Reactive Case Detection for <i>Plasmodium vivax</i> Malaria Elimination in Rural Amazonia

<div><p>Background</p><p>Malaria burden in Brazil has reached its lowest levels in 35 years and <i>Plasmodium vivax</i> now accounts for 84% of cases countrywide. Targeting residual malaria transmission entrenched in the Amazon is the next major challenge for ongoing elimination efforts. Better strategies are urgently needed to address the vast reservoir of asymptomatic <i>P</i>. <i>vivax</i> carriers in this and other areas approaching malaria elimination.</p><p>Methods</p><p>We evaluated a reactive case detection (RCD) strategy tailored for <i>P</i>. <i>vivax</i> transmission in farming settlements in the Amazon Basin of Brazil. Over six months, 41 cases detected by passive surveillance triggered four rounds of RCD (0, 30, 60, and 180 days after index case enrollment), using microscopy- and quantitative real-time polymerase chain reaction (qPCR)-based diagnosis, comprising subjects sharing the household (HH) with the index case (n = 163), those living in the 5 nearest HHs within 3 km (n = 878), and individuals from 5 randomly chosen control HHs located > 5 km away from index cases (n = 841). Correlates of infection were identified with mixed-effects logistic regression models. Molecular genotyping was used to infer local parasite transmission networks.</p><p>Principal findings/Conclusions</p><p>Subjects in index and neighbor HHs were significantly more likely to be parasitemic than control HH members, after adjusting for potential confounders, and together harbored > 90% of the <i>P</i>. <i>vivax</i> biomass in study subjects. Clustering patterns were temporally stable. Four rounds of microscopy-based RCD would identify only 49.5% of the infections diagnosed by qPCR, but 76.8% of the total parasite biomass circulating in the proximity of index HHs. However, control HHs accounted for 27.6% of qPCR-positive samples, 92.6% of them from asymptomatic carriers beyond the reach of RCD. Molecular genotyping revealed high <i>P</i>. <i>vivax</i> diversity, consistent with complex transmission networks and multiple sources of infection within clusters, potentially complicating malaria elimination efforts.</p></div

Absolute and relative frequency of symptomatic and asymptomatic <i>Plasmodium vivax</i> infections diagnosed by conventional microscopy among members of index, neighbor, and control households (HHs) in four rounds of reactive case detection (RCD) in Acrelândia, Brazil, 2013.

<p>Absolute and relative frequency of symptomatic and asymptomatic <i>Plasmodium vivax</i> infections diagnosed by conventional microscopy among members of index, neighbor, and control households (HHs) in four rounds of reactive case detection (RCD) in Acrelândia, Brazil, 2013.</p

Schematic representation of the study design.

<p>For each index case detected by routine passive surveillance and enrolled in the study we invited the following individuals to participate: (a) all subjects who shared the household (HH) with the index case (index HH, represented in red), (b) all members of the five nearest neighbors within a radius of 3 km from the index HH (neighbor HHs, represented in orange), and (c) randomly chosen subjects living in five control HHs > 5 km away from the index HH but still in the same or nearby locality (control HHs, represented in black). Four visits were made to each HH, the first at the time of index case diagnosis (day 0) and the following 30, 60, and 180 days later.</p

Distribution of the six most common <i>Plasmodium vivax</i> multilocus genotypes (that together accounted for 30.1% of all local infections) across seven study localities in Acrelândia, Brazil, 2013.

<p>Distribution of the six most common <i>Plasmodium vivax</i> multilocus genotypes (that together accounted for 30.1% of all local infections) across seven study localities in Acrelândia, Brazil, 2013.</p

Shared and unique <i>Plasmodium vivax</i> multilocus genotypes within cluster #3 from Gleba Porto Luiz, Acrelândia, Brazil, during four rounds of reactive case detection (days 0, 30, 60, and 180).

<p>We used the same color code as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005221#pntd.0005221.g001" target="_blank">Fig 1</a> to indicate the index HH (red), neighbor HHs (orange), and control HHs (black). Predominant or only genotypes found in each sample are identified with numerals; those recovered from the index case are underlined. Predominant genotypes shared by ≥ 2 parasite samples within the cluster were circled; for example, two index HH members shared the same predominant genotype (#81) on day 0. Identical superscript letters indicate groups of samples harboring parasite lineages (e.g., parasites recovered on day 60 from the index case—whose major genotype is #53—and one neighbor HH member [whose major genotype is #94]) that were considered genetically similar when analyzing both major and minor alleles although not identical (see main text for definition).</p

Frequency distribution of <i>Plasmodium vivax</i> blood-stage density classes (parasites per μL of blood in log scale).

<p>Parasitemias were estimated by quantitative real-time polymerase chain reaction (qPCR) and shown according to household (HH) type (panel A) and presence or absence of malaria-related clinical symptoms and signs (fever, headache, arthralgia, myalgia, and lower back pain) (panel B). The color code in panel A is the same as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005221#pntd.0005221.g001" target="_blank">Fig 1</a> to indicate index HHs, neighbor HHs, and control HHs.</p

Mixed-effects logistic regression analysis of the association between household (HH) type and risk of <i>Plasmodium vivax</i> infection diagnosed by microscopy or quantitative real-time polymerase chain reaction (qPCR) in Acrelândia, Brazil, 2013.

<p>Mixed-effects logistic regression analysis of the association between household (HH) type and risk of <i>Plasmodium vivax</i> infection diagnosed by microscopy or quantitative real-time polymerase chain reaction (qPCR) in Acrelândia, Brazil, 2013.</p

Inferring transmission pathways of <i>Plasmodium vivax</i> genotypes within malaria clusters.

<p>Circles represent individual study subjects carrying identical parasite genotypes; red and orange circles represent index and neighbor household (HH) members, respectively. Infections B1 and C, both diagnosed on day 0, have a common source (A) that had not been included in the study. D is a secondary infection with known source within the cluster (namely, B1). B2 and B3 have been diagnosed in the same subject as B1, 30 and 180 days later. If B1 was treated with chloroquine-primaquine (as all microscopy-positive infections were in our study), B2 may be either a recrudescence (due to chloroquine failure) or an early homologous relapse (due to primaquine failure to clear liver-stage hypnozoites); if B1 was missed by onsite microscopy and left untreated during our study, B2 may also represent a chronic, untreated blood-stage infection. B3 may represent either a chronic untreated infection (if B1 and B2 were left untreated) or a late homologous relapse (if chloroquine has successfully cleared blood-stage parasites but primaquine failed to clear hypnozoites).</p

Absolute and relative frequency of symptomatic and asymptomatic <i>Plasmodium vivax</i> infections diagnosed by quantitative real-time polymerase chain reaction (qPCR) among members of index, neighbor, and control households (HHs) in four rounds of reactive case detection (RCD) in Acrelândia, Brazil, 2013.

<p>Absolute and relative frequency of symptomatic and asymptomatic <i>Plasmodium vivax</i> infections diagnosed by quantitative real-time polymerase chain reaction (qPCR) among members of index, neighbor, and control households (HHs) in four rounds of reactive case detection (RCD) in Acrelândia, Brazil, 2013.</p

Shared and unique <i>Plasmodium vivax</i> multilocus genotypes within cluster #9 from Reserva Porto Dias, Acrelândia, Brazil, during four rounds of reactive case detection (days 0, 30, 60, and 180).

<p>The index, neighbor, and control HHs are shown in red, orange, and black, respectively; predominant or only genotypes were identified with numerals; the one recovered from the index case was underlined. Genotype #37 (circled) was recovered from four parasite samples, while genotype #75 was shared by an index HH member and a control HH member outside the cluster.</p