School of Pathology (Journal Articles)
Permanent URI for this collectionhttps://hdl.handle.net/10539/37877
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Item Switching from Sucrose-Formulated rFVIII to Octocog Alfa (BAY 81-8973) Prophylaxis Improves Bleed Outcomes in the LEOPOLD Clinical Trials(Dove Medical Press Limited, 2023-06) Mahlangu, Johnny; Kenet, Gili; Moulton, Thomas; Wicklund, Brian M.; Ahuja, Sanjay P.; Escobar, MiguelIntroduction: Previous clinical trials established the efficacy and safety of sucrose-formulated recombinant factor (F) VIII (rFVIIIFS/Kogenate FS®/Helixate FS®) and octocog alfa (BAY 81–8973/Kovaltry®; LEOPOLD trials). Aim: To report the results of a post hoc subgroup analysis assessing efficacy and safety outcomes in patients with hemophilia A who were receiving rFVIII-FS prior to enrolling into the LEOPOLD I Part B and LEOPOLD Kids Part A clinical trials and switching to octocog alfa. Methods: LEOPOLD I Part B (NCT01029340) and LEOPOLD Kids Part A (NCT01311648) were octocog alfa Phase 3, multinational, open-label studies in patients with severe hemophilia A aged 12–65 years and ≤12 years, respectively. Annualized bleeding rate (ABR) was the efficacy endpoint for both studies. Safety endpoints included adverse events (AEs) and development of FVIII inhibitors. Results: Of the 113 patients in both LEOPOLD trials, 40 (35.4%) patients received rFVIII-FS prophylaxis pre-study and had data available for pre-study total ABR. In LEOPOLD I Part B (n = 22, 35.5%), median (Q1; Q3) total ABR decreased from 2.5 (0.0; 9.0) pre-study to 1.0 (0.0; 6.8), and from 1.0 (0.0; 6.0) pre-study to 0.0 (0.0; 6.02) in LEOPOLD Kids Part A (n = 18, 35.3%). Octocog alfa was well tolerated, and no patients had drug-related serious AEs or inhibitors. Conclusion: Treatment with octocog alfa prophylaxis appeared to have a favorable risk–benefit profile compared with rFVIII-FS and thus could be an effective and improved alternative strategy for individualized treatment for children, adolescent and adult patients with severe hemophilia A currently on rFVIII-FS treatmentItem Dopamine Transporter Deficiency Syndrome (DTDS): expanding the clinical phenotype and precision medicine approaches(MDPI, 2023-06) Waddington, Simon N.; Ng, Joanne; Barral, Serena; Kurian, Manju A.Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.Item Advancing HIV Drug Resistance Technologies and Strategies: insights from South Africa’s Experience and Future Directions for Resource-Limited Settings(MDPI, 2023-06) Steegen, Kim; Hans, Lucia; van Zyl, Gert U.; Claassen, Mathilda; Khan, Aabida; Pillay, Melendhran; Govender, Subitha; Bester, Phillip A.; van Straaten, Johanna M.; Kana, Vibha; Cutler, Ewaldé; Kalimashe, Monalisa N.; Lebelo, Ramokone L.; Moloi, Mokopi B. H.Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.Item Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing(Nature Research, 2019-04) Olwagen, Courtney P.; Adrian, Peter V.; Madhi, Shabir A.Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofuidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detect 11 bacterial pathogens, 55 pneumococcal serotypes and 6 serotypes of H. infuenzae. The Fluidigm assay results were compared to conventional qPCR and culture. All reactions in the Fluidigm assay efectively amplifed their respective targets with high sensitivity and specifcity compared to qPCR. There was excellent concordance between qPCR and Fluidigm for detection of carriage prevalence (kappa>0.75) and density (Rho>0.95). Fluidigm identifed an additional 7 (4.2%) serotypes over those detected by qPCR. There was a modest concordance between culture and Fluidigm for the majority of reactions detecting S. pneumoniae serotypes/serogroups (kappa>0.6), with Fluidigm identifying an additional 113 (39.1%) serotypes. Discordant results between the three methods were associated with a low carriage density. The Fluidigm assay was able to detect common pneumococcal serotypes, H. infuenzae serotypes, and other common nasopharyngeal bacterial organisms simultaneously. Deployment of this assay in epidemiological studies could provide better insight into the efect of PCV immunization on the nasopharyngeal microbiota in the communityItem Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis(Nature Research, 2019-03) Ealand, Christopher S.; Asmal, Rukaya; Mashigo, Lethabo; Campbell, Lisa; Kana, Bavesh D.Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracyclineresponsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.Item Evaluation of automated malaria diagnosis using the Sysmex XN-30 analyser in a clinical setting(BioMed Central, 2019-01) Pillay, Evashin; Bezuidenhout, Belinda C.; Coetzer, Thérèsa L.; Khodaiji, Shanaz; Litshie, MonwabisiBackground: Early and accurate diagnosis of malaria is a critical aspect of eforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the frst clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fuorescence fow cytometry to directly detect and quantitate parasite-infected red blood cells. Results: Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specifcity. Measurements were reproducible and storage of samples at room temperature did not afect the parameters. Several Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identifed the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample. Conclusions: The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identifed, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.Item Opportunistic bacterial pathogens in bioaerosols emitted at municipal wastewater treatment plants, South Africa(Nature Research, 2025-03) Poopedi, Evida; Pierneef, Rian; Singh, Tanusha; Gomba, AnnancietarAeration tanks at wastewater treatment plants (WWTPs) emit significant amounts of bioaerosols containing potentially hazardous infectious material. Occupational exposure to airborne pathogens can pose health risks to WWTP workers. Bioaerosol samples collected at aeration tanks of two typical municipal WWTPs that use different aeration modes were analysed to investigate the composition and diversity of airborne bacteria in wastewater environments, using the Illumina MiSeq platform. Thirty-six potential airborne bacterial pathogens were identified in the air samples, and these were dominated by Bacillus, Enterococcus, Clostridium, Streptococcus, Acinetobacter, Enterobacter, Pseudomonas, Bacteroides fragilis, Acinetobacter baumannii, and Escherichia/Shigella. Bioaerosols from mechanical aeration tanks (72%, 26/36) had a relatively higher richness and diversity of airborne bacterial pathogens than diffused aeration tanks (17%, 6/36). Furthermore, most of the identified airborne bacterial pathogens (78%, 28/36) were classified as Risk Group 2 according to the revised South African Regulation for Hazardous Biological Agents, 2022, and up to 70% of these were gram-negative bacteria. The presence of potentially pathogenic bacteria in the ambient air at WWTPs suggests an elevated risk of bioaerosol exposure for workers. Therefore, further research and site-specific risk assessments are recommended to guide the implementation of effective bioaerosol strategies to protect workers’ health, with special attention paid to WWTPs that use mechanical aerators.Item Long-term inhibition of Hepatitis B virus gene expression by a primary microrna expressing ancestral adeno-associated viral vector(BioMed Central, 2025-02) Mnyandu, Njabulo Ziphezinhle; Limani, Shonisani Wendy; Ely, Abdullah; Arbuthnot, Patrick; Maepa, Mohube Betty; Wadee, ReubinaCurrent treatments for chronic infection with the hepatitis B virus (HBV) rarely cure carriers from the disease. Previously reported use of serotype 8 adeno-associated viral (AAV8) vectors to deliver expression cassettes encoding anti-HBV artificial primary microRNAs (apri-miRs) has shown promise in preclinical studies. A recently designed synthetic ancestral AAV (Anc80L65) with high liver transduction efficiency is a promising new addition to the anti-HBV vector toolbox. This study engineered Anc80L65 to express HBx-targeting apri-miRs. Single dose administration of the vectors to cultured cells and HBV transgenic mice effected reductions of secreted HBV surface antigen (HBsAg). Circulating HBV particles and HBV core antigen (HBcAg) were also significantly diminished in mice receiving the anti-HBV apri-miR-expressing ancestral AAVs. Downregulation of HBV biomarkers occurred over a period of 12 months. Absence of inflammatory responses or liver toxicity indicated that the vectors had a good safety profile. These data suggest that a single dose of apri-miR-expressing Anc80L65 is safe and capable of mediating durable suppression of HBV gene expression. Targeting HBx, which is required for transcriptional activity of covalently closed circular DNA of HBV, makes this Anc80L65-derived vector a promising candidate for functional cure from chronic HBV infection.Item A histopathological snapshot of bladder cancer: a Johannesburg experience of 1480 histopathology reports(Springer, 2025-03) Jonosky, Jaclyn; Adam, AhmedPurpose: To evaluate the histopathological characteristics of bladder cancer in patients presenting to Johannesburg hospitals over a13-year period (2010–2023). Methods: Following ethical clearance, a retrospective observational, descriptive review of histopathological reports over 13 years was conducted in Johannesburg. Inclusion criteria was bladder biopsies, TURBT specimens, and radical cystectomy (RC) specimens positive for bladder cancer. Exclusion criteria was non-primary bladder cancers (prostate, cervical, colon) and urothelial carcinoma of upper tract origin (N=970). Of the initial specimens N=2450), 1480 met the inclusion criteria, representing 858 patients, owing to multiple transurethral resections of bladder tumours (TURBT). Categorical variables were summarised as counts and percentages, while numerical variables were reported as means with standard deviations or medians with interquartile ranges, depending on data distribution and tested via the Shapiro‒Wilk test. Statistical comparisons were performed using Fisher’s exact test (sex), one-way ANOVA, or the Kruskal‒Wallis test (age). Statistical signifi cance was set at p<0.05. Results: Urothelial carcinoma accounted for 88.8% of bladder cancer, squamous cell carcinoma (7.7%), adenocarcinoma (1.5%), and other malignancies (2%). High-grade urothelial carcinoma was predominant at 75%. Non-muscle invasive disease accounted for 72% of these cases, while 28% were muscle invasive. Data from radical cystectomies showed a high proportion of aggressive and advanced disease. Conclusions: The study highlights the predominance of high-grade non-muscle invasive bladder cancer in Johannesburg, consistent with global trends. The findings suggest a shift in bladder cancer trends in Johannesburg away from assumed squamous cell carcinoma towards urothelial carcinoma.Item Resistance mutations that distinguish HIV-1 envelopes with discordant VRC01 phenotypes from multi-lineage infections in the HVTN703/HPTN081 trial: implications for cross-resistance(Elsevier, 2025-02) Cohen, Paula; Lambson, Bronwen E.; Mkhize, Nonhlanhla N.; Morris, Lynn; Moore, Penny L.; Moodley, Chivonne; Yssel, Anna E. J.; Moyo-Gwete, Thandeka; York, Talita; Gwashu-Nyangiwe, Asanda; Ndabambi, Nonkululeko; Thebus, Ruwayhida; Williamson, CarolynThe Antibody Mediated Prevention (AMP) trials showed that passively infused VRC01, a broadly neutralizing antibody (bNAb) targeting the CD4 binding site (CD4bs) on the HIV-1 envelope protein (Env), protected against neutralization-sensitive viruses. We identified six individuals from the VRC01 treatment arm with multi-lineage breakthrough HIV-1 infections from HVTN703, where one variant was sensitive to VRC01 (IC50 < 25 ug/mL) but another was resistant. By comparing Env sequences of resistant and sensitive clones from each participant, we identified sites predicted to affect VRC01 neutralization and assessed the effect of their reversion in the VRC01-resistant clone on neutralization sensitivity. In four pairs, a single mutation restored partial or full sensitiv ity to VRC01, whereas in the fifth participant, transfer of the entire β23-V5 loop was required. No VRC01 resistance mutations could be identified in the sixth participant, with the discordant clones differing by >100 amino acids. Mutations responsible for the differential neutralization phenotypes occurred at distinct sites across Env, including residues in loop D, the CD4-binding loop, and between the β23 and V5 loops. Analysis of deep sequencing env data showed that VRC01 resistance was likely the property of the acquired virus, rather than occurring through post-acquisition evolution. Although VRC01-resistant parental clones generally retained sensitivity to other CD4-binding site bNAbs, they were less potently neutralized than the VRC01-sensitive clones. In conclu sion, VRC01 resistance mutations occurred through multiple mutational pathways, but sensitivity to second-generation CD4bs bNAbs was retained even in VRC01-resistant transmitted viruses, confirming the potential of these bNAbs for HIV-1 prevention studies.