Metagenomic Detection and Genetic Characterization of Human Sapoviruses among Children with Acute Flaccid Paralysis in Nigeria.

Using a metagenomic sequencing approach on stool samples from children with Acute Flaccid Paralysis (AFP), we describe the genetic diversity of Sapoviruses (SaVs) in children in Nigeria. We identified six complete genome sequences and two partial genome sequences. Several SaV genogroups and genotypes were detected, including GII (GII.4 and GII.8), GIV (GIV.1), and GI (GI.2 and GI.7). To our knowledge, this is the first description of SaV infections and complete genomes from Nigeria. Pairwise identity and phylogenetic analysis showed that the Nigerian SaVs were related to previously documented gastroenteritis outbreaks with associated strains from China and Japan. Minor variations in the functional motifs of the nonstructural proteins NS3 and NS5 were seen in the Nigerian strains. To adequately understand the effect of such amino acid changes, a better understanding of the biological function of these proteins is vital. The identification of distinct SaVs reinforces the need for robust surveillance in acute gastroenteritis (AGE) and non-AGE cohorts to better understand SaVs genotype diversity, evolution, and its role in disease burden in Nigeria. Future studies in different populations are, therefore, recommended.

High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria.

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017-2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.

Evaluation of performance testing of different rapid diagnostic kits in comparison with EIAs to validate detection of hepatitis B virus among high risk group in Nigeria.

BACKGROUND: Hepatitis B virus (HBV) causes chronic liver-associated diseases and its early detection is of high public health importance. Its diagnosis is mainly based on immunological assays among which Enzyme-Linked Immunosorbent Assay (ELISA) and rapid tests are the most common and widespread methods. However, a major challenge is the discordance of results of any two laboratory assays which cannot be easily resolved. Therefore, this study was designed to evaluate the validity and reliability of commercially available five rapid test kits in comparison with two Enzyme Immunoassays (EIAs) in Nigeria using hepatitis B surface antigen as a reference marker. METHODS: A total of 100 sera of previously diagnosed consenting HBV-positive patients from private diagnostic laboratories in Ibadan between March and August, 2011 were tested using two EIA and five rapid commercially available HBV test kits in Nigeria. Data were analyzed by SPSS version 15, while bivariate and multivariate analyses were carried out to identify associations at P < 0.05 considered significant. RESULTS: Overall, the sensitivity rates of the two EIA kits were 100% and 99.9% (95% confidence interval [CI] = 98.9-99.7) with specificity of 100% and 99.9% (95% CI = 98.9-99.7), respectively. The sensitivity of the five rapid test kits ranged from 97.5% (95% CI = 96.4-97.6) to 98.9% (95% CI = 97.9-99.9) with specificity of 80% (95% CI = 79.3-80.9) to 90% (95% CI = 89.2-91.0). Also, the positive predictive value ranged from 88% (95% CI = 88.2-89.9) to 89% (95% CI = 88.2-89.9), while the negative predictive value ranged from 80% (95% CI = 79.3-80.9) to 90% (95% CI = 89.2-91.0) for the five rapid kits. However, that of the two EIAs ranged from 99.9% (98.9-99.7) to 100%. Further analysis showed significant (P = 0.033) variations in the sensitivity and specificity of the EIAs and rapid test kits. CONCLUSIONS: The results from this study have clearly revealed the challenges of diagnosis of HBV infections in Nigeria. This study has also demonstrated that the sensitivity of most of the rapid test kits may not be adequate when compared with EIA for early detection of HBV infections. The implications of possible misdiagnosis on the various intervention strategies that rely predominantly on correct HBV status of an individual are enormous. Therefore, there is the need to further compliment the use of rapid test kits with EIAs for HBV control in Nigeria.

Extending the utility of the WHO recommended assay for direct detection of enteroviruses from clinical specimen for resolving poliovirus co-infection.

OBJECTIVES: In a polio-free world there might be reduced funding for poliovirus surveillance. There is therefore the need to ensure that enterovirologist globally, especially those outside the global polio laboratory network, can participate in poliovirus surveillance without neglecting their enterovirus type of interest. To accomplish this, assays are needed that allow such active participation. RESULTS: In this study we describes a sensitive and specific utility extension of the recently recommended WHO RT-snPCR assay that enables independent detection of the three poliovirus types especially in cases of co-infection. More importantly, it piggy-backs on the first round PCR product of the WHO recommended assay and consequently ensures that enterovirologists interested in nonpolio enteroviruses can continue their investigations, and contribute significantly and specifically to poliovirus surveillance, by using the excess of their first round PCR product.

High prevalence of HIV p24 antigen among HIV antibody negative prospective blood donors in Ile-Ife, Nigeria.

Blood transfusion service centers in Nigeria screen donated blood for markers of HIV infection using antibody- (Ab) based rapid test and in some centers, positives are re-tested using Ab-based ELISA. Paucity of data exists on p24 antigen prevalence among HIV Ab-negative donors in Nigeria. This study aims at detecting HIV p24 antigen among prospective blood donors in Osun State, Nigeria. Prospective blood donors negative for HIV antibodies using Determine test kit were re-tested using BIORAD GENSCREEN Ultra Ag-Ab ELISA kit, a fourth-generation ELISA kit that detects HIV antibodies/p24 antigen. Of the 169 HIV Ab-negative prospective donors, 10 (5.9%) were positive for HIV p24 antigen and 70% (7/10) of them were in the age range 18-30 years. Results of this study show that blood transfusion is still one of the major routes of HIV transmission in Nigeria and a higher proportion is among youth. Inclusion of p24 antigen testing into the blood donor screening will help reduce transfusion associated HIV in Nigeria if Nucleic Acid Testing (NAT) of all blood donor samples is not affordable; also, HIV enlightenment programs tailored toward youth may help reduce this rate among donors since more young people donate blood in low/middle-income countries than in high-income countries.

Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay.

Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape.

Detection of hepatitis B virus isolates with mutations associated with immune escape mutants among pregnant women in Ibadan, southwestern Nigeria.

Perinatal transmission of hepatitis B virus (HBV) and its associated immune escape mutants (IEMs), is the major vehicle through which a population of chronically infected people who serve as infectious HBV reservoirs is maintained in communities. Therefore, to assess the risk of perinatal transmission, 272 pregnant women attending ante-natal clinics in Ibadan metropolis, southwestern, Nigeria, were screened for HBsAg using ELISA technique. Samples positive for HBsAg were subjected to HBV DNA detection by PCR amplification of the S-gene and amplicon sequencing. Isolates were genotyped and subtyped using a combination of molecular techniques. Fifteen (5.5%) of the pregnant women were positive for HBsAg of which HBV DNA was detected in seven. Five of the isolates were typed as genotype E subtype ayw4 using amino acid residues at positions 122, 127, 134 and 160. Another could only be typed as genotype E subtype ayw4 by further phylogenetic analysis. The remaining one isolate did not belong to any of genotypes A - H. Three of the HBV isolates including the untypable, had mutations in the 'a' determinant associated with IEMs. This study confirms the endemicity of HBV, the risk of perinatal transmission and the circulation of genotype E subtype ayw4 in Nigeria. It further demonstrates the presence of IEMs in Nigeria.

HBV Infection Among HIV-Infected Cohort and HIV-Negative Hospital Attendees in South Western Nigeria.

BACKGROUND: Prevalence, association and probable mode of acquisition of HBV and HIV dual infections have not been fully explored. Thus, HBV intervention plan and services are sometimes exclusively targeted towards HIV-infected population. We investigated HBV infection among HIV-infected cohort in comparison with HIV-negative hospital attendees to ascertain dual infectivity pattern; thereby encouraging appropriate allotment of intervention services. MATERIALS AND METHODS: A total of 349 (M=141; F=208; Mean=33.98 years; Range= 0.33-80 years) plasma specimens from two virus diagnostic laboratories in south-western Nigeria were analysed. These include 182 HIV-positive and 167 HIV-negative specimens from ART and GDV laboratories respectively. The specimens were initially screened for detectable HIV antigen/antibody, and subsequently HBsAg by ELISA technique. RESULTS: Overall, HBsAg was detected in 20.92% (95% CI: 16.65-25.19%) of the patients. Also, 24.82% (95% CI: 17.69-31.95%) and 18.27% (95% CI: 13.02-23.52%) HBsAg positivity was recorded for males and females respectively. CHI square analysis showed no association (P=0.14) between gender and prevalence of HBsAg. Similarly, comparison of prevalence of HBsAg by age groups shows no significant difference (P=0.24). Overall, no significant difference (P=0.59) was observed in the prevalence of HBsAg among the HIV-infected cohort and HIV-negative hospital attendees. CONCLUSIONS: Results of the study confirm endemicity and comparable rates of HBV infection independent of HIV-status.

Polio virus neutralizing antibody dynamics among children in a north-central and South-Western Nigeria state.

Northern Nigeria accounts for the highest number of confirmed wild polio viruses globally. Transmission to neighboring countries is worrisome after the country failed to meet several deadlines for polio eradication. Most studies on neutralizing antibody have focused on the Northeastern and Northwestern regions. This study measured polio virus neutralizing antibody levels among children in North-central and South-western Nigeria. Children between the ages of 10 months and 13 yr were randomly selected from Abanishe-lolu Hospital Ilorin (North-central) and Oni Memorial and Adeoyo Hospitals in Ibadan (South-west) Nigeria. The alpha neutralization method was employed. Herd immunity was 1.4% in Ilorin, 36.6% in Oni Memorial Hospital, and 49.5% in Adeoyo Hospital. Out of 299 children studied, 49 (16.4%) children had no protection against all poliovirus serotypes while 100 (33.4%) were fully protected against all three serotypes. Five (7.9%) children in Ilorin and 5 (3.4%) children in Oni Memorial Hospital Ibadan had no detectable neutralizing antibody. A significant difference was observed (p=0.000) when the GMT against poliovirus 1, 2, and 3 was compared. A significant proportion of children were not fully protected. Sero-surveillance is recommended for effective monitoring of vaccination efforts to guide health policy formulators.

Occult Hepatitis B Virus Infection among HIV Positive Patients in Nigeria.

HIV has been known to interfere with the natural history of hepatitis B virus (HBV) infection. In this study we investigate the prevalence of occult hepatitis B virus infection (OBI) among HIV-infected individuals in Nigeria. Overall, 1200 archived HIV positive samples were screened for detectable HBsAg using rapid technique, in Ikole Ekiti Specialist Hospital. The HBsAg negative samples were tested for HBsAg, anti-HBc, and anti-HCV by ELISA. Polymerase chain reaction was used for HBV DNA amplification and CD4 counts were analyzed by cytometry. Nine hundred and eighty of the HIV samples were HBsAg negative. HBV DNA was detected in 21/188 (11.2%) of patients without detectable HBsAg. CD4 count for the patients ranged from 2 to 2,140 cells/ µ L of blood (mean = 490 cells/ µ L of blood). HCV coinfection was detected only in 3/188 (1.6%) of the HIV-infected patients (P > 0.05). Twenty-eight (29.2%) of the 96 HIV samples screened were positive for anti-HBc. Averagely the HBV viral load was <50 copies/mL in the OBI samples examined by quantitative PCR. The prevalence of OBI was significantly high among HIV-infected patients. These findings highlight the significance of nucleic acid testing in HBV diagnosis in HIV patients.

High prevalence of anti-hepatitis A virus immunoglobulin G antibody among healthcare facility attendees in Osogbo, Nigeria.

The objectives of this study were to determine the prevalence of anti-hepatitis A virus (HAV) immunoglobulin G (IgG) antibody and associated factors among residents of Osogbo, a town in Nigeria with inadequate environmental sanitation and a shortage of potable water. This is a health facility-based study. Ninety one consenting, asymptomatic attendees of public healthcare facilities in Osogbo, Osun State, Nigeria, were consecutively selected for this study. Plasma samples of the study participants were tested for the presence of anti-HAV IgG using a HAV Ab Competitive Enzyme Immunoassay test kit. Ninety (98.9%) of the participants were seropositive for anti-HAV IgG antibody; group-specific prevalence was also high, but association of participants' variables with the prevalence could not be obtained due to limited sample size and high group-specific prevalence. Since the hepatitis A vaccine is not currently used in Nigeria, the chance is high that the HAV IgG antibody-positive individuals were naturally infected; consequently Osogbo can be described as highly endemic for HAV infection.

Hepatitis B core IgM antibody (anti-HBcIgM) among hepatitis B surface antigen (HBsAg) negative blood donors in Nigeria.

BACKGROUND: Transfusion associated Hepatitis B virus (TAHBV) continues to be a major problem despite mandatory screening for Hepatitis B surface Antigen (HBsAg). Presence of HBsAg is the common method for detecting hepatitis B infection. Unfortunately, this marker is not detected during the window period of the infection. Nigeria being a developing country cannot afford DNA testing of all collected units of blood which serve as the only possibility of achieving zero risk of transfusion associated HBV. Five different serological makers of hepatitis B virus (HBV) infection were therefore assessed to evaluate the reliability of using HBsAg marker alone in diagnosis of HBV infection among blood donors and to detect the serological evidence of the infection at the window period. This will preclude the possibility of transmitting hepatitis B through transfusion of Hepatitis B surface antigen (HBsAg) negative blood in Nigeria. METHODS: Between July and August 2009, 92 blood donors were enrolled for the study. The prevalence of 5 different markers of Hepatitis B virus infection was detected using Enzyme Linked Immunosorbent Assay (ELISA). Demographic factors were assessed during the study. RESULTS: HBsAg and its antibody (anti-HBs) was detected in 18 (19.6%) and 14(15.2%) of the 92 blood donors respectively. Anti-HBc IgM was found in 12(13.0%) of the 92 blood donors while Hepatitis B envelope antigen (HBeAg) and its antibody (anti-HBe) were detected in 4(8.9%) and 12(26.7%) respectively from 45 donors sampled. HBeAg is a marker of high infectivity and appears after HBsAg. At least one serological marker was detected in 30(32.6%) of the blood donors. Five (5.4%) of the 92 donors had anti-HBc IgM as the only serological evidence of hepatitis B virus infection. CONCLUSIONS: The result of this study shows that five donors have anti-HBcIgM as the only serological evidence of HBV infection. Inclusion of anti-HBcIgM in routine screening of blood donors in Nigeria should be encouraged. This is the first study to assess anti-HBcIgM in the country.