Jonathan J. Juliano

Background: Antimalarial drug resistance (AMDR) continues to threaten malaria control and elimination, underscoring the need for timely and reliable malaria molecular surveillance. Data generation through therapeutic efficacy studies (TES) and independent surveys is critical, yet limited guidance exists to support policymakers and National Malaria Control Programs (NMCPs) in selecting appropriate laboratory techniques for different surveillance objectives and operational contexts. This descriptive technical review aimed to assess the currently used laboratory techniques based on their technical and operational characteristics, identify knowledge gaps and provide decision support for Malaria Molecular Surveillance (MMS) of Plasmodium falciparum . Methods: The literature search was conducted in PubMed and supplemented by citation searching. Following title and abstract screening, data were extracted on predefined performance, operational, and technical characteristics. A non-exclusionary assessment of reporting quality was also performed. The results were reviewed by field experts, who added additional information, based on the findings. Results: Thirty-nine studies published between 1998 and 2025 met the inclusion criteria. Performance varied between PCR-based and sequencing-based techniques, with analytical sensitivity ranging from 1–1000 parasites/µL for PCR-based techniques, and 1–2000 parasites/µL for sequencing-based techniques. Clinical sensitivity and specificity were high across all techniques, though highest for sequencing-based methods. PCR-based techniques provided rapid results and required less infrastructure for the targeted detection of known SNPs but could not identify novel markers. Sequencing-based techniques offered high multiplexing potential and could often detect minority variants and discover novel markers. However, they required greater capital investment, more extensive training, and substantial bioinformatics capacity. Cost data were highly variable and often incompletely reported, highlighting a knowledge gap. We developed a structured decision-support framework, illustrated through three hypothetical country scenarios reflecting differing surveillance needs, laboratory capacities, and resource constraints. These examples demonstrate that no single method is universally optimal and that technique selection should be tailored to specific surveillance objectives, epidemiological context, existing infrastructure, budget, and long-term capacity-building goals. Conclusion: This review provides an updated overview of available techniques and practical guidance to support stakeholders in selecting accurate, feasible, and sustainable approaches for monitoring AMDR through MMS, while also highlighting key knowledge gaps.

Background: Larval source management (LSM) has been a core strategy of malaria control but is difficult to implement in holoendemic regions of rural sub-Saharan Africa. More targeted approaches that reduce the burden of effort but maintain impact are needed. Methods: The prospective cohort study examined the spatial relationship between mosquito breeding sites and malaria cases to guide targeted LSM. After enrolling households across 3 villages, we recorded malaria cases from neighboring health facilities and conducted larval and adult mosquito surveillance. Breeding site density was assessed within different search radii from malaria cases using GIS. Households were grouped by environmental similarity using factor analysis of mixed data and k-means clustering, and clusters were compared in terms of malaria incidence and mosquito abundance. Results: We identified and surveyed 69 breeding sites (March–May 2023, 90% larva positive) in the study area and documented 56 clinical malaria cases among 366 households (April–June 2023). Malaria incidence was higher within 200 m of breeding sites than in households outside 200 m (0.20 vs 0.09 cases per household, P = .005). When the search spread outward from cases, the number of covered breeding sites increased rapidly, reaching 60% within 200 m and 75% within 300 m before plateauing. Malaria risk and mosquito abundance varied across clusters, particularly near the hydroelectric station spillway and in areas with different house construction. Conclusions: The findings support the potential of reactive LSM by targeting breeding sites near malaria cases. Further research is needed to assess its effectiveness and its added value in malaria control programs.

Background: In settings where malaria transmission has declined, subpatent malaria due to low-density Plasmodium falciparum infection remains prevalent. Low-density parasitemia persisting over time may represent dynamic turnover of parasite genetic variants. Methods: We performed targeted parasite sequencing in 330 persons with asymptomatic malaria in Bagamoyo, Tanzania from 2018 to 2021. A subset of 286 with subpatent parasitemia (RDT-negative, PCR-positive) were followed over 4 weeks to examine outcomes and parasite genetic make-up within individuals in the absence of treatment. Infectiousness to mosquitoes was assessed using direct skin feeding assays. Results: Parasite diversity based on microhaplotypes at 4 hypervariable markers (CSP, AMA1, SERA2, TRAP) was high, with a mean of 1.7 (range 1–8) unique microhaplotypes per sampling. While roughly half of RDT-negative parasitemias were transient, in those with persistent subpatent carriage, parasite strain turnover was frequent, with 91% of those sampled 3 times showing new microhaplotypes at subsequent visits. Among the 28% (79/286) of subpatent carriers who developed RDT positivity within 4 weeks, 94% (15/16 with paired genotyping) were associated with turnover in the major haplotype in at least one marker, whereas persistence of the major haplotype was more common in those with persistent subpatent carriage. Infectiousness appeared to increase in those who developed patent malaria but not in those with persistent carriage. Conclusions: In coastal Tanzania, subpatent malaria is often transient while persistent carriage involves dynamically changing parasite variants and low-level infectivity to mosquitoes. Patent malaria with higher transmission potential develops in the setting of newly inoculated or ascendant genotypes.

Aedes mosquitoes are widely distributed across the Democratic Republic of Congo (DRC), and are major vectors of dengue (DENV), Zika, chikungunya (CHIKV), and yellow fever (YFV) viruses. While the high burden of malaria in the DRC receives considerable attention, arboviruses remain understudied. In the setting of recent CHIKV and YFV outbreaks in southwestern DRC, we collect Aedes mosquitoes in three areas of Kimpese, DRC, near the Angola border, to investigate their virome. Metagenomic and targeted sequencing of eight randomly selected field mosquito pools, comprising 155 mosquitoes from three collection sites, confirm high-confidence DENV reads and human blood meals in six (75%) and eight (100%) pools, respectively. We find diverse mosquito viromes including other known and putative human and animal viruses. Our findings provide strong evidence of endemic DENV transmission along the DRC-Angola border and illustrate the potential of wild-caught mosquitoes for xenosurveillance of emerging pathogens.

Malaria remains a major public health challenge in the Democratic Republic of Congo. To address the lack of contemporary entomological data, this four-year study characterized key entomological parameters in Kinshasa Province. We conducted a prospective observational cohort study (2018–2022) in three health areas. Adult mosquitoes were sampled using CDC light traps and pyrethrum spray catches. Sporozoite rates were estimated using ELISA, and trap collections were used as proxy measures of host-seeking mosquito activity to estimate light trap rates and annualized entomological inoculation rate (EIR) estimates. Insecticide susceptibility of adult females was assessed using WHO tube tests. A generalized estimating equations model evaluated the effects of key variables on mosquito abundance. A total of 1,449 Anopheles were collected, with 50% from rural, 49% from peri-urban, and 1% from urban areas. The species captured were An. gambiae s.l. (85%), An. paludis (11%), and An. funestus (4%). An. gambiae s.l. was significantly more abundant indoors and after 10 PM. The overall EIR estimate was 4.7 infective mosquitoes per trap per year, with An. gambiae s.l. contributing the highest transmission intensity. A localized increase in the transmission intensity associated with An. paludis was observed in one site during the rainy season, although mosquito numbers were small. Resistance to DDT and pyrethroids was widespread, with an estimated 0.94 frequency of the kdr L1014F mutation. Bendiocarb and pirimiphos-methyl remained effective. This study found that An. gambiae s.l. remains the predominant malaria vector in Kinshasa. The findings also suggest a possible contribution of An. paludis to malaria transmission in some rural settings, although additional longitudinal studies with larger sample sizes are needed. Because sporozoite detection was based on ELISA, additional molecular confirmation studies would be valuable. Continued entomological surveillance is essential to track biting behavior and resistance patterns and ensure that selected interventions remain effective.

PfSMARRTer- An updated version of PfSMARRT This is a multiplex PCR protocol for amplifying ama1, crt, mdr1, dhfr, dhps, and k13. The PCR primers were adopted from: AMA1 - PMID:29246158 CRT - designed for this protocol MDR1 - PMID:15132750 (MDR184 designed for this protocol) DHFR & DHPS - PMID:15273102 and Am J Trop Med Hyg. 2008 Jun; 78(6): 892–894 K13 – designed for this protocol HeOME: Aranda-Díaz A, Vickers EN, Murie K, Palmer B, Hathaway N, Gerlovina I, et al. Sensitive and modular amplicon sequencing of diversity and resistance for research and public health. bioRxiv. 2024.

Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP), an antifolate drug with antimalarial and antibiotic activity, reproducibly improves birthweight across sub-Saharan Africa and the Western Pacific. This clinical protection is independent of SP’s original malaria indication: it is not diminished by widespread antimalarial resistance or reduced transmission, and SP outperforms more potent non-antibiotic antimalarials (e.g., dihydroartemisinin-piperaquine, DP) for fetal growth. The biological mechanism is unexplained. We previously showed that gestational weight gain (GWG) is a significant component of this mechanism and mediates two-thirds of SP’s overall birthweight benefit ( NCT03009526 ). In the first longitudinal characterization of antifolate antibiotic effects on the pregnant gut microbiome, we show that ∼45% of SP’s GWG advantage over DP is explained by gut microbial changes consistent with its pharmacology. Microbiome-mediated GWG coincided with 126g higher birthweight in SP but not DP recipients (95%CI 22.6-229.3g; p=0.019). Relative to DP, SP suppressed gastrointestinal pathobionts and enriched anaerobic commensals with recognized roles in mucosal immunity and host metabolism, a microbiome-sparing pattern distinct from conventional antibiotic-associated dysbiosis.

BACKGROUND: Malaria caused by Plasmodium malariae is geographically widespread and sometimes associated with prolonged infection, yet little is known about its genomic epidemiology. METHODS: We performed hybrid capture and whole-genome sequencing of 77 isolates collected from Cameroon ( n = 7), the Democratic Republic of the Congo ( n = 16), Nigeria ( n = 4), and Tanzania ( n = 50) between 2015 and 2021, analyzing parasite genetic population structure and demography. RESULTS: There is no evidence of geographic population structure. Nucleotide diversity was significantly lower than in colocalized P . falciparum isolates, while linkage disequilibrium was significantly higher. Genome-wide selection scans identified no erythrocyte invasion ligands or antimalarial resistance orthologs as top hits; however, targeted analyses of these loci revealed evidence of selective sweeps around 4 erythrocyte invasion ligands and 6 antimalarial resistance orthologs. Demographic inference modeling suggests that African P . malariae is recovering from a bottleneck. CONCLUSION: P . malariae is genomically atypical among human Plasmodium spp. and lacks strong population structure in Africa. The low diversity has potential impacts on understanding persistent versus new infection through genomic epidemiology. FUNDING: Bill & Melinda Gates Foundation (grant 002202), USAID/PMI through Jhpiego and CDC, NIH (T32AI007151, T32AI070114, R01AI107949, R01AI129812, R21 AI148579, R01AI137395, R21AI152260, R01AI132547, and K24AI134990), and the DELTAS Africa initiative (DELGEME grant 107740/Z/15/Z).

Background: Effective malaria case management relies on accurate diagnosis and appropriate treatment. However, rapid diagnostic test (RDT) performance varies, and presumptive treatment remains common. We quantified malaria misdiagnosis and inappropriate treatment and assessed adherence to treatment guidelines in a high-burden setting. Methods: We leveraged samples collected during a cohort study of children and adults from 2015 to 2022 across 7 sites in Kinshasa Province, Democratic Republic of the Congo (DRC). We estimated the cumulative incidence and prevalence of false-positive and false-negative RDT results using polymerase chain reaction (PCR) as the gold standard and identified correlates of misdiagnosis and inappropriate treatment using Kaplan–Meier and generalized estimating equations. Results: Among 2269 participants, the 1-year cumulative incidences of false-positive and false-negative RDTs were 23.1% and 17.8%, respectively; by 7 years, these rose to 77.7% and 83.4%. Misdiagnosis (false-positive or false-negative RDT) was associated with age, recent treatment, transmission intensity, and parasite density—factors that could inform refinements to treatment guidelines. About 58% of RDT-negative participants received antimalarial treatment at a clinic visit, while 4% of RDT-positive participants did not. Fever significantly reduced adherence to test-and-treat guidelines. Conclusions: Most participants experienced malaria misdiagnosis by RDT over the course of the study, underscoring the limitations of currently used RDTs and opportunities for improved clinical decision-making. While RDTs remain essential for malaria case management, clinicians should consider contextual factors when interpreting results. Efforts to improve adherence to guidelines, which require a positive diagnostic test result before treatment, and management of non-malarial febrile illness are needed in the DRC and similar high-burden settings.