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Haiti is endemic for lymphatic filariasis (LF) and malaria, two mosquito-transmitted parasitic diseases targeted for elimination. The World Health Organization recommends a transmission assessment survey (TAS-1) to determine if LF prevalence is significantly beneath putative transmission thresholds (<2% antigen prevalence in Haiti, where Culex is the primary vector for Wuchereria bancrofti) to stop mass drug administration (MDA). Repeated TASs (TAS-2 and TAS-3) are recommended at 2-3-year intervals during post-treatment surveillance. From 2017 to 2022, The Carter Center assisted the Haitian Ministry of Public Health and Population in conducting 15 TASs in 11 evaluation units (EUs) encompassing 54 of the country's 146 districts. Children 6-7 years old were assessed for circulating filarial antigen (CFA) by Filariasis Test Strip: n = 5,239 in TAS-1; n = 11,866 in TAS-2; and n = 1,842 in TAS-3, of whom eight (0.15%), 20 (0.17%), and eight (0.43%) tested positive, respectively. The number of positive results in children was less than the threshold in each EU. When available, participants (n = 16,663) were also tested for malaria by rapid diagnostic test, with 31 (0.19%) children testing positive for Plasmodium falciparum. Integrated TASs provided an efficient means to collect epidemiological data for LF and malaria in Haiti. Results indicated thresholds for stopping and maintaining the halt of MDA for LF have been achieved in all EUs, with the halt of MDA for 571,358 people in four districts and the first TAS-3 surveys conducted in Haiti. Investigations are needed to assess the potential of ongoing LF transmission, especially in areas where CFA-positive samples were detected in TAS-3.

Background: Due to low numbers of active infections and persons presenting to health facilities for malaria treatment, case-based surveillance is inefficient for understanding the remaining disease burden in low malaria transmission settings. Serological data through the detection of IgG antibodies from previous malaria parasite exposure can fill this gap by providing a nuanced picture of where sustained transmission remains. Study enrollment at sites of gathering provides a potential approach to spatially estimate malaria exposure and could preclude the need for more intensive community-based sampling., Methods: This study compared spatial estimates of malaria exposure from cross-sectional school- and community-based sampling in Haiti. A total of 52,405 blood samples were collected from 2012 to 2017. Multiplex bead assays (MBAs) tested IgG against P. falciparum liver stage antigen-1 (LSA-1), apical membrane antigen 1 (AMA1), and merozoite surface protein 1 (MSP1). Predictive geospatial models of seropositivity adjusted for environmental covariates, and results were compared using correlations by coordinate points and communes across Haiti., Results: Consistent directional associations were observed between seroprevalence and environmental covariates for elevation (negative), air temperature (negative), and travel time to urban centers (positive). Spearman's rank correlation for predicted seroprevalence at coordinate points was lowest for LSA-1 (ρ = 0.10, 95% CI: 0.09-0.11), but improved for AMA1 (ρ = 0.36, 95% CI: 0.35-0.37) and MSP1 (ρ = 0.48, 95% CI: 0.47-0.49)., Conclusions: In settings approaching P. falciparum elimination, case-based prevalence data does not provide a resolution of ongoing malaria transmission in the population. Immunogenic antigen targets (e.g., AMA1, MSP1) that give higher population rates of seropositivity provide moderate correlation to gold standard community sampling designs and are a feasible approach to discern foci of residual P. falciparum transmission in an area., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Estimation of malaria prevalence in very low transmission settings is difficult by even the most advanced diagnostic tests. Antibodies against malaria antigens provide an indicator of active or past exposure to these parasites. The prominent malaria species within Haiti is Plasmodium falciparum, but P. vivax and P. malariae infections are also known to be endemic., Methodology/principal Findings: From 2014-2016, 28,681 Haitian children were enrolled in school-based serosurveys and were asked to provide a blood sample for detection of antibodies against multiple infectious diseases. IgG against the P. falciparum, P. vivax, and P. malariae merozoite surface protein 19kD subunit (MSP119) antigens was detected by a multiplex bead assay (MBA). A subset of samples was also tested for Plasmodium DNA by PCR assays, and for Plasmodium antigens by a multiplex antigen detection assay. Geospatial clustering of high seroprevalence areas for P. vivax and P. malariae antigens was assessed by both Ripley's K-function and Kulldorff's spatial scan statistic. Of 21,719 children enrolled in 680 schools in Haiti who provided samples to assay for IgG against PmMSP119, 278 (1.27%) were seropositive. Of 24,559 children enrolled in 788 schools providing samples for PvMSP119 serology, 113 (0.46%) were seropositive. Two significant clusters of seropositivity were identified throughout the country for P. malariae exposure, and two identified for P. vivax. No samples were found to be positive for Plasmodium DNA or antigens., Conclusions/significance: From school-based surveys conducted from 2014 to 2016, very few Haitian children had evidence of exposure to P. vivax or P. malariae, with no children testing positive for active infection. Spatial scan statistics identified non-overlapping areas of the country with higher seroprevalence for these two malarias. Serological data provides useful information of exposure to very low endemic malaria species in a population that is unlikely to present to clinics with symptomatic infections., Competing Interests: The authors have declared that no competing interests exist.

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.

Background: Serological 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., Results: A 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., Conclusions: When 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.

The island of Hispaniola remains the last location with endemic malaria in the Caribbean region, and ongoing elimination efforts aim to achieve zero cases from local transmission by the year [...]

Background: Plasmodium blood-stage infections can be identified by assaying for protein products expressed by the parasites. While the binary result of an antigen test is sufficient for a clinical result, greater nuance can be gathered for malaria infection status based on quantitative and sensitive detection of Plasmodium antigens and machine learning analytical approaches. Methods: Three independent malaria studies performed in Angola and Haiti enrolled persons at health facilities and collected a blood sample. Presence and parasite density of P. falciparum infection was determined by microscopy for a study in Angola in 2015 (n = 193), by qRT-PCR for a 2016 study in Angola (n = 208), and by qPCR for a 2012–2013 Haiti study (n = 425). All samples also had bead-based detection and quantification of three Plasmodium antigens: pAldolase, pLDH, and HRP2. Decision trees and principal component analysis (PCA) were conducted in attempt to categorize P. falciparum parasitemia density status based on continuous antigen concentrations. Results: Conditional inference trees were trained using the known P. falciparum infection status and corresponding antigen concentrations, and PCR infection status was predicted with accuracies ranging from 73–96%, while level of parasite density was predicted with accuracies ranging from 59–72%. Multiple decision nodes were created for both pAldolase and HRP2 antigens. For all datasets, dichotomous infectious status was more accurately predicted when compared to categorization of different levels of parasite densities. PCA was able to account for a high level of variance (>80%), and distinct clustering was found in both dichotomous and categorical infection status. Conclusions: This pilot study offers a proof-of-principle of the utility of machine learning approaches to assess P. falciparum infection status based on continuous concentrations of multiple Plasmodium antigens. [ABSTRACT FROM AUTHOR]

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites. [ABSTRACT FROM AUTHOR]

Background: As in most eliminating countries, malaria transmission is highly focal in Haiti. More granular information, including identifying asymptomatic infections, is needed to inform programmatic efforts, monitor intervention effectiveness, and identify remaining foci. Easy access group (EAG) surveys can supplement routine surveillance with more granular information on malaria in a programmatically tractable way. This study assessed how and which type of venue for EAG surveys can improve understanding malaria epidemiology in two regions with different transmission profiles.Methods: EAG surveys were conducted within the departments of Artibonite and Grand'Anse (Haiti), in regions with different levels of transmission intensity. Surveys were conducted in three venue types: primary schools, health facilities, and churches. The sampling approach varied accordingly. Individuals present at the venues at the time of the survey were eligible whether they presented malaria symptoms or not. The participants completed a questionnaire and were tested for Plasmodium falciparum by a highly sensitive rapid diagnostic test (hsRDT). Factors associated with hsRDT positivity were assessed by negative binomial random-effects regression models.Results: Overall, 11,029 individuals were sampled across 39 venues in Artibonite and 41 in Grand'Anse. The targeted sample size per venue type (2100 in Artibonite and 2500 in Grand'Anse) was reached except for the churches in Artibonite, where some attendees left the venue before they could be approached or enrolled. Refusal rate and drop-out rate were < 1%. In total, 50/6003 (0.8%) and 355/5026 (7.1%) sampled individuals were hsRDT positive in Artibonite and Grand'Anse, respectively. Over half of all infections in both regions were identified at health facilities. Being male and having a current or reported fever in the previous 2 weeks were consistently identified with increased odds of being hsRDT positive.Conclusions: Surveys in churches were problematic because of logistical and recruitment issues. However, EAG surveys in health facilities and primary schools provided granular information about malaria burden within two departments in Haiti. The EAG surveys were able to identify residual foci of transmission that were missed by recent national surveys. Non-care seeking and/or asymptomatic malaria infections can be identified in this alternative surveillance tool, facilitating data-driven decision-making for improved targeting of interventions. [ABSTRACT FROM AUTHOR]

Microscopy is the gold standard for malaria epidemiology, but laboratory and point-of-care (POC) tests detecting parasite antigen, DNA, and human antibodies against malaria have expanded this capacity. The island nation of Haiti is endemic for Plasmodium falciparum (Pf) malaria, though at a low national prevalence and heterogenous geospatial distribution. In 2015 and 2016, serosurveys were performed of children (ages 6–7 years) sampled in schools in Saut d'Eau commune (n = 1,230) and Grand Anse department (n = 1,664) of Haiti. Children received malaria antigen rapid diagnostic test and provided a filter paper blood sample for further laboratory analysis of the Pf histidine-rich protein 2 (HRP2) antigen, Pf DNA, and anti-Pf IgG antibodies. Prevalence of Pf infection ranged from 0.0–16.7% in 53 Saut d'Eau schools, and 0.0–23.8% in 56 Grand Anse schools. Anti-Pf antibody carriage exceeded 80% of students in some schools from both study sites. Geospatial prediction ellipses were created to indicate clustering of positive tests within the survey areas and overlay of all prediction ellipses for the different types of data revealed regions with high likelihood of active and ongoing Pf malaria transmission. The geospatial utilization of different types of Pf data can provide high confidence for spatial epidemiology of the parasite. [ABSTRACT FROM AUTHOR]

In our aim to eliminate malaria, more sensitive tools to detect residual transmission are quickly becoming essential. Antimalarial antibody responses persist in the blood after a malaria infection and provide a wider window to detect exposure to infection compared to parasite detection metrics. Here, we aimed to select antibody responses associated with recent and cumulative exposure to malaria using cross-sectional survey data from Haiti, an elimination setting. Using a multiplex bead assay, we generated data for antibody responses (immunoglobulin G) to 23 Plasmodium falciparum targets in 29,481 participants across three surveys. This included one community-based survey in which participants were enrolled during household visits and two sentinel group surveys in which participants were enrolled at schools and health facilities. First, we correlated continuous antibody responses with age (Spearman) to determine which showed strong age-related associations indicating accumulation over time with limited loss. AMA-1 and MSP-119 antibody levels showed the strongest correlation with age (0.47 and 0.43, p < 0.001) in the community-based survey, which was most representative of the underlying age structure of the population, thus seropositivity to either of these antibodies was considered representative of cumulative exposure to malaria. Next, in the absence of a gold standard for recent exposure, we included antibody responses to the remaining targets to predict highly sensitive rapid diagnostic test (hsRDT) status using receiver operating characteristic curves. For this, only data from the survey with the highest hsRDT prevalence was used (7.2%; 348/4,849). The performance of the top two antigens in the training dataset (two-thirds of the dataset; 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. [ABSTRACT FROM AUTHOR]

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 inter-plate variability while ensuring data quality. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys. Methods: Individuals' blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey. Results: There was a sigmoidal dose-response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD. Conclusions: Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration. [ABSTRACT FROM AUTHOR]

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Background: Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting submicroscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay. Methods: Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidinerich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRTPCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/μL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission. Results: Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4-41 %. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission. Conclusions: The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them. [ABSTRACT FROM AUTHOR]

Background Plasmodium vivax infections, while quite prevalent throughout South and Central America, are virtually non-existent in Haiti, where P. falciparum infections are detected in over 99% of malaria cases. Historically, few cases of P. vivax have been reported in Haiti; all of which were identified by microscopy and none were confirmed by molecular diagnostics. To further examine the transmission of P. vivax in Haiti, a cross-sectional seroepidemiological study was conducted. Methods Whole blood was collected from 814 community members and school children ranging in age between 2 and 80 years-of-age from four locations in the Ouest and Sud-Est Departments of Haiti. After separation of serum, samples were screened for antibodies toward P. vivax apical membrane antigen (AMA-1) and merozoite surface protein-1 19 (MSP-1) using an indirect enzyme-linked immunosorbent assay (ELISA). Results Of all participants screened, 4.42% (36/814) were seropositive for AMA-1, 4.55% (37/814) were seropositive for MSP-1, 7.99% (65/814) were seropositive to either antigen, and only 0.98% (7/814) were seropositive for both antigens. Seroconversion rates (SCR) for AMA-1, MSP-1, either AMA-1 or MSP-1, and for both AMA-1 and MSP-1 estimated from the cross-sectional seroprevalence indicated rates of P. vivax transmission of less than 1% per year. Conclusion Given the lack of historical evidence of P. vivax infections on the island of Hispaniola, the sparse serological evidence of antibodies toward P. vivax identified in the current study further support the notion that the transmission of P. vivax malaria might be extremely low or even completely absent in Haiti. [ABSTRACT FROM AUTHOR]