Association of alpha-thalassemia and Glucose-6-Phosphate Dehydrogenase deficiency with transcranial Doppler ultrasonography in Nigerian children with sickle cell anemia.

BACKGROUND: Stroke is a devastating complication of sickle cell anemia (SCA) and can be predicted through abnormally high cerebral blood flow velocity using transcranial Doppler Ultrasonography (TCD). The evidence on the role of alpha-thalassemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the development of stroke in children with SCA is conflicting. Thus, this study investigated the association of alpha-thalassemia and G6PD(A- ) variant with abnormal TCD velocities among Nigerian children with SCA. METHODS: One hundred and forty-one children with SCA were recruited: 72 children presented with normal TCD (defined as the time-averaged mean of the maximum velocity: < 170 cm/s) and 69 children with abnormal TCD (TAMMV ≥ 200 cm/s). Alpha-thalassemia (the α-3.7 globin gene deletion) was determined by multiplex gap-PCR, while G6PD polymorphisms (202G > A and 376A > G) were genotyped using restriction fragment length polymorphism-polymerase chain reaction. RESULTS: The frequency of α-thalassemia trait in the children with normal TCD was higher than those with abnormal TCD: 38/72 (52.8%) [α-/ α α: 41.7%, α -/ α -: 11.1%] versus 21/69 (30.4%) [α-/ α α: 27.5%, α -/ α -: 2.9%], and the odds of abnormal TCD were reduced in the presence of the α-thalassemia trait [Odds Ratio: 0.39, 95% confidence interval: 0.20-0.78, p = 0.007]. However, the frequencies of G6PDA- variant in children with abnormal and normal TCD were similar (11.6% vs. 15.3%, p = 0.522). CONCLUSION: Our study reveals the protective role of α-thalassemia against the risk of abnormal TCD in Nigerian children with SCA.

Carriage of Mutant Dihydrofolate Reductase and Dihydropteroate Synthase Genes among Plasmodium falciparum Isolates Recovered from Pregnant Women with Asymptomatic Infection in Lagos, Nigeria.

OBJECTIVE: To assess N51I, C59R and S108N polymorphisms of dihydrofolate reductase (dhfr) and A437G and K540E of dihydropteroate synthase (dhps) genes of P. falciparum isolates recovered from pregnant women with asymptomatic malaria in a coastal setting in Nigeria. SUBJECTS AND METHODS: A total of 107 consenting and consecutively enrolled pregnant women (mean age ± standard deviation, 26.6 ± 4.5 years) attending antenatal care at the Iru/Victoria Island Primary Health Centre, Lagos, were screened for peripheral malaria by microscopy, by a histidine-rich protein-2-based rapid diagnostic test (RDT) and by polymerase chain reaction (PCR) using finger-pricked and dot blood samples. DNA was extracted from the blood and used for dhfr and dhps gene polymorphism analyses by PCR and restriction fragment length polymorphism. The sociodemographic and parasite data obtained were analysed. RESULTS: Of the 107 patients, 34 (31.8%), 46 (43%) and 40 (37.4%) were found to be P. falciparum infected using microscopy, RDT and corrected RDT-PCR, respectively (p < 0.05). The prevalence of P. falciparum isolates with mutant and mixed genotypes of dhfr at codons 51, 59 and 108 was 70, 75 and 80%, respectively, and the triple mutation in the homozygous form was 35%. The prevalence of the homozygous quintuple dhfr plus dhps mutant was 5%, while that of the P. falciparum isolates with mutant or mixed genotypes of dhps at codons 437 and 540 was 37.5 and 22.5%, respectively. CONCLUSION: This study revealed the emergence of the K540E mutation among the parasite population in Lagos. However, it supports the implementation of the intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine with continuous effectiveness monitoring in the study area.

Zooming into the structure-function of RING finger proteins for anti-cancer therapeutic applications.

Cancer is one of the most common and widely diagnosed diseases worldwide. With an increase in prevalence and incidence, many studies in cancer biology have been looking at the role pro-cancer proteins play. One of these proteins is the Really Interesting New Gene (RING), which has been studied extensively due to its structure and functions such as apoptosis, neddylation, and its role in ubiquitination. The RING domain is a cysteine-rich domain known to bind Cysteine and Histidine residues. It also binds two zinc ions that help stabilize the protein in various patterns, often with a 'cross-brace' topology. Different RING finger proteins have been studied and found to have suitable targets for developing anti-cancer therapeutics. These identified candidate proteins include Parkin, COP1, MDM2, BARD1, BRCA-1, PIRH2, c-CBL, SIAH1, RBX1 and RNF8. Inhibiting these candidate proteins provides opportunities for shutting down pathways associated with tumour development and metastasis.

Improving the Understanding of the Immunopathogenesis of Lymphopenia as a Correlate of SARS-CoV-2 Infection Risk and Disease Progression in African Patients: Protocol for a Cross-sectional Study.

BACKGROUND: The COVID-19 pandemic, caused by SARS-CoV-2, continues to impact health systems throughout the world with serious medical challenges being imposed on many African countries like Nigeria. Although emerging studies have identified lymphopenia as a driver of cytokine storm, disease progression, and poor outcomes in infected patients, its immunopathogenesis, as well as environmental and genetic determinants, remain unclear. Understanding the interplay of these determinants in the context of lymphopenia and COVID-19 complications in patients in Africa may help with risk stratification and appropriate deployment of targeted treatment regimens with repurposed drugs to improve prognosis. OBJECTIVE: This study is designed to investigate the role of vitamin D status, vasculopathy, apoptotic pathways, and vitamin D receptor (VDR) gene polymorphisms in the immunopathogenesis of lymphopenia among African people infected with SARS-CoV-2. METHODS: This cross-sectional study will enroll 230 participants, categorized as "SARS-CoV-2 negative" (n=69), "COVID-19 mild" (n=32), "hospitalized" (n=92), and "recovered" (n=37), from two health facilities in Lagos, Nigeria. Sociodemographic data, travel history, and information on comorbidities will be obtained from case files and through a pretested, interview-based structured questionnaire. Venous blood samples (5 mL) collected between 8 AM and 10 AM and aliquoted into EDTA (ethylenediaminetetraacetic acid) and plain tubes will be used for complete blood count and CD4 T cell assays to determine lymphopenia (lymphocyte count <1000 cells/µL) and CD4 T lymphocyte levels, as well as to measure the concentrations of vitamin D, caspase 3, soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble Fas ligand (sFasL) using an autoanalyzer, flow cytometry, and ELISA (enzyme-linked immunosorbent assay) techniques. Genomic DNA will be extracted from the buffy coat and used as a template for the amplification of apoptosis-related genes (Bax, Bcl-2, BCL2L12) by polymerase chain reaction (PCR) and genotyping of VDR (Apa1, Fok1, and Bsm1) gene polymorphisms by the PCR restriction fragment length polymorphism method and capillary sequencing. Total RNA will also be extracted, reverse transcribed, and subsequently quantitated by reverse transcription PCR (RT-PCR) to monitor the expression of apoptosis genes in the four participant categories. Data analyses, which include a test of association between VDR gene polymorphisms and study outcomes (lymphopenia and hypovitaminosis D prevalence, mild/moderate and severe infections) will be performed using the R statistical software. Hardy-Weinberg equilibrium and linkage disequilibrium analyses for the alleles, genotypes, and haplotypes of the genotyped VDR gene will also be carried out. RESULTS: A total of 45 participants comprising 37 SARS-CoV-2-negative and 8 COVID-19-recovered individuals have been enrolled so far. Their complete blood counts and CD4 T lymphocyte counts have been determined, and their serum samples and genomic DNA and RNA samples have been extracted and stored at -20 °C until further analyses. Other expected outcomes include the prevalence and distribution of lymphopenia and hypovitaminosis D in the control (SARS-CoV-2 negative), confirmed, hospitalized, and recovered SARS-CoV-2-positive participants; association of lymphopenia with CD4 T lymphocyte level, serum vitamin D, sVCAM-1, sFasL, and caspase 3 levels in hospitalized patients with COVID-19; expression levels of apoptosis-related genes among hospitalized participants with COVID-19, and those with lymphopenia compared to those without lymphopenia; and frequency distribution of the alleles, genotypes, and haplotypes of VDR gene polymorphisms in COVID-19-infected participants. CONCLUSIONS: This study will aid in the genotypic and phenotypic stratification of COVID-19-infected patients in Nigeria with and without lymphopenia to enable biomarker discovery and pave the way for the appropriate and timely deployment of patient-centered treatments to improve prognosis. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/21242.

Asymptomatic falciparum Malaria and its Effects on Type 2 Diabetes Mellitus Patients in Lagos, Nigeria.

BACKGROUND: Asymptomatic malaria (ASM) constitutes a reservoir of malaria parasites that sustain transmission and threaten elimination efforts. Studies have also shown a significant relation between insulin resistance and malaria infection. However, data on the clinical effects of ASM and its patterns of carriage among adult malaria patients is limited. OBJECTIVES: To determine the prevalence of ASM due to Plasmodium falciparum among adult type 2 diabetes (T2DM) patients in Lagos, Nigeria; to assess the diagnostic performance of light microscopy and histidine-rich protein 2 rapid diagnostic test (HRP-2 RDT); and to determine the effects of ASM on glycemic control and anemia. MATERIALS AND METHODS: This cross-sectional study enrolled 208 afebrile, nonobese, nonhypertensive T2DM patients, aged 40-70 years, undergoing treatment (adherence, ≥95%) at six private health facilities in Lagos, Nigeria, between March and August 2015. Sociodemographic data were obtained using a semi-structured questionnaire and clinical case files. Venous blood samples were collected and processed for fasting blood sugar estimation, packed cell volume determination and malaria parasite detection by HRP2-RDT, light microscopy and polymerase chain reaction (PCR). RESULTS: The mean age of the patients was 54.5 years. ASM was diagnosed in 16.8%, 7.2% and 4.3% of the patients by PCR, light microscopy and HRP2-RDT, respectively. ASM was significantly (P < 0.05) associated with poor glycemic control, anemia and insulin resistance. The overall parasitemia ranged from 85 to 3789 parasites/µL (median, 1580 parasites/µL). Benchmarking against the PCR results, light microscopy and rapid diagnostic tests were found to have a sensitivity (95% confidence interval) of 42.9% (26.5-59.3) and 22.9% (12.1-39), respectively, in diagnosing ASM. CONCLUSION: This study revealed that T2DM patients in Lagos, Nigeria, are potential reservoirs of asymptomatic Plasmodium falciparum, which has a significantly negative effect on glycemic control and anemia. The study also found PCR to be the most effective diagnostic method.

Transition-transversion mutations in the polyketide synthase gene of Aspergillus section Nigri.

This study determined the transition-transversion mutation in the pks gene of Aspergillus section Nigri in order to gain insight into the patterns of nucleotide base substitution and the process of molecular evolution using standard recommended techniques. Results obtained depict frequent occurrence of transition (23 ± 0.96) than transversion (11.37 ± 1.38) (p < 0.05) with C/T being the most frequently observed transitional base substitution and C/A the most frequently occurring transversional base change. The number of single base insertions (56 ± 1.00) were significantly higher than the observed single base deletions (38 ± 2.00) (p < 0.05) while varying degrees of two or more base deletions and insertions were also observed both inside and outside the open reading frame. The maximum likelihood value estimated for the pks gene was calculated to be -9458.80 in 423 positions of the final dataset while the transition-transversion ratio was estimated to be 0.50. The Tajima's neutrality test approaches seven (7) with the nucleotide diversity estimated to be approximately 65%. Evolutionary test depicts positive selection as ratio of non synonymous to synonymous divergence was found to be greater than ratio of the number of non synonymous to synonymous polymorphisms. The proportion of substitution driven by positive selection was calculated to be approximately 96.2%. This research therefore provides an insight into the understanding of pks gene mutation patterns as some of the observed indels resulted in frame shift mutations.

Typhoid Fever: Tracking the Trend in Nigeria.

Typhoid fever continues to pose a serious health challenge in developing countries. A reliable database on positive blood cultures is essential for prompt interventions. To generate reliable data on Salmonella enterica serovar Typhi (S. Typhi)-positive blood culture trends in typhoidal Salmonella in Nigeria alongside changing contextual factors and antimicrobial resistance patterns, a retrospective cohort study was conducted in two hospitals in Lagos between 1993 and 2015. Medical records of typhoid patients were reviewed for positive culture and antibiogram, using standard procedures and analyzed. Additional data were retrieved from a previous study in seven facilities in Abuja and three hospitals in Kano from 2008 to 2017 and 2013 to 2017, respectively. A declining trend in percent positivity of S. Typhi was observed in Abuja with more erratic trends in Lagos and Kano. In Lagos, more than 80% of the isolates from the entire study period exhibited multiple drug resistance with a generally increasing trend. Of the chosen contextual factors, improvements were recorded in female literacy, access to improved water supply, diarrheal mortality in children younger than 5 years, gross domestic product, and poverty while access to improved sanitation facilities decreased over time nationally. Typhoid fever still poses a serious health challenge in Nigeria and in antibiotic resistance, and is a major health security issue. A combined approach that includes the use of typhoid vaccines, improvements in sanitation, and safe water supply is essential.

Occurrence of extended-spectrum and AmpC ß-lactamases in multiple drug resistant Salmonella isolates from clinical samples in Lagos, Nigeria.

PURPOSE: Salmonella spp. are important foodborne pathogens exhibiting increasing resistance to antimicrobial drugs. Resistance to broad-spectrum ß-lactams, mediated by extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase enzymes is fast spreading and has had negative impacts on the clinical outcomes, particularly on third-generation cephalosporins. This study investigated the carriage of AmpC gene among multidrug-resistant Salmonella spp. from Lagos, Nigeria. METHODS: Forty Salmonella spp. from clinical samples (S. typhi = 13; S. typhimurium = 10; S. enteritidis = 8; S. choleraesuis = 5; S. paratyphi = 4) were subjected to in vitro susceptibility test by disk diffusion methods. Isolates that were resistant to cefoxitin and third-generation cephalosporins were screened for ESBL (Double Disk Synergy Test Method) and AmpC enzyme (AmpC disk test) production. Detection of AmpC fox gene was carried out by polymerase chain reaction. RESULTS: Thirty-two (80%) of the Salmonella isolates were cefoxitin resistant. Plasmid-mediated AmpC ß-lactamase and ESBL enzymes were recorded in 10/40 (25%) and 16/40 (40%) of the Salmonella isolates, respectively. Specifically, 16/40 (40%) of the Salmonella isolates possessed 380 bp AmpC fox gene, with the highest occurrence found in S. typhi strains (43.8%) followed by S. typhimurium (25%). There was no AmpC fox gene detected in S. paratyphi strains. Interestingly, coproduction of enzymes occurred in some of the isolates, raising fears of resistance to a multitude of antibiotics in the treatment of bacterial infections. CONCLUSION: Emergence of AmpC ß-lactamase-producing Salmonella isolates in our environment was recorded for the first time, raising concern on increased antibiotic resistance among strains of Salmonella serovars in Lagos. Further genotypic study of the isolates could answer the questions on strain sources, clonal relatedness, and mechanism of spread.

Toll-like receptor (TLR4) Asp299Gly and Thr399Ile polymorphisms in relation to clinical falciparum malaria among Nigerian children: a multisite cross-sectional immunogenetic study in Lagos.

INTRODUCTION: This study determined the association of TLR4 Asp299Gly and Thr399Ile with uncomplicated and severe malaria among Nigerian children of similar ethnic background in Lagos. The association of these SNPs with high parasite density, malnutrition, hyperpyrexia and anaemia was also investigated. METHODS: Genomic DNA of the study participants was screened for the genotypes of TLR4 Asp299Gly and Thr399Ile by PCR-RFLP. Anthropometric measurement was performed on the Pf infected children stratified into asymptomatic malaria (control), uncomplicated and severe malaria (case). Parasites were detected by light microscopy and Hardy Weinberg Equilibrium (HWE) of SNP genotypes was also determined. RESULTS: A total of 279 children comprising 182 children (62.1 % male; mean ± SEM age, 57.3 ± 1.7 months) with clinical falciparum malaria and 97 children (55.7 % male; mean ± SEM age, 55.6 ± 2.5 years) with asymptomatic falciparum malaria were enrolled. All the genotypes of both TLR4 SNPs were found in the study population with their minor alleles: 299Gly and 399Ile, found to be 17.6 % and 14.7 % in severe malaria children. Unlike in asymptomatic population, the genotype distribution of TLR4 Asp299Gly SNP was not in HWE in the clinical malaria group but did not condition susceptibility. However, Asp299Gly and Thr399Ile polymorphisms were found to increase the risk of severe malaria 3-fold and 8-fold respectively (P < 0.05). They also increased the risk of severe anaemia, high parasite density and severe malnutrition 3.8 -5.3-fold, 3.3 - 4.4-fold and 4-fold respectively. CONCLUSIONS: Based on the above findings, we conclude that TLR4 Asp299Gly and Thr399Ile polymorphisms may modulate susceptibility to severe malaria among Nigerian children of Yoruba ethnic background.

Seroprevalence of parvovirus B19 antibodies and evidence of viremia among Nigerian patients with sickle cell anemia.

Clinical, biochemical and molecular evidence for the sickle cell anemia (SCA) crisis in Nigerian patients arising from parvovirus b19 infection remains inadequate. This study determined the prevalence and correlates of anti-parvovirus b19 antibodies in a population of SCA patients and non-SCA healthy controls in Lagos, Nigeria. In this prospective cross-sectional study, we enrolled 73 confirmed SCA patients from 5 district hospitals in Lagos and 81 sex and age-matched non-SCA healthy controls. Serum sample from each study participant was screened for anti-parvovirus b19 by ELISA and PCR techniques. Standard biomedical assays were also done. Anti-parvovirus b19 IgM and IgG antibodies were detected in 22 (14.3%) and 97 (62.9%) of the 154 sera screened, 13 (17.8%) and 45 (61.6%) in SCA patients; 9 (11.1%) and 52 (64.2%) in non-SCA controls. The overall seronegativity rate was 19.5%. Parvovirus B19 DNA was found in 2 (11.1%) of the 18 IgM seropositive SCA serum samples screened. On the whole, parvovirus b19 infection was more commonly asymptomatic in non-SCA controls but caused significant elevation in liver enzymes in infected SCA patients (P < 0.05). The risk of acute parvovirus b19 infection increased 65 times during unsteady state among the SCA patients. Although no deaths of infected patients were recorded during the study, age below 12 years, hospitalization and overcrowded environment were risk factors for infection. We conclude that parvovirus b19 is common in SCA patients, incurring greater susceptibility to infections.

A study on the association between parvovirus B19 infection, serum tumour necrosis factor and C-reactive protein levels among Nigerian patients with sickle cell anaemia.

INTRODUCTION: Microbial burden involving parvovirus B19 infection has been recognised as a major cause of morbidity and mortality in sickle cell anaemia (SCA) patients. Given the recent reports of parvovirus B19 infection in Nigeria and the role of inflammation in sickle cell crisis, knowledge of the relationship between the two may be essential for deploying appropriate interventions in infected patients. This study determined the serum levels of tumour necrosis factor alpha (TNF-α) and C-reactive protein (CRP) as inflammatory markers in Nigerian SCA patients with and without parvovirus B19 infections. METHODS: A total of 64 SCA patients aged 5-25 years and 41 age-matched apparently healthy volunteers with haemoglobin genotypes AA or AS were enrolled with consent into the study. Parvovirus B19 infection and serum levels of TNF-α and CRP were determined by the ELISA method. RESULTS: The overall prevalence rate of parvovirus B19 infection in the study subjects was 13.3%. This rate further showed gender variation and negative correlation with age. Significant (p < 0.05) increases in serum CRP and TNF-α levels, with further elevation in unsteady state SCA patients, were observed in comparison with the control. Unlike the control, 29.6% and 21.9% of the SCA patients elicited TNF-α and CRP above threshold levels, respectively. Parvovirus B19 infection was found to elicit greater increases in these inflammatory markers than in infected non-SCA controls. CONCLUSION: We conclude that parvovirus B19 infection is common in this environment, and that serum TNF-α and CRP are predictors of clinical inflammatory episodes in infected SCA patients.

Bacteriologic and plasmid analysis of etiologic agents of conjunctivitis in Lagos, Nigeria.

BACKGROUND: Conjunctivitis, an inflammation of the conjunctiva, is one of the most common eye problems affecting all age groups in Nigeria. A better understanding of its epidemiology and the antibiotic susceptibility of etiologic bacterial agents is crucial for the initiation of preventive and therapeutic measures. This study determined the distribution and patterns of bacterial infections in Nigerian patients with conjunctivitis. Antibiotic resistance patterns and the plasmid profiles of these pathogens were also investigated. METHODOLOGY: A total of 83 consecutive and non-duplicate conjunctival specimens were collected from patients attending eye clinics at three different hospitals in Lagos, Nigeria, between February and September 2010. Specimens were cultured on standard bacteriologic media and the recovered isolates speciated using standard techniques. Susceptibility of pathogens to antibiotics and plasmid DNA extraction were carried out by disk diffusion and alkaline lysis methods. Conjugation experiment was done with rifampicin-resistant Escherichia coli DH5α as the recipient cell. Data were analyzed using the chi-square test. RESULTS: All the specimens were culture-positive, yielding a total of 155 bacterial isolates. Gram-positive cocci comprising Staphylococcus aureus (27.7%) and coagulase-negative Staphylococcus sp. (22.6%) accounted for 50.3% (78 of 155) of conjunctivitis cases, followed by Gram-positive bacilli (22.6%), Gram-negative bacilli (21.3%), and Gram-negative cocci (4.5%). Corynebacterium spp. were the most commonly isolated Gram-positive bacilli accounting for 16.1% of conjunctivitis cases. Pseudomonas aeruginosa topped with 9.7% as the most commonly isolated Gram-negative bacilli. Other Gram-negative bacilli in order of their isolations were E. coli (6.5%), Proteus sp. (3.2%), Klebsiella sp. (1.9%), and Enterobacter aerogenes (1.9%). Moraxella spp. were the only Gram-negative cocci isolated, and they accounted for 4.5% of the total conjunctival infections. Further analysis of the complexity of infections showed that 25 specimens elicited mono-infections, while cases of polymicrobial infections caused by two pathogens and three or more pathogens constituted 51.8% and 18.1% of conjunctivitis specimens screened, respectively. The disparity in the percentage contribution of three infection patterns was significant (P < 0.05). Antibiotic susceptibility testing revealed chloramphenicol and ofloxacin as the least and most active antibiotics tested as 99 (63.9%) and 149 (96.1%) of the 155 recovered isolates were sensitive to them. On the whole, the least susceptible pathogen was P. aeruginosa with sensitivities ranging from 20% to 80%, while Moraxella sp. represented the most sensitive pathogen with sensitivities ranging from 71.4% to 100%. Other bacterial isolates also elicited antibiotic sensitivities in the range of 33.3-100%. A total of 101 isolates were screened for plasmids, of which 45 harbored plasmids, yielding a plasmid frequency of 44.6%. Conjugal transfer of resistance to chloramphenicol, ampicillin, and streptomycin was detected in the transconjugants after the mating experiment. The antibiotic resistances were transferred either singly or in combination from six of the seven selected donor strains. The antibiotic resistance pattern transferred by these donor strains was partial and was associated with the transfer of R plasmids of sizes 21.3, 15.2, and 5.0 kb from three of the six transferable strains. The frequencies of transfer of antibiotype or R plasmids to the transconjugants ranged from 1.8 × 10(-7) to 1.4 × 10(-5) transconjugants per donor strain. CONCLUSION: Conjunctivitis as an eye problem in Lagos is polymicrobial with infections associated with transferable R plasmids for chloramphenicol, ampicillin, and streptomycin. Continuous surveillance of conjunctivitis in relation to etiology, drug susceptibility, and plasmid transferability in the study area is therefore recommended.