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Background: Malaria is the parasitic disease with the highest morbidity and mortality worldwide. Angola is one of the five sub-Saharan African countries with the highest malaria burden. Real-time PCR diagnosis in endemic areas has not been implemented due to its high cost and the need for adequate infrastructure. Dried blood spots (DBSs) are an alternative for collecting, preserving, and transporting blood samples to reference laboratories. The objective of the study was to assess the efficacy of DBS as a sampling method for malaria research studies employing real-time PCR., Methods: The study was divided into two phases: (i) prospective study at the Hospital Universitario Vall d'Hebron (HUVH) to compare real-time PCR from whole blood or DBS, including 12 venous blood samples from patients with positive real-time PCR for Plasmodium spp. and 10 quality control samples (nine infected samples and one negative control). Samples were collected as DBSs (10, 20, 50 µl/circle). Samples from both phases of the study were analyzed by generic real-time PCR (Plasmodium spp.) and the subsequent positive samples underwent species-specific real-time PCR (Plasmodium species) and (ii) cross-sectional study conducted at the Hospital Nossa Senhora da Paz, Cubal (Angola), including 200 participants with fever. For each patient, a fresh capillary blood specimen [for thin and thick blood films and rapid diagnostic test (RDT)] and venous blood, collected as DBSs (two 10-µl circles were combined for a total volume of 20 µl of DBS), were obtained. DBSs were sent to HUVH, Barcelona, Spain., Results: (i) Real-time PCR from whole blood collection was positive for 100% of the 21 Plasmodium spp.-infected samples, whereas real-time PCR from DBSs detected Plasmodium spp. infection at lower proportions: 76.19% (16/21) for 10 µl, 85.71% (18/21) for 20 µl, 88.24% (15/17) for 50 µl and 85.71% (18/21) for 100 µl DBSs. (ii) Field diagnosis (microscopy and/or RDT) showed a 51.5% (103/200) positivity rate, while 50% (100/200) of the DBS samples tested positive by real-time PCR. Using field diagnosis as the reference method, the sensitivity of real-time PCR in DBS samples was 77.67% with a specificity of 79.38%. Plasmodium species were identified in 86 samples by real-time PCR: 81.40% (16/86) were caused by Plasmodium falciparum, 11.63% (10/86) were coinfections of P. falciparum + P. malariae, 4.65% (4/86) were P. falciparum + P. ovale, and 2.33% (2/86) were triple coinfections., Conclusions: The DBS volume used for DNA extraction is a determining factor in the performance of real-time PCR for Plasmodium DNA detection. A DBS volume of 50-100 µl appears to be optimal for malaria diagnosis and Plasmodium species determination by real-time PCR. DBS is a suitable method for sample collection in Cubal followed by real-time PCR analysis in a reference laboratory., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Vall d'Hebron University Hospital Drug Research Ethics Committee [PR(AG)79/2024] and by the Ethics Committee of the Angolan Ministry of Health (39 C.E/MINSA.INIS/2022). Each participant signed an informed consent form (in the case of minors, parental/guardian consent was obtained) after being informed of the objectives of the study. All procedures were performed according to the ethical guidelines of the 2013 Declaration of Helsinki. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: 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)., Methods: 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., Results: 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., Conclusions: 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., (© 2024. The Author(s).)

Background: Malaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory., Methods: A total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format., Results: In field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%., Conclusions: This is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas., (© 2023. BioMed Central Ltd., part of Springer Nature.)