Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories

Abstract

Objective. To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods. We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results. The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman’s test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions. The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

Molecular Method Is Essential to Identify Asymptomatic Malaria Reservoirs: A Successful Experience in the Malaria Elimination Program in Iran

“…The blood samples were transferred to the laboratory of the molecular medicine center of Hormozgan University of Medical Sciences under standard conditions for detection by PCR method. The sensitive molecular techniques yields more accurate result, according to the findings of earlier investigations [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. The most sensitive method is nested-PCR, which detects Plasmodium parasite levels in blood at 2–5 parasites per microliter [ 30 , 31 ].…”

“…Many studies have emphasized that the application of several malaria detection methods simultaneously, with a focus on molecular methods to detect asymptomatic reservoirs, is critical in global malaria control and elimination efforts. This significant strategy is being employed in different parts of the world, and in all investigations a sensitive molecular technique has been used alongside conventional methods [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Quantitative molecular tools (qPCR) and nested-PCR are robust and sensitive methods in malaria diagnosis; in addition, some researchers showed that PET-PCR is effective in field conditions for molecular screening and examining a large number of samples [ 38 , 39 , 40 ].…”

“…Several studies in Iran and other countries have examined the efficiency of molecular detection in malaria control and elimination programs. Most researchers have suggested that the use of molecular techniques to detect asymptomatic parasitic reserves is essential in malaria elimination programs [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].…”

Advanced Techniques and Unusual Samples for Malaria Diagnosis

“…Polymerase Chain Reaction is an advanced molecular technique that has been known to be used in the diagnosis of genetic and infectious diseases including malaria, the technique is usually applied for the confirmation of different Plasmodium species in a given sample [42], especially when the outcomes of other conventional technique (e.g Microscopy and or RDTs) indicate negative results [43] and is more sensitive compared other conventional techniques [44], especially when the parasitaemia level is very low [45]. Some studies have shown that PCR has recorded a sensitivity and specificity of 100% each by detecting 1-3 Plasmodium parasite/μL of blood samples and it also has a good ability to detect mixed infection [46].…”

Performance analysis of polymerase chain reaction in relation to rapid diagnostic test (RDT) kit (SD bio line malaria ag p.f (05fk50)) for malaria diagnosis

“…The disease is transmitted to humans through the bite of an infected female Anopheles mosquito during a blood meal [3]. Five Plasmodia species, namely Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and P. knowlesi [4] are the parasites responsible for the actual disease condition with P. falciparum being the most lethal [2,5] and is the most predominant specie in Nigeria and other sub-Saharan African countries [6]. Over half of the world's population lives in malaria-prone areas [7], and according to World Health Organisation (WHO), in 2017 there were about 219 million clinical cases of malaria and about 435,000 malaria deaths in 87 countries in the world, and Nigeria accounts for 25% of these cases [6].…”