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.
A pharmacokinetic study of rifabutin and its interaction with antiretrovirals in African patients with TB-HIV co-infection.
(2012) Naiker, Suhashni.; Pym, Alexander S.; McIlleron, Helen.
The management of HIV-associated tuberculosis (TB) is complicated by the pharmacokinetic interactions between rifampicin (RMP) and co-administered protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors. Rifabutin (RBT) is an alternative rifamycin, preferred in patients requiring PIs. Recent studies suggest the current recommended dose of RBT in combination with boosted lopinavir (LPV/r) is suboptimal and there are insufficient pharmacokinetic data evaluating the interaction between RBT coadministered with efavirenz (EFV) and nevirapine (NVP). Pharmacogenomic studies have shown that RMP concentrations are lower in patients from sub-Saharan Africa with polymorphisms of the SLCO1B1gene but there is currently no data on the pharmacogenetic determinants of RBT exposure. The pharmacokinetics of RBT were evaluated at two different doses in HIV co-infected patients before and after the introduction of LPV/r, EFV and NVPbased antiretroviral therapy (ART). After six weeks of standard TB therapy, RBT 300 mg daily was started for four weeks. Thereafter patients were randomized to receive either RBT 150 mg daily or RBT 150 mg three times a week (TPW) with LPV/r, RBT 300mg or 450mg with NVP or RBT- 450mg or 600mg with efavirenz. After four weeks on the first RBT dose, patients switched to the alternate dose and continued until the end of TB treatment. Serial RBT and 25-O-desacetylrifabutin (dRBT) concentrations were measured during a dose interval before patients switched RBT doses. The median AUC0-24 and Cmax, of RBT in patients taking 150mg RBT TPW was significantly reduced when compared to the other treatment arms. 86% of patients whilst on this intermittent RBT arm had an AUC0-24 < 4.5 μg.h/mL, level that has been associated with acquired rifamycin resistance. Rifabutin exposure was maintained within the range of AUCs that have been shown to prevent acquired rifamycin resistance (ARR) with 150mg daily dosing in combination with LPV/r. In addition, the combination of RBT with NVP 300mg resulted in significantly increased exposure of RBT, with significantly higher exposure observed with 600mg RBT. However, the combination of RBT 450mg with EFV resulted in RBT exposure lower than 300mg RBT given alone in the same patients, whereas RBT 600mg plus NVP results in bioavailability of RBT equivalent to 300mg given alone. Rifabutin was well tolerated at all doses. Only three grade 4 laboratory toxicities, elevated transaminases, neutropenia, and uveitis, possibly related to RBT were reported in patients taking NVP. SLCO1B1 rs4149032 C>T polymorphism occurs frequently in African patients in Durban and may be associated with low RBT bioavailability. These findings support recommendations for the higher dose of RBT in combination with LPV and EFV but not with NVP.
Development of novel reagents for tuberculosis detection.
(2013) Ngubane, Nqobile Angel Cebile.; Pym, Alexander S.; Khati, Makobetsa.; Rubin, Eric.
Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics.
Host induced microevolution of ESX secretion systems of M. Tuberculosis.
(2013) Sukkhu, Melisha.; Pym, Alexander S.
The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines.
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.
The role of APOBEC3G in acute and early HIV-1 subtype C infection.
(2014) Reddy, Kavidha.; Winkler, Cheryl Ann.
Introduction APOBEC3G and other related cellular cytosine deaminase family members have potent antiviral activity. In the absence of HIV-1 Vif, APOBEC3G mutates the viral DNA during viral reverse transcription. Our knowledge of the Vif-APOBEC3G interaction in human populations infected with subtype C HIV-1 is limited. Investigation of interactions between HIV and its host is crucial as it can ultimately be exploited in vaccine and therapy design. We hypothesised that certain APOBEC3G haplotypes and/or their expression in peripheral blood mononuclear cells of seroconverters affect viral setpoint and CD4+ T cell counts. We also hypothesised that certain APOBEC3G genetic variants are associated with increased frequency of G to A hypermutations during primary HIV-1 infection and that Vif variability influences disease progression and its ability to neutralise APOBEC3G haplotypes. Methods Our South African study cohort consisted of females at high risk for HIV-1 infection and women with known recent HIV-1 infection. We used quantitative real-time PCR to measure APOBEC3G expression in HIV- and HIV+ samples during primary infection. APOBEC3G variants were identified by DNA sequencing and TaqMan Genotyping. The HIV-1env gene was sequenced to assess Env diversity and the extent of APOBEC3G induced hypermutations. Vif variability was assessed by plasma derived clonal Vif sequences (n= 10-20 per patient) and Vif function was assessed by APOBEC3G degradation assays and HIV-1 infectivity assays. Results We found no correlation between APOBEC3G expression levels and plasma viral loads (r=0.053, p=0.596) or CD4+ T cell counts (r=0.030, p=0.762) in 32 seroconverters. However, APOBEC3G expression levels were significantly higher in HIV- individuals compared to HIV+ individuals (p<0.0001) including matched pre- and post-infection samples from the same individuals (n=13, p<0.0001). Twenty five single nucleotide polymorphisms (SNPs) were identified within the APOBEC3G region. SNP 186R/R was associated with significantly higher viral loads (p=0.0097) and decreased CD4+ T cell levels (p=0.0081), indicating that 186R/R has a negative effect on HIV restriction. Overall HIV-1 env sequences contained a higher number of APOBEC3F compared to APOBEC3G-induced hypermutations and the number of APOBEC3F-induced hypermutations correlated negatively with viral load (r= -0.6, p=0.006) and positively with CD4 T cell counts (r=0.6, p=0.004). We cloned and sequenced a total of 392 subtype C Vifs, which showed an interpatient diversity of 6.2% to 19.2% at the amino acid level. Interestingly, Vif sequence comparison showed a strong preference for a Lysine or a Serine at position 36 for APOBEC3G 186R/R and APOBEC3G 186H/H individuals, respectively. Selected natural subtype C Vif alleles had greater ability to counteract wild type APOBEC3G 186H as compared to the APOBEC3G 186R variant as shown by both functional and HIV infectivity assays. Conclusions In conclusion, APOBEC3G expression in peripheral blood mononuclear cells does not correlate with viral loads or CD4+ T cell counts during primary HIV-1 subtype C infection. However, genetic variants of APOBEC3G may affect HIV-1 pathogenesis. Amino acid changes in Vif may influence its anti-APOBEC3 activity. HIV-1 subtype C Vifs may have adapted to counteract the more active wild type APOBEC3G as compared to the less active APOBEC3G 186R variant. These studies have improved our understanding of viral-host interactions in African populations and HIV-1 subtype C 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.
Susceptibility of Trichomonas vaginalis to metronidazole and other compounds.
(2015) Naidoo, Sarita.; Sturm, Adriaan Willem.
Trichomonas vaginalis is the most common sexually transmitted infection caused by a single known organism worldwide; and has been associated with an increased risk of HIV acquisition and transmission. Despite its high prevalence in South Africa, limited information is available on the extent of T. vaginalis metronidazole resistance and genotypic variation in this setting. We therefore tested the susceptibility of local T. vaginalis isolates against metronidazole and drugs prescribed in combination in the context of syndromic management of vaginal discharge syndrome. Susceptibility testing of 40 isolates demonstrated that metronidazole as well as some of the other drugs tested showed inhibiting effect on T. vaginalis. We recommend that these drugs be tested for synergistic effect with metronidazole. In a different set of 160 isolates the minimum inhibitory concentrations (MIC) of metronidazole ranged from 1.1 μg/ml to > 34.2 μg/ml (6.25 μM to > 200 μM) in the aerobic assay. Interpretation of these MICs differed based on the different resistance breakpoints applied. There was no correlation between MIC and treatment outcome in the subset of 56 patients that returned for follow-up. The expected association between MIC and clinical outcome was only observed in one of eight patients with unsatisfactory treatment outcome. This patient‘s isolate had the highest MIC. In the remaining seven patients with unsatisfactory treatment outcome, no relation with the susceptibility test result was found. A possible reason for the poor correlation may be inadequate concentration of metronidazole at the site of infection. In view of this, we assessed a self-administered and collected vaginal tampon specimen for the investigation of metronidazole concentration in the vagina of five healthy volunteers, using high performance liquid chromatography (HPLC). Maximum values of metronidazole concentrations detected in both serum and vaginal fluid were obtained at two hours following oral administration of 2 g of the drug. This method can be applied in future clinical studies to correlate treatment outcome and MICs with metronidazole concentration at the site of infection. This may lead to the development of susceptibility assays and interpretation criteria that are better able to predict treatment outcome than the current methods. Another reason for the poor correlation between treatment outcome and in vitro resistance may be early reinfection. We used PCR-RFLP, targeting a 650-bp repeat region in the T. vaginalis genome, to genotype T. vaginalis isolates. Four genotypes were found in 100 T. vaginalis isolates using this method. Both the vaginal secretion of metronidazole and the strain typing methodology needs to be further investigated before a comprehensive study as outlined above can be executed.
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.
Immunopathogenesis of vulvo-vaginal candidiasis in human immunodeficiency virus infected women.
(2014) Apalata, Teke.; Moodley, Prashini.
Vulvovaginal candidiasis (VVC) is an important cause of lower genital tract infections in women. There are currently numerous clinical observations linking increased cases of symptomatic VVC to the progression of HIV epidemic. While the pathogenesis of other commonly encountered mucosal candidiasis (oral and oesophageal) in the context of HIV infection has been well studied, gaps in our knowledge remain regarding candida vaginitis. With increasing degree of immunosuppression, symptomatic VVC in HIV infected women is frequent, severe, recurrent and less responsive to conventional anti-fungal therapy. The quality of life is greatly diminished for women who experience recurrent episodes of symptomatic VVC. Furthermore, the high prevalence of HIV infected women adds to the burden of healthcare. In this study, we sought to further understand the pathogenesis of symptomatic VVC and the associated host defense mechanisms in HIV infected women. The results of this study are presented as a collection of 5 papers, 4 of which are published in peer reviewed journals, 1 is still under review. The initial chapter of this thesis, ‘Introduction and Background’, is followed by the 5 manuscripts which are grouped into 3 consecutive result chapters as follows: I. Impact of HIV on symptomatic VVC. II. Impact of symptomatic VVC on HIV RNA levels in plasma and genital secretions. III. Plasma and vaginal-associated immune responses in women with symptomatic VVC. The final chapter, ‘Discussion and Conclusions’, rounds up the thesis.
The impact of sexually transmitted infections (STI) and genital tract inflammation on HIV-1 acquisition and rate of disease progression in subtype C infected women.
(2014) Mlisana, Koleka Patience.; Kharsany, Ayesha Bibi Mahomed.; Passmore, Jo-Ann Shelley.
Introduction: Women carry more than half the burden of HIV disease globally and this burden is even higher in sub-Saharan Africa (SSA). Young women, in particular, are at disproportionate risk of HIV infection in South Africa. Understanding risk behaviours and factors associated with ability to negotiate safe sex and condom use is one of the key elements in curbing the spread of HIV. Sexually transmitted infections (STI) and bacterial vaginosis (BV), which cause female genital tract inflammation, have been identified as key drivers of the HIV epidemic. This inflammation, which is also present in the absence of symptoms, is associated with increased susceptibility to HIV infection. Although syndromic management of symptomatic STIs or BV at the first encounter with a health care provider is an important public health measure, its effectiveness is minimised because a substantial proportion of individuals have either asymptomatic infections or fail to recognise signs and symptoms of STI and are therefore excluded. Most new HIV infections in SSA occur among young people and particularly among young girls. Prompt diagnosis of acute HIV infection (AHI) is critical and benefits the individual as well as providing opportunities for public health intervention. In South Africa, the majority of HIV infections are due to infection with HIV-1 subtype C for which there is limited data compared to subtype-B HIV-1 infections. The overall aim of this study was to assess the impact of BV, STIs, and associated genital tract inflammation on acquisition of HIV-1 subtype C infections; and evaluate the rate of subsequent disease progression in women. The objectives were: i. to investigate STIs and genital tract inflammation as risk factors for HIV infection in high risk women and adequacy of syndromic management; ii. to evaluate the challenges associated with diagnosing recently acquired (acute) HIV infection in a subtype C prevalent population; iii. and to evaluate the relationship between clinical disease progression and genital and or systemic inflammation in high-risk women who became infected with HIV. We assessed the adequacy of syndromic diagnosis of STIs, compared with laboratory diagnosis of STIs, and evaluated the association between STI diagnosis and the risk of HIV acquisition in a cohort of high-risk women. Genital cytokine profiles and the degree of inflammation associated with common STIs and bacterial vaginosis were assessed. The most common signs and symptoms of acute HIV infection (AHI) were described and a clinical algorithm to identify acute HIV cases was developed. We investigated rates of HIV disease progression of subtype C–infected South African women. Methods: The CAPRISA 002 study was a prospective cohort study established to examine the pathogenesis and natural history of HIV-1 subtype C infection and to describe the immunologic, virologic and clinical characteristics of acute and early infection in KwaZulu Natal, South Africa. A total of 775 high-risk women were screened for HIV infection, and 245 HIV-uninfected women were enrolled into the study. At each monthly visit for a total of 24 months behavioural and clinical data were collected. Cervico-vaginal lavage (CVL) samples were collected at enrolment and at each six month follow-up visits and were tested for STI pathogens (including Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus type 2 (HSV-2) and Trichomonas vaginalis) and bacterial vaginosis. Forty-two cytokines were measured from the CVL and 13 from the plasma samples at enrolment. All women received monthly HIV-1 antibody and RNA testing and were assessed for AHI. Signs and symptoms at the visit with HIV-1 antibody or HIV-1 RNA positive test were compared to HIV negative visits. Logistic regression identified clinical predictors of AHI and a model-based score was assigned to each predictor to create a risk score for every woman. All women who seroconverted were followed up for more than five years and monitored for HIV disease progression. Rapid disease progression was defined as CD4+ T cell count decline to <350 cells/μl within two years post-infection. Serial clinical and laboratory assessments were compared using survival analysis and logistic regression models. CAPRISA had established several cohorts of HIV negative women to determine the feasibility of establishing cohorts and sites for HIV biomedical prevention trials. Women seroconverting in these studies were referred to the CAPRISA 002 study for longitudinal follow-up for HIV post seroconversion. Results: In this study, the HIV-1 prevalence at screening was 59.6% (95% CI: 55.9% to 62.8%). During a total of 390 person-years of follow-up, 28 new infections occurred, yielding a seroincidence rate of 7.2 (95% CI: 4.5 to 9.8) per 100 person-years. A total of 62 participants, including seroconvertors from other CAPRISA cohorts, were enrolled into the acute HIV infection Women from the HIV-1 negative cohort generally demonstrated a high level of knowledge on HIV/AIDS, and 60.3% reported use of condoms. Reported condom use at last sexual encounter varied slightly by partner type (57.0% with steady versus 64.4% with casual partners; p = 0.36), whilst self-perceived ability to choose to use a condom was significantly lower with steady partners compared to casual partners (20.8% versus 53.9%; p=0.01). An important finding was that vaginal discharge was a poor predictor of laboratory-diagnosed STIs, as only 12.3% of women (25/204) who had a laboratory-confirmed discharge causing pathogen had clinical evidence of a discharge (yielding a sensitivity of 12.3% and a specificity of 93.8%). CVL cytokine concentrations did not differ between women with asymptomatic or symptomatic STIs; and were elevated in women with either asymptomatic or symptomatic STIs compared to women with no STIs or BV. Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 of the cytokines measured in CVL being up-regulated compared with women with no infections, respectively. While BV was associated with elevated pro-inflammatory cytokine concentrations in CVL, women with BV had lower levels of chemokines and haematopoietic cytokines than women with no infections or BV. HSV-2 reactivation was inflammatory, but yielded a comparatively lower level of inflammation than Chlamydia or gonnorhoea. Trichomoniasis, despite being relatively common in this cohort, did not cause significant changes in genital tract cytokine concentrations compared to women with no infections or BV. Genital infections did not influence plasma cytokine concentrations, suggesting that the compartments were not linked with respect to cytokine responses. Although laboratorydiagnosed STIs were associated with increased risk of HIV infection [hazard ratio, 3.3 (95% confidence interval, 1.5 – 7.2)], clinical symptoms were not. Of the women who became infected, factors predictive of AHI included age, 25 years (OR = 3.2; 1.4 – 7.1), rash (OR = 6.1; 2.4 –15.4), sore throat (OR = 2.7; 1.0 – 7.6), weight loss (OR = 4.4; 1.5 – 13.4), genital ulcers (OR = 8.0; 1.6 – 39.5) and vaginal discharge (OR = 5.4; 1.6 – 18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p = 0.001). The 62 acutely infected women were identified at a median of 42 days post-infection (IQR = 34 – 59). Mean CD4 count dropped by 39.6% at 3 months and 46.7% at 6 months post-infection in women with pre-infection measurements. CD4 decline to <350 cells/μL occurred in 31%, 44%, and 55% of women at 1, 2, and 3 years post-infection, respectively, and to <500 cells/μL in 69%, 79%, and 81% at equivalent time-points. Predictors of rapid progression were CD4 count at 3 months post-infection (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.31–3.28; P = .002), setpoint viral load (HR, 3.82; 95% CI, 1.51–9.67; P = .005), and hepatitis B coinfection (HR, 4.54; 95% CI, 1.31–15.69; P = .017). Conversely, presence of any of HLAB*1302, B*27, B*57, B*5801, or B*8101 alleles predicted non–rapid progression (HR, 0.19; 95% CI, .05–.74; P = .016). Discussion/Conclusion: This study showed that syndromic STI diagnosis, which is dependent on clinical signs of vaginal discharge, was poorly predictive of laboratory-diagnosed STIs or BV and missed a significant proportion of women with asymptomatic infections. However, the level of genital inflammation, as measured by cytokine concentrations in CVL, was similar in women with symptomatic and asymptomatic infections and therefore place women at increased risk for HIV infection. While laboratory-diagnosed STIs and the presence of inflammatory cytokines in the genital tract were associated with increased susceptibility to HIV acquisition, vaginal discharge was not. Chlamydial infection was associated with the highest genital cytokine concentrations, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, targeted screening of populations at risk for STIs is critical and urgently needed. Recognition of signs and symptoms of AHI is important for early diagnosis of HIV infection. The proposed algorithm of risk-stratifying individuals for AHI provides a useful clinical tool especially in resource-limited settings where there are limited or no routinely available tests for AHI. However, validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care HIV antigen or viral load testing is needed, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission. This cohort showed high rates of rapid disease progression, with nearly half of these subtype C–infected women progressing to a CD4+ T cell count of below 350 cells/μL within 2 years of infection. Implementing 2013 World Health Organization treatment guidelines (CD4+ T cell counts less than 500cells/μL) would require most individuals to start antiretroviral therapy within 1 year of HIV infection. The economic and health systems planning implicated by these findings need to be explored and addressed to guide policy makers as countries adopt the current WHO guidelines.
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.

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