Moses Olubusuyi Adewumi

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.

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.

Preponderance of enterovirus C in RD-L20B-cell-culture-negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria

Recently, a reverse transcriptase semi-nested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses in clinical specimens. In this study, we use this assay and a modification thereof to screen acute flaccid paralysis (AFP) samples that had previously tested negative for enteroviruses by the RD-L20B algorithm. Thirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples had previously tested negative for enteroviruses in the polio laboratory in accordance with the WHO-recommended RD-L20B-cell-culture-based algorithm. Two samples that had previously been found to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, the RT-snPCR assay and a modified version of the RT-snPCR. All amplicons were sequenced, and enteroviruses were identified using the enterovirus genotyping tool and phylogenetic analysis. Amplicons were recovered from the two controls and 50% (15/30) of the samples screened. Fourteen were successfully typed, of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were enterovirus (EV) -A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and coxsackievirus (CV) -A1, both of which belong to the species Enterovirus C. In one sample, poliovirus serotype 2 was detected and found to have the VP1 ILE143 variation and was therefore identified as a vaccine strain. The results of this study showed that significant proportion of enterovirus infections (including some with Sabin PV2) are being missed by the RD-L20B-cell-culture-based algorithm, thus highlighting the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-C in Nigeria was also confirmed.

Patterns of serologic markers of hepatitis B virus infection and the risk of transmission among pregnant women in southwestern Nigeria

ABSTRACT Hepatitis B virus (HBV) infection is a major health concern in developing countries that has a high morbidity and mortality rate. Vertical transmission of HBV from mother to child has been identified as a major factor leading to chronicity with attendant liver conditions, especially in poor socioeconomic settings. This study aims to evaluate the prevalence of serological HBV markers among pregnant women in Ibadan southwestern Nigeria and to determine the implications for perinatal HBV transmission. This study revealed the presence of varied HBV serological patterns of infection or immunity among pregnant women in Ibadan, Nigeria, and thus the risk of mother to child transmission.

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.

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.

Identification of previously untypable RD cell line isolates and detection of EV-A71 genotype C1 in a child with AFP in Nigeria.

We previously attempted to identify 96 nonpolio enteroviruses (EVs) recovered on RD cell culture from children <15 years with acute flaccid paralysis (AFP) in Nigeria. We succeeded in identifying 69 of the isolates. Here, we describe an attempt to identify the remaining 27 isolates. Twenty-six (the 27th isolate was exhausted) isolates that could not be typed previously were further analyzed. All were subjected to RNA extraction, cDNA synthesis, enterovirus 5‟-UTR– VP2 PCR assay and a modified VP1 snPCR assay. Both the 5’-UTR – VP2 and VP1 amplicons were sequenced, isolates identified and subjected to phylogenetic analysis. Twenty of the 26 isolates analyzed were successfully identified. Altogether, 23 EV strains were recovered. Thesebelong to 11 EV (one EVA, nine EVB and one EVC) types which were EVA71 genotype C1 (1 strain), CVB3 (7 strains), CVB5 (1 strain), E5 (2 strain), E11 (3 strains), E13 (2 strain), E19 (1 strain), E20 (1 strain), E24 (2 strains), EVB75 (1 strain) and EVC99 (2 strains). Of the 11 EV types, the 5’-UTR-VP2 assay identified seven while the VP1 assay identified 10. Both assays simultaneously detected 7 of the 11 EV types identified in this study with 100% congruence. In this study we identified 20 of 26 samples that were previously untypable. In addition, we provided evidence that suggests that a clade of EVA71 genotype C1 might have been circulating in sub-Saharan Africa since 2008. Finally, we showed that the 5’-UTR-VP2 assay might be as valuable as the VP1 assay in EV identification.

Characterization of Coxsackievirus A20 from a child with acute flaccid paralysis in Nigeria

In light of the ongoing cVDPV2 outbreak in Nigeria, we describe the draft genome of a CVA20 strain from a child with AFP. The non-structural region of this genome unambiguously unveiled the source of such regions in recombinant cVDPV2s (JX275140 and KX162716) found in Nigeria in 2008 and 2015, respectively.

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

ABSTRACT 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

ObjectivesIn 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.ResultsIn 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.