Browsing by Author "Price, Brendon"

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    Electron microscopic morphometry of podocyte foot process effacement as a tool to distinguish primary from secondary focal segmental glomerulosclerosis (FSGS)
    (2024) da Costa, Nelson; Price, Brendon; Singh Shivani; Davidson, Bianca; Ikumi, Nadia
    Background: Focal segmental glomerulosclerosis (FSGS) is a histological pattern of glomerular injury and one of the most common causes of end-stage renal disease in adult patients. Two major subtypes of FSGS (primary oand secondary) have been identified, with differences in clinical presentation, electron microscopy (EM) foot process width (FPW) and effacement (FPE), and treatment options. Primary FSGS commonly presents as nephrotic syndrome (NS), shows diffuse FPE with a FPW >1500nm, and is generally responsive to steroid therapy. Secondary FSGS does not present with NS (non-nephrotic), shows focal FPE and a FPW <1500nm, and does not respond to steroids. Sometimes, it may be clinically very difficult to differentiate between the two. In these scenarios, EM becomes of paramount importance. In this study, we evaluated podocyte FPW and FPE in FSGS patients to investigate whether a significant difference exists between primary and secondary FSGS. Methods: Cases histologically diagnosed as FSGS in adult native renal biopsies over a 5-year period at Groote Schuur Hospital and Livingstone Hospital were reviewed. Using the 2021 KDIGO guidelines, cases were classified as nephrotic syndrome (NS) or non-nephrotic syndrome (NNS). Using EM and imaging software, podocyte FPWs were calculated and the extent of FPE determined for each case. The results were analysed and correlated with multiple variables. Results: Of a total sample size of 35, 32 cases of FSGS and 3 controls were reviewed. 23 patients presented with NS while 9 patients did not meet the criteria for NS. The NS group had a calculated median FPW of 3076nm, while the NNS group had a median FPW of 1322nm (p=0.003). 83% of the NS group had diffuse FPE, whereas 78% of the NNS group had focal FPE (p=0.003). Logistic regression revealed a FPW threshold value of 2550nm correlated with an 80% probability of a NS diagnosis. A strong correlation between primary FSGS and oedema (p=0.006), proteinuria (p=0.0005), cholesterol (p=0.003) and albumin (p=0.0002) were found. A strong correlation existed between FPW and proteinuria (p=0.0021), eGFR (p=0.017), and albumin (p=0.012). No differences were identified with regards to age, gender, and HIV status. Unsupervised hierarchical cluster analysis with no a priori assumptions identified three clusters, one NNS cluster and two NS clusters, the latter demonstrating 2 separate populations differing with respect to uPCR, creatinine, and FPW. The significance of this finding will be investigated in follow up studies. Conclusion: This is the first study, to our knowledge, in the sub-Saharan African setting to use EM to calculate FPW and determine FPE in FSGS patients. The current study results align with those of previously published international studies. In this study, primary FSGS is generally associated with diffuse FPE and FPW>3000nm, whereas secondary FSGS is associated with focal FPE and FPW <1500nm. In clinically difficult scenarios, EM FPW calculation and FPE determination remains an important adjunct to histopathology and clinical parameters in the differentiation of primary from secondary FSGS in the South African population. The significance of making this distinction lies in completely different patient management regimens between the two groups.
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    Human Papillomavirus DNA extraction and genotype analysis by multiplex real time polymerase chain reaction from formalin fixed paraffin wax-embedded cervical carcinoma specimens
    (2019) Price, Brendon; Govender, Dhirendra; Naidoo, Richard
    Introduction: Cervical squamous cell carcinoma is most commonly caused by persistent infection by high risk human papillomavirus (hrHPV) genotypes. The exact type of hrHPV varies geographically and is the basis for HPV–based vaccination for cervical squamous cell carcinoma prevention. Little is known regarding local hrHPV genotypes within the Western Cape population of South Africa. Aims and objectives: This was a pilot study aiming to extract of high quality genomic DNA from archival FFPE cervical squamous cell carcinoma cases and identify hrHPV genotypes by multiplex real time PCR (RT-PCR). Materials and methods: A retrospective search identified a total of 57 cases of cervical squamous cell carcinoma for the period 2004-2014. This was reduced to a final number of 23 that exhibited sufficient tumour burden for DNA extraction. The most common age group was 40-49 years. HIV status was as follows: two HIV-positive, 14 HIV-negative and 7 HIV unknown. DNA was extracted from archival FFPE cervical squamous cell carcinoma samples using QIAGEN QIAamp® DNA FFPE Tissue kit. Housekeeping genes were detected by endpoint PCR using standard primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences to determine the quality and integrity of extracted DNA for downstream PCR amplification experiments. HrHPV DNA amplification was optimised using a touchdown PCR technique with L1 consensus gene GP5+/GP6+ primers. HrHPV genotypes were detected using a four colour multiplex hrHPV genotyping kit. Samples showing positive results in overlapping probe filter detection spectra were subjected to DNA Sanger sequencing for final confirmation of specific hrHPV genotype. Results: Standard xylene DNA extraction methods using QIAamp® system yielded adequate amounts of DNA with average final concentration of 463.2 ng/l and A260/A280 ratio of 1.86. Housekeeping genes were successfully detected in all samples, confirming that no significant DNA degradation of target sequences occurred within the archival time range of 2004-2014. HPV L1 detection via GP5+/GP6+ primers with endpoint PCR was not achieved via standard cycling conditions and required the use of a touchdown technique with gradually decreasing annealing temperatures. This method successfully identified HPV L1 sequences in 22 out of 23 cases. Multiplex RT-PCR with four colour hydrolysis probes identified hrHPV genotypes in 22 of 23 cases with relative frequencies of HPV genotypes: 16>>18=39=45>33. Most cases showed infection with a single hrHPV genotype (HPV 16 and one case with HPV 33) with four cases demonstrating two genotypes (two with HPV 16&18, one with 16&33 and one with 39&45) and one case with three genotypes (HPV 16, 39, 45). Interestingly, none of the HIV-positive cases showed multiple hrHPV genotype infection. Four hrHPV cases with overlapping spectra for HPV 18/31 and 45/59 were subjected to Sanger sequencing for confirmation of genotype. Three of four cases showed 100% match for genotypes 18 and 45 with the final case demonstrating only co-infective HPV 16.Conclusion: Commercial DNA extraction kits yield adequate amounts of intact, amplifiable DNA in archival FFPE cervical carcinoma specimens. Touchdown PCR is necessary for HPV detection in extracted FFPE DNA cases using GP5+/GP6+ L1 primers. RT-PCR using multicolour hydrolysis probes is a rapid, sensitive technique for hrHPV genotype screening of cervical squamous cell carcinoma specimens. A three colour detection system rather than four colour kit is recommended for future studies in order to avoid extra cost in DNA sequencing cases with overlapping spectra. This pilot study demonstrates hrHPV genotype prevalence similar to that in other populations and suggests that vaccination with currently available formulations would provide a sufficiently wide coverage of HPV genotypes. Future studies will include application of the FFPE DNA extraction, endpoint PCR and RT-PCR techniques to the remainder of the cases in the original cohort.