Masters Degrees (Medical Microbiology)

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Migration of Treponema pallidum through a keratinocyte layer.
(2010) Naidoo, Kavitha.; Sturm, Adriaan Willem.
Treponema pallidum is the causative agent of the sexually transmitted disease, syphilis. The organism can not be cultured in vitro, which has inhibited the understanding of the pathogenesis of syphilis. There has been no evidence of a treponemal toxin but adherence of large numbers of treponemes is able to destroy cell monolayers of different cell types (Fitzgerald et al, 1982). Non-pathogenic treponemes failed to adhere to cultured cells and this suggests that adherence is associated with virulence of T. pallidum (Fitzgerald et al, 1977). In this study we explored the interaction of T. pallidum with HaCaT cells which are immortalized human keratinocytes with characteristics equivalent to their natural counterpart. The adhesion assay confirmed binding of the organism to HaCaT cell monolayers. Migration assays and electron microscopy revealed that T. pallidum migrates through a confluent keratinocyte layer and western blotting experiments that differentiate between soluble and insoluble occludin confirmed that T. pallidum does not loosen the tight junctions. It is concluded that T. pallidum passes through the keratinocyte layer by trans-cellular rather than inter-cellular migration.
Pathogenic effect of Trichomonas vaginalis on various cell lines in vitro.
(2010) Bhojraj, Neetha.; Sturm, Adriaan Willem.
Trichomoniasis has been linked to pelvic inflammatory disease, cervical cancer, increased HIV transmission, infertility as well as co-infections with other STIs. In 2002, an association was found with Trichomonas vaginalis and PID in HIV positive women. Therefore, the question arose whether T. vaginalis is able to invade the upper genital tract of HIV infected women. A prerequisite for invasion of the upper genital tract is the capability of the organism to adhere to the cells of the organs involved. This study therefore investigated the interaction between T. vaginalis and vaginal, cervical and endometrial cells. In comparing adhesion and cytotoxicity of T. vaginalis to cells of the upper and lower genital tract at different pH, immortalized vaginal (VK2), cervical (ME 180) and endometrial (KLE) cells were exposed to a standardized inoculum of trichomonads at pH 4.5 to 7.0. Adhesion was measured microscopically after acridine orange staining and cytotoxicity was established by measuring LDH release using a commercial kit. Adhesion of the ME-180 and VK2 cell lines was found to be pH dependent. However, the KLE cell line was not. As the pH increased, adherence to the vaginal and cervical cells decreased. Adhesion to endometrial cells was minimal at neutral pH but marked adhesion was found at lower pH. For the vaginal cell line, cytotoxicity was minimal at pH 4.5 but substantial (30 to 60%) at higher pH. In contrast, cytotoxicity on cervical and endometrial cells was highest at lower pH. The pronounced toxicity of vaginal epithelial cells at pH 5 and pH 5.5 is in keeping with the pH range found in patients with vaginitis. The observations on the cervical epithelium suggest toxic effect on the ecto-cervical epithelium immediate after acquisition of the infection. Adhesion of trichomonads to the endometrial cell line suggests that T. vaginalis is capable of colonization of the upper genital tract. At pH values applicable to the in vivo situation, toxicity was very low.
Microbiology of vaginal discharge with emphasis on gardnerella vaginalis.
(1990) Kharsany, Ayesha Bibi Mahomed.; Van den Ende, Jan.; Moodley, Jagidesa.
The microbiological aetiology of vaginal discharge was studied in 208 women attending various outpatient clinics at King Edward VIII Hospital. Specimens from the lower genital tract were collected for microscopy and culture. Vaginal wet smear examination, amine liberation test and vaginal pH estimation were performed and assessed for their reliability for the rapid diagnosis of vaginal infections. Vaginal and endo-cervical infections were present in 163 (78,4%) women. G. vaginalis (65,4%), T. vaginalis (37,9%), genital yeasts (37,0%), M. hominis (59,6%), g. urealyticum (48,1%), anaerobic bacteria (32,6%), N. gonorrhoeae (11,1%), f. trachomatis (22,1%) and Herpes simplex virus (0,9%) were detected. Of the 104 women in • whom vaginal infections were detected, bacterial vaginosis was present as the sole infection in 32 (22,2%), I. vaginalis in 35 (24,3%) and C. albicans in 23 (15,9%). Bacterial vaginosis occurred concurrently with T. vaginalis and f. albicans in 24 (16,5%) and 11 (7,5%) women respectively; whilstT. vaginalis and f. albicans occurred concurrently in 14 (9,7%) women. In 6 (4,1%) women all three infections were present. No vaginal or endo-cervical pathogens were detected in 45 (21,6%) women. Women with bacterial vaginosis were found to be significantly colonised with G. vaginalis, M, hominis, anaerobic bacteria and curved Gram-negative bacilli (p < 0,05). Vaginal wet smear microscopy detected T.. vaginalis in 29% and "clue" cells in 41,3% of smears. The presence of "clue" cells (91,8%) and a positive amine test (76,7%) was significantly associated with bacterial vaginosis. Although a raised vaginal pH was also significantly associated with bacterial vaginosis, this test was less specific (65,2%) than "clue" cells (85,9%) and the amine test (95,5%). The vaginal Gram stain, as performed in this study, was found to be unreliable for the detection of "clue" cells. G. vaginalis biotypes 1 and 5 were significantly associated with bacterial vaginosis, however the serotyping scheme did not distinguish between strains isolated from women with and without bacterial vaginosis. The antimicrobial susceptibility pattern of 93 strains of G. vaginalis was not typical of either Gram-positive or Gram-negative bacteria. Serological tests revealed reactive syphilis serology in 47 (22,6%) and the presence of hepatitis B surface antigen in 16 (7,7%) women. Antibody to human immunodeficiency virus was detected in 4 (1,9%) women attending the colposcopy clinic. This study clearly demonstrates the high prevalence of vaginal and/or endo-cervical infections in women locally, the majority of whom were asymptomatic. The high frequency of concurrent infections is of concern and there is a need for the recognition, and appropriate management of such infections.
Microbiology of vaginal discharge with emphasis on gardnerella vaginalis.
(1990) Kharsany, Ayesha Bibi Mahomed.; Van den Ende, Jan.; Moodley, Jagidesa.
The microbiological aetiology of vaginal discharge was studied in 208 women attending various outpatient clinics at King Edward VIII Hospital. Specimens from the lower genital tract were collected for microscopy and culture. Vaginal wet smear examination, amine liberation test and vaginal pH estimation were performed and assessed for their reliability for the rapid diagnosis of vaginal infections. Vaginal and endo-cervical infections were present in 163 (78,4%) women. Q. vaginalis (65,4%), I. vaginalis (37,9%), genital yeasts (37,0%), ~. hominis (59,6%), g. urealyticum (48,1%), anaerobic bacteria (32,6%), ~. gonorrhoeae (11,1%), f. trachomatis (22,1%) and Herpes simplex virus (0,9%) were detected. Of the 104 women in • whom vaginal infections were detected, bacterial vaginosis was present as the sole infection in 32 (22,2%), I. vaginalis in 35 (24,3%) and C. albicans in 23 (15,9%). Bacterial vaginosis occurred concurrently with I. vaginalis and f. albicans in 24 (16,5%) and 11 (7,5%) women respectively; whilst I. vaginalis and f. albicans occurred concurrently in 14 (9,7%) women. In 6 (4,1%) women all three infections were present. No vaginal or endo-cervical pathogens were detected in 45 (21,6%) women. Women with bacterial vaginosis were found to be significantly colonised with G. vaginalis, M, hominis, anaerobic bacteria and curved Gram-negative bacilli (p < 0,05). Vaginal wet smear microscopy detected T. vaginalis in 29% and "clue" cells in 41,3% of smears. The presence of "clue" cells (91,8%) and a positive amine test (76,7%) was significantly associated with bacterial vaginosis. Although a raised vaginal pH was also significantly associated with bacterial vaginosis, this test was less specific (65,2%) than "clue" cells (85,9%) and the amine test (95,5%). The vaginal Gram stain, as performed in this study, was found to be unreliable for the detection of "clue" cells. G. vaginalis biotypes 1 and 5 were significantly associated with bacterial vaginosis, however the serotyping scheme did not distinguish between strains isolated from women with and without bacterial vaginosis. The antimicrobial susceptibility pattern of 93 strains of G. vaginalis was not typical of either Gram-positive or Gram-negative bacteria. Serological tests revealed reactive syphilis serology in 47 (22,6%) and the presence of hepatitis B surface antigen in 16 (7,7%) women. Antibody to human immunodeficiency virus was detected in 4 (1,9%) women attending the colposcopy clinic. This study clearly demonstrates the high prevalence of vaginal and/or endo-cervical infections in women locally, the majority of whom were asymptomatic. The high frequency of concurrent infections is of concern and there is a need for the recognition, and appropriate management of such infections.
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.
Evaluation of the MTT and MABA assays for rapid screening of the in vitro activity of synthetic chalcones against Mycobacterium tuberculosis.
(2014) Moodley, Suventha.; Pillay, Manormoney.
Background: The chalcone scaffold (1,3-diaryl-2-propen-i-ones) has the advantage of easy chemical modification and has been shown to possess biological activity against a variety of organisms, including a wide range of anti-TB activity. The focus of this study was 2-fold: firstly, to compare the performance of the colorimetric MTT and MABA assays for screening synthetic chalcones, and secondly, to evaluate the activity of fluorinated and non-fluorinated chalcones against drug susceptible and resistant clinical strains of M. tuberculosis. Materials and methods: Twenty seven chalcones and chromenochalcones were screened against the laboratory strain M. tuberculosis H37Rv, using a microtitre plate MTT assay at 7 days. The MIC for 20 active compounds was subsequently determined using the MABA, MTT and the macroscopic broth assays at 7, 14 and 21 days, extracellular activity against clinical isolates of varying drug susceptibility patterns and genotypes using the MTT assay, intracellular activity in a macrophage model and eukaryotic cytotoxicity using Vero cells. Results and discussion: No significant difference in the MICs, or increase in the MICs was observed over time between the MABA (p = 0.209) and the MTT (p = 0.207) assays, in contrast to the gold standard, the macroscopic broth assay (p = 0.000). Fluorinated and non-fluorinated chalcones displayed moderate activity (32- 128 μg/mL) against MDR- and XDR-TB isolates, no significant activity against intracellular H37Rv and low selectivity for M. tuberculosis. The elevated MICs and lack of intracellular activity may be explained by the precipitation of the compounds indicating low solubility, with the exception of IV and XVI. Conclusions: The MTT assay is a more cost effective drug susceptibility testing method than the MABA assay for the rapid in vitro screening of the activity of chalcones against M. tuberculosis. Compound XIX and XI have the most potential for reformulation to improve their biological activity to yield a more potent drug candidate.
Interaction of HIV and syphilis at the keratinocyte level.
(2014) Moeketsi, Relebohile.; Sturm, Adriaan Willem.
Introduction Syphilis is a sexually transmitted disease, and once acquired, progresses through a series of overlapping stages. Data from a series of surveillance studies indicate that the acquisition of syphilis may be altered by co-infection with HIV, suggesting that HIV infection decreases the chances for infection with Treponema pallidum at the muco-cutaneous level. This study, through the inoculation of keratinocytes with HIV and/ or T.pallidum, investigated the effect of HIV infection of keratinocytes, on the transmigration abilities of T.pallidum across a keratinocyte monolayer. Methods Using magnetically labelled antibodies specific for antigens in the viral envelope, infectious HIV virions were isolated from blood. T.pallidum was harvested from the testes of rabbits in which the organism was propagated. HaCaT cells were cultured on collagen-coated transwell inserts, in 24-well tissue culture plates. Upon confluency, cells in one experiment were inoculated first with HIV, and three days later with T.pallidum; cells in a second experiment were exposed to T.pallidum first, and three days later inoculated with HIV; and cells in the third experiment were exposed to both HIV and T.pallidum at the same time. The media below the inserts, which contained the treponemes that passed through, was harvested at vii different time points (24, 48, and 72 hours). For experiments one and two, post-inoculation time points only took effect after inoculation with the second organism. DNA was extracted using Probetec lysis buffer and quantitation was done by real-time PCR. Results The number of treponemes that passed through prior HIV and T.pallidum infected monolayers indicated little difference between the two culture conditions. The treponeme numbers indicated an initial drastic decline, followed by a remarkable increase between 24 and 48 hours and a plateauing at 72 hours. However, transmigration through T.pallidum and HIV exposed keratinocytes (experiment three), displayed a slight initial decline followed by a drastic continuous increase in quantity till 48 hours post-infection, reaching significantly higher levels, compared with experiment 1 and 2. Conclusion The results suggest that at the time HIV enters the keratinocytes, changes in the cell membrane structure occur thus allowing for better adhesion and intake of T.pallidum; therefore a higher transmigration rate. This observation may explain both a decrease in primary syphilis in HIV endemic areas as well as the reported rapid progression to secondary syphilis in patients with concurrent HIV infection. patients with concurrent HIV infection.
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.
Perceived ethionamide resistance in isoniazid susceptible isolates of mycobacterium tuberculosis.
(2015) Msibi, Zama Princess N.; Sturm, Adriaan Willem.
In Mycobacterium tuberculosis, resistance to ethionamide (ETH) is usually combined with isoniazid (INH) resistance due to a number of mutations in genes that are involved in the biosynthesis of mycolic acids. ETH resistance in INH susceptible isolates is rare. Ten such isolates were identified from patients participating in other studies. Genotyping by means of IS6110 was performed to compare the relatedness of these isolates to each other. In attempts to identify the molecular basis for the resistance to ETH, the ethA, mshA and mshC genes were amplified and the amplicons sequenced using an ABI 3730 DNA Analyser. INH and ETH minimum inhibitory concentrations (MICs) were determined alone and in combination by means of checkerboard titrations in Middlebrook 7H9 broth, using the 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and microplate alamarblue assays (MABA) for detection of growth. Seven isolates were not related to each other and their INH susceptibility was confirmed. No mutations were observed in all the sequenced genes. One out of seven isolates was found to be co-resistant to INH nad ETH. The MIC for the remaining isolates was 1μg/ml for ETH. MABA revealed a paradoxical susceptibility of the isolates to ETH, where mycobacterial growth was observed in ETH concentrations higher than the MIC for the six isolates. For combination of the two drugs, MABA revealed an antagonism between INH and ETH, where the isolates grew in high ETH concentrations regardless of the concentration of INH. The paradoxical effect of ETH and antagonism between ETH and INH in our isolates does not result from mutations in ethA, mshA or mshC.
Mutation frequency in drug-susceptible clinical isolates of Mycobacterium tuberculosis under aerobic and anaerobic conditions.
(2015) Joseph, Lavania.; Sturm, Adriaan Willem.
Abstract not available.
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.
Microfluidic technologies for capturing and concentrating human immunodeficiency virus-1 (HIV-1) particles.
(2016) McArthur, Chanelle Crystal.; Balagaddé, Frederick.
HIV-1 RNA assays are routinely used in developed countries to monitor the effectiveness of antiretroviral therapy (ART). These assays require well-trained operators, expensive equipment and reagents, and established laboratory infrastructure. These requirements limit their usefulness in resource-limited settings where people are most afflicted by the HIV-1 epidemic. Recent advances in microfluidics and nanotechnology offer new approaches for rapid, low-cost, robust and simple HIV-1 viral load monitoring systems. Here we describe an approach within a microfluidic device to directly detect HIV-1 virus particles using an immune sandwich assay that includes anti-gp120 antibodies -conjugated to polystyrene microspheres and fluorescently labelled goat anti-HIV-1 FITC detection antibodies. The anti-gp120 antibody-conjugated microspheres were employed to capture and concentrate HIV-1 particles, whereas the FITC detection antibodies were used to generate fluorescent signal that represented the number of captured viruses. In the presence of HIV-1 particles, addition of microspheres and FITC detection antibody led to the formation of a microsphere/HIV-1 particle/FITC detection antibody complex. This complex was measured by analysing the fluorescence intensity produced by the FITC detection antibody bound to the HIV-1 particle within the complex. We demonstrated the utility of an in-house microfluidic device and assay in detecting 1x106 virus particles/μl with a significance of (p≤0.01). This assay was completed within 3.8 hours, without any pre- or post- treatment of reagents.
Investigating the effect of sex hormones on the immune response to TB and HIV.
(2015) Mabhula, Amanda Nomakhosi.; Leslie, Alasdair.
Background: Global incidence rates for tuberculosis (TB) indicate a gender-bias to the disease, with almost twice as many men actively infected than women (male/female ratio of 1.9). Sex hormones are known to regulate immune function and their role in tuberculosis has been suggested experimentally in animal models. However the role of sex hormones in human TB infection and whether there is any synergistic effect in HIV and TB co-infection is unknown. In addition, there is data to suggest that manipulation of sex hormones with progestin-based injectable contraceptives, particularly DMPA, increases susceptibility to both HIV and TB. Quantitation of sex hormones has relied on antibody based immunoassays, such as ELISA, which suffer both from a lack of specificity and sensitivity. Aims: Consequently, the purpose of this study was to develop a method for quantitation of sex hormones and their contraceptive analogues utilizing a targeted LC-MS/MS approach that has been demonstrated to increase the accuracy of hormone quantitation and to determine the effect of sex hormones and hormonal contraceptives on the immune response to TB and HIV. Method: Sex hormone levels were measured in plasma samples from three separate cohorts, using MRM run in positive ESI mode, on a ABSciEx Q-TRAP 5500 mass spectrometer. The method was validated and DMPA levels were measured in the FRESH cohort (n= 62) and CAPRISA CAP004 study (n= 38) to determine effects on DMPA on increased risk in HIV acquisition. Testosterone, progesterone and DMPA levels were measured in the HIV chronic patients from the Cryptococcal cohort (n = 271) and sex hormone associated changes in phenotypic expression and immunological responses using intracellular cytokine staining were determined by flow cytometry analysis. Results: We validated our assays and were able to identify and quantitate levels of DMPA in two blinded studies. In the FRESH cohort, we were able to correctly identify and quantitate DMPA levels in Depo-Provera users and injectable progestin-based (IPC) contraceptive use was associated with high risk of HIV acquisition (p = 0.0142). IPC users were found to have significant increase in CCR5+CD4+ T cells in the cervix as well as increased CCR5 expression. Preliminary data in the CAP004 study, showed differential expression of mucosal proteins in the cervicovaginal lavage associated with DMPA use. In the Cryptococcal study, we found, as expected, significant differences in testosterone and progesterone levels between male and female patients, p <0.0001 and p=0.0001 respectively. Given that only 3 female patients reported to be using the contraceptive DMPA, we identified 42 of the total number of 172 females to have significant levels of DMPA (greater than LOQ = 0.064ng/ml) and these females had significantly lower progesterone levels than females not using DMPA (p <0.0001). This large under-reporting of contraceptive use indicates the value in direct measurement as opposed to self-reporting. As expected negative correlation was observed between progesterone and DMPA levels (p = 0.0016, r = -0.2445). However, individual response profiles are highly variable and decay rates of DMPA in longitudinal samples vary greatly between individuals. We hypothesis this will impact the immunomodulatory effect of DMPA, again suggesting the need for direct measurement. In this small sample we find no significant differences in activation and exhaustion of CD4 and CD8 T cells (determined using HLA-DR, CD38 and PD1 expression), as well as T regulatory cells (FoxP3 expression) when comparing female patients with high progesterone, low progesterone, and injectable contraceptive DMPA users, as well as males with high testosterone and low testosterone levels. However, females with high progesterone levels generally had higher CD38 expression, though non-significant. Also, we observe no significant differences in cytokine expression (TNFα, IFNγ, and IL-2) as well as markers, CD107a and Mip-1β, upon stimulation with SEB, PPD, pp65 CMV and HIV peptides. Conclusion: We successfully optimized and validated a method for quantitation of sex hormones using LC-MS/MS and were able to detect and quantify levels of testosterone, progesterone and DMPA. This method has the potential in clinical studies, to eliminate the need to rely on self-reported information, as exogenous hormones or contraceptive analogues can be detected with high sensitivity and specificity. Changes in the female genital tract which may be associated with increased risk of HIV were found in injectable progestinbased contraceptive users, particularly DMPA. However, no significant immunological effects of hormone levels on immune response and phenotype expression were found in blood.
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.
Technologies for a user-friendly microfluidic system for portable applications.
(2015) Kunota, Tafara Takunda Remigio.; Balagaddé, Frederick.
In the same way that the HIV virus subdues the human immune system, the HIV/AIDS epidemic has severely overloaded the health service infrastructure in resource limited countries and threatens to systematically suppress societies’ capacity to cope with killer diseases. The epidemic has also directly impacted the health workforce, causing absenteeism, attrition (due to illness and death), and increased demand for provider time and skills. Advanced and miniaturized microfluidic systems can perform complex biotechnological functions such as growing bacteria, sequencing DNA and identifying disease causing pathogens. As a technology, microfluidics offers so many advantages but it also suffers from a variety of technological drawbacks that limit its wide spread practical application in hospitals and patient setting. Microfluidic systems require a lot of time (6 hours to an entire work-day) to set up and the set-up process requires the meticulous attention of highly trained personnel. We proposed the development of an automated, time conservative and user-friendly fluid-transport system (off-chip to on-chip) for Microfluidic Large Scale Integration platform based microfluidic devices. Using multilayer soft-lithography, micro-electric actuators and a LabVIEW graphical user-interface, a user-friendly automated microfluidic fluid transport system was developed. In comparison to the conventional manual loading system, the developed system can save at least 60% of the total chip preparation time required during the off-chip to on-chip fluid loading process. This system can be extended and made compatible with other devices that require complex off-chip to on-chip loading processes in microfluidic large scale integration platform based systems.
Microfluidic technologies for genomic interrogation of mycobacterium tuberculosis clinical isolates using the polymerase chain reaction (PCR) and high resolution melting analysis (HRMA).
(2015) Mandizvo, Tawanda.; Balagaddé, Frederick.
Background: A number of Mycobacterium tuberculosis (Mtb) genes have been shown to be under positive selection pressure in the presence of anti-TB therapy. This results in the selection of drug resistant phenotypes associated with genetic changes—which can be point mutations, deletions and/or insertions. Some mutations from multiple genes have been documented to be associated with reduced susceptibility to anti-TB drugs such as rifampicin, ethambutol, carpreomycin and fluoroquinolones. The list is continuously updated as new mutations are discovered and validated. In principle therefore, there is an urgent need to design robust molecular diagnostics and more efficacious therapeutic strategies that are able to indicate diverse genetic mechanisms behind drug resistance in individual isolates Materials and Methods: We used the LightForge system we developed at K-RITH. This LightForge system is a fluorescence detection based, highly scalable microfluidic platform. It interrogates Mycobacterium tuberculosis strains using Real-Time PCR and High Resolution Melt Analysis (HRMA) on a chip. Results and Discussion: We have used this LightForge system to identify clinical Mtb strains resistant to rifampicin—a frontline drug used to treat tuberculosis, relative to a susceptible strain H37RV, based on mutations in the rpoB gene. This system has the potential to contribute towards a low-cost solution to diagnosis of multidrug resistant tuberculosis—a current critical global healthcare challenge. The interrogation of clinical Mtb isolates—including R35, KZN 605 and Tkk 01-062—using the LightForge system has detected mutations linked to rifampicin resistance including single nucleotide polymorphisms (SNPs) in a congruous manner with commercial systems. Conclusions: In preparation for diagnosis of clinical samples, this LightForge approach is now being expanded to incorporate detection of genetic markers linked with resistance to other TB drugs that include fluoroquinolones and isoniazid based on mutations in gyrA, katG and Mab-inhA regions of the Mtb genome. The scalability of LightForge can also be harnessed to conduct digital PCR (dPCR), a critical tool for detecting genetic heterogeneity in Mtb.
Light forge : a microfluidic high throughput platform for rapid and affordable detection of drug resistant strains of tuberculosis.
(2015) Mbano, Ian Maheti.; Balagaddé, Frederick.
Tuberculosis is one of the most deadly infectious diseases currently plaguing the global community. Unfortunately, lack of accessible, reliable and affordable diagnostic tools in the high disease burden, and resource poor regions such as Sub-Saharan Africa has hampered efforts to eradicate the epidemic. This study documents the development of a microfluidic platform called Light Forge, which is capable of detecting genetic drug resistance signatures in M.tuberculosis DNA. The first phase of this study involved a molecular drug susceptibility assay on 7 strains of M.tuberculosis using the high resolution melt analysis at the rpoB, katG, mab-inhA and gyrA loci with the Light Cycler96 . These findings compared with phenotypic drug susceptibility testing and Sanger sequencing. The results from the preliminary tests showed that the commercial system could detect positive strains at sensitivity estimates of 86%, 17% , 0% and 100% for rpoB, katG, mab-inhA and gyrA respectively. Detection of non-synonymous mutation in gyrA region for all test strains halted further testing. The rpoB gene was selected for on chip profiling with the Light Forge system due to the higher sensitivity. The results from the Light Forge showed that the system was capable of detecting test strains with 100% sensitivity, with modest reproducibility and correspondence with the phenotypic drug susceptibility profiles and the sequencing results. A microfluidic TB assay based on the Light Forge system is on the horizon based on the findings of the study. However, more work is required to incorporate other genes and ultimately design the best-equipped device for the clinical setting.
Identification of M. tuberculosis pncA gene single nucleotide polymorphisms conferring resistance to pyrazinamide.
(2016) Maharaj, Kashmeel.
Abstract available in PDF file.
The isolation of antibiotic survivors in mycobacterium tuberculosis using fluorescence activated cell sorting (FACS).
(2016) Shumba, Patience.; Pym, Alexander S.
Abstract available in PDF file.
The role of mycobacterium tuberculosis pili in pathogenesis : growth and survival kinetics, gene regulation and host immune response, and in vitro growth kinetics.
(2016) Nyawo, Georgina Rumbidzai.; Pillay, Manormoney.
Abstract available in PDF file.

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