Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone

Abstract

Background: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone.Methods: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species.Results: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens.

Comparison of capillary and venous blood for malaria detection using two PCR based assays in febrile patients in Sierra Leone.

“…There were three samples fitting this description in the current study, independent of whether venous or capillary blood was used. However, this MMSR test result is also consistent with detection of other non-P. falciparum malaria species (for example P. malariae as previously observed [20]). In addition, there were 11 venous samples and nine capillary samples that were Plasmodium spp.-positive but P. falciparum-and P. vivax/P.…”

“…Depending on the assay and material tested, at least one malaria marker was detected in 36-52% of the tested samples (Tables 1 and 2). This result was in line with previous reports of malaria positivity among febrile individuals in this location as detected by PCR-based methods [18][19][20]. Table 1 Comparison of malaria detection by GFP and MMSR assays (n = 103 samples).…”

“…ovale in the GFP assay or Plasmodium spp. only in both assays provide further evidence for circulation of P. ovale and P. malariae in the tested population as previously reported [20].…”

“…Samples containing P. ovale may, however, be detected as malaria positive by MMSR with a Plasmodium spp. positive and P. falciparum-negative test outcome [20]. There were three samples fitting this description in the current study, independent of whether venous or capillary blood was used.…”

A novel deep learning-based point-of-care diagnostic method for detecting Plasmodium falciparum with fluorescence digital microscopy

“…Detection of Plasmodium spp. nucleic acid with nucleic acid amplification tests (NAATs) has superior analytical sensitivity compared to other methods, especially for mixed infections [9], and allows quantification of parasitaemia (by real-time quantitative polymerase chain reaction; qPCR) but is more technically demanding, expensive and therefore not widely available [4], although certain NAAT-methods, such as loop-mediated isothermal amplification (LAMP) show promise as a field-usable techniques [10]. Consequently, the World Health Organization (WHO) currently recommends microscopy-based methods to confirm diagnosis in suspected cases of malaria [3].…”