Exploring the host factors affecting asymptomatic Plasmodium falciparum infection: insights from a rural Burkina Faso study.

BACKGROUND: Asymptomatic Plasmodium falciparum parasitaemia forms a reservoir for the transmission of malaria disease in West Africa. Certain haemoglobin variants are known to protect against severe malaria infection. However, data on the potential roles of haemoglobin variants and nongenetic factors in asymptomatic malaria infection is scarce and controversial. Therefore, this study investigated the associations of iron homeostasis, inflammation, nutrition, and haemoglobin mutations with parasitaemia in an asymptomatic cohort from a P. falciparum-endemic region during the high transmission season. METHODS: A sub-study population of 688 asymptomatic individuals (predominantly children and adolescents under 15 years, n = 516) from rural Burkina Faso previously recruited by the NOVAC trial (NCT03176719) between June and October 2017 was analysed. Parasitaemia was quantified with conventional haemocytometry. The haemoglobin genotype was determined by reverse hybridization assays targeting a selection of 21 HBA and 22 HBB mutations. Demographics, inflammatory markers (interleukins 6 and 10, hepcidin), nutritional status (mid upper-arm circumference and body mass index), and anaemia (total haemoglobin, ferritin, soluble transferrin receptor) were assessed as potential predictors through logistic regression. RESULTS: Malaria parasites were detected in 56% of subjects. Parasitaemia was associated most strongly with malnutrition. The effect size increased with malnutrition severity (OR = 6.26, CI95: 2.45-19.4, p < 0.001). Furthermore, statistically significant associations (p < 0.05) with age, cytokines, hepcidin and heterozygous haemoglobin S were observed. CONCLUSIONS: According to these findings, asymptomatic parasitaemia is attenuated by haemoglobin S, but not by any of the other detected genotypes. Aside from evidence for slight iron imbalance, overall undernutrition was found to predict parasitaemia; thus, further investigations are required to elucidate causality and inform strategies for interventions.

PlyAB Nanopores Detect Single Amino Acid Differences in Folded Haemoglobin from Blood.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible.

Haemoglobin variants, iron status and anaemia in Sri Lankan adolescents with low red cell indices: A cross sectional survey.

Iron deficiency complicates the use of red cell indices to screen for carriers of haemoglobin variants in many populations. In a cross sectional survey of 7526 secondary school students from 25 districts of Sri Lanka, 1963 (26.0%) students had low red cell indices. Iron deficiency, identified by low serum ferritin, was the major identifiable cause occurring in 550/1806 (30.5%) students. Low red cell indices occurred in iron-replete students with alpha-thalassaemia including those with single alpha-globin gene deletions. Anaemia and low red cell indices were also common in beta-thalassaemia trait. An unexpected finding was that low red cell indices occurred in 713 iron-replete students with a normal haemoglobin genotype. It is common practice to prescribe iron supplements to individuals with low red cell indices. Since low red cell indices were a feature of all forms of α thalassaemia and also of iron deficiency, in areas where both conditions are common, such as Sri Lanka, it is imperative to differentiate between the two, to allow targeted administration of iron supplements and avoid the possible deleterious effects of increased iron availability in iron replete individuals with low red cell indices due to other causes such as α thalassaemia.

Haemoglobin Ottawa, sickle cell trait and vaso-occlusive crises.

A girl with a sickle cell trait had severe VOCs (vaso-occlusive crises), her father also had a sickle cell trait but mild VOCs, and her mother had no symptoms. Electrophoresis on agarose gel under alkaline conditions showed haemoglobin AS (HbAS) in the girl and in her father, with an S band increased more than expected (46.2% and 41.2% respectively), and a band migrating at C (16.8% and 8.9% respectively) in both. There was a band at S (19.6 %) in her mother. The C band was attributed to a hybrid tetramer with haemoglobin S (HbS) and a Hb variant. A homozygous c.46G>C mutation (Hb Ottawa, the Hb variant) was detected by Sanger sequencing in the girl. Heterozygosity for Hb Ottawa by Sanger sequencing was shown in both the father and the mother. The father, with HbAS and heterozygous for Hb Ottawa, had mild VOCs. Heterozygosity only for Hb Ottawa did not produce any abnormality in the mother. A sister and two brothers of the index patient presented a Hb variant, probably Hb Ottawa, migrating to the S zone (all 20%) at electrophoresis, without HbS. These last three were asymptomatic. We conclude that Hb Ottawa, an α-globin variant, contributes along with haemoglobin S (HbS) to VOC symptoms.

Impact of haemoglobin variants on the diagnostic sensitivity of glycated haemoglobin (HbA1c) assay methodologies in sub-Saharan Africa: a laboratory-based method validation study.

Introduction: the utility of glycated haemoglobin (HbA1c) for the diagnosis and monitoring of diabetes in sub-Saharan Africa is uncertain due to limited data on the performance of the available HbA1c assay methods in this population, which has a high prevalence of haemoglobin variants. We aimed to compare the diagnostic accuracy of the major HbA1c methodologies (Boronate Affinity, Capillary Electrophoresis, High Performance Liquid Chromatography, Immunoassay) in an African population, and assess the impact of the common haemoglobin variant HbAS (sickle cell trait). Methods: whole blood samples were obtained from 182 individuals living with type 2 diabetes in Uganda. HbA1c values for each method were compared to average glucose measured over 14 days by continuous glucose monitoring (CGM). To determine concordance, the three HbA1c assay methods were compared to the capillary electrophoresis method. Results: there was a strong correlation between CGM average glucose levels and all four HbA1c methodologies (r=0.81-0.89) which did not differ in those with and without HbAS (present in 37/182 participants). The presence of HbAS did not alter the relationship between HbA1c and CGM glucose for any assay (p for interaction >0.2 for all methods). Diagnostic accuracy for CGM average glucose thresholds of 7 and 10mmol/L was similar across methods (area under the receiver operating characteristic curve 0.80-0.84 and 0.76-0.84 respectively). The maximum bias between the HbA1c assay methodologies was 2 mmol/mol (2.07%). Conclusion: all major HbA1c technologies offer accurate and comparable HbA1c measurement even in this population with high prevalence of haemoglobin variants.

An assessment of the effect of haemoglobin variants on detection by faecal immunochemical tests.

Background Faecal immunochemical tests (FIT) for haemoglobin (Hb) are being used in the investigation of colorectal cancer. These tests use antibodies raised to the globin moiety of human Hb. Where the globin structure is abnormal or reduced, it is possible that antibody binding, and thus Hb-detection may be affected. Methods Lysates prepared from whole blood samples of patients with known variants were diluted in manufacturer-specific buffer to 10, 100 and 500 µg Hb/g faeces. These samples were analysed on four FIT analysers and the results compared with samples with no known variant present (normal samples). Results The results from this study show that of 20 variants tested, three showed a decrease in detection by all four analysers. These were ß-thalassaemia major and two fetal cord blood samples. Conclusions Of 20 common Hb variants studied, 17 did not affect detection of Hb by the FIT systems tested. Hb variants leading to a reduction in the presence of a globin chain caused a reduction in Hb detection; in such cases, cancers could be missed.

Co-Incidence or Co-Existence? Acute Lymphoblastic Leukaemia in HbE-alpha Thalassaemia: A Case Report with Review of Literature.

Haemoglobin E (HbE) is a Haemoglobin variant that commonly occurs in many places in Asia. As ß thalassaemia and α thalassaemia also occur in the same regions, the co-inheritance of these conditions leads to various phenotypic forms. HbE α thalassaemia is less common and of a milder phenotype than HbE ß thalassaemia. Though malignancies are one of the complications in thalassaemia, occurrence of leukaemia is a rare event. Here we present a case of a two-year-old male child co-presenting with pre B acute lymphoblastic leukaemia (ALL) with MLL rearrangement and HbE alpha thalassaemia. The child is on remission 12 months post-therapy with standard ALL high risk protocol with no minimal residual disease (MRD). Haematological and oncological conditions coexisting at presentation is a challenge to therapy. This case is described for its rarity. Informed consent has been obtained from the parents.

Evaluation of high performance liquid chromatography (HPLC) pattern and prevalence of beta-thalassaemia trait among sickle cell disease patients in Lagos, Nigeria.

INTRODUCTION: Sickle cell disease (SCD) is the most common inherited disorder of haemoglobin worldwide. This study evaluated the chromatographic patterns and red blood cell indices of sickle cell patients to determine the co-inheritance of other haemoglobin(Hb) variants and ß-thalassaemia trait. METHODS: Red cell indices, blood film, sickle solubility test, Hb electrophoresis using alkaline cellulose acetate membrane, and chromatographic patterns using Bio Rad HPLC Variant II were evaluated for 180 subjects. RESULTS: Based on low MCV <76fL and MCH<25 pg, in the presence of elevated A2 >4.0% on HPLC and Hb variants eluting outside the S and C windows, at least four haemoglobin phenotypes (SS: 87.7%; SC: 1.1%; SD Punjab: 0.6%; Sß-thalassemia: 10.6%) were identified. Mean Hb F% was 8.1±5.1 (median 7.65) for Hb SS and 6.03±5.2 (median 3.9) for Hb Sß-thalassemia trait. Majority of Hb SS (69.1%) had Hb F% less than 10 while 27.6% had 10-19.9 and 3.2% had ≥ 20. Mean Hb F% was higher in female Hb SS (9.55±5.09; mean age 7.4±3.8 years) than the males (7.63±4.80; mean age 6.9±3.8 years) (P=0.02). A borderline significant negative correlation between age and Hb F levels among Hb SS subjects (r= -0.169 P=0.038) was also observed. CONCLUSION: Our data suggests that α and ß- thalassaemia traits, and other haemoglobin variants co-exist frequently with SCD in our population.

Interference of the most frequent haemoglobin variants on quantification of HbA1c: comparison between the LC-MS (IFCC reference method) and three routinely used methods.

AIM: Assaying HbA1c in patients with haemoglobin variants has long been a technical challenge, despite methodological advances that have progressively limited the problem. The purpose of this study was to evaluate the impact of the most frequent haemoglobin variants on three routine separation methods compared with the IFCC reference method. PATIENTS: Blood samples from heterozygous patients (AS, AC, AD, AE) were analyzed using the IFCC reference method (LC-MS), and the results compared with those obtained by capillary electrophoresis (CAPILLARYS 2 Flex Piercing, Sebia) and two HPLC methods using cation-exchange (Variant II, Bio-Rad) and affinity chromatography (Ultra(2), Primus). RESULTS: HbA1c values obtained by the IFCC reference method were comparable to those obtained by the three tested methods whatever the haemoglobin variant. Mean relative biases did not exceed the threshold of 7% (above which differences are generally considered clinically significant), although some individual values were above this limit with Variant II in samples with HbS and for all three methods in samples with HbE. CONCLUSION: This comparative study of the LC-MS reference method and three field methods has demonstrated that these assays are not clinically influenced by the presence of the most common haemoglobin variants. The present results also confirm that the interpretation of HbA1c values in patients with Hb variants remains complex and depends on the assays used and should, in some cases, take into account parameters other than analytical ones (such as differences in glycation rates and half-lives of haemoglobin variants).