Antimicrobial resistance and virulence genes in Salmonella enterica serovars isolated from droppings of layer chicken in two farms in Nigeria.

AIM: This study aimed to investigate the isolation rate, antibiotic resistance and virulence genes of Salmonella enterica serovar from two commercial farms in Nigeria. METHODS AND RESULTS: Salmonella isolation was performed according to the United States Food and Drug Agency (USFDA) method. Serotyping, antimicrobial susceptibility testing, detection of resistance and virulence genes were done using the Kauffman-White Scheme, disc diffusion, minimum inhibitory concentration and real-time polymerase chain reaction techniques. Salmonella serovars were isolated from only farm A at 22/50 (44.0%) while none were isolated from farm B. Salmonella Typhi, 9 (40.9%); Salmonella Typhimurium, 2 (9.1%), Salmonella Enteritidis, 2 (9.1%), Salmonella Pullorum, 1 (4.5%), Salmonella Kentucky, 4 (18.2%) were identified while 4 (18.2%) were untypable. Sixteen isolates (72.7%) showed multiple drug resistance and 17 different resistance profile types with AMP-CHL-TRM-SXT as the most prevalent pattern. Resistance genes (blaTEM, 12/22 (54.5%) and virulence genes (InvA, sopB, mgtC and spi4D, 22/22 (100.0%), ssaQ, 16/22 (72.7%) and spvC, 13/22 (59.1%) were found, while blaSHV, blaCTX-M, floR, tetA, tetB, tetG and LJSGI-1 genes were absent. CONCLUSION: Pathogenic Salmonella were isolated from the chicken droppings in this study. Most of these strains were resistant to antibiotics and possessed characteristics of virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: Chicken droppings from this study area contained pathogenic strains of Salmonella and a rare occurrence of Salmonella Typhi. The study revealed that the environment and the food chain could be at risk of contamination of highly virulent and antimicrobial-resistant strains of Salmonella. These could affect the profitability of the poultry industry and food consumption. There is a need for caution in indiscriminate disposal of poultry waste and the use of uncomposted chicken droppings in soil amendment.

Paper-based microchip electrophoresis for point-of-care hemoglobin testing.

Nearly 7% of the world's population live with a hemoglobin variant. Hemoglobins S, C, and E are the most common and significant hemoglobin variants worldwide. Sickle cell disease, caused by hemoglobin S, is highly prevalent in sub-Saharan Africa and in tribal populations of Central India. Hemoglobin C is common in West Africa, and hemoglobin E is common in Southeast Asia. Screening for significant hemoglobin disorders is not currently feasible in many low-income countries with the high disease burden. Lack of early diagnosis leads to preventable high morbidity and mortality in children born with hemoglobin variants in low-resource settings. Here, we describe HemeChip, the first miniaturized, paper-based, microchip electrophoresis platform for identifying the most common hemoglobin variants easily and affordably at the point-of-care in low-resource settings. HemeChip test works with a drop of blood. HemeChip system guides the user step-by-step through the test procedure with animated on-screen instructions. Hemoglobin identification and quantification is automatically performed, and hemoglobin types and percentages are displayed in an easily understandable, objective way. We show the feasibility and high accuracy of HemeChip via testing 768 subjects by clinical sites in the United States, Central India, sub-Saharan Africa, and Southeast Asia. Validation studies include hemoglobin E testing in Bangkok, Thailand, and hemoglobin S testing in Chhattisgarh, India, and in Kano, Nigeria, where the sickle cell disease burden is the highest in the world. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. Study design, methods, and results are presented according to the Standards for Reporting Diagnostic Accuracy (STARD). HemeChip correctly identified all subjects with hemoglobin S, C, and E variants with 100% sensitivity, and displayed an overall diagnostic accuracy of 98.4% in comparison to reference standard methods. HemeChip is a versatile, mass-producible microchip electrophoresis platform that addresses a major unmet need of decentralized hemoglobin analysis in resource-limited settings.

Salmonella Bacteremia Among Children in Central and Northwest Nigeria, 2008-2015.

BACKGROUND: Etiologic agents of childhood bacteremia remain poorly defined in Nigeria. The absence of such data promotes indiscriminate use of antibiotics and delays implementation of appropriate preventive strategies. METHODS: We established diagnostic laboratories for bacteremia surveillance at regional sites in central and northwest Nigeria. Acutely ill children aged <5 years with clinically suspected bacteremia were evaluated at rural and urban clinical facilities in the Federal Capital Territory, central region and in Kano, northwest Nigeria. Blood was cultured using the automated Bactec incubator system. RESULTS: Between September 2008 and April 2015, we screened 10,133 children. Clinically significant bacteremia was detected in 609 of 4051 (15%) in the northwest and 457 of 6082 (7.5%) in the central region. Across both regions, Salmonella species account for 24%-59.8% of bacteremias and are the commonest cause of childhood bacteremia, with a predominance of Salmonella enterica serovar Typhi. The prevalence of resistance to ampicillin, chloramphenicol, and cotrimoxazole was 38.11%, with regional differences in susceptibility to different antibiotics but high prevalence of resistance to readily available oral antibiotics. CONCLUSIONS: Salmonella Typhi is the leading cause of childhood bacteremia in central Nigeria. Expanded surveillance is planned to define the dynamics of transmission. The high prevalence of multidrug-resistant strains calls for improvement in environmental sanitation in the long term and vaccination in the short term.

Vitamin D Metabolites in Mother-Infant Dyads and Associated Clinical Outcomes in a Population of Nigerian Women.

Low levels of vitamin D in maternal and cord blood have been associated with neonatal sepsis. This study assessed the association of vitamin D metabolites (25(OH)D, 3-epi-25(OH)D3, and 24,25(OH)2D3) levels in maternal and cord blood with newborn sepsis evaluation in Nigerian mother-infant dyads. Maternal and cord blood from 534 mothers and 536 newborns were processed using liquid chromatography-tandem mass spectrometry. Spearman correlation was used to compare continuous variables, Mann-Whitney for dichotomous variables, and Kruskal-Wallis for two or more groups. High cord percent 3-epi-25(OH)D3 levels were positively associated with newborn evaluation for sepsis (p = 0.036), while maternal and cord 25(OH)D and 24,25(OH)2D3 levels were not. Being employed was positively associated with maternal and newborn 3-epi-25(OH)D3 concentrations (p = 0.007 and p = 0.005, respectively). The maternal 3-epi-25(OH)D3 and percent 3-epi-25(OH)D3 were positively associated with vaginal delivery (p = 0.013 and p = 0.012, respectively). Having a weight-for-age Z-score ≤ -2 was positively associated with newborn percent 3-epi-25(OH)D3 levels (p = 0.004), while a weight-for-length Z-score ≤ -3 was positively associated with maternal and newborn percent 3-epi-25(OH)D3 levels (p = 0.044 and p = 0.022, respectively). Our study highlights the need to further investigate the biological role of 3-epi-25(OH)D3 and its clinical significance in fetal growth and newborn outcome.

Antimicrobial resistance and virulence genes of invasive Salmonella enterica from children with bacteremia in north-central Nigeria.

Objectives: Bacteremia due to invasive Salmonella enterica has been reported earlier in children in Nigeria. This study aimed to detect the virulence and antibiotic resistance genes of invasive Salmonella enterica from children with bacteremia in north-central Nigeria. Method: From June 2015 to June 2018, 4163 blood cultures yielded 83 Salmonella isolates. This is a secondary cross-sectional analysis of the Salmonella isolates. The Salmonella enterica were isolated and identified using standard bacteriology protocol. Biochemical identifications of the Salmonella enterica were made by Phoenix MD 50 identification system. Further identification and confirmation were done with polyvalent antisera O and invA gene. Antimicrobial susceptibility testing was done following clinical and laboratory standard institute guidelines. Resistant genes and virulence genes were determined using a real-time polymerase chain reaction. Result: Salmonella typhi 51 (61.4%) was the most prevalent serovar, followed by Salmonella species 13 (15.7%), choleraesuis 8 (9.6%), enteritidis 6 (7.2%), and typhimurium 5 (6.1%). Fifty-one (61.4%) of 83 Salmonella enterica were typhoidal, while 32 (38.6%) were not. Sixty-five (78.3%) of the 83 Salmonella enterica isolates were resistant to ampicillin and trimethoprim-sulfamethoxazole, followed by chloramphenicol 39 (46.7%), tetracycline 41 (41.4%), piperacillin 33 (33.9%), amoxicillin-clavulanate, and streptomycin 21 (25.3%), while cephalothin was 19 (22.9%). Thirty-nine (46.9%) of the 83 Salmonella enterica isolates were multi-drug resistant, and none were extensive drug resistant or pan-drug resistant. A blaTEM 42 (50.6%), floR 32 (38.6%), qnrA 24 (28.9%), tetB 20 (20.1%), tetA 10 (10.0%), and tetG 5 (6.0%) were the antibiotic resistance genes detected. There were perfect agreement between phenotypic and genotypic detection of antimicrobial resistance in tetracycline, ciprofloxacin, and chloramphenicol, while beta-lactam showed κ = 0.60 agreement. All of the Salmonella enterica isolates had the virulence genes invA, sopB, mgtC, and sip4D, while 33 (39.8%), 45 (51.8%), and 2 (2.4%) had ssaQ, spvC, and ljsGI-1, respectively. Conclusion: Our findings showed multi-drug resistant Salmonella enterica in children with bacteremia in northern Nigeria. In addition, significant virulence and antimicrobial resistance genes were found in invasive Salmonella enterica in northern Nigeria. Thus, our study emphasizes the need to monitor antimicrobial resistance in Salmonella enterica from invasive sources in Nigeria and supports antibiotic prudence.

Phenotypic and molecular characterization of beta-lactam resistant Multidrug-resistant Enterobacterales isolated from patients attending six hospitals in Northern Nigeria.

Infections caused by multi-drug resistant Enterobacterales (MDR-E) are difficult to treat and cause significant mortality, especially in developing countries. This study characterized the phenotypic and genotypic profiles of 49 randomly selected beta-lactam resistant MDR-E previously isolated from patients being managed in hospitals in Nigeria using whole genome sequencing. The study isolates exhibited 85.5% resistance to 3rd generation cephalosporins and 65.3% resistance to carbapenems. The blaTEM-1B (29, 59.2%), blaCTX-M-15 (38, 77.6%), and blaNDM-1 (17, 51.5%) were the most common penicillinase, ESBL, and carbapenem resistant genes across isolates, respectively. Seventeen (45%) of blaCTX-M-15 was carried on the insertion sequence ISEc9 while blaNDM-1 (11, 64.7%) were associated with ISEc33. None of the 21 plasmids detected were associated with ß-lactamase genes. Higher resistance rates were found in E. coli ST-88 (n = 2) and the high-risk ST-692 (n = 2). For Klebsiella species, the high-risk clones ST-476 (n = 8) and ST-147 (n = 3) predominated and had higher phenotypic resistance rates and higher number of AMR genes. The mechanisms and pattern of antibiotic resistance differ from patterns previously described with isolates harbouring a wide range of AMRGs. The detection of several chromosomally mediated carbapenemases in our study also represents a significant finding that warrants further investigation to better understand its' implications for clinical practice and public health. The selected MDR-Es were found to be pan-susceptible to tigecycline and had very low resistance to fosfomycin, suggesting a potential for these as empiric treatments. A surveillance approach incorporating both conventional laboratory techniques and modern molecular techniques is essential for the comprehensive characterization of the emergence and dissemination of antimicrobial resistance in Enterobacterales infections within Nigeria.

Molecular characterization of Streptococcus pneumoniae causing disease among children in Nigeria during the introduction of PCV10 (GSK).

Streptococcus pneumoniae (pneumococcus) is a leading vaccine-preventable cause of childhood invasive disease. Nigeria has the second highest pneumococcal disease burden globally, with an estimated ~49 000 child deaths caused by pneumococcal infections each year. Ten-valent pneumococcal conjugate vaccine (GSK; PCV10) was introduced in December 2014 in a phased approach. However, few studies have characterized the disease-causing pneumococci from Nigeria. This study assessed the prevalence of serotypes, antibiotic susceptibility and genomic lineages using whole genome sequencing and identified lineages that could potentially escape PCV10 (GSK). We also investigated the potential differences in pneumococcal lineage features between children with and without sickle cell disease. A collection of 192 disease-causing pneumococcal isolates was obtained from Kano (n=189) and Abuja (n=3) states, Nigeria, between 1 January 2014 and 31 May 2018. The majority (99 %, 190/192) of specimens were recovered from children aged 5 years or under. Among them, 37 children had confirmed or traits of sickle cell disease. Our findings identified 25 serotypes expressed by 43 Global Pneumococcal Sequence Clusters (GPSCs) and 85 sequence types (STs). The most common serotypes were 14 (18 %, n=35), 6B (16 %, n=31), 1 (9 %, n=17), 5 (9 %, n=17) and 6A (9 %, n=17); all except serotype 6A are included in PCV10 (GSK). PCV10 (SII; PNEUMOSIL) and PCV13 formulations include serotypes 6A and 19A which would increase the overall coverage from 67 % by PCV10 (GSK) to 78 and 82 %, respectively. The pneumococcal lineages were a mix of globally spreading and unique local lineages. Following the use of PCV10 (GSK), GPSC5 expressing serotype 6A, GPSC10 (19A), GPSC26 (12F and 46) and GPSC627 (9L) are non-vaccine type lineages that could persist and potentially expand under vaccine-selective pressure. Approximately half (52 %, 99/192) of the pneumococcal isolates were resistant to the first-line antibiotic penicillin and 44 % (85/192) were multidrug-resistant. Erythromycin resistance was very low (2 %, 3/192). There was no significant difference in clinical manifestation, serotype prevalence or antibiotic resistance between children with and without traits of or confirmed sickle cell disease. In summary, our findings show that a high percentage of the pneumococcal disease were caused by the serotypes that are covered by currently available vaccines. Given the low prevalence of resistance, macrolide antibiotics, such as erythromycin, should be considered as an option to treat pneumococcal disease in Nigeria. However, appropriate use of macrolide antibiotics should be vigilantly monitored to prevent the potential increase in macrolide resistance.

Community acquired bacteremia in young children from central Nigeria--a pilot study.

BACKGROUND: Reports of the etiology of bacteremia in children from Nigeria are sparse and have been confounded by wide spread non-prescription antibiotic use and suboptimal laboratory culture techniques. We aimed to determine causative agents and underlying predisposing conditions of bacteremia in Nigerian children using data arising during the introduction of an automated blood culture system accessed by 7 hospitals and clinics in the Abuja area. METHODS: Between September 2008 and November 2009, we enrolled children with clinically suspected bacteremia at rural and urban clinical facilities in Abuja or within the Federal Capital Territory of Nigeria. Blood was cultured using an automated system with antibiotic removing device. We documented clinical features in all children and tested for prior antibiotic use in a random sample of sera from children from each site. RESULTS: 969 children aged 2 months-5 years were evaluated. Mean age was 21±15.2 months. All children were not systematically screened but there were 59 (6%) children with established diagnosis of sickle cell disease and 42 (4.3%) with HIV infection. Overall, 212 (20.7%) had a positive blood culture but in only 105 (10.8%) were these considered to be clinically significant. Three agents, Staphylococcus aureus (20.9%), Salmonella typhi (20.9%) and Acinetobacter (12.3%) accounted for over half of the positive cultures. Streptococcus pneumoniae and non-typhi Salmonellae each accounted for 7.6%. Although not the leading cause of bacteremia, Streptococcus pneumoniae was the single leading cause of all deaths that occurred during hospitalization and after hospital discharge. CONCLUSION: S. typhi is a significant cause of vaccine-preventable morbidity while S. pneumoniae may be a leading cause of mortality in this setting. This observation contrasts with reports from most other African countries where non-typhi Salmonellae are predominant in young children. Expanded surveillance is required to confirm the preliminary observations from this pilot study to inform implementation of appropriate public health control measures.

Molecular characterization of invasive Enterobacteriaceae from pediatric patients in Central and Northwestern Nigeria.

BACKGROUND: Bacteremia is a leading cause of mortality in developing countries, however, etiologic evaluation is infrequent and empiric antibiotic use not evidence-based. Here, we evaluated the patterns of ESBL resistance in children enrolled into a surveillance study for community acquired bacteremic syndromes across health facilities in Central and Northwestern Nigeria. METHOD: Blood culture was performed for children aged less than 5 years suspected of having sepsis from Sept 2008-Dec 2016. Blood was incubated using the BACTEC00AE system and Enterobacteriacea identified to the species level using Analytical Profile Index (API20E®). Antibiotic susceptibility profile was determined by the disc diffusion method. Real time PCR was used to characterize genes responsible for ESBL production. RESULT: Of 21,000 children screened from Sept 2008-Dec 2016, 2,625(12.5%) were culture-positive. A total of 413 Enterobacteriaceae available for analysis were screened for ESBL. ESBL production was detected in 160 Enterobacteriaceae, high resistance rates were observed among ESBL-positive isolates for Ceftriaxone (92.3%), Aztreonam (96.8%), Cefpodoxime (96.3%), Cefotaxime (98.8%) and Trimethoprim/sulfamethoxazole (90%), while 87.5%, 90.7%, and 91.9% of the isolates were susceptible to Imipenem, Amikacin and Meropenem respectively. Frequently detected resistance genes were blaTEM-83.8% (134/160), and, blaCTX-M 83.1% (133/160) followed by blaSHVgenes 66.3% (106/160). Co-existence of blaCTX-M, blaTEM and blaSHV was seen in 94/160 (58.8%), blaCTX-M and blaTEM in 118/160 (73.8%), blaTEM and blaSHV in 97/160 (60.6%) and blaCTX-M and blaSHV in 100/160 (62.5%) of isolates tested. CONCLUSION: Our results indicate a high prevalence of bacteremia from ESBL Enterobacteriaceae in this population of children. These are resistant to commonly used antibiotics and careful choice of antibiotic treatment options is critical. Further studies to evaluate transmission dynamics of resistance genes could help in the reduction of ESBL resistance in these settings.

Sickle-Cell Disease in Nigerian Children: Parental Knowledge and Laboratory Results.

BACKGROUND: Sickle-cell disease (SCD) is the most common inherited genetic disorder in sub-Saharan Africa, and it is associated with early mortality and lifelong morbidity. Early diagnosis is essential for instituting appropriate care and preventive therapy. OBJECTIVE: To compare parental knowledge or perception of their offspring's hemoglobin phenotype prior to testing and actual validated laboratory test results. METHODS: In a prospective community-based survey, we assessed parental knowledge of their children's hemoglobin phenotype and corroborated this with the results from a laboratory confirmatory test determined by high-performance liquid chromatography. RESULTS: We screened 10,126 children aged less than 5 years. A total of 163 (1.6%) parents indicated that their offspring had been previously tested and had knowledge of the child's hemoglobin genotype. However, 51 (31.2%) of 163 parents of children who had been previously tested did not know the result of their offspring's test, and 18 (35.3%) of these 51 children were found to have SCD. Of those who claimed previous knowledge, 25 (15.3%) of 163 reported incorrect results. Overall, we identified 272 (2.76%) new cases from 9,963 children who had not been previously tested. CONCLUSION: There is the need to promote public awareness about SCD and the benefit of early diagnosis, quality assurance in laboratory diagnosis and institution of sustainable patient care pathways.

Molecular Surveillance Identifies Multiple Transmissions of Typhoid in West Africa.

BACKGROUND: The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children. METHODS: A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance. RESULTS: Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi. CONCLUSIONS: These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid.