Malaria species prevalence among asymptomatic individuals in four regions of Mainland Tanzania

Abstract

Recent studies point to the need to incorporate non-falciparum species detection into malaria surveillance activities in sub-Saharan Africa, where 95% of malaria cases occur. Although Plasmodium falciparum infection is typically more severe, diagnosis, treatment, and control for P. malariae, P. ovale spp., and P. vivax may be more challenging. The prevalence of these species throughout sub-Saharan Africa is poorly defined. Tanzania has geographically heterogeneous transmission levels but an overall high malaria burden. In order to estimate the prevalence of malaria species in Mainland Tanzania, 1,428 samples were randomly selected from 6,005 asymptomatic isolates collected in cross-sectional community surveys across four regions and analyzed via qPCR to detect each Plasmodium species. P. falciparum was most prevalent, with P. malariae and P. ovale spp. detected at lower prevalence (<5%) in all four regions. P. vivax was not detected. Malaria elimination efforts in Tanzania will need to account for these non-falciparum species.

Tanzania has one of the highest malaria burdens in the world, accounting for 4.1% of global malaria deaths in 2021[1]. While most malaria cases in Tanzania and elsewhere in sub-Saharan Africa are caused by Plasmodium falciparum, four other Plasmodium species (P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri) are present to varying degrees. There is also data to suggest that these species have higher prevalence than previously known, and may become more prevalent as P. falciparum is controlled and ultimately eliminated[2–6] in line with the WHO goal of a 90% reduction in global malaria burden by 2030[7]. Non-falciparum malaria may require different control measures, due to major differences in biology, including different anopheline vectors with different seasonal peaks[8], relapse and/or chronic infections[9,10], lower parasitemia[11], and higher rates of asymptomatic infection[8].

Previous work in Mainland Tanzania has characterized non-falciparum prevalence in schoolchildren (5–16 years)[6] and non-falciparum positivity rates among symptomatic patients[12]. In schoolchildren, P. ovale spp. prevalence (24%) was similar to that of P. falciparum (22%)[6], while in symptomatic patients, P. falciparum was much more abundant than non-falciparum malaria, although P. ovale spp. positivity rates surpassed 5% in seven of ten regions[12]. In both studies, P. malariae was less common than either P. falciparum or P. ovale spp., and P. vivax was rare[6,12]. In this study, we characterize the prevalence of all malaria species among asymptomatic individuals across all ages in three regions with moderate and high malaria transmission intensity, and in children under five in one region with high transmission.

The study protocol was approved by the Tanzanian Medical Research Coordinating Committee (MRCC) of the National Institute for Medical Research (NIMR) and involved approved standard procedures for informed consent and sample deidentification. Additional details are described elsewhere[13]. Deidentified samples were considered non-human subjects’ research at the University of North Carolina and Brown University.

A random subset of 1,428 dried blood spot (DBS) samples were drawn from a total of 5,860 asymptomatic samples. 694 samples were drawn from a total of 2,647 collected from all age groups during cross-sectional community surveys in Kigoma (n=252/878, high transmission), Ruvuma (n=186/741, high transmission), and Tanga (n=256/1,028, moderate transmission) regions during the Molecular Surveillance of Malaria in Tanzania (MSMT) project in 2021[13]. The random subset was representative of the regional sample distribution (X² (3,2) = 2.43, p (2 df) = 0.3, Table S1), but not representative of the age group distribution (X² (3,2) = 10.46, p (2 df) = 0.005, Table S2), or the sex distribution (X² (2,2) = 43.65, p (1 df) < 0.001, Table S3). An additional 734 samples were drawn from 3,213 collected from children under five during cross-sectional household surveys for the group antenatal care project (GANC)[14,15] in Geita in 2021.

The molecular analyses used to detect Plasmodium spp. in each sample are described in detail elsewhere[12]. Briefly, we performed a separate 18S qPCR assay for each species, which allows for both the detection of each species as well as a semi-quantitative parasitemia estimate. For each region, we calculated prevalence for each species, including both single-species and mixed-species infections. Regional-level maps of prevalence for each species were created using the R package sf (version 1.0–9) based on shape files available from GADM.org and naturalearthdata.com accessed via the R package rnaturalearth (version 0.3.2)[16]. Variation in species-specific prevalence by region and age group (young children <5 years, school-aged children 5–16 years, and adults >16 years, as previously described[12]) was assessed for significance with generalized linear models or ANOVA, as appropriate, in R.

Excluding the Geita participants who were all under five and whose ages were not recorded, the median age of the remaining 694 participants was 20 years (IQR 8–47) with a range of 6 months to 87 years. Including the Geita participants, children (≤16 years old) constituted 74.2% of participants (n=1,060), while adults (>16 years old) constituted the remaining 25.8% (n=368). Young children (<5 years old) comprised 77.9% (n=826) of the child participants, while the remaining 22.1% (n=234) were school-aged (5–16 years old). Sex identifications were available for 694 participants and female-skewed, with 505 female (72.8%) and 189 male participants (27.2%). 21.1% (n=301) of sampled individuals were RDT-positive.

Among all 1,428 samples analyzed, P. falciparum was detected in 34.2% (n=488, 95% CI: 31.7%–36.7%), P. malariae in 1.5% (n=22, 95% CI: 0.99%–2.4%), and P. ovale spp. in 3.4% (n=49, 95% CI: 2.6%–4.5%). P. vivax was not detected. P. malariae infections were nearly evenly split between single-species infections (45.5%, n=10/22) and mixed-species infections with P. falciparum (40.9%, n=9/22), with the remaining three (13.6%) being triple infections with P. falciparum and P. ovale spp. (Table 1). In contrast, most P. ovale spp. infections were mixed with P. falciparum (65.3%, n=32/49), with single-species infections being less common (28.6%, n=14/49) and triple infections comprising the remainder (Table 1). P. malariae had the highest median parasitemia at 164,080 p/μL (IQR 9,942–1,333,100 p/μL), followed by P. falciparum at 55,200 p/μL (2,910–775,000 p/μL) and P. ovale spp. at 11,868 p/μL (1,271–70,840 p/μL). However, there was no significant difference in parasitemia by species (ANOVA p=0.9).

Table 1.

Infection composition proportions for isolates infected with P. malariae and P. ovale spp.

Infection Type	Count	Proportion
Pm	10	45.5%
Pm/Pf	9	40.9%
Pm/Pf/Po	3	13.6%
All Pm Infections	22
Po	14	28.6%
Po/Pf	32	65.3%
Pm/Pf/Po	3	6.1%
All Po Infections	49

^*Sum of proportions may not equal 100% due to rounding.

All three species were detected in each region (Figure 1). Geita and Tanga had the highest P. falciparum prevalence (37.8% and 36.7%, n=278/734 and 94/256 respectively, Table S4). P. malariae was relatively rare in all four regions, with the highest prevalence in Tanga (3.1%, n=8/256) and the lowest in Geita (0.7%, n=5/734, Table S4). P. ovale spp. prevalence was slightly higher than P. malariae (3.2%−4.0%, n=6/186–10/252) in all regions except Tanga (2.3%, n=6/256, Table S4). There was significant variation (by ANOVA) in prevalence between regions for P. falciparum (p<0.001) and P. malariae (p=0.04) but not for P. ovale spp.

Figure 1 –

Maps of Tanzania showing A) location of study regions, B) P. falciparum regional prevalence, C) P. malariae regional prevalence, and D) P. ovale spp. regional prevalence. P. vivax was not detected, so is not mapped.

While age was a significant (GLM p<0.001) determinant of P. falciparum infection, there was no significant effect of age for either P. malariae or P. ovale spp. Age group was a significant (ANOVA p<0.05) determinant of infection likelihood for both P. falciparum and P. malariae, but not P. ovale spp. (Figure 2). While children were significantly (p<0.05) more likely than adults to have P. falciparum, adults were more likely to have P. malariae (p<0.05, Figure 2). There was no significant interaction between age group and region for either P. falciparum or P. malariae, but the interaction was nearly significant (p=0.055) for P. ovale spp.

Figure 2 –

Tukey Analysis of Malaria Species Prevalence by Age Group. A total of 826, 234, and 368 were in the Young Children (<5 years), Schoolchildren (5–16 years), and Adult (>16 years) groups, respectively. The total number of samples per group for each species is shown in the X-axis labels while the number of positive samples for each group is shown in the bar labels. Comparisons marked with a * are significant at the p<0.05 level. Panel A shows P. falciparum prevalence by age group. Significant pairwise comparisons are marked, while the other is insignificant. Panel B shows P. malariae prevalence by age group. One significant pairwise comparison is marked, while the others are insignificant. Panel C shows P. ovale spp. prevalence by age group. No pairwise comparisons are significant.

This study builds on previous research with schoolchildren and clinic patients to describe the prevalence of different malaria species within four regions of Mainland Tanzania. Although P. falciparum is the most prevalent species, both P. malariae and P. ovale spp. prevalence surpasses 3% in at least one region, and could increase as P. falciparum is locally eliminated. In contrast to a 2017 study on schoolchildren, which found P. ovale spp. prevalence to be similar to P. falciparum[6], we found P. ovale spp. prevalence to be much lower than that of P. falciparum. In addition, the schoolchildren survey mostly identified P. ovale spp. as single-species infections and P. malariae as mixed with P. falciparum[6], whereas we found similar proportions of single-species and mixed-species P. malariae infections and most of our P. ovale spp. samples were mixed with P. falciparum. However, our sample sizes are small and may not necessarily be representative of the full picture, particularly in Geita where samples were only collected from children under 5 years old. In addition, our study only includes four regions, only one of which (Tanga) overlaps with the previous study, and we include a wider age range.

In Tanga, we may have found lower P. ovale spp. prevalence than the previous study due to the inclusion of adults, who are less likely to test positive for this species, whereas schoolchildren are a major asymptomatic infectious reservoir[17–19], although we did not replicate a significant difference in this study. In addition, the disruptions to malaria control caused by the COVID-19 pandemic, particularly in 2020–2021[20], could have an increase in P. falciparum prevalence. Indeed, there was no difference in the malaria prevalence in the villages of Magoda, Mamboleo, and Mpapayu between 2019 (24.9%) and 2021 (24.5%, unpublished data). However, the prevalence dropped to 6.4% in 2022 (unpublished data), so further longitudinal study may clarify the impact of resumed intense P. falciparum control. However, the malaria prevalence in Tanga dropped from 34.8% to 26.2% between 2020 and 2021 (unpublished data), so P. falciparum control in this region was likely effective during the course of this study.

Unlike in our study of symptomatic patients[12], we did not find schoolchildren to be significantly more likely to test positive for either P. malariae or P. ovale spp. Although not significant, this trend is present in P. ovale spp. prevalences in this study, so the lack of significance in these species is likely an artifact of small sample sizes (Table 1, Figure 2, Figure S1). Our finding that P. malariae was significantly more prevalent among adults likely reflects the presence of chronic infections[10].

Although P. falciparum remains the most prevalent species in these four regions, P. malariae and P. ovale spp. are present in all four, whereas P. vivax is not detected. Achieving malaria elimination in Tanzania will require ongoing surveillance of these species. While standard treatments successfully clear P. falciparum, the 3.4% of patients in this study with P. ovale spp. may relapse. This study serves as a complement to previous studies focusing on schoolchildren and symptomatic patients to paint a full picture of the non-falciparum malaria landscape for communities in Mainland Tanzania. Ongoing analysis of samples collected in 2022 and 2023 will allow us to detect temporal trends in prevalence, and forthcoming genomic analysis of P. malariae and P. ovale spp. isolates from Tanzania will inform our understanding of population structure and diversity in these species.

Data availability:

Data is available upon reasonable request to the corresponding author.