Background
HIV and malaria geographically overlap in Ghana [
1,
2]. HIV co-infection has been shown to increase the vulnerability of the population to malaria [
3‐
5]. Out of the 9248 AIDS deaths reported in Ghana in 2014, 1295 (14%) were children; and 31.6% of the children who died from AIDS were under 5 years old. This age group is also considered a malaria-vulnerable group [
6]. A mathematical model has been used to postulate that HIV increases the biomass of
Plasmodium and results in an increase in emergence of anti-malarial resistance [
7]. This was based on the assumption that biomass increment will increase the rate of mutations, if the probability of de novo resistance mutations is evenly distributed among all parasites [
7]. Hence, drug administration will select for genetic mutations and spread drug resistance [
7]. As such, a substantial contribution to malaria transmission and drug resistance might originate from this sub-population of patients living with HIV. It is imperative that missed malaria infections in this population are minimal, in order to control malaria transmission and reduce drug resistance.
Improved rapid diagnostic tests (RDT), which are faster, cheaper and easier compared to the gold standard expert microscopy, are complementing malaria control efforts in resource-limited sub-Saharan African countries such as Ghana [
8]. In Ghana, most of the rapid diagnostic test kits that are available are based on histidine-rich protein-2 (HRP2) [
9‐
11], which is an antigen exclusive to
P. falciparum [
12]. However, recent reports have shown that
hrp-
2 deletions in parasite genes (
pfhrp2), which previously prevailed only in the South Americas, have now been detected in African countries like Ghana, Democratic Republic of Congo, Eritrea, Uganda and Rwanda, regardless of level of transmission [
8]. The lack of
pfhrp2 expression can prevent the detection of parasites by HRP2-based RDTs and give false negative results [
8].
In spite of the preponderance of HRP2-based RDTs in Ghana, very limited studies have been conducted on the circulation of
pfhrp2 deletion in parasites in the Ghanaian population. An assessment of diagnostics in the Bongo District of northern Ghana showed that health workers in the Community-based Health Planning and Services (CHPS) had challenges with inaccurate or invalid RDT results, which were HRP2-based [
13]. However, whilst there was no further investigation into possible circulation of parasites with
pfhrp2 gene deletions, another study found the mutation in Central (40%) and Greater Accra (22.4%) regions of Ghana [
9]. Missed malaria infections, as a result of inability of RDTs to detect parasites, can result in increased morbidity and mortality, especially in children under 5 years old [
8]. This could be more severe in people living with HIV. This study, therefore, looked at the performance of an HRP2-based RDT on the Ghanaian market and expert light microscopy, in under 5 year olds who are living with HIV in two anti-retroviral therapy (ART) centers in Atibie (Eastern region) and Accra (Greater Accra region), Ghana.
Methods
Study site and population
Data was collected at two ART clinics, the Ridge Regional Hospital and Kwahu Government Hospital. The Ridge Regional Hospital is located in the Greater Accra region whilst the Kwahu Government Hospital is located in the Eastern region of Ghana. Malaria prevalence in Greater Accra is relatively lower at 4–10%, whilst that in the Eastern region is relatively higher at 22% [
10]. The vegetation in Eastern region is forest, whilst that in Greater Accra is coastal savannah. Malaria transmission is perennial in both study sites.
Both clinics provide health services to patients living with HIV. They also provide free access to anti-retroviral drugs, counseling and prophylaxis. HIV negative participants were recruited from the out-patient department (OPD) of the main hospital. For this category, only patients who had their HIV-negative status clearly indicated in their folders were included in this study.
Study design and participant selection
This was a cross-sectional study, performed from May (beginning of the major raining season) to July 2015. The study was explained to parents of all children under 5 years old before enrollment. Consecutive patients attending the ART clinics (for HIV positive participants) and OPD of the main hospital (for HIV negative participants) were recruited into the study. Patient information such as age, sex, signs and symptoms were recorded. After malaria screening, participants were classified into asymptomatic or symptomatic groups. Those who had fever (axillary temperature ≥ 37.5 °C) with any of the following; chills and sweating, general malaise, headache, vomiting, nausea or diarrhea, were placed in the symptomatic category.
Study procedure
Malaria screening
Finger-prick capillary blood of participants was taken for three different test procedures. Blood was dropped on First Response
® Malaria Ag
P. falciparum (HRP2) malaria rapid diagnostic test kit and results recorded according to the instructions of the manufacturer. This RDT has passed the lot testing according to WHO-FIND guidelines, which subjects available test kits on the market to 3–6 rigorous rounds of testing [
14]. One of the reasons why it is recommended for this country is because of its stability despite adverse weather conditions [
14].
Thick and thin blood smears were prepared for malaria parasite detection and identification. Thin smears were fixed in 100% methanol after air-drying. Both were stained for 10 min with 10% Giemsa; and examined by two expert microscopists. Enumerating 200 high-powered visual fields, samples were recorded as positive if any amount of asexual parasites was observed per 200 white blood cells and negative if none was observed. A third microscopist resolved discordant results. Participants who tested positive for either RDT or blood smear were referred to attending doctor for appropriate treatment.
Three drops of blood were placed on filter paper (Whatman grade 3). These were dried, stored and later shipped for PCR analysis at the KIT Biomedical Research Institute in Amsterdam, the Netherlands. DNA was extracted using the Boom method [
15] and nested PCR was performed for
P. falciparum identification (see [
16]). Samples that were RDT negative but PCR positive for
P. falciparum were further tested for
pfhrp2/3 deletion, by amplifying the histidine-rich central repeat region using primers for the conserved 5′ and 3′ regions of
hrp2 and
hrp3 genes. The flanking genes for
hrp2 and
hrp3 were also amplified. A 25 µl total volume was used for PCR reactions, containing 5 µl DNA template, 10× buffer, 25 mM MgCl
2, 10 mM each dNTP, 100 mM of each primer and 5 units of Taq (Roche diagnostics, Mannheim, Germany). Refer to Additional file
1 for primers used and PCR conditions. All PCR products were resolved on 2% agarose gel electrophoresis.
Statistical analysis
Data was entered in Microsoft Office Excel
® 2010 (Microsoft Corporation, USA), and statistically analysed with XLSTAT statistical ad-in. Diagnostic tools were compared using McNemar’s test. A p value ≤ 0.05 was considered as statistically significant. Kappa coefficients were used to test agreement between the diagnostic tools RDT and microscopy. Moderate agreement is indicated when kappa coefficient is between 0.4 and 0.6, substantial agreement when between 0.6 and 0.8 and excellent agreement when 0.8 or more [
17].
The sensitivity and specificity of RDT and microscopy were calculated using PCR as the gold standard. The proportion of RDT and microscopy positives, individually, divided by the total positives determined by PCR was recorded as sensitivity. Specificity was calculated as the proportion of individual RDT and microscopy negatives divided by the total negatives in the PCR. The number of RDT (or microscopy) true positives divided by the number of all RDT (or microscopy) positive test results (true positive plus false positives) was recorded as positive predictive value (PPV); whilst negative predictive value (NPV) was recorded as the number of all RDT (or microscopy) true negatives, divided by all RDT (or microscopy) negative test results (true negatives plus false negatives).
Discussion
It has been well established before that malaria and HIV interact manifold with each other [
1‐
4]. The study population for this study had been chosen in the context of improving care for under-5-year-old HIV patients in whom, as with all children in endemic areas, malaria continues to constitute a major health threat and frequent cause of fever. The problem identified and discussed here, namely
pfhrp2, 3 depletions leading to false negative malaria RDT results, is not considered specific to the HIV-positive population, or the particular young age group.
This study has demonstrated that the sensitivity and specificity of both HRP2-based RDT and expert light microscopy are generally high in rural and urban Ghana when tested in children with HIV under 5 years old; and microscopy more so than RDT. Missed infections from microscopy were few, but generally higher in rural Atibie (Kwahu). The HRP2-based RDT used in this study missed 1/6 malaria infections in HIV positive and 12/56 in HIV negative participants. The parasite densities of the missed infections were high (minimum of 11,107 p/µl); therefore, low parasitaemia could not have been the reason for lack of detection with RDT. Further PCR test of these false negatives for pfhrp2, pfhrp3 and their flanking genes indicated the presence of gene deletions in circulation.
Missed infections from microscopy were fewer than those from RDT. The false negatives from microscopy were higher in rural Atibie than in urban Accra. This might be attributed to the subjectivity of expert microscopy to individual bias. However, state-of-the-art RDTs should be able to rectify that; but false negatives were observed in the HRP2-based RDT used. The circulation of
pfhrp2 deleted malaria parasites has been established as one of the possible reasons for false negatives in RDTs in malaria-endemic areas. One of the very few prior studies done in Ghana has shown the deletions to be present in parasites in Accra and Cape-Coast [
9]; whereas in other West African countries, several studies have found them to abound [
8,
18,
19]. In this study, they were found to circulate in both the rural highland and the urban lowland. The circulation of these mutants in the population has dire implications for malaria control in Ghana. Among the malaria-vulnerable population of people living with HIV, the consequence of missed infections may be more serious; and even exceedingly so when they are under 5 years old. As has been established in the literature, malaria morbidity and mortality is worse in children under 5 years old [
8,
20‐
22] and in people living with HIV [
3,
23]; a combination of these two factors is even more severe [
3,
4]. It is, therefore, imperative that alternative RDTs are used in Ghana, a good option will be ones which detect non-HRP2 antigens. However, those that are currently WHO pre-qualified or meet the WHO recommended procurement criteria are insufficient [
8]. The development of RDTs that detect a combination of antigens might also be beneficial, so that mutant parasites with deletions will not be missed.
The contribution of
Plasmodium parasitaemia in people living with HIV to anti-malarial drug emergence has been shown in other studies [
7]. Perhaps low malaria prevalence, as found in this HIV population under 5 years of age, might not influence parasite biomass and contribute to de novo resistance mutations. Yet, rather than use parasite count of peripheral blood as an indication of parasite biomass since it does not take into consideration sequestered parasite load, there have been suggestions to use plasma
Pfhrp2 concentrations as estimates [
24]. However, in the light of the circulation of
Pfhrp2 deletions in this study and others, this might not be an accurate estimation after all.
In a study in other parts of Ghana by Adu-Gyasi et al. [
25], malaria prevalence in HIV positive patients was lower than the average national malaria prevalence. This is similar to the present study results; and may be attributed to co-trimoxazole (CTX) prophylaxis (given with anti-retroviral therapy as recommended by the Ghana National Aids Control Programme) [
26]. All the under-5-year-olds living with HIV in this study were on prophylactic CTX. This has been shown to reduce opportunistic infections and protozoan infections like malaria [
27‐
29].
Even though 40% of the children living with HIV in this study had symptoms that were indicative of malaria, such as fever, chills and sweating, headache, general malaise, vomiting, diarrhea or nausea, PCR showed that only 4% of that category actually had malaria. In a similar study in people living with HIV in Brong-Ahafo and Ashanti regions in Ghana, 38.1% of study participants had malaria symptoms yet only 4.4% were laboratory confirmed [
25]. This suggests that the adoption of the WHO recommendation for laboratory diagnosis of malaria instead of the erstwhile clinical diagnosis by Ghana is a step in the right direction. It will reduce prescription of anti-malarials to only when it is necessary.
Ideally, subject to financing, an expansion of this study to other regions in Ghana should reveal the pattern of distribution of the pfhrp2 mutants. Nonetheless, this is one of the first studies in Ghana that has looked at the performance of popular diagnostic tools in a malaria vulnerable population with the added vulnerability of HIV. It has also detected the circulation of parasites with pfhrp2 deletions in this malaria-vulnerable population in the rural highland and urban lowland. This has shed light on the malaria situation of the malaria vulnerable sub-population and may contribute to adaptation of improved control measures.
Authors’ contributions
EDAO, MPG and PM conceived and designed this study. Data acquisition, analysis and interpretation was done by EDAO, SKD, CB, MPG and PM. EDAO wrote the first draft of the article, which was critically revised by MPG, PM and CB for important intellectual content. All authors read and approved the final manuscript.