Prevalence of Plasmodium falciparum and Non- falciparum Infections by Photo-Induced Electron Transfer-PCR in a Longitudinal Cohort of Individuals Enrolled in a Mass Drug Administration Trial in Southern Province, Zambia

Abstract

Malaria burden in Zambia has significantly declined over the last decade because of improved coverage of several key malaria interventions (e.g., vector control, case management, bed net distributions, and enhanced surveillance/responses). Campaign-based mass drug administration (MDA) and focal MDA (fMDA) were assessed in a trial in Southern Province, Zambia, to identify its utility in elimination efforts. As part of the study, a longitudinal cohort was visited and tested (by PCR targeting the 18s rRNA and a Plasmodium falciparum-specific rapid diagnostic test [RDT] from SD Bioline) every month for the trial duration (18 months). Overall, there was high concordance (> 97%) between the PCR and RDT results, using the PCR as the gold standard. The RDTs had high specificity and negative predictive values (98.5% and 98.6%, respectively) but low sensitivity (53.0%) and a low positive predictive value (53.8%). There was evidence for persistent antigenemia affecting the low specificity of the RDT, while false-negative RDTs were associated with a lower parasite density than true positive RDTs. Plasmodium falciparum was the dominant species identified, with 98.3% of all positive samples containing P. falciparum. Of these, 97.5% were mono-infections and 0.8% coinfections with one other species. Plasmodium malariae was found in 1.4% of all positive samples (50% mono-infections and 50% coinfections with P. falciparum), whereas Plasmodium ovale was found in 1.1% of all positive samples (90% mono-infections and 10% coinfections with P. falciparum). Although MDA/fMDA appeared to reduce P. malariae prevalence, P. ovale prevalence appeared unchanged.

Figure 1.

Parasite density of Plasmodium falciparum photo-induced electron transfer–PCR–positive samples stratified by the rapid diagnostic test result (A) or the microscopy result (B). Diagnostic results are shown as positive (red) or negative (gray). Microscopy positive samples (A) are shown as solid squares, while microscopy negative or samples not assessed by microscopy are shown as solid circles.

Figure 2.

Plasmodium falciparum infections identified by both the rapid diagnostic test (RDT) and photo-induced electron transfer–PCR for all samples where both test results are available for two or more consecutive months. Combined results for the current month are shown as true negatives (gray) or positives by the RDT (false positive) or PCR (false negative) or PCR and RDT (true positive) (orange), stratified into the same groups for the previous month. Samples are expressed as a percentage of the column total, with inset figures showing the number of samples.

Figure 3.

Plasmodium falciparum infections identified by both the rapid diagnostic test (RDT) and photo-induced electron transfer-PCR for all samples where both test results are available for two or more consecutive months. The breakdown for the current month, true negative^curr (A), false positive^curr (B), false negative^curr (C), or true positive^curr (D) is shown in each of the 4 panels. Bars are colored as false negatives (pink), false positives (gray), or true positives (red) for the previous month, and expressed as a percentage of the total (excluding true negative^prev), with inset figures showing the number of samples.

Figure 4.

Plasmodium malariae (A) and Plasmodium ovale (B) infections identified by photo-induced electron transfer-polymerase chain reaction per month by trial arm in the cohort for the duration of the study. Infections are shown as rapid diagnostic test (RDT)–positive (red) or RDT-negative (gray), and the size of the square denotes the number of infections (small = 1, large = 2). No P. ovale infections were found in the focal mass drug administration arm of the trial.

Figure 5.

Schematic of assumed infection progression and associated diagnostic outcomes. Early in the infection, HRP2 concentration is below the RDT limit of detection (LOD), whereas Plasmodium falciparum DNA is above the PCR LOD, giving a false-negative result. Later in the infection, sufficient HRP2 has accumulated to yield a positive rapid diagnostic test (RDT), yielding a true positive. Finally, in the posttreatment/clearance phase, parasite DNA is absent, whereas HRP2 persists, giving a false-positive RDT result. Note that progression of an infection through the above stages is not linear or absolute, that is, a false-negative infection may never develop to a true positive.