Performance of rapid diagnostic test, blood-film microscopy and PCR for the diagnosis of malaria infection among febrile children from Korogwe District, Tanzania

Despite a remarkable reduction in Plasmodium falciparum transmission across sub-Saharan Africa, malaria still remains a public health problem, especially among children aged under 5 years [1, 2]. In line with the decline, there is an urgent need for improved diagnostic tools in order to prepare for resurgence [3]. It is crucial to understand the performance of different malaria diagnostic tests in different settings during this new epidemiological context.

Fever has been the major complaint amongst children presenting at outpatient clinics, with malaria being the possible aetiology for such febrile illnesses [4]. Integrated management of childhood illness (IMCI) guidelines was developed by the World Health Organization (WHO) to improve clinical management of febrile illnesses in developing countries [5]. However, in 2010 as a result of over-diagnosis coupled with over-prescription of anti-malarials, WHO updated the guidelines specifically to improve diagnosis of malaria infection by restricting anti-malarial treatment to patients with positive test results [6, 7]. Despite this recommendation the performance of malaria diagnostic tools is affected by several factors, including low transmission, low parasite density and lack of qualified technicians for microscopy [8, 9]. This underscores the need to investigate further accurate diagnosis of malaria infection in febrile patients towards optimal use of anti-malarials, case management of asymptomatic cases and disease surveillance.

Despite its low sensitivity and limited availability, microscopy still remains the gold standard for malaria diagnosis. Malaria microscopy is time consuming, requires skilled laboratory technicians and is often subject to unreliable results from different laboratories [10]. With the availability of malaria rapid diagnostic testing (RDT), WHO recommended its use for prompt and accurate confirmation of P. falciparum infection in settings with limited laboratory facilities [11]. Both microscopy and malaria RDT have limited detection threshold, especially in situations with low parasitaemia [12, 13]. Polymerase chain reaction (PCR) is regarded as one of few most sensitive molecular techniques for detecting parasites at limits of 0.01–0.2 parasites/μL of blood [14, 15]. As with microscopy and malaria RDT, there have been different PCR assays reporting varying sensitivities and specificities [16, 17]. Nonetheless, despite being highly sensitive, PCR is expensive and cannot be utilized in routine practice in resource-limited settings.

A total of 1380 sick children attending the outpatient clinic at KDH from January to October 2013 were screened; 867/1380 (62.8 %) met the inclusion criteria and were enrolled. The median age was 15.1 months (interquartile range (IQR): 8.6–29.9) and girls accounted for 408/867 (47.1 %) of patients (Table 1). Samples from all patients were tested for malaria infection using HRP-2-based RDT and 85/867 (9.8 %, 95 % CI, 7.9–12.0 %) were positive. Seventy-two samples (8.3 %, 95 % CI, 6.5–10.1 %) were positive for P. falciparum by microscopy with geometric mean parasitaemia of 37,635 (4715–12,959) parasites/μL. Dried blood spots samples on filter paper for PCR analysis were prepared and performed from 677/867 (78.1 %) patients, of which 79/677 (11.7 %, 95 % CI, 9.3–14.3 %) were positive for P. falciparum. Samples from 190/867 (21.9 %) patients were not available for PCR analysis; 153/190 (80.5 %) patients did not have sufficient blood for spotting on a pre-made filter paper and 37/190 (19.5 %) patients had low yield DNA extracted from dried blood spots on filter paper. The complete flow of the performed diagnostic tests and results are outlined in Fig. 1.

The trends of malaria positivity by age group were similar between the three diagnostic methods, and the odds ratio of being diagnosed positive increased significantly by age (p < 0.001 for all three diagnostic tools), Fig. 2. The odds ratio in age group 2–12 months was 4.4 (95 % CI, 1.99–9.74 %) while that of children aged 36–59 months was 11.76 (95 % CI, 5.0–27.65 %) compared to that of children under 12 months old.

This study evaluated the performance of different diagnostic tools in detecting malaria infection among febrile children attending outpatient clinic at Korogwe District. PCR performed better than blood slide expert microscopy and HRP-2-based malaria RDT. Overall, malaria prevalence was low and significantly associated with age where the majority of patients were over 12 months old. Infants appear to be relatively protected from malaria (and other infections) for the first 6 months of life, mainly due to acquiring maternal antibodies and the presence of foetal haemoglobin [22‐24]. Three-quarters of malaria patients had low levels of parasitaemia (<200,000 parasites/μL).

The use of HRP-2-based malaria RDT in the current study resulted in over-diagnosis of malaria infection in 15 % of patients. This might suggest the presence of recent malaria infection. When compared with expert microscopy and PCR, the sensitivity value of malaria RDT was below the ≥95 % threshold recommended by WHO. These findings are similar to those reported by Shakely and colleagues on a hospital-based study conducted in Zanzibar, an area of low malaria transmission [25]. HRP-2-based malaria RDT is known for detecting malaria antigens that continue to circulate in blood almost 2 months after treatment of a malaria episode [26]. This is the most probable explanation for the false-positive results and sub-microscopic infections that were observed in the study. In the current study, eight patients had false-negative malaria RDT results despite having moderate to high levels of parasitaemia. This could be due to malaria parasites expressing low level of target antigen, deleted pfhrp2 gene, prozone effect or other factors, which could not be established [27‐32]. However, current information from WHO indicates that in most settings, genetic mutations (deletion of pfhrp2/pfhrp3) in parasites are not likely to be the main cause of false-negative results [33]. Therefore, further research is required to determine the true prevalence of these mutations. Despite these limitations, HRP-2-based malaria RDTs remain the preferred choice, mainly in settings with limited microscopy facilities, due to easy availability and low cost [34]. There are continued efforts to improve sensitivity and specificity of malaria RDTs for rapid and better management of malaria cases. Clinicians are advised to investigate other causes of febrile episodes despite a positive malaria RDT test. Different types of infections are common in children from resource-limited areas that lack adequate sanitation and clean water supply. Therefore, it is very likely that children attending outpatient clinics from rural settings present with multiple infections [35].

Identification of sub-microscopic infection by PCR has demonstrated that expert microscopy can miss detection of malaria in patients having low parasite densities hence leading to false-negative results. As malaria transmission declines, cases of sub-microscopic infection in both symptomatic and asymptomatic persons is likely to increase [36]. PCR and other molecular techniques are indicated to be the most sensitive diagnostic tool than microscopy and RDT [37, 38]. However, its utility in routine practice remains a concern especially at this era towards malaria elimination.

The study had a number of limitations. Firstly, some of the patients may have been on anti-malarial therapy despite verbal confirmation provided by their parents/guardians on the prior use of anti-malarial drugs. This would have contributed to the false-positive RDT results. Secondly, the malaria RDT and PCR diagnostic tools used in the current study could not detect other species of Plasmodium apart from P. falciparum. This could have missed a few malaria infections of other species, such as P. malariae and P. ovale that have been reported in the country [29, 39, 40]. Both P. malariae and P. ovale occur more commonly as mixed infections with P. falciparum [40‐42]. Lastly, the study may have biased the estimated prevalence of sub-microscopic infections due to the unavailability of nearly a quarter of patient samples for PCR analysis.

The prevalence of malaria infection was low among outpatient children from Korogwe District. Diagnosis of malaria infection is still a challenge due to variation in results among diagnostic methods. HRP-2 malaria RDT and microscopy were less sensitive than PCR. Diagnostic tools with high sensitivity are required in areas of low malaria transmission.