Masters Degrees (Medical Microbiology)

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Activation of silent biosynthetic gene clusters and profiling of secondary metabolites secreted by Endophytic Fungi for use as potential anti-HIV agents.
(2022) Makhwitine, John Phuti.; Ndlovu, Sizwe Innocent.; Mkhwanazi, Nompumelelo Prudence.
The continuous burden of Human Immunodeficiency Virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated five endophytic fungi isolated from Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Single-round fractionated extracts of treated P.chrysogenum showed potent anti-HIV activity with an IC₅₀ of 5.90 μg/mL and a 5-fold increase compared to the untreated fraction. The active fractionated extracts were subjected to gas chromatography-mass spectrometry (GCMS), and more bioactive compounds were detected in treated P.chrysogenum fractions than in untreated fractions. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites as therapeutic compounds
Antimicrobial properties of traditional medicine used for treatment of HIV/AIDS and its opportunistic infections.
(2012) Jwara, Nhlanhla David L.; Sturm, Adriaan Willem.; Gqaleni, Nceba.
This study was conducted to establish the scientific basis of the reported ethnomedicinal use of Ihlamvu laseAfrika (IHL) against Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Virus (AIDS) related infections. IHL is believed to have a positive effect on AIDS however this has neither been clinically nor laboratory proven. Such effect can either be directly due to IHL’s inhibition of the virus causing AIDS or indirectly by the inhibition of organisms causing opportunistic infections. Experiments were carried out to test for the effect of IHL against Cryptococcus neoformans, Candida albicans, Herpes Simplex Virus (HSV), Mycobacterium tuberculosis (MTB) and HIV. The toxicity of IHL was determined by means of three assays. Using the Trypan Blue Dye exclusion test, an aqueous mixture of IHL was tested on Vero cells (African Green Monkey) for acute toxicity at two concentrations. Cell membranes compromised by IHL would take up dye and eventually spill their contents. Vero cells that were exposed to 1μg/mL and 100μg/mL concentrations of IHL for 7 hours resulted in (8.9±0.15) % and (98.7±0.84) % cell viability (n=3), respectively. When the duration of incubation increased to 48 hours, percentage cell viability of 1μg/mL and 100μg/mL concentrations were (98.3±0.50) and (98.2±0.50) respectively. The second cytotoxicity test involved incorporation an aqueous mixture of IHL onto 3- (4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT). Cells were incubated in IHL for 24 and 48 hours resulting in a decrease in cell viability in a dose-dependent manner. At the lowest IHL concentration (0.1μg/mL) the cell viability was 80% and 78.5% after 24 and 48 hours incubations, respectively whereas at the highest concentration (1000μg/mL) was used in 24 and 48 hours incubation, cell viability was 50% and 80% respectively. The third cytotoxicity test called glutathione (GSH) focused on antioxidant level. The aim was to determine the highest concentration at which cells starts dying, concentrations used were 0.23; 0.46; 0.94; 1.88; 3.75; 7.50; 15.0 and 30.0 mg/mL. The results showed that the antioxidants levels were reduced in proportions relative to IHL concentration levels. The safe and effective dose of IHL obtained was 1.88mg/mL. The second objective of the study was to determine IHL’s active principle that is capable of inhibiting growth of C. albicans and C. neoformans, HSV, MTB and HIV. Solvents such as methanol, ethanol and acetone were utilized including an aqueous extract to extract it. The most suitable extract to inhibit the proliferation of the aforementioned organisms needed to be established. Upon its establishment, it was then used to determine the minimum inhibitory concentration (MIC). This was done in all susceptibility tests except for HIV whereby a ‘neat substance’ was used. In the case of HSV a causative agent for herpes, its susceptibility towards several IHL extracts was assessed with real-time polymerase chain reaction (RT-PCR). PCR attenuates specific site of DNA and quantifies viral load and the focus was the UL30 position which is targeted by most drugs. When comparing all solvent extracts as well as an aqueous extract of similar concentration, it was found that the methanol extract emerged as the strongest viral inhibitor with the lowest viral yield, and its threshold value, Ct = 18.4± 0.86 while the IHL concentration was 1.88mg/mL. The MIC of the methanol extract was 1.25mg/mL and Ct=18.9±1.14. An acetone extract proved to be the weakest thus its viral load was the highest, its Ct= (8.50±1.33) whilst IHL concentration was 1.88mg/mL. Cryptococcus neoformans known for causing meningitis and encephalitis in AIDS patients and C. albicans a causative agent for vaginal and oral thrush were two opportunistic infections tested for susceptibility towards IHL. The disk diffusion method was used for both fungal organisms. The best suited solvent extract was established and then used to determine the MIC. An aqueous extract showed the best activity with the inhibition zones of (10.5±1.642) mm when tested against C. albicans followed by ethanol extract (9.2±0.676) mm while acetone extract (8.80 ±1.21) mm had the lowest activity. The MIC of IHL’s aqueous extract was 1.0mg/mL and the corresponding zone of inhibition was (10.6±1.34) mm. When C. neoformans was tested for susceptibility against various IHL solvent extracts, the IHL’s aqueous extract had inhibition zones of (21.1±2.40) mm thus emerged as the strongest followed by methanol extract (10.3±0.43) mm while ethyl acetate extract was least active (7.13±0.33) mm. The MIC of the aqueous extract was 1.0mg/mL and its corresponding zone of inhibition was (11.4±0.55) mm. Furthermore, the growth inhibition of both C. neoformans and C. albicans by IHL’s aqueous extract were confirmed in liquid media with broth microdilution method. This technique tends to mimic what is likely to happen in a biological fluid. The results obtained depicted a dose-dependent response and both organisms shared a common MIC of 2.0mg/mL. From the broth microtitre plate aliquots samples were plated onto agar and used to further determine the minimum lethal concentration (MLC). The MLC essentially determines the antifungal concentration of an agent at which no colonies displayed visible growth. The MLC’s of IHL towards C. albicans and C. neoformans were 32 and 8 mg/mL respectively. IHL proved fungicidal at higher concentrations and fungistatic at low concentrations. Further susceptibility tests of IHL extracts were carried out on bacterial pathogens such as the MTB, a causative agent for Tuberculosis with 1% proportion method. This method seeks to determine if isolates are resistant if colonies grown in the presence of drugs are greater or equal to 1% of colonies grown in drug-free control quadrant. The best solvent extract was determined and then used to determine the MIC. Acetone extract results were 0.2% meaning that it strongly inhibited growth of MTB better than ethyl acetate (5%) and others the worst results were that of an aqueous extract (113%). A confirmation exercise was done with an agar dilution method. All extracts were incorporated onto agar and MTB colonies growing relative to negative controls after 21 days of incubation meant resistance while no growth meant susceptible. The MTB strain again proved susceptible towards the acetone extract but resistant towards methanol, ethanol, and aqueous extracts. The dichloromethane and ethyl acetate extracts seemed to have damaged the polypropylene plates rendering results null and void. Using agar dilution method, an MIC of an acetone extract was 16mg/mL. An aqueous extract was used for assessing HIV for susceptibility towards IHL. The quantitation of viral results were carried out on a spectrophotometer and a second generation tetrazolium dye (XTT) was used. The results showed that approximately -3.29 dilution of the aqueous extract did not protect cells. On the contrary, it proved to be toxic to both uninfected and infected cells. Moreover at low doses the extract demonstrated 50% protection towards uninfected cells. The third objective entailed the assessment of reproducibility of IHL that is routinely prepared by the Traditional Health Practitioner (THP). Batch to batch reproducibility is always a concern especially since traditional medicine is manufactured without any traceable set of standards. Two IHL samples that were prepared on different dates were assessed. Using a thin layer chromatography (TLC) a striking resemblance in the two samples was established visually by way of fractions produced. However, since TLC is a qualitative tool, it was incumbent that an instrument that doesn’t separate sample’s chemical constituents was used. The results produced by nuclear magnetic resonance (NMR) confirmed similarities in the two batch of IHL samples produced on different dates as it was the case with TLC. Peak intensity and the number of peaks in the chromatogram was a mirror image of the other thus confirming consistency in IHL preparation. The susceptibility tests of IHL towards viruses, bacteria and fungal pathogens present reasons why IHL is regarded as a non-specific repressor of pathogens people living with AIDS (PLWA) present with. The fourth objective of the study entailed the establishment of active principles responsible for the aforementioned activities. The acquisition of chemical fingerprints and their analysis was carried out on an Ultra Performance Liquid Chromatography Mass Spectrometer (UPLC-MS). The substances thought to be responsible for antimicrobial activities included:- thalebanin B, methyillukumbin A, kuguacin J, mauritine H, 2-methyl-3-(piperidin-1- yl) naphthalene-1,4-dione, isoferuloyllpeol, diosindigo A, kuguacin R, verbascoside, kuguacin B and nuciferin. Further confirmation studies are needed on fractions to identify their chemical makeup as well as their activities on all of the aforementioned microorganisms.
Aspects of sexually transmitted diseases at King Edward VIII hospital.
(1988) Hoosen, Anwar Ahmed.; Van den Ende, Jan.
Abstract available in PDF.
Causes of meningitis in the era of HIV.
(2021) Ramjathan, Praksha.; Swe Swe-Han, Khine.
No abstract available.
The characterization of a putative DNA repair protein in Mycobacterium Tuberculosis, encoded rv2414c.
(2023) Maleke, Dieketseng Palesa.; Senzani, Sibusiso.
Mycobacterium tuberculosis (MTB) is a causative agent of the communicable disease tuberculosis (TB), which is regarded as one of the top ten causes of death worldwide. Globally, TB accounts for over 10 million infections and over 1.8 million deaths annually. These statistics are subject to a constant increase due to the emergence of drug resistant strains. Although, the recent use of next generation sequencing technology has generated complete genome sequences and functional genomic data for various organisms (MTB included), to this point, the biological functions of several proteins encoded for in the MTB genome are not known or characterized hence they are called hypothetical proteins. Characterization of these hypothetical proteins is essential, as they could be involved in key regulatory processes of MTB, which is essential for the pathogen to retain a successful life cycle and disease progression. For successful invasion of the host and disease progression, it is important for MTB to retain genomic stability. Therefore, the degree of survival of MTB in the host environment is largely dependent on the bacterium’s ability to retain genomic stability. DNA repair mechanisms protect bacterial DNA from damage that can be induced by numerous stress factors. The hypothetical protein rv2414c encodes a gene amongst the MTB immunogenic protein identified in a study by Chiliza et al., (2019) which is closely associated with genes involved in MTB DNA repair, suggesting a possible role in DNA repair pathways. Therefore, the present study is aimed at characterizing a conserved hypothetical protein encoded rv2414c in Mycobacterium tuberculosis to elucidate the proteins biological function. For in-silico characterization, three bioinformatics tools were used, namely; Mycobroswer, I-TASSER and STRING online tools. Thereafter, a CRISPR-cas9 gene silencing mechanism was developed to elucidate the biological role of rv2414c. CRISPR system entails the co-expression of the silenced form of RNA-guided DNA endonuclease from the type II CRISPR system (dCas9) and a small guide RNA specific to a target sequence, leading to the DNA recognition complex resultant in transcription interference of corresponding DNA sequence. This CRIPSR mechanism was achieved by generating knockdown mutants. Phenotypic characterization of the mutants was accomplished by monitoring the mutants’ growth kinetics and biological assays were done to assign a possible biological function to rv2414c. Bioinformatics analysis suggests rv2414c is involved in DNA repair based on the structural networks it forms with 3 genes (dprA, recA and cinA) involved in MTB DNA repair and 3 proteins (rv3242c, rv3737 and rv2897c) that are involved in mainly aiding in the mechanism of DNA repair. However, the growth kinetics showed that rv2414c has no impact on the MTB growth rate, as all strains grew in a similar growth pattern with no statistical significance (p > 0.05) observed at the different time points. Additionally, UV biological assay showed that rv2414c is not a major role player in DNA repair, as UV exposure did not have an effect on bacterial survival rate even in the knockdown strain. A slight decrease in cell survival rate was noticed after addition of Mitomycin C (MMC) between Δrv2414c (100 ng/ml) + MMC and Δrv2414c + MMC, however, the difference was not significant. This implies that rv2414c is not involved in MTB DNA repair.
Characterization of immunoglobulin (Ig) isotypes, IgG subclasses and cytokines in the blood and genital tracts of HIV infected and healthy women from an observational cohort study (CAPRISA 082)
(2020) Zuma, Mandisa Nokukhanya.; Archary, Derseree.; Sorbia, Parveen.
Background: Heterosexual transmission remains the dominant route of HIV infections in women. Immune responses that predict HIV acquisition during pre-exposure prophylaxis (PrEP) remain undefined. We hypothesized that increased genital tract antibodies and cytokines pre-HIV infection predict HIV acquisition in seroconverters compared to non-seroconverters irrespective of PrEP use. Methods: Plasma and Softcup specimens were collected from n=12 seroconverters (cases) and n=48 non-seroconverters (controls) in the CAPRISA 082 study at five time points. Of 12 cases-, nine took PrEP, while 29 of 48 controls took PrEP. IgG1, IgG2, IgG3, IgG4, IgM and IgA, and nine cytokines: MIP-1􀄮, MIP-1􀈕, IP-10, MCP-1, and IL-8, TNF-􀄮, IL-1􀄮, IL-1􀈕, and IL-6 pre- and post-HIV infection, were measured using multiplexed technology. Results: Baseline levels of IgG subclasses, Ig isotypes, and mucosal cytokines were similar between cases and controls. Over time within the cases, plasma IgA significantly declined, in controls, plasma IgG2, IgG3, and IgM significantly declined over time (p<0.05). In cases and controls on PrEP, plasma IgG3 trended higher compared to no PrEP (p<0.1). Relative to baseline, only within the controls, mucosal IgG1, IgG2, IgG3, IgG4, IgM, and IgA declined significantly. Mucosal IgM significantly predicted four-fold increased HIV risk (p=0.01). Eight of nine cytokines in the genital tract were significantly elevated in the cases compared to controls (all p<0.05). In cases and controls who used PrEP relative to no PrEP, IP-10 was significantly lower (p=0.04 and p=0.009). Baseline mucosal IL-8 significantly correlated with mucosal IgG1, IgG2, total IgG, and IgM (p<0.001 for all). Conclusions: Although no significant elevated genital antibodies or cytokines pre-HIV infection were found, significantly different patterns of antibodies and cytokines were observed in this cohort. Plasma IgG3, one of the most effective of the IgG􀂶s eliciting diverse antibod􀁜 functions, was increased in PrEP users. Mucosal IgM was associated with increased HIV-acquisition risk, while pleiotropic IP-10, a reported risk factor was modulated in PrEP users among cases. Collectively, these data suggest that PrEP use may modulate or preserve specific immune responses that can modify HIV risk. As PrEP uptake increases, its effect on mucosal and systemic immunity is important for informing on prevention strategies where PrEP may be given alone or in combination with HIV vaccine for added efficacy.
Characterization of the function of RV1268c, an ATP binding cassette transporter in Mycobacterium tuberculosis.
(2023) Hallom, Pumelela.; Senzani, Sibusiso.
Tuberculosis (TB) is an epidemic disease that is caused by a bacterium called Mycobacterium tuberculosis (Mtb). This disease infects and kills millions of people globally. Anti-Tuberculosis (anti-TB) drugs such as isoniazid, ethambutol, pyrazinamide, and fluoroquinolones have been discovered and produced for TB treatment but regardless, TB persists because of the resistance to these drugs, leading to the development of multidrug-resistant (MDR) Mtb and extensively-drug resistant (XDR) Mtb. One of the key areas to focus on for the development of new effective anti- TB drugs are efflux systems, because they transport molecules outside cells and have a role in the resistance against TB treatment. This study aimed to identify the biological function of the Mtb protein, Rv1268c. RV1268c was one of the identified proteins in a study that was done by Chiliza et al., 2019 where a few protein biomarkers that are recognized by both active TB and latent TB patient antibodies. Some of these biomarkers that were studied are TreY, Bfr, and TrpG, which are biomarkers that are specific to ATP and play an important role in pathogenesis. Other biomarkers included MoaE, PonA1, and NarG, which are specific to latent TB and play a role in dormancy. The Rv1268c protein of Mtb is classified as a hypothetical membrane protein of the cell envelope and its associated proteins are Rv1267c and RV1269c, which are regulatory protein and a conserved putatively exported protein, respectively. The Rv1268c protein is hypothesised to be an ATP-binding cassette (ABC) transporter. The nucleotide and protein sequences of Rv1268c were v downloaded from the database of Mtb H37Rv using Mycobrowser. To create a knock down strain, annealed oligos were ligated to PLJR965 plasmid and transformed into XL10-Gold ultracompetent cells and grown on kanamycin-containing plates. Extracted plasmids were electroporated into Mtb, and after 4 weeks of incubation, the colonies were screened to check if they carried the knock down plasmid . DNA was then extracted to characterize the function of the Rv1268c Mtb protein. The results showed that Rv1268c had no effect on the in vitro growth of Mtb, while the Ethidium bromide (EtBr) assay displayed a difference on the extrusion of EtBr as the knock down and the knock down with anhydrotetracycline (aTc) had lower fluorescence as compared to the wild type which implies that Rv1268c is not an ABC transporter. The statistical analysis showed that the was no significant difference on the drug susceptibility between the wild type, knock down, and the knock down with aTc strains . The growth of neither the wild type or knock down strains was completely inhibited by either of the drugs tested. Keywords:
Characterizing the function of the Rv3218 gene in Mycobacterium tuberculosis.
(2023) Canham, Khumbuzile.; Senzani, Sibusiso.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a primordial affliction that continues to torment humankind since its known history and prehistory. TB is among the major causes of ill-health and death in the world with an estimated 1.8 million cases of death recorded yearly. The situation is worsened by the emergence of the strains of TB that are regarded as resistant. Recently, Mycobacterium bovis bacillus Calmette-Guerin (BCG), has been the only available vaccine for TB. An intense understanding of Mtb’s biology, should reveal new perceptions that can lead to the improved treatment, diagnostics, vaccines and highly needed control measures. Throughout infection, Mtb produces some proteins into the host environment to play critical role in pathogen host interactions. Close to half of the Mtb genome consists of genes with unknown functions. Among those genes is Rv3218 gene which was identified in the study by Chiliza et al., 2019. The Rv3218 gene is hypothesised to have a Diacylglycerol kinase activity. This study aimed at characterising the function of Rv3218 gene in Mtb with the purpose of coming up with ideas of how that can be used in the development of more effective and convenient diagnostic tools, therapeutics, or the total elimination of TB. There is a vast amount of molecular techniques that are currently used to characterise unknown genes. Here we employed a CRISPRi dCas9 system for the silencing of the Rv3218 gene in Mtb. We also used a number of Bioinformatics tools for in silico analysis of the gene and construction of all relevant primers necessary for this molecular cloning. The Rv3218 knockdown repressed by Anhydrotetracycline (ATc) was constructed for assaying the effect of this gene silencing compared to the MtbH37Rv wild type. We then conducted growth curves and MICs (Minimum Inhibitory Concentrations) to check if this gene has an impact on antimicrobial susceptibility and growth of Mtb. We also tested its activity as a diacylglycerol kinase via osmolarity assay as it is said that dgk mutants do not grow well on nutrient media of low osmolarity. On bioinformatics analysis, we found that the gene has cell wall and transcription regulatory functions and possesses a similar structure as diacylglycerol kinase. However, the in vitro analysis was contradictory to these findings. We found that the Rv3218 gene has no impact on the growth of Mtb and it’s susceptibly to the antimicrobial drugs that were used in this study. On the osmolarity assay, there was no observable difference between the growth of the wild type and the knockdown strain in all the concentrations of osmolarity. Judging from these findings, we then concluded that this gene does not function as a diacylglycerol kinase. We then suggested that, more advanced experimental studies still need to be conducted in order to confirm this hypothesis as we were unable to do them due to the short time frame for this study.
Cytokine immune response profiles during 5 intestinal helminths and Mycobacterium 6 tuberculosis coinfection: An in vitro and human ex vivo study in KwaZulu-Natal.
(2023) Bhengu, Khethiwe Nomcebo.; Mkhize-Kwitshana, Zilungile Lynette.; Singh, Ravesh.; Naidoo, Pragalathan.
Background: There is a striking geographic overlap between helminths and tuberculosis (TB), particularly in developing countries like Africa. Underprivileged communities are more susceptible to these illnesses due to poverty, poor sanitation, and other environmental factor Helminth and tuberculosis infections exhibit distinct immune responses, which may be antagonistic in coinfected hosts and lead to poor prognosis. Helminth infections induce anti422 inflammatory Th2/Treg responses contrary to the pro-inflammatory Th1 responses triggered by Mycobacterium tuberculosis (Mtb) infection. Reduced TB protection has been associated with a strong Th2 response. Uncertainty exists on how helminth infection affects the host’s resistance to TB. This necessitates further investigation of immune responses in helminths and TB coinfection cases, particularly in KwaZulu-Natal (KZN). Aim: To determine the cytokine response profiles during intestinal helminth and TB coinfection using lymphocytic Jurkat and monocytic THP-1 cell lines for the in vitro study and TB and helminth coinfected South African adults for the human ex vivo study. Methods: Lymphocytic Jurkat and monocytic THP-1 cell lines were stimulated for 24 and 48 hours with Mtb H37Rv and Ascaris lumbricoides (A. lumbricoides) excretory-secretory protein extracts for the in vitro study. A cross-sectional study on consenting adult participants (≥18 years) (n = 414) recruited from primary health care clinics was conducted between March 2020 and August 2021 in Durban, KwaZulu Natal, for the pilot human ex vivo study. Blood and stool samples were collected from the recruited participants. The Kato-Katz and Mini-Parasep faecal parasite concentration techniques were used to detect intestinal parasite infections in stool samples. Blood samples were analysed to determine A. lumbricoides-specific immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) levels to improve microscopy sensitivity. In this study, cytokine analysis was undertaken for 164 participants; 96 were HIV infected and had to be excluded, leaving 68 eligible participants. The eligible individuals were subdivided into uninfected controls (no helminth and TB infection) (n = 18), helminth only infected (n = TB only infected (n = 6), and TB and helminth co-infected (n = 6) groups. Thereafter, for both the in vitro and ex vivo study, the gene expression profiles of the T helper type 1(Th1) and transcription factors [Interferon-γ (IFN-γ), Tumour necrosis factor-α (TNF-α), Interleukin-2 (ILxvii 2), Nuclear factor of activated T cells 2 (NFATC2), Eomesodermin 446 (eomes), T helper 2 (Th2) and transcription factors (Interleukin-4 (IL-4), Interleukin5 (IL-5, Transforming growth factor-β (TGF-β), T helper type 17 (Th17) (Interleukin-17 (IL-17), immune protein and proteases (Granzyme B, Perforin), Regulatory T cells (Tregs) (Interleukin-10 (IL-10) and Fork head box P3 (FoxP3)] and the uninfected controls, TB alone, helminth alone and coinfected groups were determined using RT-qPCR. Results: (i) In vitro study: TB-stimulated Jurkat cells had significantly higher levels of IFN-γ, TNF-α, Granzyme B, and perforin compared to unstimulated controls, LPS, A. lumbricoides, and A. lumbricoides plus TB costimulated cells (p<0.0001). IL-2, IL-17, Eomes, and NFATC2 levels were also higher in TB-stimulated Jurkat cells (p<0.0001). TB alone stimulated cells had lower IL-5 and IL-4 levels compared to A. lumbricoides alone stimulated and TB plus A. lumbricoides costimulated Jurkat and THP-1 cells (p<0.0001. A. lumbricoides alone stimulated cells had higher IL-4 levels compared to TB plus A. lumbricoides costimulated Jurkat and THP- 1 cells (p<0.0001). TGF-β levels were also lower in TB alone stimulated cells compared to TB plus A. lumbricoides costimulated cells. IL-10 levels were lower in TB stimulated Jurkat and THP-1 cells compared to TB plus A. lumbricoides costimulated cells (p<0.0001. (ii) Ex vivo study: Similar results were noted for both the in vitro and the ex vivo study, although the human study had a smaller sample size. Conclusion: Data suggest that helminths induce a predominant anti-inflammatory Th2 and Treg response which may downregulate critical proinflammatory Th1 responses crucial for TB protection.
Cytokine production by ME-180 cells and VK2 E6/E7 cells on exposure to Neisseria gonorrhoeae, HIV, N. gonorrhoeae and HIV.
(2018) Govindasami, Merusha.; Sturm, Adriaan Willem.
Gonorrhoea is a sexually transmitted disease caused by Neisseria gonorrhoeae. Women are more at risk in developing secondary complications due to asymptomatic infections. In 2001, a study was done on the different responses of epithelial cells from three different regions of the lower female genital tract exposed to N. gonorrhoeae. Upregulation of cytokines found in cervical and vaginal secretions has been linked with human immunodeficiency virus 1 infection. In vitro studies of the immune response following exposure to multiple STI pathogens are relevant as mixed infections are common and not many studies have been done. The aim of this study was to determine the cytokine response in a co-infection model with N. gonorrhoeae and HIV using two genital epithelial cell lines. ME-180 cervical cells and VK2 E6/E7 vaginal cells were infected with N. gonorrhoeae and HIV only and with both organisms in different sequence. Infected cells were incubated at 37 °C in 5 % CO2 for 72 h. The supernatant was assayed for cytokines TNF-α, RANTES, IL-1β, IL-4, IL-6, IL-8, and IL-10 by means of the Bio-Plex Pro Cytokine, Chemokine, and Growth Factor Assay kit. The spontaneous cytokine release was higher in VK2 E6/E7 cells than in the ME-180 cells. On exposure to single organisms the response to N. gonorrhoeae was stronger than to HIV in both cells for IL-10, IL-8 and IL-6. For infection with N. gonorrhoeae the VK2 E6/E7 cells had a stronger cytokine response than ME-180 but this was not so for HIV. The response the cells had to exposure to both organisms was independent of the sequence of exposure. Further studies should be done on mixed infections of N. gonorrhoeae and HIV with additional STI pathogens.
The cytotoxic effects of fumonisin B1 in human kidney cells and the ability of allicin to ameliorate these effects.
(2019) Mahlalela, Nomalungelo Nothando Felicity.; Khan, Rene Bernadette.
Fumonisin B1 (FB1) is a widespread contaminant of crops and is produced as a secondary metabolite of fungi. It has been found to disrupt sphingolipid metabolism, cause epigenetic modifications and induce cellular toxicity that can manifest through oxidative stress and apoptosis. Although implicated in animal toxicity including kidney cancer in rats, FB1 effects in the human kidney have not been explored. Allicin is a biological component of garlic that has been widely studied for its health benefits including its anti-cancer and antioxidant properties. This study evaluated allicin as a possible therapeutic measure for FB1-induced cytotoxicity in Hek293 cells. Both FB1 and allicin decreased cellular viability (MTT assay) in a dose-dependent manner and generated IC50 values of 215 μM and 3.905 μM respectively. Three 24 h treatments (FB1, allicin and combined FB1+allicin) were compared to untreated cells for induction of apoptosis and oxidative stress. Luminometry was used to determine cytotoxicity (lactate dehydrogenase assay), caspase activity and mitochondrial toxicity (apoptosis), and quantify intracellular ROS (iROS) and glutathione (GSH) (oxidative stress). Free radical production was estimated by the TBARS and NOS assays respectively, while DNA damage was evaluated using the comet assay. Western blotting confirmed the expression of various antioxidant and apoptotic proteins, and superoxide dismutase 2 (SOD2) transcripts were quantified using qPCR. ATP concentration was increased for all treatments, but mitochondrial toxicity was increased in the allicin treatment. While lipid peroxidation decreased, luminometry results indicate that iROS was increased and was accompanied by a corresponding decrease in SOD2 and catalase protein expression. Depletion of GSH was consistent with increased GPx1, HSP70 and Nrf2 protein expression suggesting the presence of oxidative stress. SOD2 transcripts were only increased in the allicin treatment. Apoptosis was initiated as indicated by increased caspases 8 and 9, and pro-apoptotic Bax protein expression, but caspase 3/7 was not activated for the FB1 treatment. However, DNA fragmentation and cPARP were increased for all treatments suggesting that apoptosis was executed. Overall, FB1 and allicin individually and combined induced oxidative stress by increasing ROS and decreasing antioxidants. Apoptosis was also induced, although in a caspase independent manner in the FB1 treatment. Overall, allicin did not ameliorate the effects of FB1 in Hek293 cells.
Defining the role of high-dose isoniazid in the treatment of multi-drug resistance tuberculosis: isoniazid resistant profiling.
(2021) Ngema, Senamile Lale.; Dookie, Navisha.; Naidoo, Kogieleum.
Background: High-dose isoniazid is recommended in short-course regimens for multidrug-resistant tuberculosis (MDR-TB). However, there is no substantial evidence supporting its use in the presence of INH resistant mutations. Therefore, this study aimed to establish the efficacy of INH in the presence INH resistance associated mutations. Methods: We selected 94 clinical isolates obtained from 65 patients from the IndEX (CAP020) study specimen biorepository. Isolates were selected based on whole genome sequencing results showing evidence of INH resistant conferring mutations. Twenty-one isolates had inhA promoter gene and/ inhA coding region mutations, 35 had katG mutations, and 20 had both inhA promoter and/ inhA coding region plus katG mutations. Additionally, 18 INH susceptible clinical isolates were included in this analysis. Minimum inhibitory concentrations (MICs) were done in different concentration ranges depending on the mutation present. INH susceptible and H37Rv (0.016-0.256) μg/ml, inhA (0.256-4.0) μg/ml, katG (1.0-16.0) μg/ml and inhA plus katG (4.0-16) μg/ml. Results: Among 94 isolates, 36 were excluded: 11 MPT64 antigen negative, 23 non-growers and two were contaminated. Fifty-eight isolates from 55 patients were left for analysis. Eleven isolates had inhA mutations, 23 katG mutations, 12 had double mutations in inhA and katG, and 12 were INH susceptible. MICs obtained varied within isolates ranging from 0.016 to >64.0 μg/ml. InhA, katG, inhA plus katG mutations and INH susceptible isolates had median INH MIC of 8.0 (4.0-64.0), 4.0 (95% CI, 4.0-8.0), 64.0 (95% CI, 64.0-64.0), and 0.48 (95% CI, 0.32-1.0) μg/ml, respectively, confirming the association between INH MICs and genotypic profile. The MDR-TB and pre/XDR-TB had median INH MIC of 8.0 (95% CI, 8.0-32.0) and 48.0 (4.0-64.0) μg/ml, respectively. We found association between cavitary disease and increase in INH MICs for inhA mutants, median of 64.0 (64.0-64.0) μg/ml, and previous TB history and increased INH MICs (8.0[95% CI, 8.0-64]. Conclusion: This study demonstrated highly variable MIC range with significant overlap in MIC range among the mutant groups. Furthermore, inhA mutants demonstrated unexpectedly high MICs raising a concern for the ongoing use of the high-dose INH in our setting. Our findings suggest that the current one-size-fits all approach to MDR-TB short-course regimen requires urgent review.
A descriptive analysis of the routine use of genotype MTBDRsl in a high HIV/TB prevalent region in South Africa.
(2020) Lutchminarain, Keeren.; Mvelase, Nomonde Ritta.; Swe Swe-Han, Khine.
Background. Since the implementation of shortened drug regimens for the management of drug resistant tuberculosis (TB), there is a growing need for rapid detection of resistance to second line antimycobacterial drugs. The GenoType MTBDRsl Version 2 allows for the rapid molecular detection of resistance conferring mutations to fluoroquinolones (FQ) and second line injectable drugs (SLIDs). Although the GenoType MTBDRsl is recommended for use directly on smear positive and smear negative clinical specimens, the feasibility of using this assay routinely within a programmatic setting in high HIV/TB endemic areas requires exploration. Objectives. To assess the feasibility of routinely using the GenoType MTBDRsl in a high HIV/TB prevalent region and to describe the various circulating resistance patterns detected by this test in KwaZulu-Natal. Methods. A retrospective data analysis of all GenoType MTBDRsl results in newly diagnosed rifampicin resistant (RR-TB)/multidrug resistant TB (MDR-TB) specimens was performed. The assays performance on direct testing of smear positive and smear negative specimens was compared. The various mutation patterns for FQ and SLIDs identified by this test was observed. 21 Results. Of 1873 RR-TB/MDR-TB, 37,4% were smear negative and 62,5% was smear positive. In smear negative specimens, the GenoType MTBDRsl showed an inconclusive rate of 61,2%, whilst amongst the smear positive specimens, an inconclusive rate of only 6,6% was observed. The commonest mutation pattern observed for FQ occurred at the gyrA gene at codon 90 (A90V) 61/158(38,6%) followed by the D94G mutation 31/158 (19,6%). Heteroresistance for FQ was observed in the gyrA gene for 6/158 (10,1%) isolates. For SLIDs, the commonest mutation occurred in the rrs region specifically A1401G, 71/108 (65,7%) followed by C1402T at 20/108 (18.5%). Conclusion. The routine use GenoType MTBDRsl Version 2 assay is more feasible in smear positive specimens as compared to smear negative specimens in high HIV/TB settings. To improve future development of the assay, further studies looking at the various resistance patterns are required.
The developement of a Real-Time Polymerase Chain Reaction using TaqMan probes to determine the burden of multi-drug resistant tuberculosis (MDR-TB) in KwaZulu-Natal.
(2008) Ganas, Anura.; Pym, Alexander S.
Abstract available in PDF.
The development and application of a high throughput methodology to determine MICs of Mycobacterium tuberculosis isolates against antimicrobial agents.
(2014) Rampersad, Tashmin.; Sturm, Adriaan Willem.
Chapter 1 of this dissertation entails the introduction, aims, objectives and the literature review. Drug susceptibility testing of Mycobacterium tuberculosis is time consuming and expensive. Multi-point inoculation offers the advantage of testing multiple isolates on a series of solid media with a single breakpoint concentration of a drug in each plate or a series of different drug concentrations of one drug. We aimed to determine the reproducibility of MIC determination for anti-TB drugs of M. tuberculosis isolates using agar dilution with multi-point inoculation and thereafter validating the results by comparing it to classic agar dilution on quadrant plates and the MTT assay. Chapter 2 contains the manuscript that has been submitted for publication. This manuscript contains a brief introduction with the aim and objectives, and a detailed description of the methodology, results and discussion. Thirty M. tuberculosis isolates were grown in Middlebrook 7H9 broth with 20% Tween until mid-log phase was reached. Agar dilution MICs were determined on Middlebrook 7H10 agar for 11 anti-TB drugs at concentrations ranging from 128 to 0.125 mg/L. The agar plates were inoculated using a multi-point inoculation device with 36 points each delivering 1μL of a suspension of 1×104 cfu/ml. For the quadrant plate method and the MTT assay 100 μl of the same suspension was used. All tests were done 3 times in triplicate. Agar dilution with multi-point inoculation was found to be reproducible within the 11 anti-TB drugs tested and correlated well with agar dilution on quadrant plates and the MTT assay for the three anti-TB drugs tested. Chapter 3 entails a brief summary (synthesis) of the discussion found in the above-mentioned manuscript. The multi-point inoculation method has potential for wide scale application in breakpoint drug susceptibility testing as well as MIC testing of M. tuberculosis isolates. Lastly this dissertation contains the required references and appendices.
The development of a stratified keratinocyte model for chlamydia trachomatis pathogenesis studies.
(2017) Jadoo, Sasha.; Sturm, Adriaan Willem.; Joubert, Bronwyn C.
A number of different methods to generate stratified keratinocyte layers have been published. These involved the use of normal human epidermal keratinocytes (NHEKs/NEKS), which have a better ability to stratify compared to HaCaT keratinocytes, which usually require supplemented growth factors or stromal interactions with fibroblasts to do so. This study aimed to generate a model of stratified keratinocytes, closely resembling in vivo skin, using HaCaT cells and to demonstrate the effect that C. trachomatis has on these layered keratinocytes, allowing us to gain insight on the pathophysiology of this organism. All cells and bacteria were propagated and titrated according to conventional protocols. HaCaT cells were subcultured upon confluence, seeded (1x106 cells/ml) onto collagen-coated PTFE Transwell membrane inserts and incubated at 33°C and 37°C for 24 days to allow differentiation and stratification. Once cells became confluent they were exposed to the air-liquid interface and fed with KGM Gold (Lonza) supplemented with 10% FBS and additional calcium. Thereafter, cells were fixed in 3.7% phosphate-buffered formaldehyde, embedded in a paraffin block, sectioned, stained and viewed. Hematoxylin and Eosin (H&E) staining was used to determine the resemblance to in vivo human skin. Immunofluorescence was used to detect keratin 10, keratin 14 and involucrin which are markers of keratinocyte differentiation. Stratified keratinocyte layers were infected with C. trachomatis and this was confirmed using the MicroTrak ® C. trachomatis Culture Confirmation Test Kit. Subsequent changes to the layers were also observed and recorded. It was shown that HaCaT cells grown at the air-liquid interface on collagen-coated PTFE Transwell membrane inserts were able to stratify at 33°C. However, more layers of keratinocytes were seen at 37°C after the same duration of incubation (24 days). Keratin 10, keratin 14 and involucrin were all detected in the layers grown at both temperatures, suggesting that the keratinocytes had committed to differentiation. However, the fluorescence seen at 33°C for keratin 10 and involucrin was more intense as compared to that seen at 37°. This suggests that although stratification was faster at 37°C, differentiation was quicker at 33°C. C. trachomatis was able to infect layered keratinocytes grown at both temperatures although not all layers formed at 33°C were infected. Degradation of keratinocyte layers after infection with C. trachomatis was more prominent in those grown at 37°C, which is in keeping with previous findings that the optimum growth temperature of the C. trachomatis LGV biovar is 37°C. This study provided a novel insight in suggesting the manner in which C. trachomatis is able to infect and migrate through in vivo skin, leaving room for further studies in which similar methods of generating stratified keratinocytes may be used to better understand the pathophysiology of various other organisms that affect keratinocytes.
Development of an antigen detection based point-of-care test for the diagnosis of primary syphilis.
(2011) Naicker, Meleshni.; Sturm, Adriaan Willem.
Aim: To develop an antigen detection based, point-of-care test that will rapidly exclude syphilitic infection in patients presenting with genital ulcers. Materials and Method: T. pallidum subsp pallidum, Nichols strain, was propagated by intra-testicular inoculation of rabbits. T. pallidum DNA was obtained by suspending the testicular extract in ProbeTec lysis buffer followed by heating. Crude DNA was purified and concentrated. Specific primers were used for the amplification of the gene encoding the 31 kDa T. pallidum rare outer membrane porin protein (termed “Tromp1”). The amplified gene was cloned in frame with the pET100/D-TOPO vector that carries the N-terminal Xpress epitope and polyhistidine fusion tags. A screening PCR, restriction digest and DNA sequencing were used to confirm the presence of the tromp1 insert. Isolated plasmid DNA, pET100/D/tromp1 and the pET100/D/lacZ (positive control) were transformed into BL21 (DE3) pLysS E. coli cells for expression of recombinant Tromp1 and β-galactosidase as fusion proteins. SDS-PAGE and Western blot analysis were applied for detection of the recombinant proteins. Results: The gene encoding the 31 kDa Tromp protein was successfully cloned and sequenced. Multiple sequence alignment showed 100% homology between the cloned tromp1 gene sequence and its reference sequence. In addition, a screening PCR for transformation products and restriction digest of isolated plasmid DNA confirmed the presence of the tromp1 insert. Following gene expression, SDS-PAGE gel analysis showed no difference in the banding pattern between IPTG induced and uninduced lysates. The positive control however, showed a bright and distinct band at its expected size range of ~121 kDa. A Western blot and ELISA using specific antibodies to the N-terminal Xpress epitope fusion tag confirmed the absence of recombinant Tromp1 protein. Discussion and Conclusion: The results show that the tromp1 insert was successfully cloned and maintained up until the expression level. However expression of recombinant Tromp1 in BL21 (DE3) pLysS E. coli cells, for use as antigen in the serodiagnosis of primary syphilis was not achieved, despite several attempts to optimize gene expression. Expression of the positive control gene confirmed that growth and induction were properly performed.
Differences in the susceptibility of mycobacterium tuberculosis to the 1st and 2nd line antituberculosis drugs under aerobic and anaerobic conditions.
(2015) Ngcobo, Zethembiso Brightness.; Botha, Stephanus Johannes.; Sturm, Adriaan Willem.
Although Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is now considered a facultative anaerobe, and bacilli isolated from sputum specimen possess morphologies identified from bacilli growing aerobically and under oxygen deprived conditions, most of the targets for the antituberculosis drugs are readily found on bacilli that are thriving aerobically. This raises questions on the efficiency of antituberculosis drugs on eradicating the pathogen from the host during treatment. In this study to determine whether the antituberculosis drugs that are used currently for the treatment of TB have similar effect of these different populations of this mycobacterium, we grew this organism under aerobic and oxygen deprived environments and then subjected them to the antimicrobial agents. The minimum inhibitory concentration (MICs) of these isolates against nine antituberculosis drugs were determined under aerobic conditions for the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and under both aerobic and anaerobic conditions using the Microscopic Observation Drug Susceptibility (MODS) assays. In addition the bactericidal activities of isoniazid, rifampicin, kanamycin and ofloxacin were tested and compared amongst MDR isolates that were growing aerobically and anaerobically. There were some differences in the MICs determined by the MTT assay and the MODS assay for some isolates. For the susceptible isolates the MICs from the MTT assay were higher than the MICs from the MODS assay. The reverse was true for the drug resistant isolates. The reference strain H37Rv was resistant to some of the antimicrobial agents that were tested in this study. This was under both methods. However, MICs measured under anaerobic conditions with anaerobic bacilli did not yield viable results due to absence of growth as the bacilli are known to replicate at a negligible rate under anaerobic conditions. The bacilli in the inoculum were viable as following 40 days of anaerobic incubation but upon aerobic incubation of these cultures, growth was observed. And again with the bactericidal assays that were conducted on the multidrug resistant (MDR) isolates proved this. Rifampicin was the most potent antimicrobial agent against the anaerobic M. tuberculosis as susceptibility to this antimicrobial agent increased under anaerobic conditions.
Distribution, virulence and antimicrobial resistance profile of Bacillus species from the environment of four public hospitals in South Africa.
(2021) Mbhele, Zamile Nompumelelo.; Bester, Linda Antoinette.; Zishiri, Oliver Tendayi.
Hospital-acquired infections (HAIs) are counted as the most crucial global health crisis. The hospital environment may be colonized by opportunistic pathogens, which can lead to HAIs in hospitalized patients. As a result, microbial monitoring of the hospital environment is important in managing these pathogenic organisms. This research aimed to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Bacillus spp. on hospital surfaces in public hospitals in KwaZulu-Natal (KZN), South Africa (SA). A total of 777 swabs were collected from four different public hospitals classified as A (Central), B (Tertiary), C (Regional), and D (District), within three different wards (paediatric, general ward, and intensive care unit (ICU) and 11 touchable surfaces belonging to medical devices and well used equipment. Samples were assessed for the existence of Bacillus spp in these four public hospitals. Bacillus was isolated using the microbial plating method on a selective Bacillus medium, and the biochemical characteristics confirmed, including oxidase, catalase, motility, and triple sugar iron (TSI). Molecular confirmation was also performed using polymerase chain reaction (PCR) targeting the MotB gene for Bacillus cereus and 16s rRNA gene for Bacillus subtilis. The minimum inhibitory concentration (MIC) technique evaluated the antimicrobial resistance profile using the Clinical Laboratory Standards Institute (CLSI) guidelines. Molecular characterization was conducted for seven antimicrobial resistance genes and 11 virulence factors using PCR. Genetic relatedness between the isolates across the four hospitals was evaluated by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A total of 777 samples were collected in the hospital environments, of which 135 were positive for Bacillus spp. Species identification revealed 4 % (6) as Bacillus subtilis and 96 % (129) as B. cereus. The overall prevalence of Bacillus spp. per hospital was 24 % (32/135) for Central (hospital A), 33 % (45/135) for Tertiary (hospital B), 27 % (36/135) for Regional (hospital C) and 16 % (22/135) for District hospital (hospital D). A statistically significant difference in Bacillus prevalence (p = 0.044) was found between tertiary and regional hospitals (p = 0.000).The prevalence among the wards was averaging 33 % (45/135), noting ICU with the highest prevalence of 35 % (47/135). In terms of the wards, no statistically significant differences were found (p = 0.133). The hospital and the wards had a strong correlation (r = 0.525, p = 0.000).The highest prevalence on frequently touched sites was a bed with 15 % (20/135), drip stands and sinks with 12 % (16/135) respectively, ward phones with 11 % (15/135), and nurses’ tables with 10 % (14/135). Complete resistance was observed against ampicillin (100 %; 135/135), and high resistance against ciprofloxacin (99 %; 134/135), amoxicillin (97 %; 131/135), tetracycline (82 %; 112/135), cefotaxime (51 %; 69/135), and erythromycin (43 %; 59/135). Lower resistance was observed against meropenem (20 %; 27/135) and imipenem (25 %; 26/135). A total of 43 different antibiograms were detected, with 97 % (132/135) of the isolates found to be multidrug resistance (MDR). No statistically significant difference was observed between the antibiotics tested (p ≥ 0.05). The observed resistance genes were ermB (56 %; 33/59) (conferring erythromycin resistance), tetracycline resistance-conferring genes were tetM (5 %; 5/112) and tetK (4 %; 4/112). No tetA, tetB, tet39, and the blm gene (beta-lactamase resistance-conferring gene) were detected. Different toxins produced by Bacillus are associated with the pathogenicity of Bacillus species. Prevalence of the virulence enterotoxin genes associated with diarrhea prevailed in 88 % (99/135) of hblD, 77 % (104/135) in hblA and CytK respectively, nheC 67 % (90/135), nheB 65 % (88/135), nheA 64 % (89/135), hblC 55 % (74/135), bceT 44 % (59/135), hlyII 37 % (50/135), and finally EntFM 27 % (37/135) of the isolates housed the gene. No cereulide (ces gene) causing emetic syndrome was detected. Typing using ERIC-PCR noted type clusters composed of isolates from different wards, environmental sites, and equipment and showing high genetic diversity, indicating no common infection sources. This study revealed a moderate prevalence of Bacillus spp. collected from the environment of the four public hospitals. High resistance was observed for some antibiotics that are usually effective against Bacillus; this may serve as a potential risk for effective treatment of Bacillus infections. Most isolates harboured virulence factors that cause diarrheal syndrome rather than those causing the emetic syndrome. These findings highlight the need for microbial surveillance of hospital environments to inform and improve current intervention programs for cleaning methods in public hospitals to reduce environmental contamination and transmission of pathogenic Bacillus spp. infections associated with HAI’s in hospitalised patients.
Drug susceptibility testing of second and third line anti-tuberculosis drugs used in the management of extensively drug resistant tuberculosis.
(2013) Moodley, Salona.; Moodley, Prashini.
Drug resistant tuberculosis is a major contributor to South Africa’s quadruple burden of disease. Management of this infection in a highly HIV endemic area is a constant challenge. There is a paucity of new anti-tuberculosis agents in the developmental and clinical trial phases to address the problem of extensively-drug resistant tuberculosis (XDR-TB). In an attempt to affect a cure in patients with XDR-TB, it has become necessary to re-introduce previously used anti-tuberculosis drugs, as well as antimicrobial agents designed for treatment of non-tuberculosis infections. Whilst these drugs may have previously been tested and shown efficacy in drug susceptible tuberculosis, their activity in XDR TB strains was not tested before introduction for management of XDR-TB in KwaZulu-Natal, South Africa. Drug susceptibility testing (DST) plays an integral role in the diagnosis and treatment options for tuberculosis. It is able to decrease the burden and spread of resistant tuberculosis. However DSTs methods for second line anti –TB drugs (SLDs) and third line anti-TB drugs (TLDs) have not been standardised. Critical concentrations of these anti-TB drugs remain unknown or vary within and between settings thus further hampering the control of TB.

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