increased IL-10 levels and increased activation of regulatory T cells) activity measured at inclusion. Elevated concentrations of IL-10 are associated with the majority ofP. falciparuminfections during pregnancy, but this marker alone does not identify all submicroscopic infections. Reliably identifying such occult infections will require more sensitive and specific methods.

scholarly journals Causes of false-negative rapid diagnostic tests for symptomatic malaria in the DRC

2020 ◽  
Author(s):  
Jonathan B Parr ◽  
Eddy Kieto ◽  
Fernandine Phanzu ◽  
Paul Masiangi ◽  
Kashamuka Mwandagalirwa ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Falciparum Malaria ◽  
Genome Sequencing ◽  
Diagnostic Tests ◽  
False Negative ◽  
Rapid Diagnostic Tests ◽  
Whole Genome ◽  
Cross Sectional ◽  
Symptomatic Malaria ◽  
Asymptomatic Children

Background The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. Methods We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a multiplex bead-based immunoassay; and/or whole-genome sequencing. Results Among 3,627 symptomatic subjects, we identified 427 (11.8%) RDT-/microscopy+ cases. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. Malaria prevalence was high (57%) and non-falciparum co-infection common (15%). HRP2-based RDT performance was satisfactory and superior to microscopy. Conclusions Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed in the DRC. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

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