High-resolution melting analysis reveals low Plasmodium parasitaemia infections among microscopically negative febrile patients in western Kenya

Abstract

BackgroundMicroscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting.MethodsBlood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections.ResultsThe coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials.ConclusionsThe enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions.

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

“…This will greatly enhance understanding of arboviral disease threats, resulting in improved healthcare delivery and appropriate use of the limited supply of antimalarial and antibiotic drugs in endemic areas (Kipanga et al . ).…”

“…; Kipanga et al . ) , confounding estimates of arboviral infection incidence rates and disease burden (Hotez & Kamath ). Moreover, many arboviruses that contribute to human and livestock disease may still be unknown (Junglen & Drosten ; Rosenberg et al .…”

High and Heterogeneous Prevalence of Asymptomatic and Sub-microscopic Malaria Infections on Islands in Lake Victoria, Kenya

“…Given the year-round availability of fresh water, a high density of malaria vectors is found on these islands, as well as along the lakeshore communities78. Compared to Rusinga Island910 and some mainland areas in the Lake Victoria basin where epidemiological studies have been conducted11, there is a dearth of information on malaria endemicity on Mfangano and surrounding small islands. In response, this cross-sectional study aimed to determine the prevalence and geographical distribution of malaria infections among populations on islands in Lake Victoria, Homa Bay County, Kenya.…”

The effectiveness of microscopy: Rapid diagnostic test and molecular assay in diagnosing malaria.

“…However, malaria infection cannot be diagnosed with symptoms only, it should be confirmed by laboratory diagnosis [17,18]. The aim of the study is to determine the effectiveness of three different diagnostic methods (microscopy, Rapid Diagnostic Test and molecular assay) in the diagnosis of malaria infection in patients at the Federal University of Technology Health Centre, Akure.…”