The World Health Organization (WHO) malaria report showed an estimated 216 million malaria cases worldwide, with 0.45 million deaths reported in 2017 from 91 countries. It is the second most reported disease in Pakistan, affecting 4.5 million individuals with 33 reported and 1100 estimated deaths in 2016 [1]. More than 80 % of nationwide malaria incidences previously reported were from the war-torn Federally Administered Tribal Area (FATA) [2]. FATA has the highest annual parasite incidence (17.64) in the country (followed by Balochistan and Khyber Pakhtunkhwa provinces) [3], and about 0.1 million cases were screened for malaria in 2016 [4]. Among many factors that influence malaria emergence in this region are political instability, the war against terrorism, mass population displacement within the country (IDPs) and across the border, underprivileged socioeconomic conditions, declining health infrastructure, poor diagnostic, preventive and curative services, regional tribal and sectarian strife all contributing to this huge disease burden [5]. For such remote and under-developed regions, malaria is severe both in local and focal infections [6, 7]. Plasmodium five species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi) are responsible for causing malarial infection in humans. Among the two globally predominant Plasmodium species, P. vivax is the most prevalent species in Pakistan that account for 80% of the malaria cases, while P. falciparum causes infection in an estimated 20% [8]. Incidence of double infections with both P. falciparum and P. vivax in FATA region has been reported 4–5 % of all malaria infection that is quite high as compared other parts of the country. In FATA region the rate of co-infection with P. falciparum and P. vivax is quite higher (4 % to 6 %) than other malaria endemic regions of the Pakistan [5]. Diagnosis and surveillance of malaria in a region where more than one species of Plasmodium genus is present are difficult to achieve without using a more sensitive, specific, and reliable diagnostic technique.
Even though empirical diagnosis results in overtreatment due to inaccuracy, it remains the basis for malaria diagnosis in a majority of the symptomatic patients in malaria-endemic regions, where advanced diagnostic facilities are lacking. Nevertheless, a clinical or presumptive diagnosis is the least expensive and most commonly used method that forms the basis for self-treatment in areas such as FATA [9].
A variety of diagnostic tools are available for Plasmodium identification and speciation, including microscopy (light and fluorescence), immuno-chromatographic techniques, nucleic acid amplification methods (PCR and isothermal amplification), and flow cytometry [10]. Dielectrophoretic and magnetophoretic approaches are also among the promising new techniques, but their sensitivity and specificity still has to be evaluated [11]. Laboratory diagnosis of malaria at the primary healthcare level in remote areas is limited to light microscopy. However, this technique has some advantages and shortcomings in proper malaria diagnosis [12]. To overcome the shortcomings of microscopy, a rapid diagnostic test (RDT) has been developed for malaria detection, which is claimed to be simple, accurate, and cost-effective diagnostic tests for malaria. Like microscopy, RDT also has limitations [13], particularly in differences in sensitivity among various kits [14].
During the last decade, the molecular diagnostic technique Polymerase Chain Reaction (PCR) (amplification of 18S rRNA genes) has gained popularity over conventional microscopic technique and RDT [15]. The PCR technique has demonstrated its high specificity and sensitivity in Plasmodium identification and speciation [15–18]. Besides detection and mixed-species identification [19–21], PCR has also proven its remarkable utility in follow-up studies on anti-malarial efficacy, molecular epidemiology, and drug resistance studies [7, 8, 13, 22]. PCR has been proven to be superior, but the molecular diagnostic setup is not always possible as it is expensive, time-consuming, needs expertise, supervision, and resources [23]. That's why PCR-based malaria diagnosis may not be feasible in remote rural areas like FATA [24].
As per WHO malaria guidelines, all suspected cases must undergo laboratory diagnosis using microscopy and RDT before any therapeutic care because information regarding the effectiveness of presumptive diagnosis is inadequate. This study was conducted to evaluate the performance of nested PCR in the detection of malarial parasites in positive microscopy samples in a warn-torn, remote malaria-endemic region of Pakistan.