Asymptomatic falciparum malaria and genetic polymorphisms of Pfcrt K 76 T and Pfmdr 1 N 86 Y among almajirai in northeast Nigeria

Introduction: Malaria remains a public health challenge, especially in sub-Saharan Africa where asymptomatic malaria is not uncommon. In the present study, the prevalence of asymptomatic falciparum malaria was investigated in almajirai, and the genetic polymorphisms of chloroquine (CQ) resistance biomarkers were assessed. Methodology: A total of 440 apparently healthy almajirai between 3 and 12 years of age were randomly enrolled in Maiduguri, northeast Nigeria, between July and December 2010. Parasitemia and gametocytemia were assessed by light microscopy, and polymorphisms of Pfcrt K76T and Pfmdr1 N86Y were detected by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. Results: The mean age of the subjects was 8.3 ± 4.5 years, with subjects ≤ 5 years accounting for 10.7% (47/440) of the population. Prevalence of asymptomatic falciparum parasitemia and gametocytemia were 12.7% (56/440) and 8.6% (38/440), respectively. Geometric mean parasite density (GMPD) was 240 (160–630) parasites/μL, while geometric mean gametocyte density (GMGD) was 53 (24–96) gametocytes/μL. The GMPD was higher among subjects ≤ 5 years of age (p = 0.027). Pfcrt 76T was detected in 5.4% (3/56) of the isolates, and no isolates harbored Pfmdr1 86Y mutant. Conclusions: The study reveals asymptomatic falciparum malaria in almajirai and low levels of Pfcrt 76T and Pfmdr1 86Y alleles. These findings could hinder malaria control measures, and hence almajirai should be incorporated into malaria control programs.


Introduction
Falciparum malaria remains a major public health challenge in sub-Saharan Africa, especially among pregnant women and children under five years of age [1].It contributes the largest proportion of malaria morbidity and mortality and is responsible for most cases of complicated malaria [2].In northeast Nigeria, it accounts for about 98% of malaria cases with a mean annual prevalence of 22.0%, lowest (7.5%) in April and highest (80.7%) in September [3].Asymptomatic falciparum malaria (AFM), the presence of Plasmodium falciparum in peripheral blood without presenting clinical symptoms, is common among adult populations in malaria-endemic areas of Africa, Asia, and South America [4][5][6][7][8][9].It is associated with factors such as low parasitemia, increasing age, repeated malaria episodes, and increasing gestational age [10,11].In addition, AFM has also been reported in children [9,12,13], creating a major challenge to malaria diagnosis, treatment, and control.
Almajirai (almajiri -singular) are individuals usually under 15 years of age who attend informal Islamic schools and are allowed to wander in search of alms when there are no classes [24].An estimated 10 million of these children are distributed in most cities, towns, and villages in the northern Nigeria, and are without adequate shelter.Almajirai usually sleep outside [25] and are thus exposed to mosquito bites; despite this, they are left out in most malaria control programs.Repeated malaria exposure could induce preimmunity resulting in asymptomatic infection and increased gametocyte carriage among this cohort; therefore, there is a need for malaria assessment among the almajirai.In the present study, the prevalence of AFM and Pfcrt K76T and Pfmdr1 N86Y mutations were determined in a cohort of Nigerian children, the almajirai.

Study area
This study was conducted at the University of Maiduguri Teaching Hospital (UMTH), Maiduguri, northeast Nigeria with subjects (almajirai) recruited from Maiduguri, northeast Nigeria, between July and December 2010.Maiduguri, a malaria endemic area, is the capital of Borno State, with an estimated population of 1,860,000 almajirai [26].The mean annual prevalence of malaria among the general population is 22.0% (78.9% among children ≤ 5 years of age); this is lowest in April (7.5%) and highest in September (80.7%) [3].

Study design and subject enrolment
This was a cross-sectional study aimed at determining the epidemiology of AFM among almajirai in Maiduguri.Ethical approval and research permission was obtained from the Borno State Ministry of Health and Ministry of Religious Affairs, respectively.Between July and December 2010, 440 almajirai between 3 and 12 years of age who met the inclusion criteria were enrolled after informed consent was obtained.A concise medical history of each subject was obtained using a standard case record form, and each subject underwent a comprehensive clinical examination by a physician.

Collection of samples
From finger-prick blood samples, Giemsa-stained thick and thin blood smears were prepared for malaria microscopy [27].Capillary blood samples were collected for hematocrit estimation [28], and bloodspotted filter paper (Whatman 3 MM, Whatman, United Kingdom) samples were collected for molecular analyses [29].The sampling was carried out using standard operating procedures, and all samples were stored appropriately.

Assessment of falciparum parasitemia and gametocytemia
The Giemsa-stained thick smear was used for quantification of parasitemia and gametocytemia, while thin smear was used for species identification by light microscopy.A slide was declared negative if no parasites seen after examination of 100 high power field.Parasitemia was estimated by counting asexual parasites against 200 leukocytes and gametocytemia by counting gametocytes against 1,000 leukocytes.Parasite densities (/μL blood) were calculated assuming a leukocyte count of 8,000 cells/µL blood using the formulae below [30]:

Determination of hematocrit
The blood-filled capillary samples were spun at 8,000rpm for 5 minutes using a microhematocrit centrifuge (Hawskey Ltd., High Wycombe, United Kingdom), and hematocrit was determined using a microhematocrit reader (Hawskey Ltd.).Values < 30% were adjudged to be anemic [31].

Extraction of genomic DNA
The genomic DNA (gDNA) of the 56 positive samples was extracted from the blood-spotted filter paper using a QIAamp DNA Mini Kit (QIAGEN, Valencia United States) according to the manufacturer's instructions.The gDNA were stored at -20°C until use [32].

Assessment of genetic polymorphisms of Pfcrt K76T and Pfmdr1 N86Y
The mutation at codon 76 of the Pfcrt gene was detected by nested polymerase chain reaction (PCR) (Table 1) followed by restriction fragment length polymorphism (RFLP) (Table 2), as previously described [21,33].The restriction enzyme ApoI (New England Biolabs, Beverly, United States) digests the wild allele Pfcrt K76, giving two fragments of 100 bp and 34 bp, but not the mutant allele Pfcrt 76T, which remains at 134 bp.The mutation at codon 86 of Pfmdr1 gene was detected by nested PCR (Table 3) followed by RFLP (Table 2), as previously described [21,34].The restriction enzyme AflIII (New England Biolabs, Beverly, United States) digests the mutant allele Pfmdr1 Y86, giving two fragments of 190 bp and 120 bp, but not the wild allele Pfmdr1 N86, which remains at 310 bp.The digestion products of both genes were resolved on 1.5% agarose gel containing ethidium bromide (5 μL/100 mL) and visualized under ultraviolet light.Dd2 and 3D7 clones were used as positive controls for mutant and wild alleles, respectively [21,33,34].

Data analyses
The results were analyzed using SPSS version 15.0 software.Student's t test and analysis of variance (ANOVA) were used to compare mean values, and Chi squared (χ 2 ) was used to assess proportion.Statistical significance was inferred at p ≤ 0.05.

Discussion
The present study assessed AFM among a cohort of Nigerian children (almajirai) who are usually neglected in most malaria control programs.Previously reported prevalence of asymptomatic malaria among African children varies greatly [9,35].The prevalence of 12.7% reported in this study is higher than the findings of Strom et al. [35], who reported no asymptomatic malaria parasitemia among Tanzanian children, and those of Nkoghe et al. [9], who reported rates of 1%-8.7% among Gabonese children.This discordance could be attributed to two factors, namely age and use of ITNs.Subjects in the present study were relatively older, and a very small proportion (1%) used ITNs compared with the 97.2% found in a previous study [35].Studies have shown that asymptomatic malaria increases with age as pre-immunity gradually develops [36], and that the use of ITNs is a reliable malaria preventive measure [37].In contrast, the prevalence in the present study was lower than the 43.7% reported in western Kenya [38] and could be partly attributed to consumption of herbs, as 10.9% of the subjects who participated in the present study consumed herbs that may have antiplasmodial activities [39].In addition, almajirai's lifestyle exposes them to many hardships,  including infections [25]; thus, they could have experienced sufficient malaria episodes resulting in preimmunity even at an early age.The clinical implication of this finding is that clinical examination alone may not be adequate for malaria diagnosis in this cohort and could be a challenge to malaria diagnosis and control.Malaria transmission depends largely on the presence of viable gametocytes in peripheral blood, which are picked up by anopheline mosquitoes during a blood meal [40].The gametocyte carriage of 8.6% observed in the present study is similar to the rate of 10.8% reported in Kenya [38].Gametocyte carriers are reservoirs of infection that play a key role in continuous malaria transmission [5,10,41]; thus, the almajirai could serve as a source of infection to larger populations, challenging control measures.
Point mutations at codons of Pfcrt and Pfmdr1 genes are associated with CQ resistance in P. falciparum [20,21,42].Low levels of mutant alleles (Pfcrt 76T and Pfmdr1 86Y) detected in the present study indicated high CQ sensitivity among the population.Therefore, it could be opined that CQ sensitivity may be returning to northeast Nigeria years after the withdrawal of CQ due to widespread resistance [14].This is in accordance with previous studies that have shown a decline in prevalence Pfcrt 76T allele in parts of Africa such as Mali [43], Malawi [22], Kenya [44], Tanzania [45], Senegal [46], and now Nigeria.However, this calls for a larger study to re-assess CQ sensitivity in Nigeria.

Conclusions
This study revealed the presence of AFM among almajirai in northeast Nigeria and a low prevalence of mutant Pfcrt 76T and Pfmdr1 86Y alleles.These findings have significance implications on malaria diagnosis and morbidity and could jeopardize malaria control measures.It is suggested that this cohort of Nigerian children should be incorporated into various malaria control programs to ensure the success of the fight against malaria in Nigeria at large.

Table 1 .
Conditions of nested polymerase chain reaction for amplification of Pfcrt K76T.

Table 2 .
Master mix for restriction fragment length polymorphism.

Table 3 .
Conditions of nested polymerase chain reaction for amplification of Pfmdr1 N86Y.

Table 4 .
Demographic and clinical characteristics of the subjects.

Table 5 .
Asymptomatic falciparum malaria among the subjects.