Evaluating Malaria Rapid Diagnostic Tests and Microscopy for Detecting Plasmodium Infection and Status of Plasmodium falciparum Histidine-Rich Protein 2/3 Gene Deletions in Southeastern Nigeria

Abstract

Delays in malaria diagnosis increase treatment failures and deaths. In endemic regions, standard diagnostic methods are microscopy and malaria rapid diagnostic tests (mRDTs) detecting Plasmodium falciparum histidine-rich protein 2/3 (PFHRP2/PFHRP3), but gene deletions can allow certain parasites to remain undetected. We enlisted a cohort comprising 207 symptomatic individuals, encompassing both children and adults, at a hospital in Nnewi, Nigeria. The prevalence of parasites was determined using a highly sensitive, species-specific quantitative polymerase chain reaction (SS-qPCR). Within a subset of 132 participants, we assessed the sensitivity and specificity of microscopy and HRP2-mRDTs in comparison to SS-qPCR for the detection of P. falciparum. We also investigated the prevalence of pfhrp2/pfhrp3 gene deletions. Greater sensitivity was achieved with mRDTs (95%) compared with microscopy (77%). Also, mRDTs exhibited greater specificity (68%) than microscopy (44%). The positive predictive value of mRDTs (89%) surpassed that of microscopy (80%), suggesting a greater probability of accurately indicating the presence of infection. The negative predictive value of mRDTs (82%) was far greater than microscopy (39%). Of the 165 P. falciparum-positive samples screened for pfhrp2/pfhrp3 gene deletions, one gene deletion was detected in one sample. Regarding infection prevalence, 84% were positive for Plasmodium spp. (by reverse transcription [RT]-qPCR), with P. falciparum responsible for the majority (97%) of positive cases. Thus, exclusive reliance on microscopy in endemic areas may impede control efforts resulting from false negatives, underscoring the necessity for enhanced training and advocating for high-throughput molecular testing such as RT-qPCR or qPCR at referral centers to address limitations.

Figure 1.

Geographic overview of the study sites in Nigeria. The shading represents the sampling site. This map was created using Mapchart, v. 4.2 (https://www.mapchart.net/africa.html).

Figure 2.

Demographic characteristics of the study population.

Figure 3.

Distribution of Plasmodium species composition in the study population. Each circle represents 1% of a total 100%.

Figure 4.

Venn diagram for diagnostic tests. The included data comprise only those samples for which all three diagnostic tests were conducted. The number of positive (A) and negative (B) diagnostic tests results for microscopy, malaria rapid diagnostic tests (mRDTs), and species-specific quantitative polymerase chain reaction (SS-qPCR) are given. Overlapping sections represent the agreement of the results among the different tests. TBS = thick blood smear.

Figure 5.

Qualitative agreement of thick blood smears (TBSs) and malaria rapid diagnostic tests (mRDTs) with species-specific quantitative polymerase chain reaction (qPCR) and the cycle quantification (Cq) value. The dotted line represents the threshold of positivity (Cq = 37). Samples with a greater Cq value are defined as being negative (–) for a Plasmodium spp. infection. + = positive infection.

Figure 6.

Fourplex quantitative polymerase chain reaction Plasmodium falciparum histidine-rich 2 gene assay results of P. falciparum–positive samples. Ct = cycle threshold.