Diversity of Rotavirus Strains Circulating in Botswana before and after introduction of the Monovalent Rotavirus Vaccine.

BACKGROUND: Globally, rotavirus is the leading cause of acute gastroenteritis (AGE) in children aged <5 years. Botswana introduced the monovalent rotavirus vaccine (Rotarix) in July 2012. To study the impact of this vaccine on rotavirus genotypes circulating in Botswana, a comparison of the genotypes pre-vaccination (2011-2012) and post-vaccination (2013-2018) periods was conducted. SUBJECTS AND METHODS: Residual samples from 284 children <5 years of age that tested positive for rotavirus by enzyme immunoassay were genotyped. One hundred and five samples were from the pre-vaccination period and 179 were from the post-vaccination period. Genotyping was performed using two multiplexed one-step reverse transcription polymerase chain reaction (RT-PCR) assays for the amplification and genotyping of rotavirus VP7 (G) and VP4 (P) genes. RESULTS: Prior to vaccine introduction, the predominant rotavirus circulating genotypes were G9P[8] (n = 63, 60%) and G1P[8] (n = 22, 21%). During the vaccine period, G2P[4] was the predominant genotype (n = 49, 28%), followed by G9P[8] (n = 40, 22%) and G1P[8] (n = 33, 18.5%). There was a significant decline in the prevalence of G9P[8] (p = 0.001) in the post-vaccination period. There was also a notable decline in G1P[8]. A spike in G2P[4] was observed in 2013, one year post-vaccine introduction. Rotavirus strain G3P[4] (n = 8) was only detected in the post-vaccine introduction period. In 2018 there was a marked increase in genotype G3P[8] (p = 0.0003). CONCLUSIONS: The distribution of circulating rotavirus genotypes in Botswana changed after vaccine implementation. Further studies are needed to examine whether these changes are related to vaccination or simply represent natural secular variation.

Association of pulmonary tuberculosis with increased dietary iron.

To determine whether increased dietary iron could be a risk factor for active tuberculosis, dietary iron history and human immunodeficiency virus (HIV) status were studied in 98 patients with pulmonary tuberculosis and in 98 control subjects from rural Zimbabwe. Exposure to high levels of dietary iron in the form of traditional beer is associated with increased iron stores in rural Africans. HIV seropositivity was associated with a 17.3-fold increase in the estimated odds of developing active tuberculosis (95% confidence interval [95% CI], 7.4-40.6; P<.001), and increased dietary iron was associated with a 3.5-fold increase (95% CI, 1.4-8.9; P=.009). Among patients treated for tuberculosis, HIV seropositivity was associated with a 3.8-fold increase in the estimated hazard ratio of death (95% CI, 1.0-13.8; P=.046), and increased dietary iron was associated with a 1.3-fold increase (95% CI, 0.4-6.4; P=.2). These findings are consistent with the hypothesis that elevated dietary iron may increase the risk of active pulmonary tuberculosis.

Determination of carbohydrate-deficient transferrin using capillary zone electrophoresis.

BACKGROUND: Current methods for carbohydrate-deficient transferrin (CDT) often suffer from low precision, complexity, or risk of false positives attributable to genetic variants. In this study, a new capillary zone electrophoresis (CZE) method for CDT was developed. METHODS: CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 microm (i.d.) x 47 cm] and the CEOFIX CDT buffer system with addition of 50 microL of anti-C3c and 10 microL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm. RESULTS: CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 +/- 0.05 min; monosialotransferrin, 9.72 +/- 0.05 min; asialotransferrin, 9.52 +/- 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312,000 +/- 21,000 for the mono- and 199 000 +/- 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 +/- 0.06 and 9.89 +/- 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIA (x) values by Deming regression analysis: y = 1.92x - 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%. CONCLUSIONS: CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.

Carbohydrate-deficient transferrin and chronic alcohol ingestion in subjects with transferrin CD-variants.

Carbohydrate-deficient transferrin (CDT) is widely accepted as screening test for excessive alcohol consumption. However, results from subjects with transferrin variants must be interpreted with caution since chromatography-based methods may give false-positive results. Furthermore, due to the co-elution in HPLC or the co-migration in capillary zone electrophoresis (CZE) of the di- and trisialylated C transferrins with the tetrasialylated D peak, exact measurement of CDT is impossible in CD-variants. Therefore, in this study, we tried to offer a different solution, including only the asialo-D, asialo-C, monosialo-D, monosialo-C, disialo-D and trisialo-D transferrins in the CDT calculation and referring to a different cut-off value for CDT in transferrin CD-variants. Comparison of alcohol consumers with teetotalers demonstrated area under the receiver operating characteristic curve of 0.79 and 0.76 for carbohydrate-deficient transferrin, 0.71 and 0.71 for mean corpuscular volume and 0.51 and 0.68 for gamma-glutamyltransferase in 43 subjects with transferrin CD-variants and 225 subjects with CC-phenotypes, respectively. Since false-positive carbohydrate-deficient transferrin results due to a transferrin CD-variant have major social implications, capillary electrophoresis-based or similar methods (HPLC, FPLC) should be preferred in populations carrying a high D-allele frequency.

A new method for fast haptoglobin phenotyping and hemoglobin binding capacity calculation based on capillary zone electrophoresis.

A capillary zone electrophoresis method was developed for haptoglobin (Hp) phenotyping in hemoglobin (Hb) supplemented serum. The method allows a complete resolution of the major haptoglobin phenotypes Hp 1-1, Hp 2-1, and Hp 2-2 based on the difference in charge-to-mass ratio of their Hb-Hp complexes. Identification of these phenotypes was achieved by their significant differences in migration times and their marked difference in electrophoretic pattern. Our method showed full agreement with starch gel electrophoresis. Furthermore, following neuraminidase treatment of the serum, the Hp subtypes Hp1-1FF, FS and SS could be resolved, based on the same criteria as the phenotyping. The new electrophoretic method allowed typing of the rare phenotypes Hp 2-1 modified (Hp 2-1M) and Hp Johnson. The calculated hemoglobin binding capacity of serum correlates well with the nephelometrically determined haptoglobin concentration. The new method for typing haptoglobin gives prospectives for fast haptoglobin typing and Hp 1-1 subtyping.

Transferrin polymorphism influences iron status in blacks.

BACKGROUND: Genetic variants of human transferrin (TF) have been described, but little is known about their functional differences. We studied iron status according to TF phenotype in a healthy Zimbabwean population and in subjects at risk of African iron overload. METHODS: The study population consisted of 483 nondrinkers, 31 drinking spouse pairs, and 5 family pedigrees (n = 88) with index cases of iron overload. TF phenotypes were determined using starch gel electrophoresis. To evaluate iron status, serum iron, total iron-binding capacity (TIBC), ferritin, and soluble TF receptors were measured, and the percentage of saturation and the serum iron:TF ratio were calculated. The binding of the TF variants was studied by equilibrium dialysis. RESULTS: The reference population was characterized by a high TF D allele frequency (0.050) and a complete absence of homozygous TF DD individuals. Similar allele frequencies were observed in subjects at risk of African iron overload. In the reference population, male TF CD heterozygotes had significantly lower (P <0.01) values for serum iron, TIBC, TF saturation, and serum iron:TF ratio than the TF CC homozygotes; in females, only TIBC was significantly different. Overall red blood cell indices did not differ according to TF phenotype. In the population at risk of African iron overload, only serum iron:TF ratio was consistently significantly lower in TF CD phenotypes (P <0.05). After equilibrium dialysis, the amount of iron bound by TF was significantly lower (P <0.01) in TF CD individuals. CONCLUSIONS: The present data demonstrate a functional difference between TF phenotypes in blacks.

Haptoglobin polymorphism and mortality in patients with tuberculosis.

SETTING: A rural Zimbabwean hospital and the surrounding community. OBJECTIVES: To determine whether a particular haptoglobin phenotype is associated with increased susceptibility to clinical pulmonary tuberculosis, and to determine the outcome of treatment for pulmonary tuberculosis according to haptoglobin phenotype. DESIGN: A case-control study, and a prospective cohort study. RESULTS: We studied 98 consecutive patients with sputum-positive pulmonary tuberculosis and 98 sex- and age-matched controls. The haptoglobin (Hp) phenotype distributions did not differ significantly between the tuberculosis patients and controls (P = 0.5). During the 18-month follow-up period after the start of tuberculosis treatment, 6/18 (33%) cases with Hp 2-2 phenotype died compared to 9/47 (19%) with Hp 2-1 and 3/31 (10%) with Hp 1-1. In a logistic regression model, the odds of dying were 6.1-fold greater with Hp 2-2 than with Hp 1-1 (95%CI 1.04-35.1, P = 0.04). CONCLUSION: Our results suggest that there is equal susceptibility to clinical pulmonary tuberculosis disease amongst different haptoglobin phenotypes. Nonetheless, tuberculosis patients with Hp 2-2 phenotype had a higher risk of mortality.

African iron overload.

African iron overload has been recognised in sub-Saharan Africa for seventy years. The condition is distinct from the well-characterised HLA-linked haemochromatosis described in Caucasians. Increased dietary iron intake predisposes to the condition. Recent evidence suggest that African iron overload may be caused by an interaction between increased dietary iron and a genetic defect not associated with the HLA-locus. Iron deposition is prominent both in macrophages and in hepatic parenchymal cells. Iron overload is distinct from alcoholic liver disease, although the excess dietary iron is derived from a traditional beverage that contains alcohol. African iron overload has clinical consequences. It is a cause of hepatic fibrosis and cirrhosis, and associations with diabetes mellitus, peritonitis, scurvy and osteoporosis have been described. African iron overload may be a cause of hepatocellular carcinoma. The disorder is associated with a poor outcome in tuberculosis, an infection that is highly prevalent in sub-Saharan Africa.

Reference range of serum haptoglobin is haptoglobin phenotype-dependent in blacks.

Reference values for serum haptoglobin (Hp), were established in a Black Zimbabwean population. The upper limit (2.15 g/l) is comparable to the one in Caucasians, but the lower limit (0.12 g/l) is much lower than the proposed interim international reference limit (0.3 g/l). Subjects that typed as Hp 0-0 by starch gel electrophoresis technique were retyped using high performance gel permeation chromatography. This resulted in a 32% decrease in the frequency of Hp 0-0, but an increase in Hp 2-2 and Hp 2-1M phenotype frequencies. In the Zimbabwean Blacks, the Hp 0-0 frequency was estimated to be 2.9%. Haptoglobin reference values were found to be Hp phenotype-dependent; highest values were found in Hp 1-1 (median 0.88 g/l; range 0.31-1.69 g/l) and in Hp 2-1 (median 0.90 g/l; range 0.31-2.22 g/l) and lower values (median 0.66 g/l; range 0.13-1.79 g/l) in Hp 2-2 subjects. The Hp 2-1M phenotype was characterized by low reference values (0.18-1.25 g/l) (P<0.05). In three cases of the rare variant Hp Johnson, high Hp concentrations were found (median 1. 57 g/l; range 0.98-1.57 g/l).