Investigation of carbapenem-resistant Enterobacterales isolates at a tertiary laboratory in Pretoria, South Africa.

This study aimed to investigate phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales isolates (CRE) at a tertiary laboratory in South Africa. A total of 99 CRE isolates were collected between 2019 and 2021. Carbapenemase production was tested using modified carbapenem inhibitory method. Colistin susceptibility testing was performed using the ComASP™ Colistin broth microdilution method. Conventional PCR assays were conducted for detection of mcr-1 gene and common carbapenemase genes (blaVIM, blaNDM, blaIMP, blaKPC, blaOXA-23, blaOXA-51 and blaOXA-48). Rep-PCR assay was conducted to determine the genetic relatedness of the study isolates. Majority of the isolates were Klebsiella pneumoniae (83%). Carbapenem resistant K. pneumoniae cluster was observed from ICU and surgical ward samples. Colistin resistance was observed in 13% (12/93) of the isolates namely, in 11 K. pneumoniae and one Enterobacter cloacae. The blaOXA-48 (65%) was the most prevalent gene detected followed by blaNDM (25%) and blaVIM (22%). Several K. pneumoniae isolates concomitantly carried multiple carbapenemase genes with one isolate carry up to 5 five genes blaVIM, blaNDM, blaOXA-48, blaOXA-23 and blaOXA-51. The mcr-1 gene was not detected in the isolates. Rep-PCR assay showed that most isolates matched cluster A (50%). The high prevalence of blaOXA-48, blaNDM and emerging colistin resistant isolates is of concern for patient management at this institution and needs close monitoring. Rep-PCR is a valuable tool in establishing infection clusters in resource-limited settings.

Antibacterial Activities of Monsonia Angustifolia and Momordica Balsamina Linn Extracts against Carbapenem-Resistant Acinetobacter Baumannii.

Carbapenemase-producing Acinetobacter baumannii (A. baumannii) is resistant to most of the available antibiotics and poses serious therapeutic challenges. The study investigated Monsonia angustifolia (M. angustifolia) and Momordica balsamina Linn (M. balsamina Linn) extracts for antibacterial activity against a clinical isolate of carbapenemase-producing A. baumannii using the Kirby Bauer disc diffusion and TLC coupled with bioautography. MIC determination experiments were conducted on a molecularly characterized A. baumannii isolate identified using VITEK2. Positive PCR detection of blaOXA-51 and blaOXA-23 confirmed isolate identity and the presence of a carbapenemase-encoding gene. Antibacterial activity was observed with the methanolic extract of M. balsamina Linn with a MIC of 0.5 mg/mL. Compounds with Rf values of 0.05; 0.17; 0.39 obtained from M. angustifolia hexane extract; compounds with Rf values of 0.58; 0.78; 0.36; 0.48; 0.5; 0.56; 0.67; 0.9 obtained from M. angustifolia dichloromethane extract; compounds with Rf values of 0.11; 0.56; 0.24; 0.37 obtained from M. angustifolia acetone extract and compounds with Rf values of 0.11; 0.27 obtained from M. angustifolia methanol extract demonstrated a level of antibacterial activity. M. angustifolia and M. balsamina Linn plant extracts have a clinically significant antibacterial activity against a carbapenemase-producing A. baumannii strain.

Phytochemical Screening, Antioxidant and Antibacterial Properties of Extracts of Viscum continuum E. Mey. Ex Sprague, a South African Mistletoe.

Viscum continuum E. Mey. Ex Sprague is a woody evergreen semi-parasitic shrub that grows on the branches of other trees. It is used by African traditional healers for post-stroke management. This study reports on the qualitative phytochemical screening and the antioxidant and antimicrobial activities of Viscum continuum's acetone, methanol, hexane and dichloromethane extracts. Standard protocols for the phytochemical screening of extracts were employed. TLC bio-autography was used for qualitative antioxidants analysis. Assays: 2,2-diphenyl-1-picrylhydrazyl, H2O2 free-radical scavenging and ferric chloride reducing power were carried out for quantitative antioxidant analysis. The antimicrobial potential of extracts was screened using disc diffusion, bio-autography and broth micro-dilution. The results indicate the presence of alkaloids, phenolics and tannins in all extracts. Acetone and methanol revealed significant amount of saponins, phenolics, tannins and terpenoids. The extracts exhibited significant antioxidant potential on TLC with positive compound bands at an Rf range of 0.05-0.89. DPPH, H2O2 and the reduction of Fe3+ to Fe2+ assays indicated that methanol extract has a strong antioxidant potential, followed by acetone, DCM and lastly hexane. The extracts of Viscum continuum show the potential to be antibacterial agents. It can be concluded that Viscum continuum extracts contain phytochemicals which are capable of mitigating against chronic health conditions such as cancer, stroke and stress-related and infectious diseases.

Whole-Genome Sequencing of a Colistin-Resistant Acinetobacter baumannii Strain Isolated at a Tertiary Health Facility in Pretoria, South Africa.

BACKGROUND: Acinetobacter baumannii's (A. baumannii) growing resistance to all available antibiotics is of concern. The study describes a colistin-resistant A. baumannii isolated at a clinical facility from a tracheal aspirate sample. Furthermore, it determines the isolates' niche establishment ability within the tertiary health facility. METHODS: An antimicrobial susceptibility test, conventional PCR, quantitative real-time PCR, phenotypic evaluation of the efflux pump, and whole-genome sequencing and analysis were performed on the isolate. RESULTS: The antimicrobial susceptibility pattern revealed a resistance to piperacillin/tazobactam, ceftazidime, cefepime, cefotaxime/ceftriaxone, imipenem, meropenem, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tigecycline, and colistin. A broth microdilution test confirmed the colistin resistance. Conventional PCR and quantitative real-time PCR investigations revealed the presence of adeB, adeR, and adeS, while mcr-1 was not detected. A MIC of 0.38 µg/mL and 0.25 µg/mL was recorded before and after exposure to an AdeABC efflux pump inhibitor. The whole-genome sequence analysis of antimicrobial resistance-associated genes detected beta-lactam: blaOXA-66; blaOXA-23; blaADC-25; blaADC-73; blaA1; blaA2, and blaMBL; aminoglycoside: aph(6)-Id; aph(3″)-Ib; ant(3″)-IIa and armA) and a colistin resistance-associated gene lpsB. The whole-genome sequence virulence analysis revealed a biofilm formation system and cell-cell adhesion-associated genes: bap, bfmR, bfmS, csuA, csuA/B, csuB, csuC, csuD, csuE, pgaA, pgaB, pgaC, and pgaD; and quorum sensing-associated genes: abaI and abaR and iron acquisition system associated genes: barA, barB, basA, basB, basC, basD, basF, basG, basH, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, and entE. A sequence type classification based on the Pasteur scheme revealed that the isolate belongs to sequence type ST2. CONCLUSIONS: The mosaic of the virulence factors coupled with the resistance-associated genes and the phenotypic resistance profile highlights the risk that this strain is at this South African tertiary health facility.

Adverse events following immunization reporting and impact on immunization services in informal settlements in Nairobi, Kenya: a prospective mixed-methods study.

INTRODUCTION: adverse events following immunization (AEFIs) are thought to contribute to cases of vaccine hesitancy, yet little data exists describing the state of reporting and management of AEFIs. This study investigated the occurrence and influence of AEFIs on vaccine hesitancy in an informal settlement of Nairobi. METHODS: this was a prospective mixed-methods study involving 7 focus group discussions, 8 key informant interviews and 457 face-to-face interviews with caregivers. Caregivers were recruited at/or before the 6 week clinic visit and assessed for occurrence of AEFIs in their children at the subsequent 10- and 14-week visits and a follow-up two weeks following the 14 weeks visit via phone calls. RESULTS: in this study, 12.3% (56/457) of the infants experienced an AEFI. Of these, 19 did not report for the next scheduled vaccine. Fever was the most common AEFI, for which most caregivers (66.7%) used Paracetamol as antipyretic, while 20.8% sought help from a nearby health facility. Three of the 56 AEFIs (convulsions) that occurred in study participants could be classified as severe reactions. Diphtheria, pertussis and tetanus (DPT 3) completion rate was 75.3%. Most (96.4%) caregivers considered immunization an important strategy for child survival. Vaccine hesitancy occurred among 3.6% of participants, 30% of whom attributed their hesitancy to occurrence of AEFIs. The review of health records revealed that no AEFI had been reported from any of the study facilities. CONCLUSION: cases of adverse events following immunization are not reported in Mathare Valley and they do have implications for vaccine hesitancy by some caregivers.

Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa.

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of bla OXA-51 . Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were bla OXA-51 -positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

Investigating multi-drug resistant Acinetobacter baumannii isolates at a tertiary hospital in Pretoria, South Africa.

PURPOSE: Antimicrobial resistance is now globally recognised amongst the greatest threat to human health. Acinetobacter baumannii's' (A. baumannii) clinical significance has been driven by its ability to obtain and transmit antimicrobial resistance factors. In South Africa, A. baumannii is a leading cause of healthcare associated infections (HAI). In this study, we investigated the genetic determinants of multi-drug resistant A. baumannii (MDRAB) at a teaching hospital in Pretoria, South Africa. METHODS: One hundred non repetitive isolates of A. baumannii were collected for the study. Antimicrobial susceptibility testing was performed using the VITEK2 system. The prevalence of antibiotic resistance associated genes and AdeABC efflux pump system were investigated using conventional PCR. Genetic relatedness of isolates was determined using rep-PCR. RESULTS: Seventy (70) of 100 isolates collected were confirmed multi-drug resistant and were blaOXA51positive. Phenotypically, the isolates where resistant to almost all tested antibiotics. One isolate showed intermediate susceptibility to tigecycline while all were susceptible to colistin. Oxacillinase gene blaOXA-23 was the most detected at 99% and only 1% was positive for blaOXA-40. For Metallo-betalactamases (MBL), blaVIMwas the most frequently detected at 86% and blaSIM-1 at 3% was the least detected. Fifty-six isolates had the required gene combination for an active efflux pump. The most prevalent clone was clone A at 69% of the isolates. Colistin and tigecycline are the most effective against investigated isolates. CONCLUSION: The major genotypic determinant for drug resistances is oxacillinases blaOXA-23. The study reports for the first time, blaOXA-40 and blaSIM-1 detection in A. baumannii in South Africa.

A novel hepatitis B virus recombinant genotype D4/E identified in a South African population.

BACKGROUND: Genetic diversity is a characteristic trait of the hepatitis B virus (HBV) and has been associated with different clinical outcomes. In South Africa, HBV infection is a major public health concern. Most HBV infections are caused by genotype A strains. However rare cases of infection with HBV genotype D have been reported. The purpose of this study was to investigate the molecular characteristics of a rare HBV subgenotype D4 isolate. METHODS: The full-length genome of isolate ZADGM6964 was amplified in a one-step polymerase chain reaction. The amplified product was purified and cloned into a pGEM®-T Easy Vector System to investigate the genetic diversity of the viral quasi-populations. The primary isolate and clones were then directly sequenced and analysed using an array of bioinformatics software. RESULTS: Phylogenetic analysis showed that the primary isolate and cloned sequences formed a monophyletic cluster away from subgenotype D4 reference strains. Further recombination analysis revealed that isolate ZADGM6964 was in fact a D4/E recombinant strain with breakpoints identified within the X and overlapping pre-Core/Core open reading frames with a >70% bootstrap confidence level. The recombinant genotype D4/E was found to be unique from other D/E strains archived in the genetic database, GenBank. CONCLUSION: This study represents the first ever report on the isolation and molecular characterization of an HBV D4/E recombinant strain in South Africa. The findings provide evidence of further HBV genetic diversity in South Africa than has been previously reported.

Barriers to effective uptake and provision of immunization in a rural district in Uganda.

INTRODUCTION: Hoima, one of the largest districts in mid- western Uganda, has persistently performed poorly with low immunization coverage, high immunization drop outs rates and repeated outbreaks of vaccine preventable diseases especially measles. The objectives of this study were to evaluate the state of immunization services and to identify the gaps in immunization health systems that contribute to low uptake and completion of immunization schedules in Hoima District. METHODS: This was a cross sectional mixed methods study, utilizing both qualitative and quantitative approaches. A situation analysis of the immunization services was carried out using in-depth interviews with vaccinators, focus group discussions and key informant interviews with ethno-videography. Secondary data was sourced from records at headquarters and vaccination centres within Hoima District. The quantitative component utilized cluster random sampling with sample size estimated using the World Health Organization's 30 cluster sampling technique. RESULTS: A total of 311 caretaker/child pairs were included in the study. Immunization completion among children of age at least 12 months was 95% for BCG, 96% for OPV0, 93% for DPT1, 84.5% for DPT2, 81% for DPT3 and 65.5% for measles vaccines. Access to immunization centres is difficult due to poor road terrain, which affects effectiveness of outreach program, support supervision, mentorship and timely delivery of immunization program support supplies especially refrigerator gas and vaccines. Some facilities are under-equipped to effectively support the program. Adverse Events Following Immunization (AEFI) identification, reporting and management is poorly understood. CONCLUSION: Immunization services in Hoima District require urgent improvement in the following areas: vaccine supply, expanding service delivery points, more health workers, transport and tailored mechanisms to ensure adequate communication between health workers and caretakers.

Improving skills and institutional capacity to strengthen adolescent immunisation programmes and health systems in African countries through HPV vaccine introduction.

Several African countries have recently introduced or are currently introducing the HPV vaccine, either nationwide or through demonstration projects, while some countries are planning for introduction. A collaborative project was developed to strengthen country adolescent immunisation programmes and health systems in the African Region, addressing unique public health considerations of HPV vaccination: adolescents as the primary target group, delivery platforms (e.g. school-based and facility based), socio-behavioural issues, and the opportunity to deliver other health interventions alongside HPV vaccination. Following a successful "taking-stock" meeting, a training programme was drafted to assist countries to strengthen the integration of adolescent health interventions using HPV vaccination as an entry point. Two workshops were conducted in the Eastern and Southern African Regions. All countries reported on progress made during a final joint symposium. Of the 20 countries invited to participate in either of the workshops and/or final symposium, 17 countries participated: Angola, Botswana, Ethiopia, Kenya, Malawi, Mauritius, Mozambique, Namibia, Rwanda, Seychelles, South Africa, South Sudan, Swaziland, Tanzania, Uganda, Zambia and Zimbabwe. Countries that are currently implementing HPV vaccination programmes, either nationally or through demonstration projects, reported varying degrees of integration with other adolescent health interventions. The most commonly reported adolescent health interventions alongside HPV vaccination include health education (including sexually transmitted infections), deworming and delivering of other vaccines like tetanus toxoid (TT) or tetanus diphtheria (Td). The project has successfully (a) established an African-based network that will advocate for incorporating the HPV vaccine into national immunisation programmes; (b) created a platform for experience exchange and thereby contributed to novel ideas of revitalising and strengthening school-based health programmes as delivery platform of adolescent immunisation services and other adolescent health interventions, as well as identifying ways of reaching out-of-school girls through facility and community based programmes; and (c) laid a foundation for incorporating future adolescent vaccination programmes.

Sustained favourable HIV viral load response in South African patients during concomitant HAART and cancer therapy.

HIV infection has led to an increase of both AIDS defining and non-AIDS defining cancers. Immunosuppressive cancer therapy had been noted for increased HIV viral load in cancer patients infected with HIV before the advent of highly active antiretroviral therapy (HAART). Assessing the outcome of concomitant HAART and cancer treatment in regions endemic for HIV is thus important. From a cohort of 34 cancer patients infected with HIV, 10 (six underwent radiotherapy and four chemotherapy) had at least three serial samples collected before, during and after treatment. From each sample, HIV viral load, CD4 + and CD8 + cell count was investigated. HIV genotypic drug resistance was assessed for six patients with a detectable HIV viral load at baseline. Of the 10 patients; one was HIV positive only, three were HIV and HBV co-infected and six were HIV, HBV and HCV triple infected. Four patients were HAART experienced at recruitment, while six were HAART naive with detectable HIV viral loads. A significant HIV viral load decrease (P = 0.0128) was observed in all six patients with detectable baseline HIV viral loads. The four patients on HAART at recruitment maintained lower than detectable HIV viral load status throughout cancer therapy. A positive correlation between the rise in CD4 + cell count and attaining a lower than detectable HIV viral load was also observed (P = 0.0439). This pilot study supports concomitant treatment of HIV/AIDS and cancer in HIV endemic regions irrespective of the type of cancer diagnosed, prescribed cancer therapy or other viral (HBV, HCV or both) co-infections.

Active co-infection with HBV and/or HCV in South African HIV positive patients due for cancer therapy.

Human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) share routes of transmission. There is limited data on the incidence of active co-infection with HBV and/or HCV in cancer patients infected with HIV in Africa. This was a prospective study based on 34 patients with varied cancer diagnosis, infected with HIV and awaiting cancer therapy in South Africa. HIV viral load, CD4+ cell counts, Alanine-aminotransferase and aspartate aminotransferase levels were tested. Exposure to HBV and HCV was assessed serologically using commercial kits. Active HBV and/or HCV co-infection was detected using viral specific nested PCR assays. HCV 5'-UTR PCR products were sequenced to confirm active HCV infection. Active viral infection was detected in 64.7% of patients for HBV, 38.2% for HCV, and 29.4% for both HBV and HCV. Occult HBV infection was observed in 63.6% of the patients, while seronegative HCV infection was found in 30.8% of patients. In addition, CD4+ cell count < 350 cells/µl was not a risk factor for increased active HBV, HCV or both HBV and HCV co-infections. A total of 72.7%, 18.2% and 9.1% of the HCV sequences were assigned genotype 5, 1 and 4 respectively.The study revealed for the first time a high active HBV and/or HCV co-infection rate in cancer patients infected with HIV. The findings call for HBV and HCV testing in such patients, and where feasible, appropriate antiviral treatment be indicated, as chemotherapy or radiotherapy has been associated with reactivation of viral hepatitis and termination of cancer therapy.

Identification and genetic characterization of unique HIV-1 A1/C recombinant strain in South Africa.

HIV isolates from South Africa are predominantly subtype C. Sporadic isolation of non-C strains has been reported mainly in cosmopolitan cities. HIV isolate j51 was recovered from a rural South African heterosexual female aged 51 years. Near full length amplification of the genome was attempted using PCR with primers targeting overlapping segments of the HIV genome. Analysis of 5593 bp (gag to vpu) at a bootstrap value greater than 70% found that all but the vpu gene was HIV-1 subtype A1. The vpu gene was assigned HIV-1 subtype C. The recombination breaking point was estimated at position 6035+/- 15 bp with reference to the beginning of the HXB2 reference strain. Isolate j51 revealed a unique genome constellation to previously reported recombinant strains with parental A/C backbones from South Africa though a common recombination with subtype C within the vpu gene. Identification of recombinant strains supports continued surveillance of HIV genetic diversity.

Genetic characterization of HIV before widespread testing of HIV vaccine candidates at a clinical trial site in Pretoria, South Africa.

We studied 123 samples from adult chronic HIV patients initiating HAART from various centers around a newly established clinical trial site in Pretoria. Each sample was sequenced in at least one structural gene (pol, gag, and env) or functional gene (vif, vpr, and vpu). A subset of 25 samples was subjected to near full-genome analysis. All samples were HIV-1 subtype C. Highly conserved regions within the gene sequences were observed. Overall, the gag and vif sequences showed closer similarity followed by the env, vpr, pol, and vpu. The env gene was the most difficult to sequence, resulting in only 31 sequences from 40 samples; of these, 25 were predicted to be R5 coreceptor tropic, while 6 were X4 tropic. The study asserted the predominance of HIV-1 subtype C within the catchment population.