This study provides evidence of the absence of qPCR-positive, false-negative RDT results and therefore of pfhrp2/3 deletions in P. falciparum isolates circulating in Monrovia (Liberia). Among the 1038 individuals included in the study, only 22 had a negative RDT and a positive microscopy. Sixteen of these samples tested by qPCR confirmed the absence of P. falciparum DNA, therefore indicating a false positive result by microscopy. Results of this study suggest that P. falciparum parasites circulating in Monrovia do not yet carry pfhrp2/hrp3 deletions and are, therefore, conveniently detectable using PfHRP2-based RDTs. However, continuous monitoring for the emergence of PfHRP2 deletions is needed to avoid RDT failures that could potentially compromise malaria control programs in Liberia.
The prevalence of P. falciparum infections among individuals with fever or history of fever during the preceding week was 19.6% by RDT and 21.0% by microscopy. Among those individuals who were negative by both diagnostic tests, the prevalence of P. falciparum infection by qPCR was 5.3%, indicating a moderate level of low-density malaria infections which are undetected among febrile individuals. The carriage of sub-patent infections might be higher among afebrile individuals, as observed in pregnant women at first ANC visit (9.2%), who tend to carry asymptomatic low-density infections . Overall, the low malaria positivity rates in Monrovia compared to estimates from other African countries [23, 24] might be due to the relatively lower risk of malaria infection among the population residing in Monrovia compared to the rural areas in Liberia. Positivity rate is higher among individuals reporting joint pain, vomiting, chills and shivers and weakness. In contrast, cough and nausea were associated with lower malaria positivity rates, suggesting these clinical signs may appear to be resulted by other diseases such as respiratory infections. Positivity rate was also higher among older individuals, suggesting that occupational or motility factors may contribute to increased risk of exposure to malaria parasites in areas outside Monrovia with higher transmission.
The specificity of RDT, compared to microscopy, was high (99%), with most of the false positive results being negative by qPCR, suggesting HRP2 persistence after a recently cleared P. falciparum infection . False negative results were more abundant, with 11% of the microscopy-positive subjects were negative by RDT. This is below the overall estimate of 19.9% obtained from community-based malaria surveys in 19 sub-Saharan African countries . Importantly, qPCR confirmed the absence of P. falciparum DNA in the 16 samples tested, indicating that the discordant results were due to either incorrect microscopy readings or infection of other malaria species. Independent of the reason above, this study rules out the possibility of true (qPCR-confirmed) parasitaemic cases undetected by the RDT. This thus provides evidence that none of the parasite isolates collected in this study were potential carriers of pfhrp2/hrp3 deletions.
This study has several limitations. First, a subset of dried blood spots (including 6 of the 22 which were collected from individuals with RDT-negative but microscopy-positive results) were not available for molecular testing. Second, the fees of consultation and malaria diagnostic tests in the institution of recruitment may have led to an underrepresentation of populations with low social-economic backgrounds who may be more prone to malaria infection. Finally, the qPCR is P. falciparum-specific, and does not provide molecular information on other species. This fails to conclude whether the qPCR-negative but microscopy-positive samples could be due to incorrect microscopic examinations or infections of non-falciparum parasites.