Laboratory methods

Blood counts, different from haemoglobin, were performed using a Sysmex analyser KX21N or XT-2000i (Sysmex Long Grove, IL, USA). Reticulocyte counts were estimated by microscopy on cresil blue-stained blood smears.

Albumin, prealbumin, and C-reactive protein (CRP) levels were measured with an ADVIA 2400 analyser (Siemens Healthcare, Spain). Folic acid, vitamin B12 and ferritin levels were measured with an ADVIA Centaur (Siemens Healthcare, Spain). sTfR levels were measured in a BN-II nephelometer (Dade-Siemens Healthcare, Spain) and vitamin A levels by reversed phase high-performance liquid chromatography [17]. Erythropoietin quantification was performed using the Quantikine human erythropoietin immunoassay kit (R&D Systems, USA).

Bacteria were detected in blood cultures in the BACTEC® 9050 (Becton-Dickinson, USA). In children ≤18 months of age HIV infection was defined as a HIV-1 DNA-positive result, detected using the Amplicor HIV-1 DNA-PCR kit (Roche Diagnostics, USA) and in older children it was defined as two positive rapid tests, using the Determine HIV-1/2 Rapid Test (Abbott Laboratories, USA) and confirming by the Uni-Gold Rapid Test (Trinity Biotech Co., Ireland), or as discordant rapid test results resolved by an additional HIV DNA-positive result. Epstein-Barr virus (EBV) and Parvovirus B19 (PV-B19) were identified by real time quantitative PCR (qPCR) using the Artus PCR kits (QIAGEN, Spain). Plasmodium falciparum (Pf) parasites were identified by microscopy on Giemsa-stained blood films. Pf qPCR was performed for microscopically negative samples. Schistosoma haematobium was determined by direct microscopic examination of urine sediment [18]. Intestinal parasites in stool (see Table 3 for details) were detected by microscopic examination using the merthiolate-iodine-formalin concentrations method [19].

Distribution of possible aetiological and confounding factors of anaemia among cases and community controls

¹n/N (column percentage); ²Chi-squared test; ³Fisher’s exact test; ⁴11 Streptococcus pneumoniae, 5 Staphylococcus aureus, 5 Escherichia coli, 3 Salmonella typhimurium, 10 others. ⁵1 E. coli, 1 Klebsiella spp and 1 g negative bacilli, lactose fermenting oxidase negative (these findings were considered contaminations or transient bacteraemia); ⁶the intestinal parasites are 3 Ascaris lumbricoides, 2 Giardia lamblia, 1 Strongyloides stercoralis, ⁷the intestinal parasites are 6 Ascaris lumbricoides, 2 Giardia lamblia, 1 Entamoeba hystolitica, 1 Strongyloides stercoralis, ⁸Haemoglobinopathy includes S and E

Abbreviations: EBV Epstein-Barr virus, G6PD Glucose 6 phosphate dehydrogenase, Pf Plasmodium falciparum, WAZ Weight for age Z score

Haemoglobinopathies and β-thalassaemia were assessed using the β-thalassaemia Short Program from the Variant Haemoglobin Testing System® (Bio-Rad, Hercules, USA). Detection of α-thalassaemia (3.7 kb deletion) was performed by the GAP-PCR method [20]. Glucose 6-phosphate dehydrogenase (G6PD) deficiency was determined using the Beutler fluorescent spot test [21].