Diversity of vaginal microbiota associated with bacterial vaginosis and the impact on pregnancy outcomes in HIV-infected and uninfected women.

The composition of vaginal microbiota in pregnancy is important as it may influence susceptibility to adverse pregnancy outcomes. Dysbiosis of the vaginal microbiota caused by the replacement of protective resident microorganisms such as Lactobacillus spp. by anaerobic bacteria is known as bacterial vaginosis (BV). BV is prevalent in women of African descent and may be a cause of higher rates of adverse birth outcomes in these women. Adverse birth outcomes have been reported to be higher in HIV-infected women both on treatment or treatment naïve compared to HIV-uninfected women. The relationship between the vaginal microbiome, HIV and pregnancy outcomes has not yet been established in Zimbabwean women, nor reported in sub Saharan Africa, where the burden of disease is high. Identification of specific organisms and immune factors associated with adverse pregnancy outcomes could lead to interventions or diagnostic algorithms to prevent and predict these outcomes in this population. We recruited 420 pregnant Zimbabwean women [48 (11.4%) HIV infected, 372 (88.6%) HIV uninfected], between 13-35 weeks of gestation with a median gestational age of 30 weeks. Vaginal swabs were collected at enrolment and women were followed until pregnancy outcome was determined. Bacterial DNA was extracted from the vaginal swabs using an optimized Phenol chloroform extraction method with an addition of an enzymatic cocktail step. Library preparation was done using the Universal primers (515F/806R) for the hypervariable V4 region of the 16S rRNA gene. For the first PCR KAPA Hotstart + primers (Roche Lifescience, UK), were used for amplification. After amplification AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used for cleaning of the PCR products at all stages. For the second PCR Nextera XT, Index Kit (Illumina) was used adding unique sequencing adapters to the amplicons. Quantitative PCR was used for library quantification and pooled libraries were the sequenced using Illumina MiSeq. Upstream analysis of sequencing data was done using QIIME and UPARSE and downstream analysis using custom R scripts on samples with good quality reads (>5000). Concentrations of 27 cytokines were measured in vaginal swab samples using a Bio-Plex Pro Human Cytokine 27- plex Assay (Bio-Rad Laboratories Inc., USA). Assay plates were read using a Bio-Plex Suspension Array Reader (Bio-Rad Laboratories Inc., USA). Data were analyzed using Bio-Plex manager software (version 4). Cytokine levels that were below the lower limit of detection of the assay were reported as the mid-point between zero and the lowest detectable level measured for that given cytokine. Pregnant women in this cohort had vaginal microbiota that clustered into 3 main community state types (CST): 109/356 (31%) CST1 - Lactobacillus iners dominant, 102/356 (29%) CST2 - Lactobacillus crispatus dominant and 145/356 (41%) CST3 - Gardnerella vaginalis dominant. There was a high prevalence of dysbiotic vaginal communities and L. iners was the predominant taxa found in > 90% of the women. When exploring whether the vaginal microbiota is associated with pregnancy outcomes, L. iners was highly associated low birth weight (LBW) and small for gestational age deliveries (SGA) while in contrast G. vaginalis was associated with normal birth outcome. There were no strong relationships identified between preterm birth (PTB) and vaginal microbiota. There was a strong correlation between vaginal microbiota and proinflammatory and adaptive cytokines. BV associated organisms (Gardnerella, Aerococcus, Prevotella Coriobacteriaceae and Dialister) were highly inflammatory while Lactobacillus spp were associated with low inflammation. Low levels of IL-13 and PDGF-BB cytokines were associated with LBW and PTB. We found that HIV status was highly associated with high vaginal microbial diversity. HIV-infected women had significantly higher alpha diversity in their vaginal microbiota, and they were more likely to have CST3. Surprisingly, having a diverse community type was not associated with high levels of vaginal inflammation. However low levels of IL13 and IL-I7 while high levels of TNF-α were associated with HIV status. In conclusion this study found that a highly dysbiotic vaginal environment is characteristics of Zimbabwean pregnant women and that vaginal microbiota is weakly associated with some poor pregnancy outcomes. The high prevalence of L. iners and its association with poor pregnancy outcomes suggests that vaginal microbiota may partly explain the high incidence of adverse pregnancy outcomes in African women. Low level of Th2 cytokines and growth factors may lead to adverse birth outcomes. Longitudinal studies of vaginal microbiota and soluble factors are needed in African women

Timely Detection of SARS-CoV-2 in Limited Resource Settings: The Role of the Laboratory in Zimbabwe

The recommended approach for response to severe acute respiratory syndrome coronavirus 2, was to test to enable timely detection, isolation and contact tracing so as to reduce the rapid spread of the disease. This highlighted that the laboratory as one of the core capacities of the International Health Regulations and key technical area in the International Health Security was critical in curbing the spread of the virus. Zimbabwe embarked on testing for SARS-CoV-2 in February 2020 following the guidance and support from WHO leveraging the existing testing capacity. Testing was guided by a laboratory pillar which constituted members from different organizations partnering with the Ministry of Health and Child Care. SARS-CoV-2 testing expansion was based on a phased approach using a tiered system in which laboratory staff from lower tiers were seconded to test for coronavirus using RT-PCR with National Microbiology Reference Laboratory (NMRL) being the hub for centralized consolidation of all results. As the pandemic grew nationally, there was an increase in testing per day and reduction in turnaround time as five laboratories were fully capacitated to test using RT-PCR open platforms, thirty-three provincial and district laboratories to test using TB GeneXpert and 5 provincial laboratories to use Abbott platforms

Genomic epidemiology and the role of international and regional travel in the SARS-CoV-2 epidemic in Zimbabwe: a retrospective study of routinely collected surveillance data.

BACKGROUND: Advances in SARS-CoV-2 sequencing have enabled identification of new variants, tracking of its evolution, and monitoring of its spread. We aimed to use whole genome sequencing to describe the molecular epidemiology of the SARS-CoV-2 outbreak and to inform the implementation of effective public health interventions for control in Zimbabwe. METHODS: We performed a retrospective study of nasopharyngeal samples collected from nine laboratories in Zimbabwe between March 20 and Oct 16, 2020. Samples were taken as a result of quarantine procedures for international arrivals or to test for infection in people who were symptomatic or close contacts of positive cases. Samples that had a cycle threshold of less than 30 in the diagnostic PCR test were processed for sequencing. We began our analysis in July, 2020 (120 days since the first case), with a follow-up in October, 2020 (at 210 days since the first case). The phylogenetic relationship of the genome sequences within Zimbabwe and global samples was established using maximum likelihood and Bayesian methods. FINDINGS: Of 92 299 nasopharyngeal samples collected during the study period, 8099 were PCR-positive and 328 were available for sequencing, with 156 passing sequence quality control. 83 (53%) of 156 were from female participants. At least 26 independent introductions of SARS-CoV-2 into Zimbabwe in the first 210 days were associated with 12 global lineages. 151 (97%) of 156 had the Asp614Gly mutation in the spike protein. Most cases, 93 (60%), were imported from outside Zimbabwe. Community transmission was reported 6 days after the onset of the outbreak. INTERPRETATION: Initial public health interventions delayed onset of SARS-CoV-2 community transmission after the introduction of the virus from international and regional migration in Zimbabwe. Global whole genome sequence data are essential to reveal major routes of spread and guide intervention strategies. FUNDING: WHO, Africa CDC, Biotechnology and Biological Sciences Research Council, Medical Research Council, National Institute for Health Research, and Genome Research Limited.WHO, Africa CDC, Biotechnology and Biological Sciences Research Council, Medical Research Council, National Institute for Health Research, and Genome Research Limite

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Analysis of virulence factors and antibiotic resistance genes in group B streptococcus from clinical samples

Background Streptococcus agalacticae (Group B Streptococcus, GBS) is one of the most important causative agents of serious infections among neonates. This study was carried out to identify antibiotic resistance and virulence genes associated with GBS isolated from pregnant women. Methods A total of 43 GBS isolates were obtained from 420 vaginal samples collected from HIV positive and negative women who were 13–35 weeks pregnant attending Antenatal Care at Chitungwiza and Harare Central Hospitals in Zimbabwe. Identification tests of GBS isolates was done using standard bacteriological methods and molecular identification testing. Antibiotic susceptibility testing was done using the modified Kirby-Bauer method and E-test strips. The boiling method was used to extract DNA and Polymerase Chain Reaction (PCR) was used to screen for 13 genes. Data was fed into SPSS 24.0. Results Nine distinct virulence gene profiles were identified and hly-scpB-bca-rib 37.2% (16/43) was common. The virulence genes identified were namely hly 97.8% (42/43), scpB 90.1% (39/43), bca 86.0% (37/43), rib 69.8% (30/43) and bac 11.6% (5/43). High resistance to tetracycline 97.7% (42/43) was reported followed by 72.1% (31/43) cefazolin, 69.8% (30/43) penicillin G, 58.1% (25/43) ampicillin, 55.8% (24/43) clindamycin, 46.5% (20/43) ceftriaxone, 34.9% (15/43) chloramphenicol, and 30.2% (13/43) for both erythromycin and vancomycin using disk diffusion. Antibiotic resistance genes among the resistant and intermediate-resistant isolates showed high frequencies for tetM 97.6% (41/42) and low frequencies for ermB 34.5% (10/29), ermTR 10.3% (3/29), mefA 3.4% (1/29), tetO 2.4% (1/42) and linB 0% (0/35). The atr housekeeping gene yielded 100% (43/43) positive results, whilst the mobile genetic element IS1548 yielded 9.3% (4/43). Conclusion The study showed high prevalence of hly, scpB, bca and rib virulence genes in S. agalactiae strains isolated from pregnant women. Tetracycline resistance was predominantly caused by the tetM gene, whilst macrolide resistance was predominantly due to the presence of erm methylase, with the ermB gene being more prevalent. Multi-drug resistance coupled with the recovery of resistant isolates to antimicrobial agents such as penicillins indicates the importance of GBS surveillance and susceptibility tests. It was also observed that in vitro phenotypic resistance is not always accurately predicted by resistance genotypes

Carriage of antibiotic-resistant Enterobacteriaceae in hospitalised children in tertiary hospitals in Harare, Zimbabwe

Abstract Background Extended-spectrum β-lactamase-producing and gentamicin resistant Enterobacteriaceae are increasingly recognised as a major cause of infection in low-income countries. We assessed the prevalence of gastrointestinal carriage of these bacteria in hospitalised children in Harare, Zimbabwe. Methods We conducted a cohort study in paediatric inpatients at two tertiary-referral hospitals between May and July 2015. Rectal swabs and faecal samples were collected within 24 h of admission and further follow-up samples were collected on alternate days during hospitalization. Disc-based, selective and enrichment methods were used to detect carriage of these two forms of resistance. Standard methods were used to confirm resistance status and determine the susceptibility of resistant isolates to other commonly-used antibiotics. Results One hundred and sixty four paediatric inpatient admissions (median age = 1.0 year, IQR = 0.2–2.2years) were enrolled, and an average of 1.9 faecal samples per patient were collected. On admission, 68/164 (41%) patients had both ESBL and gentamicin-resistant Enterobacteriaceae detected, 18 (11%) had ESBL only, 17 (10%) had gentamicin resistance only and 61 (37%) had negative screening for both forms of resistance. During hospitalisation, 32/164 (20%) patients were found to have a type of resistant organism which was not present in their admission sample. We found that faecal samples and use of a selective enrichment broth enhanced the detection of resistant organisms. Amongst resistant bacteria isolated, there were high levels of resistance to ciprofloxacin and chloramphenicol, but not ertapenem. Conclusions More than half of children had enteric carriage of a clinically-relevant form of antibiotic resistance on admission to public-sector hospitals in urban Zimbabwe. Additionally, a fifth of children acquired a further form of resistance during hospitalisation. Urgent action is needed to tackle the spread of antibiotic resistant enteric bacteria in African hospitals

Evaluation of TUBEX-TF and OnSite Typhoid IgG/IgM Combo rapid tests to detect Salmonella enterica serovar Typhi infection during a typhoid outbreak in Harare, Zimbabwe

BACKGROUND: Salmonella enterica serovar Typhi, the causative agent of typhoid, is endemic in most parts of the world especially in Africa. Reliable and rapid diagnosis of the bacterium is therefore critical for confirmation of all suspected typhoid cases. In many parts of Zimbabwe, laboratory capacity to isolate the microorganism by culture method as a way of diagnosis has limitations. In this study, two rapid serological kits, TUBEX-TF and OnSite Typhoid IgG/IgM Combo, were evaluated for possible expeditious diagnosis of Salmonella enterica serovar Typhi infection during a typhoid outbreak in Zimbabwe. METHODS: Blood was collected from patients with clinical signs and symptoms of typhoid in Harare, Zimbabwe during an outbreak. The standard culture method was used to diagnose the disease. Two rapid kits, the TUBEX-TF and OnSite Typhoid IgG/IgM Combo, were also used in parallel to diagnose typhoid according to manufacturers’ instructions. The diagnostic accuracy of the two kits was evaluated using the culture method as the gold standard. RESULTS: From all the cases diagnosed by the blood culture (n = 136), we enrolled 131 patients for the TUBEX-TF and 136 for the OnSite Typhoid IgG/IgM Combo tests. With the culture method as a reference standard, we found that TUBEX-TF test was 100% sensitive and 94.12% specific, with 63.16% positive and 100% negative predictive values (NPVs) and the OnSite Typhoid IgG/IgM Combo test was 100% sensitive and 94.35% specific, with 63.16% positive and 100% NPVs. CONCLUSION: Our results indicated that TUBEX-TF and OnSite Typhoid IgG/IgM Combo rapid tests were useful tools for the rapid diagnosis of Salmonella enterica serovar Typhi infection during typhoid outbreaks in Zimbabwe. The tests performed very well in laboratory evaluations of blood culture-confirmed typhoid cases in Harare, Zimbabwe

Effect of handrubbing using locally-manufactured alcohol-based handrubs in paediatric wards in Harare, Zimbabwe

Abstract We assessed bacterial contamination of hands of adults present in paediatric wards in two tertiary-care hospitals in Harare, Zimbabwe and the microbiologic efficacy of locally-manufactured alcohol-based hand rub (ABHR). During unannounced visits, samples were collected using hand-print and hand-rinse methods. Samples were collected from 152 individuals (16 nurses, 10 doctors, 28 students, 86 parents/guardians, 12 others). Contamination of hands with Gram-negative bacteria was found in 91% of adults tested with a mean of 14.6 CFU (hand-rinse method; IQR 3–65), representing a high risk for transmission of pathogens potentially leading to nosocomial infections. A single application of ABHR under controlled conditions achieved an average of 82% (or 0.72 log) reduction in detectable counts. Amongst 49 Enterobacteriaceae isolates from hands, 53% were resistant to gentamicin and 63% were resistant to cefpodoxime. Use of ABHR represents an attractive intervention for reducing nosocomial infections in this setting

Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009–2017

Background Typhoid fever remains a major public health problem in Zimbabwe with recurrent outbreaks reported since 2009. To provide guidance on appropriate treatment choice in order to minimise the morbidity and mortality of typhoid fever and prevent large scale outbreaks, we investigated the antimicrobial susceptibility patterns, prevalence of Salmonella enterica serotype Typhi (S. Typhi) H58 haplotype and molecular subtypes of S. Typhi from outbreak strains isolated from 2009 to 2017 in Zimbabwe and compared these to isolates from neighbouring African countries. Methods Antimicrobial susceptibility testing was performed on all isolates using the disk diffusion, and E-Test, and results were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (2017). S. Typhi H58 haplotype screening was performed on 161 (58.3%) isolates. Pulsed-field gel electrophoresis (PFGE) was performed on 91 selected isolates across timelines using antibiotic susceptibility results and geographical distribution (2009 to 2016). Results Between 2009 and 2017, 16,398 suspected cases and 550 confirmed cases of typhoid fever were notified in Zimbabwe. A total of 276 (44.6%) of the culture-confirmed S. Typhi isolates were analysed and 243 isolates (88.0%) were resistant to two or more first line drugs (ciprofloxacin, ampicillin and chloramphenicol) for typhoid. The most common resistance was to ampicillin-chloramphenicol (172 isolates; 62.3%). Increasing ciprofloxacin resistance was observed from 2012 to 2017 (4.2 to 22.0%). Out of 161 screened isolates, 150 (93.2%) were haplotype H58. Twelve PFGE patterns were observed among the 91 isolates analysed, suggesting some diversity exists among strains circulating in Zimbabwe. PFGE analysis of 2013, 2014 and 2016 isolates revealed a common strain with an indistinguishable PFGE pattern (100% similarity) and indistinguishable from PFGE patterns previously identified in strains isolated from South Africa, Zambia and Tanzania. Conclusions Resistance to first line antimicrobials used for typhoid fever is emerging in Zimbabwe and the multidrug resistant S. Typhi H58 haplotype is widespread. A predominant PFGE clone circulating in Zimbabwe, South Africa, Zambia and Tanzania, argues for cross-border cooperation in the control of this disease