Real-time PCR for malaria diagnosis and identification of Plasmodium species in febrile patients in Cubal, Angola

Malaria is the parasitic disease with the highest morbimortality worldwide. The World Health Organization (WHO) estimates that there were approximately 249 million cases in 2022, of which 3.4% were in Angola. Diagnosis is based on parasite identification by microscopy examination, antigen detection, and/or molecular tests, such as polymerase chain reaction (PCR). This study aimed to evaluate the usefulness of real-time PCR as a diagnostic method for malaria in an endemic area (Cubal, Angola). A cross-sectional study was carried out at the Hospital Nossa Senhora da Paz in Cubal, Angola, including 200 patients who consulted for febrile syndrome between May and July 2022. From each patient, a capillary blood sample was obtained by finger prick for malaria field diagnosis [microscopy and rapid diagnostic test (RDT)] and venous blood sample for real-time PCR performed at the Hospital Universitario Vall d’Hebron in Barcelona, Spain. Any participant with a positive result from at least one of these three methods was diagnosed with malaria. Of the 200 participants included, 54% were female and the median age was 7 years. Malaria was diagnosed by at least one of the three techniques (microscopy, RDT, and/or real-time PCR) in 58% of the participants, with RDT having the highest percentage of positivity (49%), followed by real-time PCR (39.5%) and microscopy (33.5%). Of the 61 discordant samples, 4 were only positive by microscopy, 13 by real-time PCR, and 26 by RDT. Plasmodium falciparum was the most frequent species detected (90.63%), followed by P. malariae (17.19%) and P. ovale (9.38%). Coinfections were detected in ten participants (15.63%): six (60%) were caused by P. falciparum and P. malariae, three (30%) by P. falciparum and P. ovale, and one (10%) triple infection with these three species. In addition, it was observed that P. falciparum and P. malariae coinfection significantly increased the parasite density of the latter. RDT was the technique with the highest positivity rate, followed by real-time PCR and microscopy. The results of the real-time PCR may have been underestimated due to suboptimal storage conditions during the transportation of the DNA eluates. However, real-time PCR techniques have an important role in the surveillance of circulating Plasmodium species, given the epidemiological importance of the increase in non-falciparum species in the country, and can provide an estimate of the intensity of infection.