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Background Current malaria diagnostic methods require blood collection, that may be associated with pain and the risk of transmitting blood-borne pathogens, and often create poor compliance when repeated sampling is needed. On the other hand, the collection of saliva is minimally invasive; but saliva has not been widely used for the diagnosis of malaria.

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The aim of this study was to evaluate the diagnostic performance of saliva collected and stored at room temperature using the OMNIgene ®•ORAL kit for diagnosing Plasmodium falciparum malaria. Results Prevalence of malaria detected by TFM, nPCR-saliva and nPCR-blood was 22, 29, and 35%, respectively. Using TFM as the gold standard, the sensitivity of nPCR-saliva and nPCR-blood in detecting P. Falciparum was 95 and 100%, respectively; with corresponding specificities of 93 and 87%. When nPCR-blood was used as gold standard, the sensitivity of nPCR-saliva and microscopy was 82 and 68%, respectively; whereas, the specificity was 99 and 100%, respectively. Nested PCR-saliva had a very good agreement with both TFM (kappa value 0.8) and blood PCR (kappa value 0.8). At parasitaemia > 10,000 parasites/µl of blood, the sensitivity of nPCR-saliva was 100%.

Nested PCR-saliva detected 16 sub-microscopic malaria infections. One year after sample collection, P. Falciparum DNA was detected in 80% of saliva samples stored at room temperature. Background Plasmodium falciparum malaria remains one of the most important infectious diseases in sub-Saharan Africa (SSA). Even with the significant reduction in malaria morbidity and mortality since the beginning of the third millennium, 214 million people (88% living in SSA) acquired malaria and 438,000 people (90% from SSA) died from malaria in 2015 []. Transmission-blocking interventions, such as insecticide-treated nets, have been hailed as significant contributors to the decrease in the number of malaria cases and deaths. The proportion of children less than 5 years old in SSA who sleep under insecticide-treated nets increased from.

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Study design and population A cross-sectional study was conducted from October 2014 to April 2015 in selected health care facilities in three regions of Cameroon, i.e., the Far North, Centre, and Northwest regions. In the Far North region, the study was conducted in the Maroua regional hospital; the Catholic Health Center Nkolbisson was the study site in the Center region; while the PMI Nkwen Bamenda and the Mother of Mary Hospital Widikum were study sites in the Northwest region. Malaria transmission is perennial in both the Center and North West regions, but peak transmission occurs at different times of the year; whereas, malaria transmission is seasonal in the Far North region. All three regions were selected as part of an on-going study aimed at characterizing the etiologies of non-malarial fevers in Cameroon. Individuals at least 2 years old with axillary temperature above 37.5 °C at presentation or complaint of fever within 24 h preceding enrollment were included in the study. Informed consent was obtained from eligible participants who were above 18 years. Parents or legal guardians of younger children gave written informed assent on behalf of their children.

Ethical approvals were obtained from the Committee on Human Subjects of the University of Hawaii (protocol number CHS 21724) and the National Research Ethics Committee of the Ministry of Public Health Cameroon (protocol Number 2014/04/442/CE/CNERSH/SP). Administrative approvals were also obtained from the directors of the various health institutions and from the Ministry of Public Health, Cameroon. Sample collection and storage After informed consent had been obtained, paired blood and saliva samples were collected from each participant. Approximately 2 ml of venous blood was drawn into an EDTA tube. Blood samples were transported in cold boxes to the laboratory where they were aliquoted and stored at − 20 °C until DNA extraction. Participants were requested to dispense approximately 1 ml of saliva into OMNIgene ®•ORAL (OM-501) kit (DNA Genotek, Ottawa, Ontario, Canada) following recommendations from the manufacturer. To avoid blood contamination of saliva, individuals with overt gum bleeding or who complained of pain in any part of their mouth were excluded.

All saliva samples were stored at room temperature from the time of collection until DNA extraction (about 2–6 weeks). After 12–13 months of storage at room temperature DNA was re-extracted from saliva aliquots of patients who original tested positive for P. Falciparum DNA in saliva. Malaria microscopy Thick blood smears were prepared, stained with 10% Giemsa and read by two trained microscopists. A slide was considered positive if one or more blood-stage malaria parasites was detected after reading at least 100 high power fields. Parasitaemia (parasites/µl of blood) was determined by counting the number of parasites against 200 white blood cells and multiplying a constant of 8000 white blood cells/µl of blood.