These are the first reports of P. falciparum Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low density P. falciparum, non-falciparum, and Pfhrp2/3-deleted malaria parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

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BackgroundSerological data indicating the presence and level of antibodies against infectious disease antigens provides indicators of exposure and transmission patterns in a population. Laboratory testing for large-scale serosurveys is often hindered by time-consuming immunoassays that employ multiple tandem steps. Some nations have recently begun using malaria serosurveillance data to make inferences about the malaria exposure in their populations, and serosurveys have grown increasingly larger as more accurate estimates are desired. Presented here is a novel approach of antibody detection using bead-based immunoassay that involves incubating all assay reagents concurrently overnight.ResultsA serosurvey in was performed in Haiti in early 2017 with both sera (n = 712) and dried blood spots (DBS, n = 796) collected for the same participants. The Luminex® multiplex bead-based assay (MBA) was used to detect total IgG against 8 malaria antigens: PfMSP1, PvMSP1, PmMSP1, PfCSP, PfAMA1, PfLSA1, PfGLURP-R0, PfHRP2. All sera and DBS samples were assayed by MBA using a standard immunoassay protocol with multiple steps, as well a protocol where sample and all reagents were incubated together overnight—termed here the OneStep assay. When compared to a standard multi-step assay, this OneStep assay amplified the assay signal for IgG detection for all 8 malaria antigens. The greatest increases in assay signal were seen at the low- and mid-range IgG titers and were indicative of an enhancement in the analyte detection, not simply an increase in the background signal of the assay. Seroprevalence estimates were generally similar for this sample Haitian population for all antigens regardless of serum or DBS sample type or assay protocol used.ConclusionsWhen using the MBA for IgG detection, overnight incubation for the test sample and all assay reagents greatly minimized hands-on time for laboratory staff. Enhanced IgG signal was observed with the OneStep assay for all 8 malaria antigens employed in this study, and seroprevalence estimates for this sample population were similar regardless of assay protocol used. This overnight incubation protocol has the potential to be deployed for large-scale malaria serosurveys for the high-throughput and timely collection of antibody data, particularly for malaria seroprevalence estimates.

BackgroundThe Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens.MethodsFrom September 2012 through February 2014, dried blood spots (DBS) were collected in Haiti from 9317 febrile patients presenting to 17 health facilities in 5 departments throughout the country as part of a bed net intervention study. All DBS from RDT positive persons and a random sampling of DBS from RDT negative persons were assayed for P. falciparum DNA by nested and PET-PCR (n = 2695 total). All PCR positive samples (n = 331) and a subset of PCR negative samples (n = 95) were assayed for three malaria antigens by a multiplex bead assay: pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and HRP2/3. Any samples positive for P. falciparum DNA, but negative for HRP2/3 antigens were tested by nested PCR for Pfhrp2 and Pfhrp3 gene deletions.ResultsOf 2695 DBS tested for Plasmodium DNA, 345 (12.8%) were originally found to be positive for P. falciparum DNA; 331 of these had DBS available for antigen detection. Of these, 266 (80.4%) were positive for pAldo, 221 (66.8%) positive for pLDH, and 324 (97.9%) were positive for HRP2/3 antigens. Seven samples (2.1%) positive for P. falciparum DNA were not positive for any of the three antigens by the bead assay, and were investigated for potential Pfhrp2/3 gene deletion by PCR. These samples either successfully amplified Pfhrp2/3 genes or were at an estimated parasite density too low for sufficient DNA to perform successful genotyping.ConclusionsMalaria positive samples in multiple Haitian sites were found to contain the HRP2/3 antigens, and no evidence was found of Pfhrp2/3 deletions. Malaria RDTs based on the detection of the HRP2/3 antigens remain a reliable P. falciparum diagnostic tool as Haiti works towards malaria elimination.

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.

D iagnosis and appropriate case management ofPlasmodium falciparum infection has greatly improved in many malaria-endemic settings through the use of rapid diagnostic tests (RDTs) that detect the histidine-rich protein 2 (HRP2) antigen (1). As the only Plasmodium species infecting humans to produce this antigen, the P. falciparum parasite expresses HRP2 in abundance and releases it into the bloodstream during blood-stage infection, making this marker a very sensitive and specific target for falciparum malaria (1,2). The pfhrp2 gene is located on chromosome 8 of the parasite genome, and a paralogous gene (pfhrp3) is located on chromosome 13. The 2 protein products share common epitopes for diagnostic antibodies, enabling the HRP3 antigen to also be detected to some extent by HRP2based RDTs (3-6).P. falciparum produces large quantities of these antigens during human blood-stage infection, but their biologic functions are not well elucidated, and pfhrp2-deleted and pfhrp3-deleted parasites still complete the human-mosquito lifecycle successfully (7). Reports of these gene deletions have increased over the past decade from multiple countries in Africa, South America, and Asia (https://apps.who.int/ malaria/maps/threats) (8). For countries that rely on HRP2-based RDTs for diagnosis of P. falciparum infection, those reports affirm the need to monitor the performance of this tool because deleted parasites could emerge and elicit false-negative results.P. falciparum infection represents ≈99.7% of all malaria cases in sub-Saharan Africa, and ≈300 million HRP2-based RDTs are used in this region annually (9). Studies in the east Africa countries of Eritrea (10) and Ethiopia (11,12) have found high prevalence of pfhrp2/pfhrp3 deletions, forcing changes away from HRP2-based RDTs to accurately diagnose P. falci-

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.

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.