Medical Microbiology

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Browsing Medical Microbiology by browse.metadata.advisor "Hoek, Kim Gilberte Pauline"

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    Genotypic and epidemiological characterization of Methicillin resistant Staphylococcus aureus (MRSA) and Coagulase Negative Staphylococcal (CoNS) strains isolated at Tygerberg Hospital
    (Stellenbosch : Stellenbosch University, 2016-12) Van Zyl, Kristien Nel; Hoek, Kim Gilberte Pauline; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology: Medical Microbiology
    Background The global burden of methicillin resistant Staphylococcus aureus (MRSA) can be largely attributed to S. aureus’ ability to acquire the resistance element, SCCmec (Staphylococcal chromosome complex mec). Classification of SCCmec types is an essential component of staphylococcal epidemiology and is based on the arrangement and classes of the mec and ccr gene complexes and Open Reading Frames (ORFs) in the joining (J) regions. At least twelve SCCmec types and numerous subtypes have been described to date. We identified potentially novel and novel variant SCCmec types in MRSA isolates from a tertiary hospital in Cape Town, South Africa. This study aimed to describe the molecular structure and possible origin of these novel elements in our setting, and to determine the prevalence of these SCCmec types. Methods We screened 87 clinical MRSA and 100 methicillin resistant coagulase negative Staphylococci (MR-CoNS) isolates using a multiplex PCR for SCCmec typing. Additional typing employed a combination of six multiplex PCRs on 3 MRSA isolates each from the novel and novel variant types. Whole genome sequencing (WGS) was performed on representative isolates using the Illumina next-generation sequencing platform. Results Among the MRSA isolates, 36% contained the novel SCCmec type (ccrC/Class A mec), followed by SCCmec IV (24%), the novel variant SCCmec type (ccrA1B1, ccrC/Class B mec) (17%) and SCCmec III (11%). Only one MR-CoNS isolate contained the novel type. Preliminary genomic analysis support the PCR findings, and show the presence of a possible truncated SCCHg element in the novel, novel variant and SCCmec III isolates. Discussion We have successfully optimized and implemented two SCCmec typing assays on MRSA and MR-CoNS isolates in our setting and in doing so, identified possible novel and novel variant SCCmec elements. The novel SCCmec type is common among local MRSA isolates, and may reflect clonal spread within the hospital. Preliminary WGS analysis showed that these isolates are composites of SCCHg and SCCmec elements, however further phylogenetic analysis is required to provide insights into how these elements emerged.
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    Investigation of the contribution of oral bacterial microbiota to health and disease in a South African cohort.
    (Stellenbosch : Stellenbosch University, 2024-02) Pekeur, Jade Chantel; Hoek, Kim Gilberte Pauline ; Whitelaw, Andrew Christopher ; Erasmus, Rajiv T. ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
    ENGLISH ABSTRACT: Background: Metabolic syndrome (MetS) is a worldwide epidemic. Risk factors for MetS include diet and lifestyle, as well as genetic predisposition; however, evidence suggests that disruption of the oral microbiota is an important emerging risk factor for MetS. Additionally, periodontal disease (PD) is more prevalent in individuals with MetS and both conditions are associated with inflammation and insulin resistance. Porphyromonas gingivalis has been linked to PD which may be a marker for MetS. The aim of the study was to compare the microbial diversity of subgingival plaque of individuals with MetS and PD to that of healthy individuals, and to determine the abundance of P. gingivalis in the oral cavity of individuals with and without PD. An additional aim was to compare amplicon sequencing to quantitative PCR of P. gingivalis in individuals with PD. Methods: Subgingival plaque samples were obtained from 119 individuals from the mixed ancestral community in Bellville South, Western Cape. Individuals were classified as having MetS and/or PD using standardised criteria. Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene was performed and sequences were clustered into operational taxonomic units based on 97% similarity, using the Human Oral Microbiome Database. Microbial community profiles of healthy and diseased groups were compared using alpha and beta diversity measures. Additionally, P. gingivalis DNA in dental plaque was quantified by a SYBR green based real-time quantitative polymerase chain reaction (qPCR). Results: There were no significant differences in microbial community composition or diversity between individuals with and without MetS and between those with PD and controls. No statistically significant health-associated and PD associated OTUs were identified. Only one OTU, classified Peptostreptococcaceae (XIII) (G-1), was significantly more abundant in the MetS group. The real-time qPCR results showed similar levels of P. gingivalis detected in healthy individuals and those with PD. Higher P. gingivalis levels were observed in the severe PD group, however, the sample set was too small to determine adequate statistical differences. Conclusions: The findings suggest that factors (such as lifestyle, environment, and host immunity) other than genus-level oral microbial community composition may be responsible for the progression of MetS or PD in the Bellville South community. The study was limited to small sample sets and therefore, subtle differences in the microbial community may not have been detected. Furthermore, a larger study could reveal the role Peptostreptococcaceae plays in metabolic syndrome. P. gingivalis was not an indicator for PD, but may be an indicator of severe PD. Further investigation is needed regarding the role that other periodontal pathogens, as well as bacterial abundance, play in the initiation and progression of PD.
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    Molecular detection of Mycobacterium tuberculosis in stool of children with suspected intrathoracic tuberculosis
    (Stellenbosch : Stellenbosch University, 2018-03) Bosch, Corne; Hoek, Kim Gilberte Pauline; Walters, Elisabetta; Warren, Robin; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
    ENGLISH SUMMARY: The bacteriological confirmation of tuberculosis in children is challenging. The current diagnostic gold standard, liquid culture of respiratory specimens, has low sensitivity in paucibacillary paediatric tuberculosis, and sputum collection in young children is relatively invasive and resource-intensive. Stool is easy to collect and may contain mycobacterial deoxyribonucleic acid (DNA) from swallowed sputum. However, the performance of polymerase chain reaction (PCR) assays, including Xpert MTB/RIF and HAIN FluoroType may be affected by PCR inhibition from stool enzymes and by instrument failure due to particulate matter blocking filters. In this study, we aimed to evaluate the diagnostic performance of stool specimens using a variety of stool pre-processing steps, including decontamination and lyophilisation; as well as various DNA extraction and molecular detection protocols. This study formed part of a larger prospective study involving children with suspected intrathoracic tuberculosis where up to 6 respiratory specimens were collected. Stool specimens were collected at enrolment where one portion was tested by a direct Xpert MTB/RIF protocol; the second portion was frozen for lyophilisation and/or DNA extraction protocols followed by PCR-based molecular detection. DNA was extracted from stools using either a manual commercial stool or soil kit. Extracted DNA was tested for the presence of mycobacterial DNA using the Xpert MTB/RIF cartridge according to standard manufacturer’s protocol and/or a modified “Tube Fill” protocol; and/or the HAIN FluoroType® MTB assay. The results were compared to a composite reference standard and a secondary reference standard (first respiratory culture), which was a better reflection of true performance in our setting. Our results indicate that the standard and Tube Fill Xpert MTB/RIF protocols, as well as the FluoroType MTB detection platforms are able to detect mycobacterial DNA from stool specimens. The Xpert MTB/RIF performed directly on decontaminated stool specimens was found to have the best diagnostic accuracy with sensitivities of 45.8% - 47.1% and specificities of 97.8% - 98.2%. This method was also found to have the lowest indeterminate rate of 3.4% - 10.3%. The other protocols investigated displayed unacceptable sensitivity and specificity combinations with high rates of indeterminate results. The high indeterminate rates were concerning and further optimisation and method simplification are required to propose stool as a non-invasive specimen type for the rapid confirmation of TB in children.
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    The molecular epidemiology of pneumocystis jirovecii in Cape Town, South Africa
    (Stellenbosch : Stellenbosch University, 2016-12) Banda, Derrick; Whitelaw, Andrew; Hoek, Kim Gilberte Pauline; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology
    ENGLISH ABSTRACT : Pneumocystis jirovecii is an opportunistic fungal pathogen that causes Pneumocystis pneumonia (PCP) in immunocompromized hosts. PCP is associated with substantial morbidity, and mortality rates range from 10% to 40%. The diagnosis of PCP relies on the microscopic detection of P. jirovecii in stained clinical samples. Polymerase chain reaction (PCR) may provide better sensitivity than microscopy; therefore, evaluation and implementation of PCR assays are required for the detection of Pneumocystis infection. P. jirovecii is not cultivatable, therefore molecular tools are used for characterizing P. jirovecii genotypes; common targets are the dihydropteroate synthase (DHPS) and mitochondrial large subunit rRNA (mtLSU rRNA) genes. DHPS is a therapeutic target; mutations may be associated with co-trimoxazole prophylaxis and treatment failure. Polymorphisms in mtLSUrRNA have been used for phylogenetic studies. Aims: 1) to evaluate a real time PCR (rtPCR) assay for diagnosis of PCP by comparing the performance to immunofluorescence (IF) and 2) to describe the molecular epidemiology of P. jirovecii isolates from Tygerberg Hospital by analyzing DHPS and mtLSU rRNA genes. Methods: Clinical samples from 305 children and adult patients at Tygerberg Hospital were collected, after testing using IF. DNA was extracted using the NucliSens easyMAG platform (Biomérieux). The rtPCR assay targeting the major surface glycoprotein (MSG) gene was evaluated to detect P. jirovecii DNA. The DHPS and mtLSU rRNA genes were amplified by nested PCR and analyzed by DNA sequencing. Results: The SYBR Green rtPCR detected P.jirovecii in 57% of samples (175/305) compared to the 7% (21/305) detected by IF. Our rtPCR had a sensitivity of 100% and specificity of 46%, although this increased if the detection threshold increased. Of the 50 negative control samples used in this study, none tested positive for P.jirovecii. There were 237 lower respiratory tract (LRT) and 58 upper respiratory tract (URT) samples. The yield of PCR in LRT samples was 55.3% (131/237) compared to 70.6% (41/58) in URT samples (p=0.03). In contrast, none of the URT samples were positive using IF, and 8.9% (21/237) of LRT samples were positive on IF. DHPS was successfully amplified in 123 (70.3%) samples; and mtLSU in 126 (72%) samples. Genotype 1 (wild type) was the predominant DHPS genotype, and a mutation rate of 42.3% was recorded for this gene. The mtLSU genotype 3 was present in 50.8% of samples, genotype 1 (42%) was the next most common genotype. Mixed genotypes were detected in 2.4% of the samples analyzed for each gene. There was no clear association between DHPS polymorphisms and mtLSU genotype. Conclusions: The SYBR Green rtPCR was more sensitive than IF for detection of P. jirovecii; especially in URT samples, which is comparable to previous studies. The DHPS mutation rate increased to 42% from 27% recorded in 2013 from our division. The increase in DHPS mutation rate may be a result of on-going co-trimoxazole use, for prophylaxis or treatment of PCP or other infections. Our findings need to be linked to clinical data to better understand transmission dynamics and potential impact of strain variation on clinical outcome, and further studies are required to better describe the local strain diversity.
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    A phenotypic and genotypic characterisation of strain types, virulence factors and agr groups of colonising Staphylococcus aureus associated with bloodstream infection
    (Stellenbosch : Stellenbosch University, 2015-03) Karayem, Karayem; Hoek, Kim Gilberte Pauline; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology
    ENGLISH ABSTRACT : Several studies investigating the molecular characteristics of Staphylococcus aureus have been conducted worldwide, however, in South Africa, most of these have focused on Methicillinresistant S. aureus (MRSA). This study investigated the phenotypic and genotypic characteristics of isolates of S. aureus collected from the blood and nasal cavity of patients admitted to Tygerberg Hospital, South Africa. Investigations included determining the association between blood and nasal isolates, describing the molecular epidemiology of the population, determining the prevalence of various virulence factor genes among the different clones and descibing the accessory gene regulator (agr) functionality of S. aureus clones. Pulsed-field gel electrophoresis (PFGE), performed on 208 blood and nasal isolates from 162 patients with S. aureus bacteraemia, showed that 93 (57.4%) of the patients were colonised with the same strain type (p =0.061). MRSA was significantly associated with endogenous bacteraemia (same strain obtained from the blood and the nose) (p = 0.042). Molecular typing of the 208 blood and nasal isolates (43.3% MRSA) revealed that the majority of strains were ST239-t37-agr I (25.5%) which harboured different SCCmec types including SCCmec type III and a potentially novel type presumed to consist of ccrC/Class A mec. ST612-MRSA-IV was the second most predominant clone (10.2%). Other MRSA clones included ST5-t045 with a potentially novel variant of SCCmec type I consisting of ccrA1B1 and a ccrC/Class B mec; and ST461-MRSA-IV, reported for the first time in South Africa. All 18 (8.7%) pvl-positive isolates were MSSA except one isolate (ST612-MRSA-IV). The identification of novel MRSA clones (ST641-MRSA-IV), MSSA STs (ST2122, ST2126), and the potentially novel SCCmec type and type I variant suggest the local emergence of new clones. Twenty-one isolates (representing nine clonal complexes (CCs)) previously characterised by Multi-locus Sequence Typing (MLST) were analysed for the prevalence of 38 virulence factor genes. There was an association between different enterotoxin gene cluster (egc) gene combinations and CC5, CC22, CC30, and CC45. Both CC15 and CC97 were negative for Superantigen (SAg) genes. The intracellular adhesion locus A (icaA) gene was common (90.4%) and detected in all CCs (except CC30) and the enterotoxin I (sei) gene was significantly more widespread in MRSA isolates (77.8% in MRSA; 25.0% MSSA; p = 0.03). Accessory gene regulator dysfunction was significantly higher amongst MRSA than MSSA isolates and was more commonly associated with ST36-MRSA-II, ST239-MRSA-III and ST239-MRSA- ccrC/Class A mec. Shifting of agr in the same host was not common. Key findings in this study relate to the likely emergence of populations at Tygerberg Hospital, as evidenced by novel STs and potentially novel SCCmec types. The identification of a circulating clone within the burns unit both illustrates the potential for organisms to spread within the hospital, as well as reinforcing the value of molecular typing for infection control purposes. The association of different agr types, agr functionality, and virulence factors with typing data has shown results consistent with other studies, as well as some unusual results. However, the clinical relevance of these associations is not yet well understood, and should form the basis of further research.