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.

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van den Hoogen et al.Plasmodium falciparum Antibodies in Elimination n = 3,204)-Etramp 5 ag 1 and GLURP-R0 (area-under-the-curve, AUC, 0.892 and 0.825, respectively)-was confirmed in the test dataset (remaining one-third of the dataset; n = 1,652, AUC 0.903 and 0.848, respectively). As no further improvement was seen by combining seropositivity to GLURP-R0 and Etramp 5 ag 1 (p = 0.266), seropositivity to Etramp 5 ag 1 alone was selected as representative of current or recent exposure to malaria. The validation of antibody responses associated with these exposure histories simplifies analyses and interpretation of antibody data and facilitates the application of results to evaluate programs.

Accurate malaria diagnosis is foundational for control and elimination, and Haiti relies on HRP2-based rapid diagnostic tests (RDTs) identifying Plasmodium falciparum in clinical and community settings. In 2017, one household and two easy-access group (EAG) surveys tested all participants (N=32,506) by conventional and high-sensitivity RDTs (cRDT/hsRDT). A subset of blood samples (n=1,154) were laboratory tested for HRP2 by bead-based immunoassay and for P. falciparum 18S rDNA by PET-PCR. Both RDT types detected low concentrations of HRP2 with sensitivity estimates between 2.6 and 14.6 ng/mL. Compared to the predicate HRP2 laboratory assay,, RDT sensitivity ranged from 86.3% to 96.0% between tests and settings, and specificity from 90.0% to 99.6%. In the household survey, the hsRDT provided a significantly higher number of positive tests, but this represented a very small proportion (<0.2%) of all participants. These data show an hsRDT may have limited utility in a malaria elimination setting like Haiti.

Measuring antimalarial antibodies can estimate transmission in a population. to compare outputs, standardized laboratory testing is required. Here we describe the in-country establishment and quality control (QC) of a multiplex bead assay (MBA) for three sero-surveys in Haiti. Total IgG data against 21 antigens were collected for 32,758 participants. Titration curves of hyperimmune sera were included on assay plates, assay signals underwent 5-parameter regression, and inspection of the median and interquartile range (IQR) for the y-inflection point was used to determine assay precision. The medians and IQRs were similar for Surveys 1 and 2 for most antigens, while the IQRs increased for some antigens in Survey 3. Levey-Jennings charts for selected antigens provided a pass/fail criterion for each assay plate and, of 387 assay plates, 13 (3.4%) were repeated. Individual samples failed if IgG binding to the generic glutathione-S-transferase protein was observed, with 659 (2.0%) samples failing. An additional 455 (1.4%) observations failed due to low bead numbers (<20/analyte). The final dataset included 609,438 anti-malaria IgG data points from 32,099 participants; 96.6% of all potential data points if no Qc failures had occurred. the MBA can be deployed with high-throughput data collection and low interplate variability while ensuring data quality.Measurement of antibody responses to malaria at the population-level can describe recent and historical transmission patterns 1-4 and is informative for malaria research and program policies 5-7 . Antibodies can be quantitatively measured by a variety of techniques including the enzyme-linked immunosorbent assay (ELISA) and multiplex bead assays (MBAs). The latter allows the simultaneous detection of antibodies to multiple antigenic targets and has been utilized now for Plasmodium serology for over a decade 8 . Since the advent of Plasmodium-specific MBAs, numerous assay optimisation and implementation studies 9-17 , as well as epidemiological application studies 18-21 , have been published by various groups.In generating responses to many antigens simultaneously, MBAs have the advantage of reducing needed reagent quantities, sample volume, and time in the laboratory compared to the ELISA 8,9 . Assays of any type require controls or standards to assess variability across runs or batches and to compare research studies, and with a broad panel of antigens in the MBA, it is potentially difficult to find standards for all targets being assayed. Recently, standardization using curves of known concentrations of total human IgG has been suggested, but this was problematic in showing insufficient reproducibility between operators 13 . Moreover, these do not allow for the assessment of antigen-specific responses which are important for quality control in assessing the stability of www.nature.com/scientificreports www.nature.com/scientificreports/ specific antibodies over time. In-house pools of hyperimmune sera are commonly employed for serological studies, and recently, a World H...

Introduction Serological methods provide useful metrics to estimate age-specific period prevalence in settings of low malaria transmission; however, evidence on the use of seropositivity as an endpoint remains scarce in studies to evaluate combinations of malaria control measures, especially in children. This study aims to evaluate the immediate effects of a targeted mass drug administration campaign (tMDA) in Haiti by using serological markers. Methods The tMDA was implemented in September–October 2018 using sulfadoxine-pyrimethamine and single low-dose primaquine. A natural quasi-experimental study was designed, using a pretest and posttest in a cohort of 754 randomly selected school children, among which 23% reported having received tMDA. Five antigens were selected as outcomes (MSP1-19, AMA-1, Etramp5 antigen 1, HSP40, and GLURP-R0). Posttest was conducted 2–6 weeks after the intervention. Results At baseline, there was no statistical difference in seroprevalence between the groups of children that were or were not exposed during the posttest. A lower seroprevalence was observed for markers informative of recent exposure (Etramp5 antigen 1, HSP40, and GLURP-R0). Exposure to tMDA was significantly associated with a 50% reduction in the odds of seropositivity for Etramp5 antigen 1 and a 21% reduction in the odds of seropositivity for MSP119. Conclusion Serological markers can be used to evaluate the effects of interventions against malaria on the risk of infection in settings of low transmission. Antibody responses against Etramp5 antigen 1 in Haitian children were reduced in the 2–6 weeks following a tMDA campaign, confirming its usefulness as a short-term marker in child populations.

Haiti is targeting malaria elimination by 2025. The Grand’Anse department in southwestern Haiti experiences one-third to half of all nationally reported Plasmodium falciparum cases. Although there are historical reports of Plasmodium vivax and Plasmodium malariae, today, non-falciparum infections would remain undetected because of extensive use of falciparum-specific histidine-rich protein 2 (HRP2) rapid diagnostic tests (RDT) at health facilities. A recent case–control study was conducted in Grand’Anse to identify risk factors for P. falciparum infection using HRP2-based RDTs (n = 1,107). Post hoc multiplex Plasmodium antigenemia and antibody (IgG) detection by multiplex bead assay revealed one blood sample positive for pan-Plasmodium aldolase, negative for P. falciparum HRP2, and positive for IgG antibodies to P. malariae. Based on this finding, we selected 52 samples with possible P. malariae infection using IgG and antigenemia data and confirmed infection status by species-specific PCR. We confirmed one P. malariae infection in a 6-month-old infant without travel history. Congenital P. malariae could not be excluded. However, our finding—in combination with historical reports of P. malariae—warrants further investigation into the presence and possible extent of non-falciparum malaria in Haiti. Furthermore, we showed the use of multiplex Plasmodium antigen and IgG detection in selecting samples of interest for subsequent PCR analysis, thereby reducing costs as opposed to testing all available samples by PCR. This is of specific use in low-transmission or eliminating settings where infections are rare.