An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Cohort profile: A Prospective Household cohort study of Influenza, Respiratory syncytial virus and other respiratory pathogens community burden and Transmission dynamics in South Africa, 2016-2018.

PURPOSE: The PHIRST study (Prospective Household cohort study of Influenza, Respiratory Syncytial virus, and other respiratory pathogens community burden and Transmission dynamics in South Africa) aimed to estimate the community burden of influenza and respiratory syncytial virus (RSV) including the incidence of infection, symptomatic fraction, and to assess household transmission. PARTICIPANTS: We enrolled 1684 individuals in 327 randomly selected households in a rural and an urban site over three consecutive influenza and two RSV seasons. A new cohort of households was enrolled each year. Participants were sampled with nasopharyngeal swabs twice-weekly during the RSV and influenza seasons of the year of enrolment. Serology samples were collected at enrolment and before and after the influenza season annually. FINDINGS TO DATE: There were 122 113 potential individual follow-up visits over the 3 years, and participants were interviewed for 105 783 (87%) of these. Out of 105 683 nasopharyngeal swabs, 1258 (1%) and 1026 (1%) tested positive on polymerase chain reaction (PCR) for influenza viruses and RSV, respectively. Over one third of individuals had PCR-confirmed influenza each year. Overall, there was influenza transmission to 10% of household contacts of an index case. FUTURE PLANS: Future planned analyses include analysis of influenza serology results and RSV burden and transmission. Households enrolled in the PHIRST study during 2016-2018 were eligible for inclusion in a study of SARS-CoV-2 transmission initiated in July 2020. This study uses similar testing frequency to assess the community burden of SARS-CoV-2 infection and the role of asymptomatic infection in virus transmission.

Genetic Basis of Antigenic Variation of SAT3 Foot-And-Mouth Disease Viruses in Southern Africa.

Foot-and-mouth disease (FMD) continues to be a major burden for livestock owners in endemic countries and a continuous threat to FMD-free countries. The epidemiology and control of FMD in Africa is complicated by the presence of five clinically indistinguishable serotypes. Of these the Southern African Territories (SAT) type 3 has received limited attention, likely due to its restricted distribution and it being less frequently detected. We investigated the intratypic genetic variation of the complete P1 capsid-coding region of 22 SAT3 viruses and confirmed the geographical distribution of five of the six SAT3 topotypes. The antigenic cross-reactivity of 12 SAT3 viruses against reference antisera was assessed by performing virus neutralization assays and calculating the r1-values, which is a ratio of the heterologous neutralizing titer to the homologous neutralizing titer. Interestingly, cross-reactivity between the SAT3 reference antisera and many SAT3 viruses was notably high (r1-values >0.3). Moreover, some of the SAT3 viruses reacted more strongly to the reference sera compared to the homologous virus (r1-values >1). An increase in the avidity of the reference antisera to the heterologous viruses could explain some of the higher neutralization titers observed. Subsequently, we used the antigenic variability data and corresponding genetic and structural data to predict naturally occurring amino acid positions that correlate with antigenic changes. We identified four unique residues within the VP1, VP2, and VP3 proteins, associated with a change in cross-reactivity, with two sites that change simultaneously. The analysis of antigenic variation in the context of sequence differences is critical for both surveillance-informed selection of effective vaccines and the rational design of vaccine antigens tailored for specific geographic localities, using reverse genetics.

Inherent biophysical stability of foot-and-mouth disease SAT1, SAT2 and SAT3 viruses.

Foot-and-mouth disease (FMD) virus (FMDV) isolates show variation in their ability to withstand an increase in temperature. The FMDV is surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot climate regions where FMD is prevalent. The three SAT serotypes, with their particularly low biophysical stability also only yield vaccines of low protective capacity, even with multiple booster vaccinations. The aim of the study was to determine the inherent biophysical stability of field SAT isolates. To characterise the biophysical stability of 20 SAT viruses from Southern Africa, the thermofluor assay was used to monitor capsid dissociation by the release of the RNA genome under a range of temperature, pH and ionic conditions. The SAT2 and SAT3 viruses had a similar range of thermostability of 48-54 °C. However, the SAT1 viruses had a wider range of thermostability with an 8 °C difference but with many viruses being unstable at 43-46 °C. The thermostable A-serotype A24 control virus had the highest thermostability of 55 °C with some SAT2 and SAT3 viruses of similar thermostability. There was a 10 °C difference between the most unstable SAT virus (SAT1/TAN/2/99) and the highly stable A24 control virus. SAT1 viruses were generally more stable compared to SAT2 and SAT3 viruses at the pH range of 6.7-9.1. The effect of ionic buffers on capsid stability showed that SAT1 and SAT2 viruses had an increased stability of 2-9 °C and 2-6 °C, respectively, with the addition of 1 M NaCl. This is in contrast to the SAT3 viruses, which did not show improved stabilisation after addition of 1 M or 0.5 M NaCl buffers. Some buffers showed differing results dependent on the virus tested, highlighting the need to test SAT viruses with different solutions to establish the most stabilising option for storage of each virus. This study confirms for the first time that more stable SAT field viruses are present in the southern Africa region. This could facilitate the selection of the most stable circulating field strains, for adaptation to cultured BHK-21 cells or manipulation by reverse genetics and targeted mutation to produce improved vaccine master seed viruses.

Hospital-based surveillance for rotavirus gastroenteritis in children younger than 5 years of age in Ethiopia: 2007-2012.

BACKGROUND: Rotavirus surveillance was initiated in Ethiopia to estimate the burden of rotavirus gastroenteritis in children <5 years of age, to generate data to assist the policy-making process for new vaccine introduction and to monitor impact of vaccination on disease burden after introduction. METHODS: Sentinel surveillance was conducted at 3 hospitals in Addis Ababa, Ethiopia using a standardized WHO surveillance protocol from August 2007 to March 2012. Children <5 years of age, hospitalized for the primary reason of treatment for acute gastroenteritis, were enrolled, stool samples were collected and tested for group A rotavirus using an enzyme immunoassay. Confirmed positive specimens were further characterized by rotavirus genotyping. RESULTS: A total of 1841 children were enrolled and 21% were rotavirus positive. Children 6-12 months of age had the highest proportion of rotavirus (36%) followed by children <6 months of age (23%). There was no significant difference between sexes. Significant differences in clinical characteristics, such as vomiting, vomiting episodes, cases with vomiting and diarrhea among rotavirus positive cases, were observed. Rotavirus circulated year round with peak prevalence from October through January. The most prevalent detected genotypes were G1P[8] (20%), G12P[8] (17%) and G3P[6] (15%), respectively. CONCLUSIONS: Rotavirus infection is common in Ethiopian children. A safe and effective intervention against the infection is needed to prevent severity of the disease. Rotavirus vaccine introduction is planned before the end of 2013. The established surveillance system and the data generated can be used to monitor the impact of rotavirus vaccination program on severe disease.

Update of rotavirus strains circulating in Africa from 2007 through 2011.

BACKGROUND: The African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the subcontinent since 1998, largely based on intercountry workshops conducted at Rotavirus Regional Reference Laboratories. This article reports on rotavirus genotypes generated at Regional Reference Laboratories, South Africa between 2007 and 2011 from 16 African countries. METHODS: Stool samples were collected from <5-year-old children with diarrhea following World Health Organization criteria of hospital-based rotavirus surveillance. Enzyme immunoassay (EIA) was performed by National Laboratories. Regional Reference Laboratories retested 10% of randomly selected EIA positives and 10% of EIA negatives from each country as part of quality control. At least 50 rotavirus EIA positives from each country per year were subjected to reverse transcriptase polymerase chain reaction based on G-/P-types. Sequencing was conducted in 5-10% of each representative G or P genotype to confirm the genotype, as well as to type some of the samples that could not be genotyped with reverse transcriptase polymerase chain reaction-based methods. RESULTS: A total of 2555 of rotavirus EIA positives were genotyped. G1 was the most predominant (28.8%), followed by G9 (17.3%), G2 (16.8%), G8 (8.2%), G12 (6.2%) and G3 (5.9%). Similarly, the P[8] strain was the most prevalent (40.6%), followed by P[6] (30.9%) and P[4] (13.9%). The top G/P combinations detected were G1P[8] (18.4%), G9P[8] (11.7%), G2P[4] (8.6%), G2P[6] (6.2%), G1P[6] (4.9%), G3P[6] (4.3%), G8P[6] (3.8%) and G12P[8] (3.1%). CONCLUSIONS: There is high genetic diversity of rotavirus strains circulating in the subcontinent. Understanding the strain diversity pre- and postvaccine introduction are important in Africa to understand the broader impact of the rotavirus vaccines on regionally circulating strains.