Clustering of subpatent infections in households with asymptomatic rapid diagnostic test-positive cases in Bioko Island, Equatorial Guinea independent of travel to regions of higher malaria endemicity: a cross-sectional study.

BACKGROUND: Prevalence of falciparum malaria on Bioko Island remains high despite sustained, intensive control. Progress may be hindered by high proportions of subpatent infections that are not detected by rapid diagnostic tests (RDT) but contribute to onward transmission, and by imported infections. Better understanding of the relationship between subpatent infections and RDT-detected infections, and whether this relationship is different from imported versus locally acquired infections, is imperative to better understand the sources of infection and mechanisms of transmission to tailor more effective interventions. METHODS: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on a sub-set of samples from the 2015 Malaria Indicator Survey to identify subpatent infections. Households with RDT(+) individuals were matched 1:4 with households with no RDT(+) individuals. The association between living in a household with an RDT(+) individual and having a subpatent infection was evaluated using multivariate hierarchical logistic regression models with inverse probability weights for selection. To evaluate possible modification of the association by potential importation of the RDT(+) case, the analysis was repeated among strata of matched sets based on the reported eight-week travel history of the RDT(+) individual(s). RESULTS: There were 142 subpatent infections detected in 1,400 individuals (10.0%). The prevalence of subpatent infections was higher in households with versus without an RDT(+) individual (15.0 vs 9.1%). The adjusted prevalence odds of subpatent infection were 2.59-fold greater (95% CI: 1.31, 5.09) for those in a household with an RDT(+) individual compared to individuals in a household without RDT(+) individuals. When stratifying by travel history of the RDT(+) individual, the association between subpatent infections and RDT(+) infections was stronger in the strata in which the RDT(+) individual(s) had not recently travelled (adjusted prevalence odds ratio (aPOR) 2.95; 95% CI:1.17, 7.41), and attenuated in the strata in which recent travel was reported (aPOR 1.76; 95% CI: 0.54, 5.67). CONCLUSIONS: There is clustering of subpatent infections around RDT(+) individual(s) when both imported and local infection are suspected. Future control strategies that aim to treat whole households in which an RDT(+) individual is found may target a substantial portion of infections that would otherwise not be detected.

Screening for Pfhrp2/3-Deleted Plasmodium falciparum, Non-falciparum, and Low-Density Malaria Infections by a Multiplex Antigen Assay.

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis. Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed. Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3. Conclusions: These are the first reports of 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 parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Malaria Parasite Density in Individuals with Different Rapid Diagnostic Test Results and Concentrations of HRP2 Antigen.

Low-density malaria infections are a source of human morbidity in endemic settings and potentially contribute to ongoing malaria transmission. Conventional rapid diagnostic tests (RDTs) were designed to detect clinically relevant parasite and antigen levels, but it is largely unknown what proportion of parasite (and antigen positive) infections are missed by conventional RDTs. Furthermore, RDTs can also provide false positives from lingering histidine-rich protein 2 (HRP2) antigenemia from a past infection. We analyzed 207 samples from Angolan outpatients with a bead-based HRP2 antigen assay and by qRT-PCR for the presence of parasite nucleic acids. Among patients HRP2 positive but negative by conventional RDT, the rate of quantitative reverse transcription-PCR (qRT-PCR) positivity was 45% (95% CI: 35-56%), with a median parasitemia of 3.4 parasites/µL (interquartile range: 0.14-4.8). Only 15% (7-26%) of HRP2-negative samples were found to have parasite nucleic acids. A substantial proportion of persons with blood HRP2 antigen concentrations not detected by the conventional RDT were found to have evidence of active infection, but at low parasite density levels.

Beyond Blood Smears: Qualification of Plasmodium 18S rRNA as a Biomarker for Controlled Human Malaria Infections.

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.