High prevalence of resistance to third-generation cephalosporins detected among clinical isolates from sentinel healthcare facilities in Lagos, Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) in bacterial pathogens is a worldwide concern that demands immediate attention. Most information on AMR originates from high-income countries and little is known about the burden in Africa, particularly Nigeria. Using four sentinel sites (General hospitals) in Lagos State, this study sought to estimate the burden of AMR. METHODS: This is a hospital-based surveillance using secondary health care centres. Four sites were randomly selected and included in the study. Clinical isolates were collected over a period of 6 months for each site from August 2020 to March 2021. All isolates were characterised and analysed for resistance to 15 antibiotics using the Kirby-Baur method. Multiplex PCR assay was used for the detection of Extended spectrum beta lactamase genes. Data analysis was done using SPSS version 27.0. RESULTS: Four hundred and ninety-nine (499) patients consented and participated in this study, consisting of 412 (82.6%) females and 87 (17.4%) males. The mean age ± SD of the participants was 33.9 ± 13.8 with a range of 1-89 years. The majority (90.8%) of the participants were outpatients. Two hundred and thirty-two (232) isolates were obtained from 219 samples, comprising of 120 (51.7%) Gram positive and 112 (48.3%) Gram negative organisms. Key bacterial pathogens isolated from this study included Staphylococcus aureus (22.8%), Escherichia coli (16.4%), Staphylococcus spp. (15.9%), Enterococcus spp. (7.3%) and Klebsiella pneumoniae (6.5%). There was high prevalence of multi-drug resistance (79.3%) among the isolates with 73.6% of Staphylococcus aureus phenotypically resistant to methicillin and 70% possessed the MecA gene. 76.5% of Enterococcus spp. isolated were Vancomycin resistant. Overall, resistance to Cephalosporins was most frequently/commonly observed (Cefotaxime 87.5%). CONCLUSION: A high incidence of AMR was identified in clinical bacteria isolates from selected general hospitals in Lagos State, highlighting the necessity for the implementation of national action plans to limit the prevalence of AMR. Surveillance via collection of isolates has a lot of promise, especially in resource-limited environments.

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.

Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria.

In an outbreak, effective detection of the aetiological agent(s) involved using molecular techniques is key to efficient diagnosis, early prevention and management of the spread. However, sequencing is necessary for mutation monitoring and tracking of clusters of transmission, development of diagnostics and for vaccines and drug development. Many sequencing methods are fast evolving to reduce test turn-around-time and to increase through-put compared to Sanger sequencing method; however, Sanger sequencing remains the gold standard for clinical research sequencing with its 99.99% accuracy This study sought to generate sequence data of SARS-CoV-2 using Sanger sequencing method and to characterize them for possible site(s) of mutations. About 30 pairs of primers were designed, synthesized, and optimized using endpoint PCR to generate amplicons for the full length of the virus. Cycle sequencing using BigDye Terminator v.3.1 and capillary gel electrophoresis on ABI 3130xl genetic analyser were performed according to the manufacturers' instructions. The sequence data generated were assembled and analysed for variations using DNASTAR Lasergene 17 SeqMan Ultra. Total length of 29,760bp of SARS-CoV-2 was assembled from the sample analysed and deposited in GenBank with accession number: MT576584. Blast result of the sequence assembly shows a 99.97% identity with the reference sequence. Variations were noticed at positions: nt201, nt2997, nt14368, nt16535, nt20334, and nt28841-28843, which caused amino acid alterations at the S (aa614) and N (aa203-204) regions. The mutations observed at S and N-gene in this study may be indicative of a gradual changes in the genetic coding of the virus hence, the need for active surveillance of the viral genome.

Laboratory Optimization Tweaks for Sanger Sequencing in a Resource-Limited Setting.

Despite various challenges that hinder the implementation of high-tech molecular methods in resource-limited settings, we have been able to implement and achieve International Organization for Standardization 15189:2012 accreditation for genotypic HIV drug resistance testing in our facility. At the Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Nigeria has recorded a high sequencing success rate and good quality sequence data. This was achieved by optimizing laboratory processes from 2008 to the current date. We have optimized sample preparation, RT-PCR, several post-PCR processes, and the cycle sequencing to improve the sensitivity of amplification even with limited plasma samples and low viral copy numbers. The optimized workflow maximizes output, minimizes reagent wastage, and achieves substantial cost savings without compromising the quality of the sequence data. Our performance at our last external quality assurance program is a testimonial to the efficiency of the workflow. For the 5-sample panel, each with 67-68 mutation points evaluated, we scored 100% for all 5 specimens. Our optimized laboratory workflow is thus documented to support laboratories and to help researchers achieve excellent results the first time and eliminate contamination while minimizing the wastage of costly sequencing reagents.