Abstract:
Background:
Microscopy 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.
Methods:
Blood 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.
Results:
The 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.
Conclusions:
The 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