The Immediate Effects of a Combined Mass Drug Administration and Indoor Residual Spraying Campaign to Accelerate Progress Toward Malaria Elimination in Grande-Anse, Haiti.

BACKGROUND: Haiti is planning targeted interventions to accelerate progress toward malaria elimination. In the most affected department (Grande-Anse), a combined mass drug administration (MDA) and indoor residual spraying (IRS) campaign was launched in October 2018. This study assessed the intervention's effectiveness in reducing Plasmodium falciparum prevalence. METHODS: An ecological quasi-experimental study was designed, using a pretest and posttest with a nonrandomized control group. Surveys were conducted in November 2017 in a panel of easy access groups (25 schools and 16 clinics) and were repeated 2-6 weeks after the campaign, in November 2018. Single-dose sulfadoxine-pyrimethamine and primaquine was used for MDA, and pirimiphos-methyl as insecticide for IRS. RESULTS: A total of 10 006 participants were recruited. Fifty-two percent of the population in the intervention area reported having received MDA. Prevalence diminished between 2017 and 2018 in both areas, but the reduction was significantly larger in the intervention area (ratio of adjusted risk ratios, 0.32 [95% confidence interval, .104-.998]). CONCLUSIONS: Despite a moderate coverage, the campaign was effective in reducing P. falciparum prevalence immediately after 1 round. Targeted MDA plus IRS is useful in preelimination settings to rapidly decrease the parasite reservoir, an encouraging step to accelerate progress toward malaria elimination.

Persistence of Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 in Relation to Symptoms in a Nationwide Prospective Study.

BACKGROUND: Assessing the duration of immunity following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a first priority to gauge the degree of protection following infection. Such knowledge is lacking, especially in the general population. Here, we studied changes in immunoglobulin isotype seropositivity and immunoglobulin G (IgG) binding strength of SARS-CoV-2-specific serum antibodies up to 7 months following onset of symptoms in a nationwide sample. METHODS: Participants from a prospective representative serological study in the Netherlands were included based on IgG seroconversion to the spike S1 protein of SARS-CoV-2 (N = 353), with up to 3 consecutive serum samples per seroconverted participant (N = 738). Immunoglobulin M (IgM), immunoglobulin A (IgA), and IgG antibody concentrations to S1, and increase in IgG avidity in relation to time since onset of disease symptoms, were determined. RESULTS: While SARS-CoV-2-specific IgM and IgA antibodies declined rapidly after the first month after disease onset, specific IgG was still present in 92% (95% confidence interval [CI], 89%-95%) of the participants after 7 months. The estimated 2-fold decrease of IgG antibodies was 158 days (95% CI, 136-189 days). Concentrations were sustained better in persons reporting significant symptoms compared to asymptomatic persons or those with mild upper respiratory complaints only. Similarly, avidity of IgG antibodies for symptomatic persons showed a steeper increase over time compared with persons with mild or no symptoms (P = .022). CONCLUSIONS: SARS-CoV-2-specific IgG antibodies persist and show increasing avidity over time, indicative of underlying immune maturation. These data support development of immune memory against SARS-CoV-2, providing insight into protection of the general unvaccinated part of the population. CLINICAL TRIALS REGISTRATION: NL8473 (the Dutch trial registry).

Comparison of diagnostics for the detection of asymptomatic Plasmodium falciparum infections to inform control and elimination strategies.

The global burden of malaria has been substantially reduced over the past two decades. Future efforts to reduce malaria further will require moving beyond the treatment of clinical infections to targeting malaria transmission more broadly in the community. As such, the accurate identification of asymptomatic human infections, which can sustain a large proportion of transmission, is becoming a vital component of control and elimination programmes. We determined the relationship across common diagnostics used to measure malaria prevalence - polymerase chain reaction (PCR), rapid diagnostic test and microscopy - for the detection of Plasmodium falciparum infections in endemic populations based on a pooled analysis of cross-sectional data. We included data from more than 170,000 individuals comparing the detection by rapid diagnostic test and microscopy, and 30,000 for detection by rapid diagnostic test and PCR. The analysis showed that, on average, rapid diagnostic tests detected 41% (95% confidence interval = 26-66%) of PCR-positive infections. Data for the comparison of rapid diagnostic test to PCR detection at high transmission intensity and in adults were sparse. Prevalence measured by rapid diagnostic test and microscopy was comparable, although rapid diagnostic test detected slightly more infections than microscopy. On average, microscopy captured 87% (95% confidence interval = 74-102%) of rapid diagnostic test-positive infections. The extent to which higher rapid diagnostic test detection reflects increased sensitivity, lack of specificity or both, is unclear. Once the contribution of asymptomatic individuals to the infectious reservoir is better defined, future analyses should ideally establish optimal detection limits of new diagnostics for use in control and elimination strategies.

Conventional and High-Sensitivity Malaria Rapid Diagnostic Test Performance in 2 Transmission Settings: Haiti 2017.

Accurate malaria diagnosis is foundational for control and elimination, and Haiti relies on histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) identifying Plasmodium falciparum in clinical and community settings. In 2017, 1 household and 2 easy-access group surveys tested all participants (N = 32 506) by conventional and high-sensitivity RDTs. A subset of blood samples (n = 1154) was laboratory tested for HRP2 by bead-based immunoassay and for P. falciparum 18S rDNA by photo-induced electron transfer polymerase chain reaction. Both RDT types detected low concentrations of HRP2 with sensitivity estimates between 2.6 ng/mL 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 high-sensitivity RDT provided a significantly higher number of positive tests, but this represented a very small proportion (<0.2%) of all participants. These data show that a high-sensitivity RDT may have limited utility in a malaria elimination setting like Haiti.

Programmatic options for monitoring malaria in elimination settings: easy access group surveys to investigate Plasmodium falciparum epidemiology in two regions with differing endemicity in Haiti.

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.

High-throughput malaria serosurveillance using a one-step multiplex bead assay.

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 shape of the iceberg: quantification of submicroscopic Plasmodium falciparum and Plasmodium vivax parasitaemia and gametocytaemia in five low endemic settings in Ethiopia.

BACKGROUND: The widespread presence of low-density asymptomatic infections with concurrent gametocytes may be a stumbling block for malaria elimination. This study investigated the asymptomatic reservoir of Plasmodium falciparum and Plasmodium vivax infections in schoolchildren from five settings in northwest Ethiopia. METHODS: Two cross-sectional surveys were conducted in June and November 2015, enrolling 551 students from five schools and 294 students from three schools, respectively. Finger prick whole blood and plasma samples were collected. The prevalence and density of P. falciparum and P. vivax parasitaemia and gametocytaemia were determined by 18S rRNA quantitative PCR (qPCR) and pfs25 and pvs25 reverse transcriptase qPCR. Antibodies against blood stage antigens apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-119) were measured for both species. RESULTS: Whilst only 6 infections were detected by microscopy in 881 slides (0.7%), 107 of 845 blood samples (12.7%) were parasite positive by (DNA-based) qPCR. qPCR parasite prevalence between sites and surveys ranged from 3.8 to 19.0% for P. falciparum and 0.0 to 9.0% for P. vivax. The median density of P. falciparum infections (n = 85) was 24.4 parasites/µL (IQR 18.0-34.0) and the median density of P. vivax infections (n = 28) was 16.4 parasites/µL (IQR 8.8-55.1). Gametocyte densities by (mRNA-based) qRT-PCR were strongly associated with total parasite densities for both P. falciparum (correlation coefficient = 0.83, p = 0.010) and P. vivax (correlation coefficient = 0.58, p = 0.010). Antibody titers against P. falciparum AMA-1 and MSP-119 were higher in individuals who were P. falciparum parasite positive in both surveys (p < 0.001 for both comparisons). DISCUSSION: This study adds to the available evidence on the wide-scale presence of submicroscopic parasitaemia by quantifying submicroscopic parasite densities and concurrent gametocyte densities. There was considerable heterogeneity in the occurrence of P. falciparum and P. vivax infections and serological markers of parasite exposure between the examined low endemic settings in Ethiopia.

Anti-malarial seroprevalence assessment during an elimination programme in Chabahar District, south-eastern Iran.

BACKGROUND: Iran has achieved a substantial decline in malaria incidence over the past decades. A common feature of malaria-endemic settings is the requirement for more sensitive techniques to describe levels of low transmission. In this study, serological and parasitological methods were used to measure transmission levels of Plasmodium falciparum and Plasmodium vivax during an elimination programme (2012) in Chabahar District, Sistan and Baluchistan Province, south-eastern Iran. METHODS: Participants were randomly selected from 64 different geographical clusters in Chabahar city and surrounding villages. Antibody responses to P. falciparum and P. vivax blood-stage antigens were assessed by ELISA, while microscopy and molecular testing were used to determine parasite carriage by species. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCR). RESULTS: There was no evidence of recent transmission in the study areas, indicated by an absence of parasite infections in all ages and low or absent serological responses to either species in young children. The best model for age P. falciparum seroconversion was one with a change in exposure 21 years before sampling was done in Chabahar city (P = 0.018) and 4 years in the villages (P = 0.039). There was a higher level of recent P. vivax transmission compared to P. falciparum, based on the SCRs, in both the city and village settings. CONCLUSION: Serological analysis identified a decline in P. falciparum transmission in the urban areas of Chabahar, consistent with a previously described decrease in malaria in the early 1990s, demonstrating the utility of this approach to reconstruct exposure history. At present, it remains unclear whether the P. vivax antibody responses reflect active transmission due to new infections or relapse infections. The absence of parasitological and serological evidence of recent malaria transmission in Chabahar District is viable evidence for certification of elimination.

Serology describes a profile of declining malaria transmission in Farafenni, The Gambia.

BACKGROUND: Malaria morbidity and mortality has declined in recent years in a number of settings. The ability to describe changes in malaria transmission associated with these declines is important in terms of assessing the potential effects of control interventions, and for monitoring and evaluation purposes. METHODS: Data from five cross-sectional surveys conducted in Farafenni and surrounding villages on the north bank of River Gambia between 1988 and 2011 were compiled. Antibody responses to MSP-119 were measured in samples from all surveys, data were normalized and expressed as seroprevalence and seroconversion rates (SCR) using different mathematical models. RESULTS: Results showed declines in serological metrics with seroprevalence in children aged one to 5 years dropping from 19 % (95 % CI 15-23 %) in 1988 to 1 % (0-2 %) in 2011 (p value for trend in proportions < 0.001) and the SCR dropping from 0.069 year(-1) (0.059-0.080) to 0.022 year(-1) (0.017-0.028; p = 0.004). The serological data were consistent with previously described drops in both parasite prevalence in children aged 1-5 years (62 %, 57-66 %, in 1988 to 2 %, 0-4 %, in 2011; p < 0.001), and all-cause under five mortality rates (37 per 1000 person-years, 34-41, in 1990 to 17, 15-19, in 2006; p = 0.059). CONCLUSIONS: This analysis shows accurate reconstruction of historical malaria transmission patterns in the Farafenni area using anti-malarial antibody responses. Demonstrating congruence between serological measures, and conventional clinical and parasitological measures suggests broader utility for serology in monitoring and evaluation of malaria transmission.

Spatial Clustering and Risk Factors for Malaria Infections and Marker of Recent Exposure to Plasmodium falciparum from a Household Survey in Artibonite, Haiti.

Targeting malaria interventions in elimination settings where transmission is heterogeneous is essential to ensure the efficient use of resources. Identifying the most important risk factors among persons experiencing a range of exposure can facilitate such targeting. A cross-sectional household survey was conducted in Artibonite, Haiti, to identify and characterize spatial clustering of malaria infections. Household members (N = 21,813) from 6,962 households were surveyed and tested for malaria. An infection was defined as testing positive for Plasmodium falciparum by either a conventional or novel highly sensitive rapid diagnostic test. Seropositivity to the early transcribed membrane protein 5 antigen 1 represented recent exposure to P. falciparum. Clusters were identified using SaTScan. Associations among individual, household, and environmental risk factors for malaria, recent exposure, and living in spatial clusters of these outcomes were evaluated. Malaria infection was detected in 161 individuals (median age: 15 years). Weighted malaria prevalence was low (0.56%; 95% CI: 0.45-0.70%). Serological evidence of recent exposure was detected in 1,134 individuals. Bed net use, household wealth, and elevation were protective, whereas being febrile, over age 5 years, and living in either households with rudimentary wall material or farther from the road increased the odds of malaria. Two predominant overlapping spatial clusters of infection and recent exposure were identified. Individual, household, and environmental risk factors are associated with the odds of individual risk and recent exposure in Artibonite; spatial clusters are primarily associated with household-level risk factors. Findings from serology testing can further strengthen the targeting of interventions.

Acceptability, Feasibility, Drug Safety, and Effectiveness of a Pilot Mass Drug Administration with a Single Round of Sulfadoxine-Pyrimethamine Plus Primaquine and Indoor Residual Spraying in Communities with Malaria Transmission in Haiti, 2018.

For a malaria elimination strategy, Haiti's National Malaria Control Program piloted a mass drug administration (MDA) with indoor residual spraying (IRS) in 12 high-transmission areas across five communes after implementing community case management and strengthened surveillance. The MDA distributed sulfadoxine-pyrimethamine and single low-dose primaquine to eligible residents during house visits. The IRS campaign applied pirimiphos-methyl insecticide on walls of eligible houses. Pre- and post-campaign cross-sectional surveys were conducted to assess acceptability, feasibility, drug safety, and effectiveness of the combined interventions. Stated acceptability for MDA before the campaign was 99.2%; MDA coverage estimated at 10 weeks post-campaign was 89.6%. Similarly, stated acceptability of IRS at baseline was 99.9%; however, household IRS coverage was 48.9% because of the high number of ineligible houses. Effectiveness measured by Plasmodium falciparum prevalence at baseline and 10 weeks post-campaign were similar: 1.31% versus 1.43%, respectively. Prevalence of serological markers were similar at 10 weeks post-campaign compared with baseline, and increased at 6 months. No severe adverse events associated with the MDA were identified in the pilot; there were severe adverse events in a separate, subsequent campaign. Both MDA and IRS are acceptable and feasible interventions in Haiti. Although a significant impact of a single round of MDA/IRS on malaria transmission was not found using a standard pre- and post-intervention comparison, it is possible there was blunting of the peak transmission. Seasonal malaria transmission patterns, suboptimal IRS coverage, and low baseline parasitemia may have limited the effectiveness or the ability to measure effectiveness.

SARS-CoV-2 Spike S1-specific IgG kinetic profiles following mRNA or vector-based vaccination in the general Dutch population show distinct kinetics.

mRNA- and vector-based vaccines are used at a large scale to prevent COVID-19. We compared Spike S1-specific (S1) IgG antibodies after vaccination with mRNA-based (Comirnaty, Spikevax) or vector-based (Janssen, Vaxzevria) vaccines, using samples from a Dutch nationwide cohort. In adults 18-64 years old (n = 2412), the median vaccination interval between the two doses was 77 days for Vaxzevria (interquartile range, IQR: 69-77), 35 days (28-35) for Comirnaty and 33 days (28-35) for Spikevax. mRNA vaccines induced faster inclines and higher S1 antibodies compared to vector-based vaccines. For all vaccines, one dose resulted in boosting of S1 antibodies in adults with a history of SARS-CoV-2 infection. For Comirnaty, two to four months following the second dose (n = 196), S1 antibodies in adults aged 18-64 years old (436 BAU/mL, IQR: 328-891) were less variable and median concentrations higher compared to those in persons ≥ 80 years old (366, 177-743), but differences were not statistically significant (p > 0.100). Nearly all participants seroconverted following COVID-19 vaccination, including the aging population. These data confirm results from controlled vaccine trials in a general population, including vulnerable groups.

Seropositivity to Nucleoprotein to detect mild and asymptomatic SARS-CoV-2 infections: A complementary tool to detect breakthrough infections after COVID-19 vaccination?

BACKGROUND: With COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination. METHODS: Multiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) without COVID-19 vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech). RESULTS: The sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80-90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50-81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 ≥ 14 days after the first dose as compared to those unexposed to SARS-CoV-2 at ≥ 7 days after the second dose (p = 0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in infection-naïve participants. CONCLUSIONS: Serological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.

Reduced Antibody Acquisition with Increasing Age following Vaccination with BNT162b2: Results from Two Longitudinal Cohort Studies in The Netherlands.

Vaccine-induced protection against severe COVID-19, hospitalization, and death is of the utmost importance, especially in the elderly. However, limited data are available on humoral immune responses following COVID-19 vaccination in the general population across a broad age range. We performed an integrated analysis of the effect of age, sex, and prior SARS-CoV-2 infection on Spike S1-specific (S1) IgG concentrations up to three months post-BNT162b2 (Pfizer/BioNTech; Comirnaty) vaccination. In total, 1735 persons, eligible for COVID-19 vaccination through the national program, were recruited from the general population (12 to 92 years old). Sixty percent were female, and the median vaccination interval was 35 days (interquartile range, IQR: 35−35). All participants had seroconverted to S1 one month after two vaccine doses. S1 IgG was higher in participants with a history of SARS-CoV-2 infection (median: 4535 BAU/mL, IQR: 2341−7205) compared to infection-naive persons (1842 BAU/mL, 1019−3116), p < 0.001. In infection-naive persons, linear mixed effects regression showed a strong negative association between age and S1 IgG (p < 0.001) across the entire age range. Females had higher S1 IgG than males (p < 0.001). In persons with an infection history, age nor sex was associated with S1 IgG concentrations. The lower magnitude of S1 antibodies in older persons following COVID-19 vaccination will affect long-term protection.

Rapid Screening for Non-falciparum Malaria in Elimination Settings Using Multiplex Antigen and Antibody Detection: Post Hoc Identification of Plasmodium malariae in an Infant in Haiti.

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.

Considerations for quality assurance of multiplex malaria antigen detection assays with large sample sets.

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.

Risk Factors for Malaria Infection and Seropositivity in the Elimination Area of Grand'Anse, Haiti: A Case-Control Study among Febrile Individuals Seeking Treatment at Public Health Facilities.

The island of Hispaniola aims to eliminate malaria by 2025; however, there are limited data to describe epidemiologic risk factors for malaria in this setting. A prospective case-control study was conducted at four health facilities in southwest Haiti, aiming to describe factors influencing the risk of current and past malaria infection. Cases were defined as individuals attending facilities with current or recent fever and positive malaria rapid diagnostic test (RDT), while controls were those with current or recent fever and RDT negative. Serological markers of recent and cumulative exposure to Plasmodium were assessed using the multiplex bead assay from dried blood spots and used for alternate case definitions. Kuldorff's spatial scan statistic was used to identify local clusters of infection or exposure. Logistic regression models were used to assess potential risk factors for RDT positivity and recent exposure markers, including age-group, gender, and recruiting health facility as group-matching variables. A total of 192 cases (RDT positive) and 915 controls (RDT negative) were recruited. Consistent spatial clusters were identified for all three infection and exposure metrics, indicating temporal stability of malaria transmission at these sites. Risk factors included remoteness from health facilities and household construction, furthermore, insecticide-treated net ownership or use was associated with reduced odds of RDT positivity. These findings indicate the malaria risk in Grand'Anse is driven primarily by location. Travel, occupation, and other behavioral factors were not associated with malaria. These data can support the National Malaria Program to refine and target their intervention approaches, and to move toward elimination.

Comparison of Commercial ELISA Kits to Confirm the Absence of Transmission in Malaria Elimination Settings.

Background: Antimalarial antibody measurements are useful because they reflect historical and recent exposure to malaria. As such, they may provide additional information to assess ongoing transmission in low endemic or pre-elimination settings where cases are rare. In addition, the absence of antibody responses in certain individuals can indicate the cessation of transmission. Commercial malaria enzyme-linked immunosorbent assays (ELISA) detect antimalarial antibodies and are commonly used to screen blood donations for possible malaria infection. However, there is no standardized test to detect antimalarial antibodies for epidemiological use. Here we compared five commercially available ELISA kits (Trinity Biotech, newbio, DiaPro, Cellabs, and NovaTec) in search of a standardized tool for supporting claims of absence of malaria transmission. For comparison, a research-based (RB) ELISA protocol was performed alongside the commercial kits. Results: The commercial kits were first compared using serum samples from known malaria-unexposed individuals (n = 223) and Toxoplasma-infected individuals (n = 191) to assess specificity and cross-reactivity against non-malaria infections. In addition, 134 samples from ≥10-year-olds collected in a hyperendemic region in the Gambia in the early 1990s were used to assess sensitivity. Three out of five kits showed high sensitivity (90-92%), high specificity (98-99%), low cross-reactivity (0-3%) and were considered user-friendly (Trinity Biotech, newbio and NovaTec). Two of these kits (Trinity Biotech and NovaTec) were taken forward for epidemiological evaluation and results were compared to those using the RB-ELISA. Samples from two pre-elimination settings (Praia, Cape Verde; n = 1,396, and Bataan, the Philippines; n = 1,824) were tested. Serological results from both the Trinity Biotech kit and the RB-ELISA concurred with recent passively detected case counts in both settings. Results from the Trinity Biotech kit reflected a significant decrease in the number of reported cases in Bataan in the 1990s better than the RB-ELISA. Results from the NovaTec kit did not reflect transmission patterns in either setting. Conclusion: The Trinity Biotech commercial ELISA kit was considered reliable for epidemiological use and accurately described transmission patterns in two (previously) malaria endemic settings. The use of this simple and standardized serological tool may aid national control and elimination programs by confirming that regions are free from malaria.

Selection of Antibody Responses Associated With Plasmodium falciparum Infections in the Context of Malaria Elimination.

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.

Quality control of multiplex antibody detection in samples from large-scale surveys: the example of malaria in 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 inter-plate variability while ensuring data quality.