Study area and population
This study was conducted in Lafia, a city located within the middle belt region in north-central Nigeria. Lafia lies within Nigeria’s Guinea savannah ecological zone and in this area malaria is known to be endemic and perennial as described previously [30, 31]. The main vectors serving as agents of malaria transmission in this region are Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus, Anopheles moucheti, Anopheles melas, and Anopheles nili [2, 32]
Children aged 9 months to 12 years who presented with symptoms compatible with uncomplicated or mild malaria at the Dalhatu Araf Specialist Hospital Lafia, between 2006 and 2011, were enrolled into the study on "determinants of disease outcome in P. falciparum-infected children" after satisfying the inclusion criteria as follows:(i) at least 2 children of the same household living under the same roof; and, ii) they must have been residing in the same house for at least 6 months. Uncomplicated or mild malaria was defined as symptomatic malaria with presentations that included chills, pyrexia at presentation (axillary temperature >37.5oC) or history of fever within the preceding 48 hours, presence of asexual forms of P. falciparum in peripheral blood smears and absence of any indication of severe illness or vital organ dysfunction.
Participation in the study was voluntary. Before being included in the study, the study protocol was explained to the parent/guardian of the children from each family, and then informed consent was obtained. Ethical approval was obtained from the Ethics Committees of the Nasarawa State Ministry of Health and the Dalhatu Araf Specialist Hospital, Lafia, Nasarawa State, Nigeria, with reference numbers S/MH/519/VOL.1/84 and DASH/ADM/MR/VOL.1/0001, respectively. There were no selection criteria in the enrolment process of children in the households as children were registered at random. The first child presented by a parent during the enrolment process, irrespective of age or sex, was documented as child 1 for that particular household; the second child presented was child 2 and so on. All malaria-confirmed cases were appropriately treated by the Hospital clinicians.
Sample collection and microscopy
Approximately 0.5 ml of venous blood samples were collected from each child for parasitological and haematological analyses. All samples were de-identified at the point of collection for the confidentiality of participants and only labelled with alphanumeric codes. Three drops of blood collected were blotted on labelled 3MM Whatman filter paper, air dried, individually sealed in plastic bags, and stored at room temperature until they were used for DNA extraction. Thick and thin blood smears were also prepared for microscopic examination. The slides were labelled and allowed to dry, after which the thin smears were fixed with methanol and subsequently allowed to dry. Slides were stained with freshly prepared 5% Giemsa stain for 20 min at room temperature  and examined under the microscope. Parasitaemia was quantified relative to 250 white blood cells (WBC) on thick films and estimated as parasites per µl assuming a mean WBC of 8,000 per µl of blood. Smears were labelled negative if no parasites were seen after examination of 200 high-power field (HPF) at 1000× magnification on a thick blood film. Blood haemoglobin levels were estimated by haematocrit measurement of packed cell volume (PCV) using the micro-haematocrit centrifuge.
Parasite DNA extraction and genotyping of Plasmodium falciparum msp-2 gene
DNA was isolated from the dried blood spots on filter paper using the QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions; 150 μl of distilled water was used to elute DNA, which was then stored at -20oC until use.
All samples were genotyped for P. falciparum msp-2 gene by nested PCR according to previously described amplification procedures . Briefly, a primary reaction and a second reaction (nested) was carried out on each sample. The primary reaction amplifies the entire coding region of the msp-2 gene using the MSP2-1 and MSA2-4 primer pairs. Two sets of nested reactions were subsequently employed to amplify the central polymorphic region of the gene using the allele-specific primers FC 27-1/FC 27-2 and 3D7-1/3D7-2 for the FC27 and 3D7 allele types, respectively (Table 1). A third nested reaction was carried out with MSP2-2 and MSP2-3 primers to assess the frequency of isolates, which may be positive for msp-2, but not specific for FC27 or 3D7 allele type due to the polymorphic nature of the central region. The primary PCR mixture consisted of a final volume of 25 µl that included 12.5 µl of Go Taq® Green Master Mix (Promega Madison, USA), 2.0 µl of each primer (10 µM) and 5 µl of genomic DNA. The reaction was performed using the following cycling condition: initial denaturation step at 94oC for 5 min followed by 35 cycles of 10 sec at 94oC, 30 sec at 57oC and 40 sec at 72oC and a final extension step of 72oC for 3 min. All nested reactions were performed in a final volume of 25 µl containing 2.0 µl of PCR product from the primary reaction, 12.5 µl of Go Taq® Green Master Mix (Promega Madison, USA) and 2.0 µl of each primer (10 µM). The PCR cycling condition was: initial denaturation step at 94oC for 5 sec followed by 30 cycles of 10 sec at 94oC, 30 sec at 57oC and 40 sec at 72oC, and a final extension step of 72oC for 3 min. Primer sequences were synthesized by Invitrogen Life Technologies, UK. All PCR assays were performed using a BIOMETRA TB1 ThermalCycler (Biotron, Göttingen Germany).
PCR products were subjected to electrophoresis on 2% agarose and visualized under ultraviolet trans-illumination after staining with SYBR® Green. Fragment sizes were visually calculated relative to a standard size (100 bp) molecular weight DNA marker (New England Biolabs GmbH, Frankfurt am Main, Germany). The DNA fragments were grouped into bins if their fragment sizes were within 20 bp intervals.
Data were analysed using XLSTAT Version 2019.1.2 . The Student’s t-test was used to compare different normally distributed continuous variables. Numerical data not conforming to normal distribution were log-transformed. The multiplicity of infection (MOI) was defined as the minimum number of parasite genotypes per infected individual and was calculated as the mean number of PCR fragments or parasite genotypes per infected child. Clonality of infection was defined as the number of distinct PCR fragments per infected child. An infection was defined as polyclonal if more than one distinct PCR fragments or parasite genotypes were present in an isolate. Statistical significance was defined as p <0.05.