Steady-state pharmacokinetics of lamivudine in end-stage kidney failure persons with detectable and undetectable HIV-1 RNA in peritoneal dialysis effluent.

BACKGROUND: Renally adjusted lamivudine dosages are effective. However, some of the kidney failure patients managed with lamivudine-containing regimens are failing to suppress HIV in peritoneal dialysis (CAPD) effluent. The steady-state lamivudine pharmacokinetics among these patients was evaluated. METHODS: This overnight open-label pharmacokinetic study enrolled participants living with HIV and managed with CAPD. Lamivudine levels in blood serum and CAPD effluent samples were quantified using liquid chromatography coupled with a mass spectrometer. Pharmacokinetic measures were obtained through non-compartmental analysis. RESULTS: Twenty-eight participants were recruited with a median antiretroviral (ARV) drug duration of 8 (IQR,4.5-10.5) years and a CAPD duration of 13.3 (IQR,3.3-31.9) months. 14.3% (4/28) had detectable unsuppressed HIV-1 viral load in CAPD effluents. The majority (78,6%,22/28) of participants received a 50 mg dose, while 10.7% (3/28), and another 10.7% (3/28) received 75 mg and 300 mg dosages, respectively. Among those treated with 75 and 300 mg, 66.7% (2/3) and 33.3% (1/3) had detectable HIV-VL in CAPD, respectively. The peritoneal membrane characteristics and CAPD system strengths were variable across the entire study population. Lamivudine exposure was increased in blood serum (50 mg-AUC0-24 h, 651.3 ng/mL; 75 mg-AUC0-24 h, 677.84 ng/mL; 300 mg-AUC0-24 h, 3135.89 ng/mL) compared to CAPD effluents (50 mg-AUC0-24 h, 384.91 ng/mL; 75 mg-AUC0-24 h, 383.24 ng/mL; 300 mg-AUC0-24 h, 2001.60 ng/mL) among the entire study population. The Cmax (50 mg, 41.5 ng/mL; 75 mg, 53.2 ng/mL; 300 mg, 199.1 ng/mL) and Cmin (50 mg, 17.8 ng/mL; 75 mg, 16.4 ng/mL; 300 mg, 76.4 ng/mL) measured in serum were within the therapeutic levels. CONCLUSIONS: Steady-state lamivudine pharmacokinetic measures were variable among the entire study population. However, the total lamivudine exposure was within the therapeutic levels.

Whole genome molecular analysis of respiratory syncytial virus pre and during the COVID-19 pandemic in Free State province, South Africa.

Respiratory syncytial virus (RSV) is the most predominant viral pathogen worldwide in children with lower respiratory tract infections. The Coronavirus disease 2019 (COVID-19) pandemic and resulting nonpharmaceutical interventions perturbed the transmission pattern of respiratory pathogens in South Africa. A seasonality shift and RSV resurgence was observed in 2020 and 2021, with several infected children observed. Conventional RSV-positive nasopharyngeal swabs were collected from various hospitals in the Free State province, Bloemfontein, South Africa, from children suffering from respiratory distress and severe acute respiratory infection between 2020 to 2021. Overlapping genome fragments were amplified and complete genomes were sequenced using the Illumina MiSeq platform. Maximum likelihood phylogenetic and evolutionary analysis were performed on both RSV-A/-B G-genes with published reference sequences from GISAID and GenBank. Our study strains belonged to the RSV-A GA2.3.2 and RSV-B GB5.0.5a clades. The upsurge of RSV was due to pre-existing strains that predominated in South Africa and circulating globally also driving these off-season RSV outbreaks during the COVID-19 pandemic. The variants responsible for the resurgence were phylogenetically related to pre-pandemic strains and could have contributed to the immune debt resulting from pandemic imposed restrictions. The deviation of the RSV season from the usual pattern affected by the COVID-19 pandemic highlights the need for ongoing genomic surveillance and the identification of genetic variants to prevent unforeseen outbreaks in the future.

A metagenomic investigation of the faecal RNA virome structure of asymptomatic chickens obtained from a commercial farm in Durban, KwaZulu-Natal province, South Africa.

BACKGROUND: Virome studies on birds, including chickens are relatively scarce, particularly from the African continent. Despite the continuous evolution of RNA viruses and severe losses recorded in poultry from seasonal viral outbreaks, the information on RNA virome composition is even scantier as a result of their highly unstable nature, genetic diversity, and difficulties associated with characterization. Also, information on factors that may modulate the occurrence of some viruses in birds is limited, particularly for domesticated birds. Viral metagenomics through advancements in sequencing technologies, has enabled the characterization of the entire virome of diverse host species using various samples. METHODS: The complex RNA viral constituents present in 27 faecal samples of asymptomatic chickens from a South African farm collected at 3-time points from two independent seasons were determined, and the impact of the chicken's age and collection season on viral abundance and diversity was further investigated. The study utilized the non-invasive faecal sampling method, mRNA viral targeted enrichment steps, a whole transcriptome amplification strategy, Illumina sequencing, and bioinformatics tools. RESULTS: The results obtained revealed a total of 48 viral species spanning across 11 orders, 15 families and 21 genera. Viral RNA families such as Coronaviridae, Picornaviridae, Reoviridae, Astroviridae, Caliciviridae, Picorbirnaviridae and Retroviridae were abundant, among which picornaviruses, demonstrated a 100% prevalence across the three age groups (2, 4 and 7 weeks) and two seasons (summer and winter) of the 27 faecal samples investigated. A further probe into the extent of variation between the different chicken groups investigated indicated that viral diversity and abundance were significantly influenced by age (P = 0.01099) and season (P = 0.00099) between chicken groups, while there was no effect on viral shedding within samples in a group (alpha diversity) for age (P = 0.146) and season (P = 0.242). CONCLUSION: The presence of an exceedingly varied chicken RNA virome, encompassing avian, mammalian, fungal, and dietary-associated viruses, underscores the complexities inherent in comprehending the causation, dynamics, and interspecies transmission of RNA viruses within the investigated chicken population. Hence, chickens, even in the absence of discernible symptoms, can harbour viruses that may exhibit opportunistic, commensal, or pathogenic characteristics.

The prevalence of HIV resistance mutations and their influence on the shedding of HIV-1 into peritoneal dialysis effluent.

HIV drug resistance mutations (HIVDRMs) are important determinants of therapeutic effects and outcomes even in end-stage kidney failure (ESKF) people living with HIV (PLWHIV). This study evaluated the prevalence of HIVDRMs and their effect on the shedding of HIV-1 into peritoneal dialysis (PD) effluents. This cross-sectional study of PLWHIV and having ESKF and managed with antiretroviral therapy (ART) and PD, collected enrolled patients' demographic information, clinical and laboratory data, and sequenced HIV-1 RNA in unsuppressed plasma and PD effluent samples. HIV viral load and HIVDRMs were determined using qualitative polymerase chain reaction (qPCR) and Stanford University HIVDRM Database, respectively. There were 60 participants recruited with a median age of 43.0 (interquartile range [IQR], 38.0-47) years and were predominantly on abacavir (88.3%), lamivudine (98.3%), and efavirenz (70%) for a median duration of 8 (IQR, 5-11) years. Among participants with detectable HIV-1 in PD effluents, the prevalence of HIVDRMs was 62.5% (5/8) compared to 7.7% (4/52) among those with undetectable HIV-1 (p = 0.001) with non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations predominating. On Spearman's correlation analysis, high plasma HIV levels (ρ = 0.649, p < 0.001), T-cell CD4 count (ρ = -0370, p < 0.004), serum creatinine (ρ = -0.396, p < 0.002), and white blood cell count (ρ = -0.294, p < 0.023) levels were significant factors correlated with the detection of HIV-1 in PD effluents. Moreover, HIVDRMs presence (ρ = 0.504, p < 0.001) particularly NNRTI resistance (ρ = 0.504, p < 0.001) were also significantly correlated with detection of HIV-1 in PD effluents. The presence of HIVDRMs, high plasma HIV viral load, and T-cell CD4 count were correlated with HIV-1 shedding into PD effluents.

High Detection Frequency of Vaccine-Associated Polioviruses and Non-Polio Enteroviruses in the Stools of Asymptomatic Infants from the Free State Province, South Africa.

Enterovirus (EV) infections are widespread and associated with a range of clinical conditions, from encephalitis to meningitis, gastroenteritis, and acute flaccid paralysis. Knowledge about the circulation of EVs in neonatal age and early infancy is scarce, especially in Africa. This study aimed to unveil the frequency and diversity of EVs circulating in apparently healthy newborns from the Free State Province, South Africa (SA). For this purpose, longitudinally collected faecal specimens (May 2021-February 2022) from a cohort of 17 asymptomatic infants were analysed using metagenomic next-generation sequencing. Overall, seven different non-polio EV (NPEV) subtypes belonging to EV-B and EV-C species were identified, while viruses classified under EV-A and EV-D species could not be characterised at the sub-species level. Additionally, under EV-C species, two vaccine-related poliovirus subtypes (PV1 and PV3) were identified. The most prevalent NPEV species was EV-B (16/17, 94.1%), followed by EV-A (3/17, 17.6%), and EV-D (4/17, 23.5%). Within EV-B, the commonly identified NPEV types included echoviruses 6, 13, 15, and 19 (E6, E13, E15, and E19), and coxsackievirus B2 (CVB2), whereas enterovirus C99 (EV-C99) and coxsackievirus A19 (CVA19) were the only two NPEVs identified under EV-C species. Sabin PV1 and PV3 strains were predominantly detected during the first week of birth and 6-8 week time points, respectively, corresponding with the OPV vaccination schedule in South Africa. A total of 11 complete/near-complete genomes were identified from seven NPEV subtypes, and phylogenetic analysis of the three EV-C99 identified revealed that our strains were closely related to other strains from Cameroon and Brazil, suggesting global distribution of these strains. This study provides an insight into the frequency and diversity of EVs circulating in asymptomatic infants from the Free State Province, with the predominance of subtypes from EV-B and EV-C species. This data will be helpful to researchers looking into strategies for the control and treatment of EV infection.

Longitudinal analysis of the enteric virome in paediatric subjects from the Free State Province, South Africa, reveals early gut colonisation and temporal dynamics.

The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut.

Genomic Analysis of Rwandan G9P[8] Rotavirus Strains Pre- and Post-RotaTeq® Vaccine Reveals Significant Distinct Sub-Clustering in a Post-Vaccination Cohort.

Although the introduction of rotavirus vaccines has substantially contributed to the reduction in rotavirus morbidity and mortality, concerns persist about the re-emergence of variant strains that might alter vaccine effectiveness in the long term. The G9 strains re-emerged in Africa during the mid-1990s and have more recently become predominant in some countries, such as Ghana and Zambia. In Rwanda, during the 2011 to 2015 routine surveillance period, G9P[8] persisted during both the pre- and post-vaccine periods. The pre-vaccination cohort was based on the surveillance period of 2011 to 2012, and the post-vaccination cohort was based on the period of 2013 to 2015, excluding 2014. The RotaTeq® vaccine that was first introduced in Rwanda in 2012 is genotypically heterologous to Viral Protein 7 (VP7) G9. This study elucidated the whole genome of Rwandan G9P[8] rotavirus strains pre- and post-RotaTeq® vaccine introduction. Fecal samples from Rwandan children under the age of five years (pre-vaccine n = 23; post-vaccine n = 7), conventionally genotyped and identified as G9P[8], were included. Whole-genome sequencing was then performed using the Illumina® MiSeq platform. Phylogenetic analysis and pair-wise sequence analysis were performed using MEGA6 software. Distinct clustering of three post-vaccination study strains was observed in all 11 gene segments, compared to the other Rwandan G9P[8] study strains. Specific amino acid differences were identified across the gene segments of these three 2015 post-vaccine strains. Important amino acid differences were identified at position N242S in the VP7 genome segment of the three post-vaccine G9 strains compared to the other G9 strains. This substitution occurs at a neutralization epitope site and may slightly affect protein interaction at that position. These findings indicate that the Rwandan G9P[8] strains revealed a distinct sub-clustering pattern among post-vaccination study strains circulating in Rwanda, with changes at neutralization epitopes, which may play a role in neutralization escape from vaccine candidates. This emphasizes the need for continuous whole-genome surveillance to better understand the evolution and epidemiology of the G9P[8] strains post-vaccination.

Cheminformatics bioprospection of selected medicinal plants metabolites against trypsin cleaved VP4 (spike protein) of rotavirus A.

Rotaviruses have continued to be the primary cause of acute dehydrating diarrhoea in children under five years of age despite the global introduction of four World Health Organization (WHO) prequalified oral vaccines in over 106 countries. Currently, no medication is approved by the Food and Drug Administration (FDA) specifically for treating rotavirus A-induced diarrhoea. Consequently, it is important to focus on developing prophylactic and curative therapeutics to combat rotaviral infections. For the first time, this study computationally screened and identified metabolites from Spondias mombin, Macaranga barteri and Dicerocaryum eriocarpum as potential novel inhibitors with broad-spectrum activity against VP5* and VP8* (spike protein) of rotavirus A (RVA). The initial top 20 metabolites identified through molecular docking were further filtered using drug-likeness and pharmacokinetics parameters. The molecular properties of the resulting top-ranked compounds were predicted by conducting density functional theory (DFT) calculations, while molecular dynamics (MD) simulation revealed their thermodynamic compatibility with a significant affinity towards VP8* than VP5*. Except for ellagic acid (-11.78 kcal/mol), the lead compounds had higher binding free energy than the reference standard (VP5* (-11.81 kcal/mol), VP8* (-14.12 kcal/mol)) with 2SG (-20.98 kcal/mol) and apigenin-4'-glucoside (-23.56 kcal/mol) having the highest affinity towards VP5* and VP8*, respectively. Of all the top-ranked compounds, better broad-spectrum affinities for both VP5* and VP8* than tizoxanide were observed in 2SG (VP5* (-20.98 kcal/mol), VP8* (-20.13 kcal/mol)) and sericetin (VP5* (-20.46 kcal/mol), VP8* (-18.31 kcal/mol)). While the identified leads could be regarded as potential modulators of the investigated therapeutic targets for effective management of rotaviral infection, additional in vitro and in vivo evaluation is strongly recommended, and efforts are on-going in this regard.Communicated by Ramaswamy H. Sarma.

Draft genomes of Aeromonas caviae from patients with cholera-like illness during the 2022-2023 cholera outbreak in Malawi.

Aeromonas caviae is an increasingly recognized etiological agent of acute gastroenteritis. Here, we report five draft genomes of A. caviae isolated from suspected cholera cases during the 2022-2023 cholera outbreak in Malawi.

Metabolites profiling and cheminformatics bioprospection of selected medicinal plants against the main protease and RNA-dependent RNA polymerase of SARS-CoV-2.

Despite the existence of some vaccines, SARS-CoV-2 (S-2) infections persist for various reasons relating to vaccine reluctance, rapid mutation rate, and an absence of specific treatments targeted to the infection. Due to their availability, low cost and low toxicity, research into potentially repurposing phytometabolites as therapeutic alternatives has gained attention. Therefore, this study explored the antiviral potential of metabolites of some medicinal plants [Spondias mombin, Macaranga barteri and Dicerocaryum eriocarpum (Sesame plant)] identified using liquid chromatography-mass spectrometry (LCMS) as possible inhibitory agents against the S-2 main protease (S-2 MP) and RNA-dependent RNA polymerase (RP) using computational approaches. Molecular docking was used to identify the compounds with the best affinities for the selected therapeutics targets. Afterwards, compounds with poor physicochemical characteristics, pharmacokinetics, and drug-likeness were screened out. The top-ranked compounds were further subjected to a 120-ns molecular dynamics (MD) simulation. Only quercetin 3-O-rhamnoside (-48.77 kcal/mol) had higher binding free energy than the reference standard (zafirlukast) (-44.99 kcal/mol) against S-2 MP. Conversely, all the top-ranked compounds (ellagic acid hexoside, spiraeoside, apigenin-4'-glucoside and chrysoeriol 7-glucuronide) except gnetin L (-24.24 kcal/mol) had higher binding free energy (-55.19 kcal/mol, -52.75 kcal/mol, -47.22 kcal/mol and -43.35 kcal/mol) respectively, against S-2 RP relative to the reference standard (-34.79 kcal/mol). The MD simulations study further revealed that the investigated inhibitors are thermodynamically stable and form structurally compatible complexes that impede the regular operation of the respective S-2 therapeutic targets. Although, these S-2 therapeutic candidates are promising, further in vitro and in vivo evaluation is required and highly recommended.Communicated by Ramaswamy H. Sarma.

Comparative whole genome analysis reveals re-emergence of human Wa-like and DS-1-like G3 rotaviruses after Rotarix vaccine introduction in Malawi.

G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 at Queen Elizabeth Central Hospital in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, 5 years after the introduction of the Rotarix rotavirus vaccine. Here, we analysed representative twenty-seven whole genome sequences (G3P[4], n = 20; G3P[6], n = 1; and G3P[8], n = 6) randomly selected each month between November 2017 and August 2019 to understand how G3 strains re-emerged in Malawi. We found four genotype constellations that were associated with the emergent G3 strains and co-circulated in Malawi post-Rotarix vaccine introduction: G3P[4] and G3P[6] strains with the DS-1-like genetic backbone genes (G3-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2), G3P[8] strains with the Wa-like genetic backbone genes (G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), and reassortant G3P[4] strains consisting of the DS-1-like genetic backbone genes and a Wa-like NSP2 (N1) gene (G3-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each ribonucleic acid (RNA) segment of the emergent G3 strains was between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intergenogroup interspecies reassortment; and VP6, NSP1, and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the emergent G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings show that multiple strains with either Wa-like or DS-1-like genotype constellations have driven the re-emergence of G3 strains. The findings also highlight the role of human mobility and genome reassortment events in the cross-border dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease-burden settings to inform disease prevention and control.

Genomic characterization of the rotavirus G3P[8] strain in vaccinated children, reveals possible reassortment events between human and animal strains in Manhiça District, Mozambique.

Mozambique introduced the rotavirus vaccine (Rotarix®; GlaxoSmithKline Biologicals, Rixensart, Belgium) in 2015, and since then, the Centro de Investigação em Saúde de Manhiça has been monitoring its impact on rotavirus-associated diarrhea and the trend of circulating strains, where G3P[8] was reported as the predominant strain after the vaccine introduction. Genotype G3 is among the most commonly detected Rotavirus strains in humans and animals, and herein, we report on the whole genome constellation of G3P[8] detected in two children (aged 18 months old) hospitalized with moderate-to-severe diarrhea at the Manhiça District Hospital. The two strains had a typical Wa-like genome constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1) and shared 100% nucleotide (nt) and amino acid (aa) identities in 10 gene segments, except for VP6. Phylogenetic analysis demonstrated that genome segments encoding VP7, VP6, VP1, NSP3, and NSP4 of the two strains clustered most closely with porcine, bovine, and equine strains with identities ranging from 86.9-99.9% nt and 97.2-100% aa. Moreover, they consistently formed distinct clusters with some G1P[8], G3P[8], G9P[8], G12P[6], and G12P[8] strains circulating from 2012 to 2019 in Africa (Mozambique, Kenya, Rwanda, and Malawi) and Asia (Japan, China, and India) in genome segments encoding six proteins (VP2, VP3, NSP1-NSP2, NSP5/6). The identification of segments exhibiting the closest relationships with animal strains shows significant diversity of rotavirus and suggests the possible occurrence of reassortment events between human and animal strains. This demonstrates the importance of applying next-generation sequencing to monitor and understand the evolutionary changes of strains and evaluate the impact of vaccines on strain diversity.

The Evolution of Post-Vaccine G8P[4] Group a Rotavirus Strains in Rwanda; Notable Variance at the Neutralization Epitope Sites.

Africa has a high level of genetic diversity of rotavirus strains, which is suggested to be a possible reason contributing to the suboptimal effectiveness of rotavirus vaccines in this region. One strain that contributes to this rotavirus diversity in Africa is the G8P[4]. This study aimed to elucidate the entire genome and evolution of Rwandan G8P[4] strains. Illumina sequencing was performed for twenty-one Rwandan G8P[4] rotavirus strains. Twenty of the Rwandan G8P[4] strains had a pure DS-1-like genotype constellation, and one strain had a reassortant genotype constellation. Notable radical amino acid differences were observed at the neutralization sites when compared with cognate regions in vaccine strains potentially playing a role in neutralization escape. Phylogenetic analysis revealed that the closest relationship was with East African human group A rotavirus (RVA) strains for five of the genome segments. Two genome sequences of the NSP4 genome segment were closely related to bovine members of the DS-1-like family. Fourteen VP1 and eleven VP3 sequences had the closest relationships with the RotaTeq™ vaccine WC3 bovine genes. These findings suggest that the evolution of VP1 and VP3 might have resulted from reassortment events with RotaTeq™ vaccine WC3 bovine genes. The close phylogenetic relationship with East African G8P[4] strains from Kenya and Uganda suggests co-circulation in these countries. These findings highlight the need for continued whole-genomic surveillance to elucidate the evolution of G8P[4] strains, especially after the introduction of rotavirus vaccination.

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

Genomic Analysis of G2P[4] Group A Rotaviruses in Zambia Reveals Positive Selection in Amino Acid Site 7 of Viral Protein 3.

The G2P[4] genotype is among the rotavirus strains that circulate commonly in humans. Several countries have reported its immediate upsurge after the introduction of rotavirus vaccination, raising concern about sub-optimal vaccine effectiveness against this genotype in the long term. This study aimed to gain insight into the evolution of post-vaccine Zambian G2P[4] group A rotavirus (RVA) strains and their overall genetic make-up by analysis of sequence alignments at the amino acid (AA) level. Twenty-nine Zambian G2P[4] rotavirus strains were subjected to whole-genome sequencing using the Illumina MiSeq® platform. All the strains exhibited the typical DS-1-like genotype constellation, and the nucleotide sequences of the 11 genome segments showed high nucleotide similarities (>97%). Phylogenetic analyses together with representative global G2P[4] RVA showed that Zambian strains clustered into human lineages IV (for VP2, VP4, VP7, NSP1, and NSP5), V (for VP1, VP3, VP6, NSP2, and NSP3), and XXIII (for NSP4). The AA differences between the lineages where the study strains clustered and lineages of global reference strains were identified and analyzed. Selection pressure analysis revealed that AA site seven in the Viral Protein 3 (VP3) genome segment was under positive selection. This site occurs in the region of intrinsic disorder in the VP3 protein, and Zambian G2P[4] strains could potentially be utilizing this intrinsically disordered region to survive immune pressure. The Zambian G2P[4] strains from 2012 to 2016 comprised the G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary G2P[4] strains. Continuous whole-genome surveillance of G2P[4] strains remains imperative to understand their evolution during the post-vaccination period.

Evaluation of extraction and enrichment methods for recovery of respiratory RNA viruses in a metagenomics approach.

Viral metagenomics is increasingly applied in viral detection and virome characterization. Different extraction and enrichment techniques may be adopted, however, reports on their effective influence on viral recovery is often conflicting. Using a three step enrichment steps, the effect of three extraction kits and the influence of DNase treatment with or without rRNA removal for respiratory RNA virus recovery from nasopharyngeal swab samples was evaluated. The viral cocktail containing six different RNA viruses pooled in equal volume were subjected to the different extraction and enrichment methods, sequenced using the Illumina MiSeq, and analysed using Genome Detective. The PureLink® Viral RNA/DNA Mini Kit (PureLink) was highly efficient with better recovery of all the viral agents in the cocktail. The use of rRNA treatment resulted in increased viral recovery with PureLink and QIAamp® Viral RNA Mini kit, while having comparable recovery rate as DNase only with the QIAamp® MinElute Virus Spin Kit. The observed low reads and genome coverage of some of the viruses could be attributed to their low abundance. Depending on sample matrix, extraction choice and enrichment strategy may influence recovery of respiratory RNA virus in metagenomics studies, therefore individual evaluation and adoption may be necessary for a robust result.

Metagenomic Analysis of Respiratory RNA Virome of Children with and without Severe Acute Respiratory Infection from the Free State, South Africa during COVID-19 Pandemic Reveals Higher Diversity and Abundance in Summer Compared with Winter Period.

Viral respiratory infections contribute to significant morbidity and mortality in children. Currently, there are limited reports on the composition and abundance of the normal commensal respiratory virome in comparison to those in severe acute respiratory infections (SARIs) state. This study characterised the respiratory RNA virome in children ≤ 5 years with (n = 149) and without (n = 139) SARI during the summer and winter of 2020/2021 seasons in South Africa. Nasopharyngeal swabs were, collected, pooled, enriched for viral RNA detection, sequenced using Illumina MiSeq, and analysed using the Genome Detective bioinformatic tool. Overall, Picornaviridae, Paramoxyviridae, Pneumoviridae, Picobirnaviridae, Totiviridae, and Retroviridae families were the most abundant viral population in both groups across both seasons. Human rhinovirus and endogenous retrovirus K113 were detected in most pools, with exclusive detection of Pneumoviridae in SARI pools. Generally, higher viral diversity/abundance was seen in children with SARI and in the summer pools. Several plant/animal viruses, eukaryotic viruses with unclear pathogenicity including a distinct rhinovirus A type, were detected. This study provides remarkable data on the respiratory RNA virome in children with and without SARI with a degree of heterogeneity of known viruses colonizing their respiratory tract. The implication of the detected viruses in the dynamics/progression of SARI requires further investigations.

Plant and Soil Core Mycobiomes in a Two-Year Sorghum-Legume Intercropping System of Underutilized Crops in South Africa.

Fungal communities form close beneficial (mutualists) or detrimental (pathogens) associations with their plant hosts. Their diversity and abundance can be affected by agricultural practices which include cropping systems such as rotations and intercropping. Despite the importance of cropping systems in increasing productivity, knowledge of the fungal mycobiome and the core inhabitants for under-utilised cereal and legume crops, particularly over a period, is still limited. The core mycobiomes in plant tissues and bulk soils of a cereal-legume intercrop were characterized over two years using high-throughput sequencing. The intercropping trial consisted of sorghum, Bambara groundnut, cowpea, dry bean, and soybean. A greater number of molecular operational taxonomic units (MOTUs) were found in plant tissues compared to those from the soils and between year one and year two. Principal coordinate analyses revealed that fungal communities for each year were relatively distinct, particularly for the soils. The core mycobiome was dominated by a Davidiellaceae sp. (Cladosporium), Didymellaceae sp. 1 (Phoma), Didymellaceae sp. 2 (Epicoccum), Fusarium sp. 2, Unidentified (Ascomycota), and Cryptococcus MOTUs that were present in all plant tissues and soils of year one and two. Other key MOTUs were only specific to a year, substrate, or crop. Although the mycobiome of sorghum were more distinct than the cores of the legumes, there were still MOTUs dominant across all of the crops. Characterization of this baseline core across two years provides insight into those fungi that are always present in these crops, and that could be utilized in improving crop performance and productivity.

Pathogen Profile of Children Hospitalised with Severe Acute Respiratory Infections during COVID-19 Pandemic in the Free State Province, South Africa.

Severe acute respiratory infections (SARI) contribute to mortality in children ≤5 years. Their microbiological aetiologies are often unknown and may be exacerbated in light of coronavirus disease 19 (COVID-19). This study reports on respiratory pathogens in children ≤5 years (n = 84) admitted with SARI during and between the second and third waves of COVID-19 infection in South Africa. Nasopharyngeal/oropharyngeal swabs collected were subjected to viral detection using QIAstat-Dx® Respiratory SARS-CoV-2 Panel. The results revealed viral positivity and negativity detection rates of 88% (74/84) and 12% (10/84), respectively. Of the 21 targeted pathogens, human rhinovirus/enterovirus (30%), respiratory syncytial virus (RSV; 26%), and severe acute respiratory syndrome coronavirus 2 (24%) were mostly detected, with other viruses being 20% and a co-infection rate of 64.2% (54/84). Generally, RSV-positive samples had lower Ct values, and fewer viruses were detected during the third wave. Changes in the circulation patterns of respiratory viruses with total absence of influenza virus could be attributed to measures against COVID-19 transmission, which may result in waned immunity, thereby increasing susceptibility to severe infections in the following season. High viral co-infection rate, as detected, may complicate diagnosis. Nonetheless, accurate identification of the pathogens may guide treatment decisions and infection control.

SARS-CoV-2-positive patients display considerable differences in proteome diversity in urine, nasopharyngeal, gargle solution and bronchoalveolar lavage fluid samples.

Proteome profile changes post-severe acute respiratory syndrome coronavirus 2 (post-SARS-CoV-2) infection in different body sites of humans remains an active scientific investigation whose solutions stand a chance of providing more information on what constitutes SARS-CoV-2 pathogenesis. While proteomics has been used to understand SARS-CoV-2 pathogenesis, there are limited data about the status of proteome profile in different human body sites infected by the SARS-CoV-2 virus. To bridge this gap, our study aims to characterize the proteins secreted in urine, bronchoalveolar lavage fluid (BALF), gargle solution, and nasopharyngeal samples and assess the proteome differences in these body samples collected from SARS-CoV-2-positive patients. We downloaded publicly available proteomic data from (https://www.ebi.ac.uk/pride/). The data we downloaded had the following identifiers: (i) PXD019423, n = 3 from Charles Tanford Protein Center in Germany. (ii) IPX0002166000, n = 15 from Beijing Proteome Research Centre, China. (iii) IPX0002429000, n = 5 from Huazhong University of Science and Technology, China, and (iv) PXD022889, n = 18 from Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905 USA. MaxQuant was used for the human peptide spectral matching using human and SARS-CoV-2 proteome database which we downloaded from the UniProt database (access date 13th October 2021). The individuals infected with SARS-CoV-2 viruses displayed a different proteome diversity from the different body sites we investigated. Overally, we identified 1809 proteins across the four sample types we compared. Urine and BALF samples had significantly more abundant SARS-CoV-2 proteins than the other body sites we compared. Urine samples had 257(33.7%) unique proteins, followed by nasopharyngeal with 250(32.8%) unique proteins. Gargle solution and BALF had 38(5%) and 73(9.6%) unique proteins respectively. Urine, gargle solution, nasopharyngeal, and bronchoalveolar lavage fluid samples have different protein diversity in individuals infected with SARS-CoV-2. Moreover, our data also demonstrated that a given body site is characterized by a unique set of proteins in SARS-CoV-2 seropositive individuals.