Browsing by Author "Paul, Lynthia"

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    Open Access
    An AraC/XylS family transcriptional regulator homologue from Bacteroides fragilis is associated with cell survival following DNA damage
    (Oxford University Press, 2008) Casanueva, Ana I; Paul, Lynthia; Patrick, Sheila; Abratt, Valerie R
    A putative transcriptional regulator of the AraC/XylS family was identified in a genomic genebank of Bacteroides fragilis Bf-1, which partially relieved the sensitivity of Escherichia coli DNA repair mutants to the DNA-damaging agents, metronidazole and mitomycin C. A homologue of this gene with the same phenotype was identified as BF638R3281 in B. fragilis 638R. Transcription of BF638R3281 was constitutive with respect to exposure to sublethal doses of metronidazole. BF638R3281 was interrupted by single cross-over gene-specific insertion mutation, and the gene disruption was confirmed by PCR and DNAsequencing analysis. The mutant grew more slowly than the wild type, and the mutation rendered B. fragilis more sensitive to metronidazole and mitomycin C. This indicates that the BF638R3281 gene product plays a role in the survival of B. fragilis following DNA damage by these agents.
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    Development of a rapid molecular method for detection of C. perfringens and other pathogens in water sources
    (2025) Savahl, Naqsha; Paul, Lynthia; Hoosain, Nisreen
    Access to clean water is a basic requirement for all. Global water scarcity necessitates the reclamation and reuse of water. Effective, rapid surveillance for pathogens in recycled water is essential, assisting pathogen detection in water intended for reuse by humans, animals, and industry. Monitoring of repurposed water is essential as it might contain residual microorganisms that are less sensitive to disinfection. Examples are Clostridium perfringens (a spore forming microorganism with a cell envelope that is less sensitive to chemical disinfection and the cause of diarrhoea via contaminated water and food) and Legionella pneumophila (a waterborne, pulmonary pathogen protected from disinfection due to its intracellular lifestyle inside Protists such as Entamoeba). Molecular detection of nucleic acids is a tool enabling more rapid detection of pathogens in water compared to culture-based methods. Molecular tests using nucleic amplification and detection via the polymerase chain reaction (PCR) method needs thermal cycling instruments and thus is limited in resource-poor environments and spaces lacking infrastructure such as electricity. A solution is to use isothermal amplification methods, requiring heated devices at a single temperature and excluding the need for thermal cycling devices. A battery-operated heated device suffices for isothermal methods such as Recombinase Polymerase Amplification (RPA) and requires less time compared to PCR. Furthermore, isothermal amplification, when combined with the detection of generated amplicons on lateral flow devices, excludes the need for detection methods requiring electrified equipment. The study described here aimed to evaluate RPA, combined with lateral flow detection of labelled amplicons, as a tool to detect deoxyribonucleic acid (DNA) from two waterborne pathogens, i.e., Clostridium perfringens (C. perfringens) and Legionella pneumophila (L.pneumophila). In-house designed primers targeting the C. perfringens phospholipase C gene (plc) and previously published primers to detect L. pneumophila mipA gene were evaluated using conventional PCR and RPA. The lowest amount of DNA needed for amplification and detection was determined using PCR and both agarose gel electrophoreses and lateral flow detection (LFD). For LFD, labelled primers were used to generate dual-labelled amplicons that were captured and detected on a commercial lateral flow device. The RPA-LFD method was then tested with DNA extracted from a limited number of water samples provided by the local municipality. Lastly, as efficient DNA extraction is a limiting step in molecular detection. Consequently, three commercial, manual DNA purification methods were evaluated for their ability to extract DNA from C. perfringens. The generated data showed successful detection of DNA from both pathogens via RPA-LFD). A minimum of at least 1x10-3 ng of pure DNA, extracted from the cultured pathogens, were efficiently amplified, and detected for both microorganisms. Future work should evaluate the method more extensively with DNA extracted from variety of real-world water samples as PCR inhibitors in water from different resources might influence the sensitivity of the method. Regarding DNA extractions, we compared three different DNA extraction and purification methods (solid phase, desalting and magnetic bead purification) to obtain fit for purpose DNA from cultured C. perfringens. Extraction kits were also compared for equipment and infrastructure needed, efficiency of extraction, cost, and ease of use. All kits generated DNA fit for downstream amplification, but consideration should be given to kits that require fewer steps to obtain pure DNA and use minimal equipment. Kits with a removal step for PCR inhibitors in water should also preferentially be used. In conclusion, the data shows that RPA-LFD could be a feasible method to monitor pathogens in water. Further development of the RPA-LFD method using dual labelled amplicons are necessary, including validation of the method with an expanded set of real-world water samples. The method should further be combined with viability testing, to confirm that DNA detected from samples originates from viable microorganisms and not cell free DNA present in water.
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    Incidence and distribution of human leptospirosis in the Western Cape Province, South Africa, (2010-2019): a retrospective study
    (2022) Gizamba, Jacob Mugoya; Odayar, Jasantha; Dlamini, Sipho; Paul, Lynthia
    Background Leptospirosis is an emerging zoonotic infection of global importance. Among humans, the infection is associated with varying clinical manifestations ranging from mild selflimiting febrile illness to severe illness mainly characterized by pulmonary hemorrhagic syndrome and acute kidney injury due to Weil's disease. In addition, leptospirosis presents with symptoms that mimic commonly known infections that cause febrile illnesses such as malaria, influenza, hepatitis, yellow fever, and viral hemorrhagic diseases. This has consequently, led to under-estimation of the burden of leptospirosis hence contributing to it neglected status. The burden of leptospirosis is reported to be substantially high in tropical regions and resource limited settings. In Africa, few countries have data and reports on human leptospirosis and research studies are scarce. In South Africa, the infection is an important underreported public health concern however information on the incidence trend and distribution of the infection is lacking. Yet such epidemiological description is essential for effective prevention of the infection. This study aimed at determining the incidence of Human Leptospirosis from 2010 to 2019, and to compare the incidence based on seasonal and demographic factors in Western Cape Province (WCP), South Africa. Methods The study was a retrospective secondary analysis of all data on ELISA IgM tests that were positive for leptospirosis between January 2010 and December 2019 in WCP, South Africa. Data was obtained from the National Health Laboratory Services (NHLS), where all serological tests on serum samples of patients who are clinically suspected to be having a leptospirosis infection are conducted. All leptospirosis positive results were grouped, and the incidence proportion of leptospirosis estimated according to sex, age, season, and year of occurrence. The provincial population sizes were used as the denominator when estimating the incidence and it was expressed as leptospirosis cases per 100,000 population. Negative binomial regression was used to estimate the effect of sex, year of occurrence and season on the incidence of human leptospirosis over the study period. The results were presented as incidence rate ratios (IRR) with 95% confidence intervals (CI). Results A total of 254 cases of human leptospirosis were recorded by the NHLS in the WCP, South Africa between 2010 and 2019. The highest number of cases was recorded in 2015 (42 cases, 16.5%) and lowest in 2012 (9 cases, 3.5%). The incidence of leptospirosis fluctuated widely across all the 10 years with the annual incidence ranging between 0.15 and 0.66 per 100,000 population and an average annual incidence of 0.40 per 100,000 population. The incidence was significantly higher among males compared to females (0.55 and 0.25 per 100,000 population respectively; incidence rate ratio (IRR) 2.2, 95% CI: 1.66,3.03) and the overall male to female ratio was 2.14:1. The average incidence of leptospirosis was highest among the 18-44-year-old age cohort (0.56 cases per 100,000 population), and lowest among the ≤17-year-old age cohort (0.07 cases per 100,000 population). The 18-44 (IRR 8.0, 95% CI: 4.65,15.15) and ≥ 45 (IRR 7.4, 95% CI: 4.17,14.17) age cohorts were more at risk of infection compared to ≤17age cohort. The incidence proportion in fall, summer and spring seasons were slightly higher compared to what was observed in winter season. However, and there was no significant association between season and incidence of leptospirosis. Conclusions The incidence of leptospirosis widely fluctuated between 2010 and 2019, with males and those above 18 years of age substantially at risk of infection. The results show that leptospirosis is an important zoonotic disease within the province and potentially disproportionately affecting males and the productive age demographic groups. These findings emphasize the need to enhance targeted prevention strategies and provoke further investigation on the importance of environmental and socioeconomic factors on the occurrence of leptospirosis within Western Cape Province and South Africa at large.
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    Molecular Characterisation Of Clostridium Perfringens Isolates From Patients At Groote Schuur Hospital
    (2023) Monki, Thembisa; Paul, Lynthia; Kullin Brian
    Clostridium perfringens is a spore-forming bacterium isolated from various mammals and birds. In humans it causes infections ranging from non-lethal diarrhoea to tissue-destructive, lethal diseases. Infection originates from spores found in environmental reservoirs such as soil, faecal-contaminated water, and food. Infection can also be caused, when the organisms disseminate from the gastrointestinal tract (GIT) of human carriers to the rest of the body. South Africa (RSA) lacks published data on the specific strains and toxin types responsible for human clostridial infections, as only identification and drug susceptibility testing are done in routine laboratories. The main aim of this study was to characterise 19 clinical isolates of C. perfringens (collected between 2017- 2020) using phenotypical and molecular methods such as PCR-based toxin typing, antimicrobial susceptibility testing and whole genome sequencing. Strains were isolated from patients with invasive clostridial disease. In agreement with other studies, toxin typing confirmed that all isolates encode the Phospholipase C (plc) gene which encodes for the tissue-destructive alpha toxin. Of the 19 isolates tested, 16⁄ 19 (84 %) belonged to Type A, i.e., these isolates only contained the alpha toxin (cpa) and not the other toxins used for profiling. The remaining 16% belonged to type F, thus code for both alpha and enterotoxin (cpe). Toxin types B, C, D, E and G, i.e., strains with PCR amplicons for the beta, epsilon, iota and NetB genes, were not found amongst this set of isolates. All isolates were susceptible to the antibiotic piperacillin (and its lactamase inhibitor partnertazobactam), the carbapenem imipenem and the fluoroquinolone moxifloxacin. Clinical resistance was observed against amoxicillin/clavulanic acid (5%) and penicillin G (10%). Overall, the least effective antibiotics were clindamycin (68%) and metronidazole (68%), with isolates exhibiting either intermediate or complete resistance. Whole genome analysis of a subset of 5 mono-and multidrug resistant (MDR) isolates did not reveal known resistance factors encoding for metronidazole resistance, but the genetic determinant for macrolide resistance (ermQ) was identified in one clindamycin resistant isolate. WGS also revealed that the genome of 1/5 isolates clustered with previously published human isolates originating from non-lethal gastroenteritis cases, although the enterotoxin (cpe) gene was not identified either via PCR-based toxin typing or whole genome analysis. The genomes of the remaining 4/5 isolates cluster with published genomes originating from the environment, birds, or unidentified animals. Multi-locus sequencing type (MSLT) reveals two isolates (GSH 46 and GSH 48) belonged to a single MLST ST274, 2 indicating that they were from the same origin.
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    Technical evaluation of a Real-time polymerase chain reaction (PCR) assay for the detection of Bartonella spp for diagnostic purposes
    (2022) Booley, Ghowa; Paul, Lynthia; Moodley, Clinton; Naicker, Preneshni
    Infective endocarditis (IE) is a rare disease affecting heart tissues. The laboratory diagnosis of culture-negative endocarditis is complicated, and largely based on the combination of nucleic acid detection methods and serological investigation. There is a paucity of published data on microbes causing culture-negative endocarditis, but a recent report indicated that the bacterium Bartonella was the commonest cause of culture-negative endocarditis at a tertiary care facility in Cape Town, South Africa. This laboratory-based, non-clinical pilot study, evaluated the utility of a previously published real-time PCR assay for detecting Bartonella spp. on cats. This will be the first time this target will be evaluated in a real-time PCR assay to detect Bartonella spp. in human samples. For this study, we constructed a plasmid vector containing an insert of 83bp, derived from the Bartonella nuoG gene. In this non-clinical, laboratory evaluation, we used one laboratory sample to amplify the nuoG bacterial DNA fragment and cloned it into a plasmid vector. Using this plasmid in a technical validation, we demonstrated that the previously described assay could detect nuoG when using the LightCycler 480 Probes Master Mix. The results indicated that the assay reliably detected as little as 1000 copies of the target DNA, and infrequently also detected 10-100 copies of the target. The study showed no amplification using some commonly encountered organisms found in our clinical setting, thus indicating 100% specificity for Bartonella. We demonstrated that a plasmid construct containing an internal fragment from the nuoG gene successfully detected the target using a real-time PCR assay. Future testing should include further optimisation to improve reaction efficiency of the assay with spiked diagnostic samples, including peripheral blood, and DNA extracted from heart valve samples. The utility of the RT-PCR for diagnostic purposes should be evaluated by comparing assay turnaround time, sensitivity, and specificity of this assay versus the conventional PCR and Sanger sequencing currently in use to detect Bartonella spp. in heart valves. We concluded that the assay exhibited strong potential for use as a diagnostic PCR using the constructed plasmid, but that further optimization to improve PCR efficiency, and work to determine the clinical sensitivity and specificity are needed before the assay can be applied to blood samples.
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    The utility of a real-time PCR to detect Leptospira in a routine diagnostic setting
    (2022) Chanda, Raphael; Paul, Lynthia; Moodley, Clinton; Naicker, Preneshni
    Leptospirosis is a neglected zoonotic infection with world-wide distribution. A paucity of leptospirosis data from the African continent exists, mainly due to limited access to diagnostics. The clinical presentation ranges from mild to severe disease with multi organ involvement, while the mild form mimics another common tropical disease i.e., malaria. The gold standard for diagnostic detection currently is an immunological test discerning the presence of specific antibodies present in the immune phase of the disease. The serological methods are hindered by the inability to distinguish past from current infection and utility limited to only the immune phase of the disease. Due to lack of sensitivity and specificity in serological methods, improved diagnostic methods are needed to aid early identification in the acute phase. Methods should also distinguish saprophyte and pathogenic species. To address this gap, we developed an inhouse PCR assay targeting the microbe's rrs and lipL32 genes, using primer sets previously reported in literature to be both sensitive and specific for pathogenic Leptospira spp. Using inhouse constructed plasmids, we did a non-clinical, technical validation employing probe-based, real time polymerase chain reaction assays and a locally available commercial kit. Although our assay needs further optimization, we demonstrated that the PCR reliably detected 100 copies and 1000 copies of Leptospira rrs and lipL32 targets respectively. To test specificity, we did real-time PCR with pure DNA from a selected set of pathogens known to be prevalent in bacteremia's in local settings and observed that the rrs target was amplified with Group B streptococci as template but no other tested pathogens, while no non-specific amplification for lipL32 was observed. The non-specific amplification had been reported previously in the literature, suggesting the rrs gene is not a good target to use, even when primers are specifically designed to only detect Leptospira rrs. Future work using the assay should include optimizing assay performance using DNA extracted from the ideal clinical samples to detect Leptospira, namely urine and blood of patients clinically suspected to have leptospirosis. However, the assay demonstrated potential for use as a diagnostic PCR using the constructed plasmid, but further optimization to improve PCR efficiency and assessing its performance in clinical setting is required
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    Towards Phage Therapy: Characterization of Clostridioides difficile Bacteriophages from a South African Strain Collection
    (2024) Golding, Cara; Paul, Lynthia; Kullin Brian
    Clostridioides difficile infection is a worldwide public health concern that affects persons with gastrointestinal dysbiosis, notably hospitalised patients on antibiotic therapy and those with other types of gastrointestinal conditions. This opportunistic infection is caused by a Grampositive, spore-forming bacillus, with conditions including diarrhoea, pseudomembranous colitis, and toxic megacolon. Antibiotic resistance has been reported for the standard of care antibiotics, while newer drugs are too costly for resource-limited clinical settings. Faecal microbiota transplantation (FMT) is highly effective but may pose a risk to immunocompromised individuals. Phage therapy is being increasingly investigated as a therapy option, notably for persons who cannot receive FMT. However, the host and regional specificity of C. difficile phages must first be characterised to ensure the correct phage cocktail is used for therapy. Although C. difficile phages have been described in other parts of the world, analysis of local South African strains has not been conducted prior to this study. This project aimed to isolate and characterise phages from a stored collection of South African C. difficile isolates, focusing on ribotype 017, which has been identified in Western Cape hospitalised patients with tuberculosis. Using a bioinformatic approach, we extracted phage genomes from previously generated C. difficile genome assemblies, assessed genomic relatedness, organised phage genomes into functional modules, and identified host defence systems. Phage induction was done using mitomycin C in liquid cultures and plaque overlay assays. Attempts were made to purify phages and generate PCR amplicons for sequence confirmation. The results of this study demonstrate the presence of phages in local C. difficile isolates and provide evidence for their classification as Caudoviricetes. Further studies are needed to determine the specific taxonomy, since recent updates have rendered previous morphology-based classification of phages inadequate.