Enhanced detection of submicroscopic malaria using qPCR during an outbreak in central Uganda: implications for surveillance and elimination

Summary

Background: In August 2025, Mubende District, Uganda, reported a cluster of unexplained childhood deaths with fever, vomiting, and rapid progression was reported to the Ministry of Health Uganda. We conducted a tiered laboratory investigation to identify the etiological agent responsible for the cluster of childhood deaths and guide public health response.

Methods: Twenty-two symptomatic household contacts of the deceased children from across three districts (Mubende, Kyankwanzi, Kakumiro) were investigated. Samples underwent routine testing, malaria diagnostics (microscopy, RDT, qPCR targeting varATS), and broad pathogen exclusion (viral hemorrhagic fevers, arboviruses, bacteria, toxicology, metagenomic sequencing).

Results: Malaria was confirmed as the predominant agent (63.6% prevalence by qPCR). Parasitemia ranged from 0.135–283,282 parasites/µL. Conventional methods (microscopy/RDT) detected only 50% of qPCR-positive infections, missing seven submicroscopic cases (31.8% of all samples). All alternative etiologies were negative.

Conclusion: Malaria caused the clustered deaths. Half of infections were submicroscopic and missed by routine diagnostics. Incorporating qPCR into tiered outbreak response enhances case detection and supports elimination efforts.

 Background

Unexplained childhood deaths in sub-Saharan Africa demand rapid investigation (1). Uganda experiences endemic malaria, arboviruses, and viral hemorrhagic fevers (VHFs), complicating differential diagnosis (2,3). Recent outbreaks include Ebola (Sudan virus) in 2022 (1), Marburg in 2017 (4), and Crimean-Congo hemorrhagic fever (5). On August 9, 2025, Mubende’s Public Health Emergency Operations Centre reported a cluster of childhood deaths presenting with acute fever, headache, vomiting, and death within 24–48 hours. While severe malaria is a leading cause of childhood mortality in Uganda (6), the rapid deterioration and clustering raised concerns about VHFs or toxic exposures.

Conventional malaria diagnostics i.e. microscopy (detection limit ~50–100 parasites/µL) and RDTs miss low-density infections (7–10). Molecular methods like qPCR targeting

varATS detect <1 parasite/µL, identifying submicroscopic infections that sustain transmission (11–13). Such infections are epidemiologically important, often asymptomatic, and contribute to ongoing transmission (14,15). In Uganda, submicroscopic infections constitute a substantial hidden reservoir, particularly in school-aged children (16). We identified the etiological agents using tiered laboratory diagnostics and assessed the added value of quantitative polymerase chain reaction (q PCR) for malaria detection.

Methods

The clustered deaths occurred in Mubende, Kyankwanzi, and Kakumiro districts (Figure 1) between August and September 2025. These rural districts have tropical climate, bimodal rainfall, and close human-animal interactions (17). The three districts had also experienced outbreaks of Ebola Virus Disease (EVD), including the 2022–2023 Sudan Ebola virus outbreak that originated in Mubende District.

We collected specimens from 22 symptomatic household contacts of the deceased children identified through active case finding. Venous blood (EDTA and serum), stool, urine, and two water samples were collected and transported at 2–8°C to Mubende Regional Referral Hospital and national reference laboratories for analysis.

Baseline testing at Mubende included complete blood count, renal and liver function tests, and bacterial cultures. Microscopy (Field’s stain), mRDT (SD Bioline Malaria Ag P.f/Pan), and qPCR targeting varATS (QIAamp DNA Mini Kit, real-time PCR, detection limit <1 parasite/µL) were performed. Samples were tested for VHFs (filoviruses, arenaviruses, bunyaviruses), arboviruses (dengue, chikungunya, Zika), bacterial pathogens, toxicological agents, and metagenomic sequencing at Mubende regional referral hospital laboratory and central Emergency Response and surveillance laboratory(CERSL).

Descriptive statistics were used. qPCR served as the reference standard. Infections were categorized as high parasitemia (≥1,000 parasites/µL), submicroscopic (<10 parasites/µL, negative by microscopy/RDT), or negative.

We conducted this investigation in response to a public health emergency. The MoH authorized this investigation and the office of the Center for Global Health, US Centers for Disease Control and Prevention determined that this activity was not human subject research and with its primary intent being for public health practice or disease control. We obtained permission to conduct the investigation from the district health authorities of Mubende region where the cases were identified. We obtained verbal consent from the guardians and assent from the respondents since all were below 18years of age.

Results

Among 22 symptomatic household contacts, complete blood count and renal function tests were generally normal. Liver function tests showed mild alkaline phosphatase elevation in some individuals, but no severe abnormalities. qPCR detected malaria in 14/22 samples (63.6% prevalence). Parasitemia ranged from 0.135 to 283,282 parasites/µL.

Seven samples (31.8%) had high parasitemia (1,177–283,282 parasites/µL) and were positive by qPCR, RDT, and microscopy (perfect concordance). Seven samples (31.8%) were submicroscopic (0.135–5.155 parasites/µL), detected only by qPCR and missed by both RDT and microscopy. Eight samples (36.4%) were negative by all methods. Conventional diagnostics detected only 7 of 14 malaria-positive cases (50%). qPCR doubled case detection, increasing measured prevalence from 31.8% to 63.6% (Table 1). All samples tested negative for VHFs, arboviruses, bacterial pathogens, toxicological agents, and novel pathogens by metagenomic sequencing.

Table 1. qPCR, mRDT, and microscopy results

Discussion

This investigation identified malaria as the primary cause of clustered childhood deaths in Mubende, Kyankwanzi, and Kakumiro districts. The 63.6% malaria prevalence among symptomatic contacts, combined with comprehensive negative results for other high-consequence pathogens, confirms malaria as the predominant agent. This aligns with Uganda’s high burden of severe malaria (6,18,19). The tiered approach demonstrated that qPCR detected low-density parasitemia missed by microscopy and RDTs, identifying seven additional infections (50% of all positives). This finding is consistent with studies from Ghana and Uganda showing qPCR sensitivity of 39.3% for microscopy and 55.7% for HRP2-based RDTs (7) . Field evaluations of the Bioline RDT in Uganda showed >91% sensitivity for high-density infections but frequent false negatives for low-density infections (9).

Perfect concordance among methods for high-density infections (≥1,000 parasites/µL) validates RDTs for point-of-care diagnosis in resource-limited settings but highlights their limitation in detecting low-parasitemia cases (8,10). Doubling of case detection through qPCR (from 31.8% to 63.6% prevalence) shows that reliance on conventional diagnostics alone would have substantially underestimated outbreak magnitude. The detection of seven submicroscopic infections (31.8% of all samples, 50% of positives) reveals a substantial hidden reservoir invisible to conventional surveillance. Submicroscopic infections are often paucisymptomatic, persistent, and contribute to ongoing transmission (14,15). Their relative infectiousness to mosquitoes is approximately one-third that of patent infections, but their long duration and high prevalence make them epidemiologically important (20).

Longitudinal studies from eastern Uganda showed qPCR detected an additional 12.1 percentage points of prevalence above microscopy, with school-aged children producing >50% of mosquito infections (21). Other studies confirm that school-aged children harbor a disproportionate burden of submicroscopic infections (16,22,23). Global meta-analyses reveal that the proportion of submicroscopic infections is highest in low-transmission settings and varies geographically (24). For elimination programs, relying solely on microscopy or RDT risks underestimating prevalence and missing persistent transmission foci (25). Sensitive molecular diagnostics are essential for identifying the hidden reservoir (17).

Current Ugandan surveillance systems mainly use microscopy and RDTs, which under detect low-density infections. In our investigation, qPCR increased case detection from 31.8% to 63.6%, demonstrating that conventional methods miss a substantial proportion of infections. For outbreak investigations, molecular diagnostics improve characterization of outbreak magnitude and transmission patterns. For elimination programs, sensitive methods are critical for identifying submicroscopic infections that sustain residual transmission (17,26,27). However, implementation is limited by cost, infrastructure, and training needs (28).

Study limitations: The sample size was small (n=22) and non-random, limiting generalizability. We could not sample deceased children (already buried), precluding direct etiological confirmation in index cases. Clinical outcome data and treatment response were not systematically assessed. We did not perform gametocyte-specific assays or mosquito feeding experiments to directly assess transmission potential. Nonetheless, the investigation achieved its primary objectives.

Conclusion: This tiered investigation identified malaria as the primary cause of clustered childhood deaths in Mubende, Kyankwanzi, and Kakumiro districts. Negative results for VHFs, arboviruses, bacteria, and toxic exposures ruled out other high-risk etiologies. qPCR detected twice as many infections as microscopy and RDTs, revealing a substantial submicroscopic reservoir missed by routine surveillance.

Recommendation: Integrate molecular diagnostics such as qPCR into tiered diagnostic strategies for malaria outbreak investigations, surveillance in elimination settings, and monitoring of control program effectiveness. While conventional diagnostics remain appropriate for clinical case management in high-transmission settings, molecular methods are essential for comprehensive case ascertainment, detection of transmission foci, and verification of elimination.

Conflict of Interest: The authors declare no conflict of interest.

Authors’ contributions: VN took the lead in conceptualizing the project, data curation, investigation analysis writing the original draft. FN, MM, KM, NM, BM,KAK were involved investigation, designing methodology, writing, reviewing the article. SG, MRE were involved in supervision and editing to ensure scientific integrity. All authors read and approved the final article.

Acknowledgements:  The authors are grateful to the Uganda Public Health Fellowship Program and the Ministry of Health for the technical guidance and support provided during this investigation. We appreciate the National Reference Laboratories for conducting laboratory testing and providing timely results. We acknowledge Mubende Regional Referral Hospital and the district health teams for their support in field coordination, case investigation, and data collection.

Copyrighting and licensing: All materials in the Uganda Public Health Bulletins is in the public domain and may be used and printed without permission. However, citation as to source is appreciated. Any article can be reprinted or published. If cited as a reprint, it should be referenced in the original form

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