Field evaluation of the Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test: Causes of Microscopy Discordance and Performance in Uganda

TL;DR

This study evaluates a malaria rapid diagnostic test in Uganda, finding it highly sensitive but with issues of false positives due to persistent antigens.

Contribution

The study quantifies the performance and causes of discordance for a combination malaria RDT in a real-world setting.

Findings

The Bioline Malaria Ag P.f/Pan RDT had high sensitivity (>91%) for detecting malaria.

False negatives were mainly due to low-density P. falciparum or non-falciparum infections.

False positives were common due to persistent antigenemia, leading to overuse of antimalarials.

Abstract

Histidine Rich Protein 2 (HRP2)/pan-Lactate Dehydrogenase (pLDH) combination Rapid Diagnostic Tests (RDTs) may address the shortcomings of RDTs that detect HRP2 alone. However, the relative contribution of the possible causes of discordant results (RDT-negative and microscopy-positive) and performance in field settings are poorly quantified.

This study utilized samples from two cross-sectional surveys conducted in 32 districts at 64 sites across Uganda between November 2021 and March 2023 that enrolled 6354 febrile participants ≥ two years of age. Discordant samples (negative by HRP2/pLDH RDT and positive by microscopy) underwent quantitative PCR (qPCR) to detect and quantify parasitemia. Those confirmed to be positive for P. falciparum at > 1 parasites/microliter (p/μL) were tested for pfhrp2 and pfhrp3 deletions using digital PCR. Those that were negative or had P. falciparum detected at ≤ 1 p/μL underwent Plasmodium species testing using nested PCR. The performance of the Bioline Malaria Ag P.f/Pan combination RDT was evaluated by comparison with microscopy and qPCR.

There were 166 (8.4%) discordant samples out of 1988 microscopy positive samples. Of these, 90/166 (54.2%) were confirmed to contain P. falciparum at levels > 1 p/μL whereas 76/166 (45.8%) were negative or had P. falciparum levels ≤ 1 p/μL. Only one P. falciparum positive sample was confirmed to have a deletion in pfhrp3. The primary reasons for RDT-negative, microscopy-positive discordance in samples testing negative for P. falciparum were non-falciparum species (37/76, 48.7%) or false positives by microscopy (31/76, 40.8%). The sensitivity of the Bioline Malaria Ag P.f/Pan combination RDT was high (> 91%) using either microscopy or qPCR as the gold standard. However, specificity was low (56.7%) when microscopy was used as the gold standard; it improved to 64.0% when qPCR was used as the gold standard.

The Bioline Malaria Ag P.f/Pan combination RDT was found to be highly sensitive in Uganda and reliable for ruling out malaria. False negative RDT results were primarily due to low density P. falciparum infections, non-falciparum infections, or incorrect microscopy results. In contrast, false positive RDT results were common due to persistent antigenemia; this may result in overuse of antimalarial drugs and missed diagnoses of non-malarial febrile illnesses.

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