Challenges in conducting population-based seroepidemiology survey of COVID-19 in Lagos State, Nigeria.

Population-based study is known to be a very essential type of study during and after a pandemic or epidemic, as it provides crucial information on the incidence, prevalence, and risk factors of the disease in question. There has been limited information about the challenges faced in conducting such surveys in Nigeria. In this paper, we will share our experience, and describe the challenges faced in conducting a population-based seroepidemiological study of COVID-19 in Lagos, Nigeria. Some challenges were peculiar to specific Local Government Areas (LGAs) while others were general. The challenges include general misconceptions of community members about health research, difficulties in mapping houses, planning for data collection, standardizing data collection, working in hard-to-reach communities when resources were limited as well as difficulty in collection of blood and naso-oropharyngeal swabs. Ways of overcoming these problems, lessons learnt, and recommendations are hereby discussed.

Low levels of HIV-1 drug resistance mutations in patients who achieved viral re-suppression without regimen switch: a retrospective study.

BACKGROUND: We identified a HIV-positive cohort in virologic failure (VF) who re-suppressed without drug switch. We characterized their drug resistance mutations (DRM) and adherence profiles to learn how to better manage HIV drug resistance. A retrospective cohort study utilizing clinical data and stored samples. Patients received ART at three Nigerian treatment centres. Plasma samples stored when they were in VF were genotyped. RESULT: Of 126 patients with samples available, 57 were successfully genotyped. From ART initiation, the proportion of patients with adherence ≥90% increased steadily from 54% at first high viral load (VL) to 67% at confirmed VF, and 81% at time of re-suppressed VL. Sixteen (28%) patients had at least one DRM. Forty-six (81%) patients had full susceptibility to the three drugs in their first-line (1 L) regimen. Thirteen (23%) were resistant to at least one antiretroviral drug but three were resistant to drugs not used in Nigeria. Ten patients had resistance to their 1 L drug(s) and six were fully susceptible to the three drugs in the recommended second-line regimen. CONCLUSION: This cohort had little drug resistance mutations. We conclude that if adherence is not assured, patients could exhibit virologic failure without having developed mutations associated with drug resistance.

Occult Hepatitis B virus infection in previously screened, blood donors in Ile-Ife, Nigeria: implications for blood transfusion and stem cell transplantation.

BACKGROUND: Hepatitis B virus (HBV) transmission through blood transfusion is reduced by screening for hepatitis B surface antigen (HBsAg). However this method cannot detect the presence of occult hepatitis B virus infection. This study sought to determine the prevalence of occult hepatitis B virus infection among blood donors in Ile-Ife, Nigeria. For the first time in Nigeria we employed an automated real-time PCR- method to investigate the prevalence of occult HBV in blood donors. METHODS: Blood donors screened with HBsAg immunochromatographic rapid test kits at the blood transfusion units of two hospitals and found to be negative were recruited into the study. Questionnaires to elicit risk factors for HBV infection were administered and then 10 ml of blood was collected from each donor. Plasma samples obtained from these HBsAg negative blood donors were screened again for HBsAg using an enzyme-linked immunosorbent assay (ELISA) method, and those found negative were screened for the presence of total antibody to the HBV core antigen (anti-HBc) using ELISA method. Those positive to anti-HBc were then tested for HBV DNA, using an automated real-time PCR method. RESULTS: Five hundred and seven blood donors found HBsAg negative by immunochromatographic rapid test kits at both blood transfusion units, were tested for HBsAg using ELISA and 5 (1 %) were HBsAg positive. The 502 found negative were tested for anti-HBc and 354 (70.5 %) were found positive implying previous exposure to HBV and 19 (5.4 %) of the 354 anti-HBc positive had HBV DNA signifying occult HBV infection. No risk factors were found to be associated with the presence of HBV DNA among those who tested positive. CONCLUSION: Occult HBV infection exists in blood donors in Ile-Ife, Nigeria and the use of HBsAg alone for screening prospective donors will not eliminate the risk of HBV transmission in blood transfusion or stem cell transplantation.

Detection of High-Risk Human Papillomavirus Genotypes Among HIV-Infected Women in Four States in Nigeria.

Introduction The World Health Organization states that almost all cervical cancer cases are linked to infection with high-risk human papillomaviruses transmitted through sexual contact. Implementing effective surveillance and preventive measures would enable the prevention of most cervical cancer cases, especially in HIV-infected women. Every year, about 12,000 women in Nigeria are diagnosed, with almost 8,000 deaths. HPV cervical cancer testing capacity is low in Nigeria. Testing scale-up and sensitization efforts across health facilities, including cervical tissue sample collection, are needed to reduce the cases of cervical cancer. This study aimed to assess the genotype-specific prevalence of clinically relevant high-risk HPV among women living with HIV in Nigeria. Methods A descriptive, cross-sectional study was conducted among adult HIV-infected women attending health facilities in four Nigerian states. From August to October 2022, cervical tissue was collected into PCR cell media, transported to the Nigerian Institute of Medical Research, and assayed for HPV presence and genotype using the Cobas 6800 System (Roche Diagnostics). Statistical analysis was conducted with Stata 2. Results A total of 4423 cervical swab samples were tested. The ages of women ranged from 18 to 72 years (mean 36.61±8.61). In our study, we found that 16.3% of participants tested positive for HPV. Among the high-risk HPV genotypes detected, HPV16 was present in 1.44% of participants, HPV18 in 1.29%, and other high-risk HPV (OHR-HPV) in 11.35%. Additionally, co-infections were observed, with 0.98% of participants testing positive for both HPV16 and OHR-HPV, 1.12% for HPV18 and OHR-HPV, and 0.12% for HPV16, HPV18, and OHR-HPV concurrently. However, 7.4% of the total results were deemed invalid. Conclusion OHR-HPV is prevalent among HIV-infected women across the north and west geopolitical zones of Nigeria. Policies and interventions geared towards curtailing the incidence of cervical cancer are fervently solicited.

Seroprevalence of SARS-CoV-2 IgG among healthcare workers in Lagos, Nigeria.

Healthcare workers (HCWs) are disproportionately infected with SARS-CoV-2 when compared to members of the general public; estimating the seroprevalence of SARS-CoV-2 antibody and SARS-CoV-2 infection rate among HCWs is therefore crucial. This study was carried out in four health facilities in Lagos Nigeria to determine the prevalence of IgG antibodies (seroprevalence) and SARS-CoV-2 active infection rate via a positive rtPCR result, the cross-sectional study was conducted between December 2020 and July 2021. Nasopharyngeal and blood samples were collected from HCWs and screened for SARS-CoV-2 infection using the rtPCR technique and antibody using the Abbott anti-SARS-CoV-2 IgG CMIA assay, respectively. Demographic and occupational exposures data were obtained and analysed using descriptive and inferential statistics, variables significant via inferential statistics were subjected to a multivariate analysis. A total of 413 participants were enrolled, with a mean age in years of 38.4±11.0. The seroprevalence was 30.9% (115/372) while 63/395 (15.9%) were actively infected with the virus. HCWs whose job role had direct contact with patients had a higher percentage of SARS-CoV-2 infection when compared with those not in direct contact, also being a health care worker was significantly associated with getting a positive COVID-19 PCR result. In conclusion the SARS-CoV-2 seroprevalence seen in this study was higher than national serosurvey estimates indicating HCWs are at higher risk of COVID-19 infection when compared to the general public. Vaccination and effective implementation of infection control measures are important to protect HCWs.

Low level SARS-CoV-2 RNA detected in plasma samples from a cohort of Nigerians: Implications for blood transfusion.

The present global pandemic triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has lingered for over a year in its devastating effects. Diagnosis of coronavirus disease 2019 (COVID-19) is currently established with a polymerase chain reaction (PCR) test by means of oropharyngeal-, nasopharyngeal-, anal-swabs, sputum and blood plasma. However, oral and nasal swabs are more commonly used. This study, therefore, assessed sensitivity and specificity of plasma as a diagnostic in comparison with a combination of oral and nasal swab samples, and the implications for blood transfusion. Oropharyngeal (OP) and nasopharyngeal (NP) swab samples were obtained from 125 individuals suspected to have COVID-19 and stored in viral transport medium (VTM) tubes. Ten millilitres of blood samples in EDTA were also obtained by venepuncture and spun to obtain plasma. Viral RNA was obtained from both swabs and plasma by manual extraction with Qiagen QIAamp viral RNA Mini Kit. Detection was done using a real time fluorescent RT-qPCR BGI kit, on a QuantStudio 3 real-time PCR instrument. Average age of study participants was 41 years, with 74 (59.2%) being male. Out of the 125 individuals tested for COVID-19, 75 (60%) were positive by OP/NP swab. However, only 6 (4.8%) had a positive plasma result for COVID-19 with median Ct value of 32.4. Sensitivity and specificity of RT-PCR SARS-CoV-2 test using plasma was 8% and 100% respectively. There was no false positive recorded, but 69 (55.2%) false negatives were obtained by plasma. SARS-CoV-2 viral RNA was detected, albeit low (4.8%) in plasma. Plasma is likely not a suitable biological sample to diagnose acute SARS-CoV-2 infection. The implication of transfusing blood in this era of COVID-19 needs further investigations.

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.

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.

Hepatitis C viral load and genotypes among Nigerian subjects with chronic infection and implication for patient management: a retrospective review of data.

INTRODUCTION: Hepatitis C Virus (HCV) is highly infectious with no currently available vaccine. Prior to treatment, it is recommended to confirm HCV infection with either quantitative or qualitative nucleic acid test. Access to these assays in Nigeria is limited but for effective management of patients, HCV viral load (VL) prior to therapy is required and genotype may be needed in some instances. This study aimed at reviewing the pattern of HCV viral load and genotype in the country, and its implication in patient management. METHODS: this was a retrospective study that involved data abstraction from an electronic database of an accredited laboratory between June 2013 and May 2017. De-linked data were abstracted from records of adult subjects with HCV VL and genotype results, these were analysed using Microsoft Excel 2010 and SPSS v20. RESULTS: within the study period, 346 subjects had baseline VL and 134 (38.7%) had genotype results available. Of these, 202/346 (58.4%) had detectable VL results with higher prevalence in males (64.7%) and ≥51years (42.5%) age group. The median VL among 202 subjects was 407,430 (IQR: 96,388 - 1,357,012) IU/mL. Distribution of genotypes showed that genotypes 1 and 4 had prevalence of 63.2% and 16.8% respectively. CONCLUSION: genotypes 1 and 4 have the highest prevalence. A greater proportion of subjects had VL values ≤800,000 IU/mL, an indication that they are more likely to respond well to available antiviral therapy hence, access to these antivirals will greatly improve management of HCV infection in Nigeria.

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.

Development and implementation challenges of a quality assured HIV infant diagnosis program in Nigeria using dried blood spots and DNA polymerase chain reaction.

Nigeria has one of the highest HIV burdens as well as mother-to-infant transmission rates in the world. A pilot program using polymerase chain reaction (PCR)-based testing of dried blood spot (DBS) specimens was implemented to enable early identification of HIV-infected infants and timely referral and linkage to care. From February 2007 to October 2008, whole blood was collected by finger prick to prepare DBS from infants <18 months presenting in six public mother-and-child health facilities in Lagos, Nigeria. The DBS were tested using the Roche Amplicor HIV-1 DNA Test, v1.5. To monitor laboratory testing quality, all of the PCR-positive and 10% of the PCR-negative DBS were retested by the same method at another reference laboratory. Three hundred and sixty-five randomly selected infants were screened using HIV rapid tests (RT) according to the national algorithm and RT-negative and PCR-positive specimens were also tested using Genscreen enzyme-linked immunosorbent assay (EIA) (Bio-Rad, France). The turnaround time (TAT) from sample collection, testing, and dispatching of results from each health facility was monitored. A total of 1,273 infants with a median age of 12.6 weeks (1 day to 71.6 weeks) participated in the program and 280 (22.0%) were PCR positive. HIV transmission levels varied greatly in the different health facilities ranging from 7.1% to 38.4%. Infants aged 48 to 72 weeks had the highest level of PCR positivity (41.1%). All PCR-positive specimens were confirmed by retesting. The mean turnaround time from DBS collection to returning of the laboratory result to the health facilities was 25 days. Three infants were found to be HIV antibody negative by rapid tests but were positive by both PCR and the fourth generation EIA. The DBS-based PCR program accurately identified all of the HIV-infected infants. However, many programmatic challenges related to the laboratory and TAT were identified.

Sero-prevalence and factors associated with Hepatitis B and C co-infection in pregnant Nigerian women living with HIV infection.

INTRODUCTION: Perinatal and horizontal transmission of Hepatitis B occur in areas of high endemicity as most infections are acquired in the first 5 years of life. Unless Hepatitis B and C infected pregnant women identified, and appropriate treatment provided, children born to these women are at high risk of chronic Hepatitis B (and C) virus infection. The objecive of this study was to determined the prevalence and the factors associated with Hepatitis B and C Virus infection in pregnant HIV positive Nigerians. METHODS: A cross sectional study among HIV Positive pregnant women seen at a large PMTCT clinic in Lagos Nigeria. The women were screened for Hepatitis B and C Virus infection at enrollment. HIV viral load, CD4 count, liver transaminases and hemoglobin levels were also determined. Data were managed with SPSS for windows version. Ethical approval was obtained from the Institutions Ethical Review Board. RESULTS: Of the 2391 studied subjects, 101(4.2%) and 37(1.5%) respectively were seropositive for Hepatitis B and C Virus infection. Twowomen (0. 08%) had triple infections. blood transfusion, (cOR: 2.3; 95% CI:1.1-4.6), history of induced abortion (cOR:2. 2;95% CI:1.3-3.6), and elevated baseline ALT (cOR:2. 2; 95%CI:2. 2;4.2) were significantly associated with HBV. History of induced abortion was the only factor found to be associated with HIV/ HCV (cOR: 1.9;95%CI:1. 3-3.9). CONCLUSION: Hepatitis B Virus infection (4.2%) is relatively common in our environment and associated with induced abortion, blood transfusion and elevated baseline transaminase. Hepatitis C Virus infection (1.5%) is less common and associated with only history of induced abortion.

Comparison of the COBAS/Ampliprep Taqman and Amplicor HIV-1 monitor tests in Lagos, Nigeria.

BACKGROUND: The use of real-time Polymerase chain reaction (PCR) technology options is increasing in resource-limited settings because they are faster, improve assay sensitivity, have higher throughput, larger dynamic ranges and reduced rates of contamination. In 2010, UNAIDS ranked Nigeria as the second highest population of people living with HIV and AIDS (2.98 million people) in the world. OBJECTIVE: The objective of this study was to compare the analytical performances of the Amplicor HIV-1 Monitor (version 1.5) and the COBAS Ampliprep/Taqman (version 2.0) used in monitoring HIV disease progression in HIV-infected individuals. METHOD: In a cross-sectional study, HIV-1 RNA values obtained with the Amplicor HIV-1 monitor version 1.5 were compared with those of the COBAS/Ampliprep TaqMan HIV-1 version 2.0 in a routine clinical setting. Between May and November 2011, 176 plasma samples collected were analysed in parallel using both techniques. Data analysis was done using statgraphics Centurion XVI and Medcalc version 12.0. RESULT: The correlation coefficient for the two assays was 0.83 and the level of agreement using a Bland-Altman plot was 94.2%. CONCLUSION: These findings suggest that the results from the two methods were comparable, hence the COBAS/Ampliprep Taqman version 2.0 is recommended for high-volume laboratories.

Experience of quality management system in a clinical laboratory in Nigeria.

ISSUES: Quality-management systems (QMS) are uncommon in clinical laboratories in Nigeria, and until recently, none of the nation's 5 349 clinical laboratories have been able to attain the certifications necessary to begin the process of attaining international accreditation. Nigeria's Human Virology Laboratory (HVL), however, began implementation of a QMS in 2006, and in 2008 it was determined that the laboratory conformed to the requirements of ISO 9001:2000 (now 2008), making it the first diagnostic laboratory to be certified in Nigeria. The HVL has now applied for the World Health Organization (WHO) accreditation preparedness scheme. The experience of the QMS implementation process and the lessons learned therein are shared here. DESCRIPTION: In 2005, two personnel from the HVL spent time studying quality systems in a certified clinical laboratory in Dakar, Senegal. Following this peer-to-peer technical assistance, several training sessions were undertaken by HVL staff, a baseline assessment was conducted, and processes were established. The HVL has monitored its quality indicators and conducted internal and external audits; these analyses (from 2007 to 2009) are presented herein. LESSONS LEARNED: Although there was improvement in the pre-analytical and analytical indicators analysed and although data-entry errors decreased in the post-analytical process, the delay in returning laboratory test results increased significantly. There were several factors identified as causes for this delay and all of these have now been addressed except for an identified need for automation of some high-volume assays (currently being negotiated). Internal and external audits showed a trend of increasing non-conformities which could be the result of personnel simply becoming lax over time. Application for laboratory accreditation, however, could provide the renewed vigour needed to correct these non-conformities. RECOMMENDATION: This experience shows that sustainability of the QMS at present is a cause for concern. However, the tiered system of accreditation being developed by WHO-Afro may act as a driving force to preserve the spirit of continual improvement.