Malaria Diagnosis

Article Citation: Al-Awadhi, M. A. (2020, October 18). Malaria Diagnosis. Kuwaiti Journal of Medical Parasitology.

Malaria is a deadly parasitic infection caused predominantly by Plasmodium falciparum and Plasmodium vivax parasites. The parasite is transmitted from an infected female Anopheles mosquito to the human host during a blood meal. Therefore, the endemicity of malaria within a region depends largely on the presence of Anopheles mosquitos, stagnant bodies of freshwater (natural or man-made) where mosquitos lay eggs, and humans in the vicinity. Worldwide, malaria causes more than 400,000 deaths annually, the vast majority of which occur in tropical and sub-tropical African countries (WHO, 2020).

Malaria is suspected when a patient’s body temperature is higher than 37.5°C and is residing in or has a travel history to a malaria-endemic country. To diagnose malaria, the WHO (2018) recommends microscopy or malaria rapid diagnostic test (RDT) for all patients with suspected malaria before treatment is administered.

Light microscopy of Giemsa-stained thick and thin blood smears is the diagnostic standard for malaria and allows species-level identification of different Plasmodium spp., the parasite stage and the quantification of parasite density when monitoring the progress of treatment. Rapid diagnostic tests (RDTs) may be used at times when the services of expert microscopists are not available, as in rural health centers, or when an urgent test result is required (e.g. ports of entry, patient in critical condition). RDTs are commonly based on Plasmodium lactate dehydrogenase (pLDH) and P. falciparum histidine-rich protein 2 (PfHRP-2) antigen detection, which are useful due to their release into the peripheral blood by P. falciparum-infected erythrocytes (Desakorn et al., 1997). A previous study in the Cameroon has demonstrated that PfHRP-2 detection in the blood of pregnant women was superior to traditional microscopy, where about 20% of malaria-positive women at delivery have shown negative results in peripheral and intervillous space thick blood smears (Leke et al., 1999). However, the main drawback of RDTs is false-positivity due to antigen circulation in the blood after parasite elimination, which could persist for 7-60 days depending on the screened antigen and type of antimalarial therapy (Dalrymple et al., 2018; Iqbal et al., 2004). Moreover, recent studies have reported RDT misdiagnosis due to PfHRP-2 and PfHRP-3 gene deletions (parasite mutation) in highly endemic African countries such as Djibouti, Ghana and Uganda (Amoah et al., 2020; Bosco et al., 2020; Iriart et al., 2020).

Various molecular techniques have been developed to increase the sensitivity of malaria diagnosis which include polymerase chain reaction (PCR), nested PCR, multiplex PCR, quantitative (q)-PCR, capture and ligation probe (CLIP)-PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and high throughput (Ht)-LAMP (Tedla, 2019). A study had recently demonstrated malachite green (MG)-LAMP as a useful tool, especially in resource-limited regions, producing less false-positives and -negatives than microscopy and RDTs (Gachugia et al., 2020). However, such techniques are usually complex to perform, require expensive instrumentation and reagents, or are more time-consuming than microscopy and RDTs. Nonetheless, molecular techniques remain important to investigate Plasmodium spp. genotypes, which are of particular importance in the parasite’s resistance mechanisms to antimalarial drugs and in monitoring the geographical distribution of drug resistant genotypes. Moreover, qPCR is more efficient in detecting and distinguishing mixed Plasmodium spp. infections than microscopy and RDTs (Wardhani et al., 2020).

Interestingly, a recent study had demonstrated fluorescent nucleic acid staining followed by flow cytometry as efficient tools in detecting Plasmodium-infected RBCs, having the capability to differentiate the parasite developmental stage in about 1 minute (Toya et al., 2020). Other studies are demonstrating new concepts which utilize machine learning (ML) and algorithms in the diagnosis of malaria and automation of blood film reporting (De Bruyne, 2020), in addition to the use of ML in predicting Plasmodium mitochondrial protein sequences for use as targets of anti-malarial drugs (Bian et al., 2020). However, cost-effectiveness remains an important issue, especially in low-income countries which suffer the highest burden of malaria-attributed morbidity and mortality. While RDTs remain the most feasible choice to diagnose malaria in highly affected countries, the continuous surveillance of ever-evolving Plasmodium genotypes and modification of RDT components accordingly are of high importance.


Amoah, L. E., Abuaku, B., Bukari, A. H., Dickson, D., Amoako, E. O., Asumah, G., Asamoah, A., Preprah, N. Y., & Malm, K. L. (2020). Contribution of P. falciparum parasites with Pfhrp 2 gene deletions to false negative PfHRP 2 based malaria RDT results in Ghana: A nationwide study of symptomatic malaria patients. PloS one15(9), e0238749.

Bosco, A. B., Anderson, K., Gresty, K., Prosser, C., Smith, D., Nankabirwa, J. I., Nsobya, S., Yeka, A., Opigo, J., Gonahasa, S., Namubiru, R., Arinaitwe, E., Mbaka, P., Kissa, J., Won, S., Lee, B., Lim, C. S., Karamagi, C., Cunningham, J., Nakayaga, J. K., … Cheng, Q. (2020). Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019. Malaria journal19(1), 300.

Dalrymple, U., Arambepola, R., Gething, P. W., & Cameron, E. (2018). How long do rapid diagnostic tests remain positive after anti-malarial treatment?. Malaria journal, 17(1), 228.

De Bruyne, S., Speeckaert, M. M., Van Biesen, W., & Delanghe, J. R. (2020). Recent evolutions of machine learning applications in clinical laboratory medicine. Critical reviews in clinical laboratory sciences, 1-22. Advance online publication.

Desakorn, V., Silamut, K., Angus, B., Sahassananda, D., Chotivanich, K., Suntharasamai, P. et al., (1997). Semi-quantitative measurement of Plasmodium falciparum antigen PfHRP2 in blood and plasma. Transactions of the royal society of tropical medicine and hygiene, 91(4), 479-483.

Gachugia, J., Chebore, W., Otieno, K., Ngugi, C. W., Godana, A., & Kariuki, S. (2020). Evaluation of the colorimetric malachite green loop-mediated isothermal amplification (MG-LAMP) assay for the detection of malaria species at two different health facilities in a malaria endemic area of western Kenya. Malaria journal19(1), 329.

Iqbal, J., Siddique, A., Jameel, M., & Hira, P. R. (2004). Persistent histidine-rich protein 2, parasite lactate dehydrogenase, and panmalarial antigen reactivity after clearance of Plasmodium falciparum monoinfection. Journal of clinical microbiology, 42(9), 4237-4241.

Iriart, X., Menard, S., Chauvin, P., Mohamed, H. S., Charpentier, E., Mohamed, M. A., Berry, A., & Aboubaker, M. H. (2020). Misdiagnosis of imported falciparum malaria from African areas due to an increased prevalence of pfhrp2/pfhrp3 gene deletion: the Djibouti case. Emerging microbes & infections9(1), 1984-1987.

Leke, R. F., Djokam, R. R., Mbu, R., Leke, R. J., Fogako, J., and Megnekou, R. (1999). Detection of the Plasmodium falciparum antigen histidine-rich protein 2 in blood of pregnant 144 women: Implications for diagnosing placental malaria. Journal of clinical microbiology, 37(9), 2992-2996.

Tedla, M. (2019). A focus on improving molecular diagnostic approaches to malaria control and elimination in low transmission settings: Review. Parasite epidemiology and control6, e00107.

Toya, Y., Tougan, T., Horii, T., & Uchihashi, K. (2020). Lysercell M enhances the detection of stage-specific Plasmodium-infected red blood cells in the automated hematology analyzer XN-31 prototype. Parasitology international, 102206. Advance online publication.

Wardhani, P., Butarbutar, T. V., Adiatmaja, C. O., Betaubun, A. M., Hamidah, N., & Aryati (2020). Performance comparison of two malaria rapid diagnostic test with real time polymerase chain reaction and gold standard of microscopy detection method. Infectious disease reports12(Suppl 1), 8731.

World Health Organization. (2018, January 18). Overview of malaria treatment.

World Health Organization. (2020, January 14). Malaria.