Lower Microscopy Sensitivity with Decreasing Malaria Prevalence in the Urban Amazon Region, Brazil, 2018-2021

Abstract

Malaria is increasingly diagnosed in urban centers across the Amazon Basin. In this study, we combined repeated prevalence surveys over a 4-year period of a household-based random sample of 2,774 persons with parasite genotyping to investigate the epidemiology of malaria in Mâncio Lima, the main urban transmission hotspot in Amazonian Brazil. We found that most malarial infections were asymptomatic and undetected by point-of-care microscopy. Our findings indicate that as malaria transmission decreases, the detection threshold of microscopy rises, resulting in more missed infections despite similar parasite densities estimated by molecular methods. We identified genetically highly diverse populations of Plasmodium vivax and P. falciparum in the region; occasional shared lineages between urban and rural residents suggest cross-boundary propagation. The prevalence of low-density and asymptomatic infections poses a significant challenge for routine surveillance and the effectiveness of malaria control and elimination strategies in urbanized areas with readily accessible laboratory facilities.

Keywords: Amazon Basin; Brazil; Plasmodium falciparum; Plasmodium vivax; South America; epidemiology; malaria; microscopy; parasites; urban; vector-borne infections.

Figure 1

Location of the municipality of Mâncio Lima, Brazil, the study site for study of microscopy sensitivity and decreased malaria prevalence in the urban Amazon Region, Brazil, 2018–2021. Mâncio Lima (dark gray shading), population 19,294 in 2022, is situated in the upper Juruá Valley region (light gray shading) of the western Brazilian Amazon, adjacent to the border with Peru, covering an area of 5,453 km². Insert map shows location of study area in South America.

Figure 2

Monthly counts of microscopy-confirmed cases and percentages of infections diagnosed by PCR for Plasmodium vivax and P. falciparum in study of microscopy sensitivity and decreased malaria prevalence in the urban Amazon Region, Brazil, January 2018–December 2021. Circles indicate Plasmodium vivax and triangles P. falciparum. Red circles and triangles represent study waves 1 (April‒May 2018) and 2 (September‒October 2018); green, waves 3 (May‒June 2019) and 4 (September‒October 2019); blue, waves 5 (October‒November 2020), 6 (April‒May 2021), and 7 (October‒November 2021). Error bars indicate 95% CIs for prevalence rates. Anonymized malaria case notifications were downloaded from the electronic database of the Ministry of Health of Brazil (http://200.214.130.44/sivep_malaria)

Figure 3

Proportion of TaqMan-detected single-species Plasmodia vivax (A) and P. falciparum (B) infections that were detected by microscopy according to parasite density (amplicon copies per microliter measured by species-specific TaqMan assays) in study of microscopy sensitivity and decreased malaria prevalence in the urban Amazon Region, Brazil, January 2018–December 2021. Lines represents the fitted logistic model trends; the shaded area indicates 95% CI for waves 1 and 2 (2018; red), waves 3 and 4 (2019; green) and waves 5–7 (2020–2021; blue). For this analysis, data from waves 1 and 2 (2018), 3 and 4 (2019), and 5–7 (2020–2021) were combined to achieve balanced sample sizes for fitting logistic models. CI could not be properly estimated for P. falciparum infections in waves 5–7 because of the small sample size (Appendix Table 2). The dashed horizontal line indicates 50% microscopic detectability at a given parasite density threshold, which for P. vivax infections in 2018 corresponds to 2,088 (95% CI 734‒14,572) amplicon copies/μL.

Figure 4

Minimal spanning trees representing the connectivity of Plasmodium vivax haplotypes from the Juruá Valley region of Brazil (264 isolates collected 2016‒2021). Circles represent haplotypes with size linearly proportional to the number of isolates sharing them. Lines connect pairs of haplotypes with <5 allele mismatches and the overall network represents the most likely haplotype genealogy, ensuring that the summed distance of all links of the tree is the minimum possible. Haplotype colors indicate the likely site of infection (Appendix Figure 2). The circle with blue and red slices indicates a haplotype that was shared by 2 parasites from different (urban and rural) origins.

Figure 5

Minimal spanning trees representing the connectivity of Plasmodium falciparum haplotypes from the Juruá Valley region of Brazil (162 isolates collected 2018 ‒2021). Similar to Figure 4, circles represent haplotypes with size linearly proportional to the number of isolates sharing them and lines connect pairs of haplotypes with <5 allele mismatches. Haplotype colors indicate the likely site of infection (Appendix Figure 2). In 7 circles (haplotypes), slices of different colors indicate that the corresponding haplotypes were shared by parasites from different geographic origins.