Whole genome sequencing and phylogenetic analysis of SARS-CoV-2 strains isolated during the COVID-19 pandemic in Nigeria.

Objectives: The emergence and spread of SARS-CoV-2 have stimulated ongoing research into the virus transmission dynamics, circulating variants, and potential mutations. This study was conducted to understand the genomic dynamics of the epidemic in Nigeria. Design: Whole genome sequencing was conducted on SARS-CoV-2 samples collected during the first and second outbreaks using the Oxford Nanopore MinION sequencing platform. Phylogenetic analysis was conducted, and genomes were grouped into different pangolin lineages. Results: The study revealed four circulating SARS-CoV-2 variants. The Alpha (B.1.1.7) variant was the most prevalent (32.7%), followed by Beta (B.1 B.1.1, L.3, and B.1.1.318) (30.8%), Eta (B.1.525) (28.9%), and Delta (B.1.617, AY.1, AY.109, and AY.36) (7.7%). Phylogenetic analysis revealed three clusters with four Nextstrain clades (20I, 20B, 21D, and 21J). The Alpha lineages (B.1.1.7) clustered with references from Italy. The Beta lineages (Clade 20B) (B.11, B.11318, and L3) and sub-lineage B.11 were distinct. Sub-lineage B.11318 is clustered with references from the USA, whereas sub-lineage L3 is clustered with references from Russia, the Philippines, Australia, and Japan. The 21D and 21J, belonging to two Pango lineages, Eta (B.1525) and Delta (B.1.617 and AY.109), showed high genetic similarity. Conclusion: The phylogenetic relatedness of the lineages suggests multiple virus introduction, which could be a source of more virulent, locally adapted variants.

Invasive bacterial disease in young infants in rural Gambia: Population-based surveillance.

Background: Invasive bacterial diseases (IBD) cause significant mortality in young infants. There are limited population-based data on IBD in young infants in Sub-Saharan Africa. Methods: We conducted population-based surveillance for IBD among infants aged 0-90 days in a demographic surveillance area in rural Gambia between 1 March 2011 and 31 December 2017. Infants admitted to health facilities within the study area had standardised clinical evaluation plus conventional microbiological investigation. We defined IBD as isolation of pathogenic bacteria from blood, cerebrospinal fluid, lung, or pleural aspirate. We determined incidence, aetiology and case-fatality of IBD. Results: A total of 3794 infants were admitted and 3605 (95%) had at least one sample collected for culture. We detected 254 (8.0%) episodes of IBD (bacteraemia 241; meningitis 14; pneumonia seven). The incidence of IBD in infants aged 0-90 days was 25 episodes/1000 person-years (95% confidence interval (CI) = 22-28), the incidence in neonates was 50 episodes/1000 person-years (95% CI = 43-58) and the incidence in infants aged 29-90 days was 12 episodes/1000 person-years (95% CI = 9-15). The most common pathogens causing IBD were Staphylococcus aureus (n = 102, 40%), Escherichia coli (n = 37, 15%), Streptococcus pneumoniae (n = 24, 9%) and Klebsiella pneumoniae (n = 12, 5%). Case-fatality was 29% (95% CI = 23-37) in neonates and 19% (95% CI = 11-29) in infants aged 29-90 days. A minimum of 7.3% of all young infant deaths in the population were caused by IBD. Conclusions: IBD are common in young infants in rural Gambia and have a high case-fatality. Strategies are needed to prevent IBD in young infants. Overcoming barriers to widespread implementation of existing vaccines and developing new vaccines against the most common pathogens causing IBD should be among top priorities for reducing the high mortality rate in young infants.

Multidrug-Resistant Kocuria rosea and Methicillin-Resistant Staphylococcus aureus Co-Infection in a Nigerian Patient with COVID-19: A Case Report.

BACKGROUND Bacterial Infections, especially, of the respiratory system, have been reported as one of the medical concerns in patients with the Coronavirus Disease-2019 (COVID-19), particularly those with multiple co-morbidities. We present a case of a diabetic patient with co-infection of multi-drug-resistant Kocuria rosea and methicillin-resistant Staphylococcus aureus (MRSA) who contracted COVID-19. CASE REPORT A 72-year-old man with diabetes presented with symptoms including cough, chest pain, urinary incontinence, respiratory distress, sore throat, fever, diarrhea, loss of taste, and anosmia and was confirmed to have COVID-19. At admission, he was also found to have sepsis. MRSA was isolated in conjunction with another organism, resembling coagulase-negative Staphylococcus, which was misidentified using commercial biochemical testing systems. The strain was finally confirmed to be Kocuria rosea by 16S rRNA gene sequencing. Both strains were highly resistant to multiple classes of antibiotics, but the Kocuria rosea was resistant to all the cephalosporins, fluoroquinolones, and macrolides tested. The use of ceftriaxone and ciprofloxacin did not improve his condition, which ultimately led to his death. CONCLUSIONS This case report shows that the presence of multi-drug-resistant bacteria infections can be fatal in patients with COVID-19, especially in patients with other co-morbidities like diabetes. This case report also shows that biochemical testing may be inadequate in identifying emerging bacterial infections and there is a need to include proper bacterial screening and treatment in the management of COVID-19, especially in patients with other co-morbidities and with indwelling devices.

Prevalence and spatiotemporal distribution of rotavirus diarrhea among children younger than five years old in Lagos, Nigeria.

Data on spatiotemporal distribution of rotavirus diarrhea are limited in many endemic settings. This study determined the prevalence and seasonal distribution of rotavirus among Nigerian children with diarrhea. Here, a total of 406 fecal samples were collected from patients attending six health facilities in Lagos between January - December 2019. Socio-demographic data of each enrolled child were collected. Rotavirus VP6 antigen was detected by enzyme-linked immunoassay (ELISA) and confirmation by VP7 gene detection by reverse transcription polymerase-chain reaction. The overall rotavirus diarrhea prevalence was 16.3% by ELISA with children above 2 years having 29.2% of this prevalence and higher occurrence in females (59.1%) than males (40.9%) (P < .05). Rotavirus diarrhea diagnosis using RT-PCR showed 100% concordance with ELISA. Cases of rotavirus diarrhea were detected from March to July and from September to November with the highest number of cases detected in May and June (22.7% each), followed by July (21.2%). The prevalence of rotavirus diarrhea remains high in Lagos with an emerging higher disease activity in children above 2. A different rotavirus transmission dynamics compared to previous studies from Nigeria and other African countries was found. VP6 ELISA may reliably be used for continuous rotavirus surveillance in Nigeria.

Significance of hematologic abnormalities in COVID-19 severity among infected patients in Lagos, Nigeria.

Background: There have been suggestions that hematologic abnormalities in COVID-19 are linked with the progression and severity of diseases and mortality. Lymphopenia, sepsis, and thrombocytopenia were highly reported in patients with COVID-19. This study investigated the significance of hematologic abnormalities in patients with COVID-19 in Lagos, Nigeria, and its potential as a diagnostic tool for COVID-19 severity. Results: This was a retrospective observational study with a total of 340 patients with COVID-19 (236 patients included in the analysis). These patients were categorized into two groups, comprising 71 patients with severe COVID-19 (SCP) and 165 patients with non-severe COVID-19 (NSCP). The majority were males in both categories (SCP 74.6% and NSCP 63.6%). The mean ± SD ages for SCP and NSCP were 52.28 ± 16.87 and 42.44 ± 17.18 years, respectively. The SCP (52.1%) and NSCP (20.0%) had underlying health conditions. The SCP exhibited significantly higher neutrophil counts (P < 0.05) and significantly lower mean hemoglobin, red blood cell (RBC), packed cell volume (PCV), and lymphocyte values (P < 0.05). Anemia and lymphocytopenia were more prominent in the SCP group than in the NSCP group (P < 0.05). Hemoglobin, RBC, PCV, and lymphocytes were inversely correlated with age-group in the SCP, while only lymphocytes and platelets were inversely correlated with age-group in the NSCP. The highest area under the ROC curve (AUC) for neutrophils was 0.739 with a sensitivity of 62.0% and specificity of 80.0%, while white blood cells had an AUC of 0.722 with a sensitivity of 73.2% and specificity of 61.2%. The AUC for neutrophil-lymphocyte ratio (NLR) was 0.766 with a sensitivity of 63.3% and specificity of 83.5%, while that for the platelet-lymphocyte ratio (PLR) was 0.695 with a sensitivity and specificity of 61.7% and 77.8%. Conclusions: COVID-19 affected the levels of hemoglobin, RBC, PCV, and lymphocytes in the blood, and the differences were significant between the SCP and NSCP. The significant changes in neutrophil and lymphocyte counts may be useful in the prognosis and management of COVID-19 severity in hospital settings. Furthermore, NLR and PLR may be used in the prognosis and management of severe COVID-19 infection, as well as provide an objective basis for early identification and management in low-resource settings.

SARS-COV-2 antibody responses to AZD1222 vaccination in West Africa.

Real-world data on vaccine-elicited neutralising antibody responses for two-dose AZD1222 in African populations are limited. We assessed baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using binding antibodies analysis coupled with pseudotyped virus neutralisation assays in two cohorts from West Africa: Nigerian healthcare workers (n = 140) and a Ghanaian community cohort (n = 527) pre and post vaccination. We found 44 and 28% of pre-vaccination participants showed IgG anti-N positivity, increasing to 59 and 39% respectively with anti-receptor binding domain (RBD) IgG-specific antibodies. Previous IgG anti-N positivity significantly increased post two-dose neutralizing antibody titres in both populations. Serological evidence of breakthrough infection was observed in 8/49 (16%). Neutralising antibodies were observed to wane in both populations, especially in anti-N negative participants with an observed waning rate of 20% highlighting the need for a combination of additional markers to characterise previous infection. We conclude that AZD1222 is immunogenic in two independent West African cohorts with high background seroprevalence and incidence of breakthrough infection in 2021. Waning titres post second dose indicates the need for booster dosing after AZD1222 in the African setting despite hybrid immunity from previous infection.

Standardization of Epidemiological Surveillance of Invasive Group A Streptococcal Infections.

Invasive group A streptococcal (Strep A) infections occur when Streptococcus pyogenes, also known as beta-hemolytic group A Streptococcus, invades a normally sterile site in the body. This article provides guidelines for establishing surveillance for invasive Strep A infections. The primary objective of invasive Strep A surveillance is to monitor trends in rates of infection and determine the demographic and clinical characteristics of patients with laboratory-confirmed invasive Strep A infection, the age- and sex-specific incidence in the population of a defined geographic area, trends in risk factors, and the mortality rates and rates of nonfatal sequelae caused by invasive Strep A infections. This article includes clinical descriptions followed by case definitions, based on clinical and laboratory evidence, and case classifications (confirmed or probable, if applicable) for invasive Strep A infections and for 3 Strep A syndromes: streptococcal toxic shock syndrome, necrotizing fasciitis, and pregnancy-associated Strep A infection. Considerations of the type of surveillance are also presented, noting that most people who have invasive Strep A infections will present to hospital and that invasive Strep A is a notifiable disease in some countries. Minimal surveillance necessary for invasive Strep A infection is facility-based, passive surveillance. A resource-intensive but more informative approach is active case finding of laboratory-confirmed Strep A invasive infections among a large (eg, state-wide) and well defined population. Participant eligibility, surveillance population, and additional surveillance components such as the use of International Classification of Disease diagnosis codes, follow-up, period of surveillance, seasonality, and sample size are discussed. Finally, the core data elements to be collected on case report forms are presented.

Childhood meningitis in rural Gambia: 10 years of population-based surveillance.

BACKGROUND: The introduction in many countries of conjugate vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis has led to significant reductions in acute bacterial meningitis (ABM) in children. However, recent population-based data on ABM in sub-Saharan Africa are limited. METHODS: Population-based surveillance for meningitis was carried out in a rural area of The Gambia under demographic surveillance from 2008 to 2017, using standardised criteria for referral, diagnosis and investigation. We calculated incidence using population denominators. RESULTS: We diagnosed 1,666 patients with suspected meningitis and collected cerebrospinal fluid (n = 1,121) and/or blood (n = 1,070) from 1,427 (88%) of cases. We identified 169 cases of ABM, 209 cases of suspected non-bacterial meningitis (SNBM) and 1,049 cases of clinically suspected meningitis (CSM). The estimated average annual incidence of ABM was high at 145 per 100,000 population in the <2-month age group, 56 per 100,000 in the 2-23-month age group, but lower at 5 per 100,000 in the 5-14-year age group. The most common causes of ABM were Streptococcus pneumoniae (n = 44), Neisseria meningitidis (n = 42), and Gram-negative coliform bacteria (n = 26). Eighteen of 22 cases caused by pneumococcal serotypes included in PCV13 occurred prior to vaccine introduction and four afterwards. The overall case fatality ratio for ABM was 29% (49/169) and was highest in the <2-month age group 37% (10/27). The case fatality ratio was 8.6% (18/209) for suspected non-bacterial meningitis and 12.8% (134/1049) for clinically suspected meningitis cases. CONCLUSIONS: Gambian children continue to experience substantial morbidity and mortality associated with suspected meningitis, especially acute bacterial meningitis. Such severely ill children in sub-Saharan Africa require improved diagnostics and clinical care.

Evaluation of respiratory tract bacterial co-infections in SARS-CoV-2 patients with mild or asymptomatic infection in Lagos, Nigeria.

Background: A common complication of any respiratory disease by a virus could be a secondary bacterial infection, which is known to cause an increase in severity. It is, however, not clear whether the presence of some opportunistic pathogens called pathobionts contributes to the severity of the disease. In COVID-19 patients, undetected bacterial co-infections may be associated with the severity of the disease. Therefore, we investigated the implications of bacterial co-infections in COVID-19 cases. Results: This is a cross-sectional study that involved archived specimens collected from nasopharyngeal samples of 150 people for COVID-19 screening in Lagos. DNA extraction from the samples was carried out to determine the presence of five respiratory bacterial pathogens using nested real-time PCR, and data were analysed using the Chi-square test. Of the 150 samples collected, 121 (80.7%) were positive for SARs-CoV-2 infection and 29 were negative. The proportion of patients with bacteria co-infection in COVID-19-negative, asymptomatic, and mild cases were 93.1%, 70.7%, and 67.5%, respectively. There was no statistically significant difference between mild COVID-19 conditions and bacteria co-infection (p = 0.097). There was also no significant difference in the nasal carriage of Staphylococcus aureus, Mycoplasma pneumoniae, and Haemophilus spp. However, there was a statistically significant increase in the carriage of Moraxella catarrhalis and Chlamydophila pneumoniae among COVID-19-negative patients when compared with the positive patients (p value = 0.003 and 0.000 for Moraxella catarrhalis and Chlamydophila pneumoniae, respectively). Conclusions: The current study shows that bacterial co-infection and superinfection with COVID-19 are not associated with mild and asymptomatic COVID-19 cases in our setting. However, given the high prevalence of Staphylococcus aureus and Mycoplasma pneumoniae among the mild COVID-19 cases seen in this study, early diagnosis and treatment of these bacterial co-infections are still encouraged to mitigate the effect on the severity of COVID-19.

Widespread sharing of pneumococcal strains in a rural African setting: proximate villages are more likely to share similar strains that are carried at multiple timepoints.

The transmission dynamics of Streptococcus pneumoniae in sub-Saharan Africa are poorly understood due to a lack of adequate epidemiological and genomic data. Here we leverage a longitudinal cohort from 21 neighbouring villages in rural Africa to study how closely related strains of S. pneumoniae are shared among infants. We analysed 1074 pneumococcal genomes isolated from 102 infants from 21 villages. Strains were designated for unique serotype and sequence-type combinations, and we arbitrarily defined strain sharing where the pairwise genetic distance between strains could be accounted for by the mean within host intra-strain diversity. We used non-parametric statistical tests to assess the role of spatial distance and prolonged carriage on strain sharing using a logistic regression model. We recorded 458 carriage episodes including 318 (69.4 %) where the carried strain was shared with at least one other infant. The odds of strain sharing varied significantly across villages (χ2=47.5, df=21, P-value <0.001). Infants in close proximity to each other were more likely to be involved in strain sharing, but we also show a considerable amount of strain sharing across longer distances. Close geographic proximity (<5 km) between shared strains was associated with a significantly lower pairwise SNP distance compared to strains shared over longer distances (P-value <0.005). Sustained carriage of a shared strain among the infants was significantly more likely to occur if they resided in villages within a 5 km radius of each other (P-value <0.005, OR 3.7). Conversely, where both infants were transiently colonized by the shared strain, they were more likely to reside in villages separated by over 15 km (P-value <0.05, OR 1.5). PCV7 serotypes were rare (13.5 %) and were significantly less likely to be shared (P-value <0.001, OR -1.07). Strain sharing was more likely to occur over short geographical distances, especially where accompanied by sustained colonization. Our results show that strain sharing is a useful proxy for studying transmission dynamics in an under-sampled population with limited genomic data. This article contains data hosted by Microreact.

The use of DIY (Do it yourself) sampling and telemonitoring model for COVID-19 qPCR testing scale up.

The first case of COVID-19 in Nigeria was recorded on February 27, 2020, being an imported case by an Italian expatriate, to the country. Since then, there has been steady increase in the number of cases. However, the number of cases in Nigeria is low in comparison to cases reported by other countries with similar large populations, despite the poor health system prevailing in the country. This has been mainly attributed to the low testing capacity in Nigeria among other factors. Therefore, there is a need for innovative ways to increase the number of persons testing for COVID-19. The aim of the study was to pilot a nasopharyngeal swab self-sample collection model that would help increase COVID-19 testing while ensuring minimal person-to-person contact being experienced at the testing center. 216 participants took part in this study which was carried out at the Nigerian Institute of Medical Research between June and July 2020. Amongst the 216 participants, 174 tested negatives for both self-collected samples and samples collected by Professionals, 30 tested positive for both arms, with discrepancies occurring in 6 samples where the self-collected samples were positive while the ones collected by the professionals were negative. The same occurred in another set of 6 samples with the self-collected samples being negative and the professional-collected sample coming out positive, with a sensitivity of 83.3% and a specificity of 96.7%. The results of the interrater analysis are Kappa = 0.800 (95% CI, 0.690 to 0.910) which implies an outstanding agreement between the two COVID-19 sampling methods. Furthermore, since p< 0.001 Kappa (k) coefficient is statistically different from zero, our findings have shown that self-collected samples can be reliable in the diagnosis of COVID-19.

Pathogens Associated With Linear Growth Faltering in Children With Diarrhea and Impact of Antibiotic Treatment: The Global Enteric Multicenter Study.

BACKGROUND: The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment. METHODS: The Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models. RESULTS: Among 8077 MSD cases analyzed, the proportion with stunting (HAZ below -1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0-11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12-23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02). CONCLUSIONS: Linear growth faltering among children aged 0-23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

Impact of the introduction of pneumococcal conjugate vaccination on invasive pneumococcal disease and pneumonia in The Gambia: 10 years of population-based surveillance.

BACKGROUND: The Gambia introduced seven-valent pneumococcal conjugate vaccine (PCV7) in August 2009, followed by PCV13 in May, 2011, using a schedule of three primary doses without a booster dose or catch-up immunisation. We aimed to assess the long-term impact of PCV on disease incidence. METHODS: We did 10 years of population-based surveillance for invasive pneumococcal disease (IPD) and WHO defined radiological pneumonia with consolidation in rural Gambia. The surveillance population included all Basse Health and Demographic Surveillance System residents aged 2 months or older. Nurses screened all outpatients and inpatients at all health facilities using standardised criteria for referral. Clinicians then applied criteria for patient investigation. We defined IPD as a compatible illness with isolation of Streptococcus pneumoniae from a normally sterile site (cerebrospinal fluid, blood, or pleural fluid). We compared disease incidence between baseline (May 12, 2008-May 11, 2010) and post-vaccine years (2016-2017), in children aged 2 months to 14 years, adjusting for changes in case ascertainment over time. FINDINGS: We identified 22 728 patients for investigation and detected 342 cases of IPD and 2623 cases of radiological pneumonia. Among children aged 2-59 months, IPD incidence declined from 184 cases per 100 000 person-years to 38 cases per 100 000 person-years, an 80% reduction (95% CI 69-87). Non-pneumococcal bacteraemia incidence did not change significantly over time (incidence rate ratio 0·88; 95% CI, 0·64-1·21). We detected zero cases of vaccine-type IPD in the 2-11 month age group in 2016-17. Incidence of radiological pneumonia decreased by 33% (95% CI 24-40), from 10·5 to 7·0 per 1000 person-years in the 2-59 month age group, while pneumonia hospitalisations declined by 27% (95% CI 22-31). In the 5-14 year age group, IPD incidence declined by 69% (95% CI -28 to 91) and radiological pneumonia by 27% (95% CI -5 to 49). INTERPRETATION: Routine introduction of PCV13 substantially reduced the incidence of childhood IPD and pneumonia in rural Gambia, including elimination of vaccine-type IPD in infants. Other low-income countries can expect substantial impact from the introduction of PCV13 using a schedule of three primary doses. FUNDING: Gavi, The Vaccine Alliance; Bill & Melinda Gates Foundation; UK Medical Research Council; Pfizer Ltd.

Comparative performance of SARS-CoV-2 real-time PCR diagnostic assays on samples from Lagos, Nigeria.

A key element in containing the spread of the SARS-CoV-2 infection is quality diagnostics which is affected by several factors. We now report the comparative performance of five real-time diagnostic assays. Nasopharyngeal swab samples were obtained from persons seeking a diagnosis for SARS-CoV-2 infection in Lagos, Nigeria. The comparison was performed on the same negative, low, and high-positive sample set, with viral RNA extracted using the Qiagen Viral RNA Kit. All five assays are one-step reverse transcriptase real-time PCR assays. Testing was done according to each assay's manufacturer instructions for use using real-time PCR platforms. 63 samples were tested using the five qPCR assays, comprising of 15 negative samples, 15 positive samples (Ct = 16-30; one Ct = 35), and 33 samples with Tib MolBiol E-gene Ct value ranging from 36-41. All assays detected all high positive samples correctly. Three assays correctly identified all negative samples while two assays each failed to correctly identify one different negative sample. The consistent detection of positive samples at different Ct/Cq values gives an indication of when to repeat testing and/or establish more stringent in-house cut-off value. The varied performance of different diagnostic assays, mostly with emergency use approvals, for a novel virus is expected. Comparative assays' performance reported may guide laboratories to determine both their repeat testing Ct/Cq range and/or cut-off value.

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.

Persistent and Emerging Pneumococcal Carriage Serotypes in a Rural Gambian Community After 10 Years of Pneumococcal Conjugate Vaccine Pressure.

BACKGROUND: The continuing impact of pneumococcal conjugate vaccines (PCVs) in regions with high pneumococcal transmission is threatened by the persistence of vaccine serotypes (VTs) and the emergence of nonvaccine serotypes (NVTs). METHODS: In 2016, we conducted a cross-sectional carriage survey (CSS5) in a community where PCV7 was first introduced in 2006 during a cluster-randomized trial conducted before nationwide introduction of PCV7 (2009) and PCV13 (2011). We estimated prevalence of PCV13 VT and NVT by age and compared these with earlier surveys before (CSS0), during (CSS1-3), and after the trial but before PCV13 (CSS4). Genomic analysis was conducted for the nontypeable pneumococci. RESULTS: Prevalence of PCV13 VT carriage decreased during the 10 years between CSS0 and CSS5 across all age groups (67.6% to 13.5%, P < .001; 59.8% to 14.4%, P < .001; 43.1% to 17.9%, P < .001; and 24.0% to 5.1%, P < .001, in <2, 2-4, 5-14, and ≥15 years, respectively). However, there was no difference between CSS4 and CSS5 in children ≥2 years and adults (children <2 years, no data). The prevalence of PCV13 NVT increased between CSS0 and CSS5 for children <2 years but decreased in older children and adults. In CSS5, serotypes 3, 6A, and 19F were the most common VT and nontypeable isolates were the most common NVT. Among nontypeable isolates, 73.0% lost the ability to express a capsule. Of these, 70.8% were from a VT background. CONCLUSIONS: The decrease in PCV13 VT that has occurred since the introduction of PCV13 appears to have plateaued. Significant carriage of these serotypes remains in all age groups.

Within-host microevolution of Streptococcus pneumoniae is rapid and adaptive during natural colonisation.

Genomic evolution, transmission and pathogenesis of Streptococcus pneumoniae, an opportunistic human-adapted pathogen, is driven principally by nasopharyngeal carriage. However, little is known about genomic changes during natural colonisation. Here, we use whole-genome sequencing to investigate within-host microevolution of naturally carried pneumococci in ninety-eight infants intensively sampled sequentially from birth until twelve months in a high-carriage African setting. We show that neutral evolution and nucleotide substitution rates up to forty-fold faster than observed over longer timescales in S. pneumoniae and other bacteria drives high within-host pneumococcal genetic diversity. Highly divergent co-existing strain variants emerge during colonisation episodes through real-time intra-host homologous recombination while the rest are co-transmitted or acquired independently during multiple colonisation episodes. Genic and intergenic parallel evolution occur particularly in antibiotic resistance, immune evasion and epithelial adhesion genes. Our findings suggest that within-host microevolution is rapid and adaptive during natural colonisation.