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Background: The parasite species Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), formerly known as Plasmodium ovale, are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species., Methods: Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC., Results: The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/μl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/μl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/μl for P. ovalecurtisi and P. ovalewallikeri, respectively., Conclusions: We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JBP reports research support from Gilead Sciences, non-financial support from Abbott Laboratories, and consulting for Zymeron Corporation, all outside the scope of the manuscript. All other authors declare no competing interests., (Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low-density infections are often submicroscopic, not detected with rapid diagnostic tests (RDTs) or microscopy but detectable by polymerase chain reaction (PCR)., Methods: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over 4 weeks., Results: Among 6076 participants, P. falciparum prevalences by RDT, microscopy, and PCR were 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female sex, dry season months, screened windows, and bed net use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over 4 weeks, 23% of submicroscopic carriers (61 of 266) became RDT positive, with half exhibiting symptoms, while half (133 of 266) were no longer parasitemic at the end of 4 weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence., Conclusions: Microheterogeneity in transmission observed at the village level appears to affect both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum . Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission., Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018., Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias , suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes., Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors., Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel., Competing Interests: SC, NB, MM, BN, OA, VG, KN, DG, ZP, CH, ZP, AM, JO, RV, SM, SS, LM, UM, AM, JL, AB, JJ, JB No competing interests declared, (© 2023, Connelly et al.)

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Achieving malaria elimination requires considering both Plasmodium falciparum and non-P. falciparum infections. We determined prevalence and geographic distribution of 4 Plasmodium spp. by performing PCR on dried blood spots collected within 8 regions of Tanzania during 2017. Among 3,456 schoolchildren, 22% had P. falciparum, 24% had P. ovale spp., 4% had P. malariae, and 0.3% had P. vivax infections. Most (91%) schoolchildren with P. ovale infections had low parasite densities; 64% of P. ovale infections were single-species infections, and 35% of those were detected in low malaria endemic regions. P. malariae infections were predominantly (73%) co-infections with P. falciparum. P. vivax was detected mostly in northern and eastern regions. Co-infections with >1 non-P. falciparum species occurred in 43% of P. falciparum infections. A high prevalence of P. ovale infections exists among schoolchildren in Tanzania, underscoring the need for detection and treatment strategies that target non-P. falciparum species.

Background: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed., Methods: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development., Results: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae., Conclusions: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum., (© 2022. The Author(s).)

Ultrasensitive PCR used in low-transmission malaria-endemic settings has revealed a much higher burden of asymptomatic infections than that detected by rapid diagnostic tests (RDTs) or standard PCR, but there is limited evidence as to whether this is the case in higher transmission settings. Using dried blood spots (DBS) collected among 319 schoolchildren in Bagamoyo, Tanzania, we found good correlation (Pearson's R = 0.995) between Plasmodium falciparum parasite densities detected by a DNA-based 18s rRNA real-time PCR (qPCR) and an RNA-based ultrasensitive reverse transcriptase (RT)-PCR (usPCR) for the same target. Whereas prevalence by usPCR was higher than that found by qPCR (37% versus 32%), the proportion of additionally detected low-density infections (median parasite density < 0.050 parasites/µL) represented an incremental increase. It remains unclear to what extent these low-density infections may contribute to the infectious reservoir in different malaria transmission settings.