In Vitro Effect of 5-Nitroimidazole Drugs against Trichomonas vaginalis Clinical Isolates.

Infections with the sexually transmitted parasite Trichomonas vaginalis are normally treated with metronidazole, but cure rates are suboptimal and recurrence rates following treatment are high. Therefore, our objective was to assess the in vitro antitrichomonas activities of three other 5-nitroimidazole drugs and compare them with metronidazole. T. vaginalis isolates (n = 94) isolated from South African women presenting with vaginal discharge syndrome at two sexually transmitted disease clinics in KwaZulu-Natal were grown from frozen stock. Twofold serial dilutions (16 to 0.25 mg/L) of metronidazole, tinidazole, ornidazole, and secnidazole were prepared in Diamond's broth. The MICs were read after 48 h of anaerobic incubation at 37°C. An MIC of <2 mg/L was defined as susceptible, an MIC of 2 mg/L was defined as intermediate, and an MIC of >2 mg/L was defined as resistant. Sixty-one percent (57/94) of the T. vaginalis isolates were susceptible to metronidazole, 80% (75/94) were susceptible to tinidazole, 75% (71/94) were susceptible to secnidazole, and 89% (84/94) were susceptible to ornidazole. Resistance levels were 11%, 2%, and 1% for metronidazole, tinidazole, and secnidazole, respectively, while no resistance was observed for ornidazole. Intermediate scores were 28% for metronidazole, 18% for tinidazole, 24% for secnidazole, and 11% for ornidazole. Isolates from a proportion of women with bacterial vaginosis (BV) had higher MICs, and no isolates from women coinfected with another sexually transmitted infectious organism were resistant to any of the antimicrobials tested. This study showed that among T. vaginalis isolates in KwaZulu-Natal, there is no in vitro resistance to ornidazole. Of the 5-nitroimidazoles, metronidazole showed the highest level of resistance. The very low levels of resistance for the other three antimicrobials indicate that all three are viable options as a replacement for metronidazole if these in vitro findings are found to correlate with clinical outcomes. IMPORTANCE Trichomonas vaginalis is the most common nonviral sexually transmitted infection associated with reproductive sequelae and HIV acquisition risk worldwide. Despite its role in reproductive health, a high prevalence in South Africa, and the reported metronidazole resistance worldwide, no alternative regimens have been tested against T. vaginalis in our setting. This study compared the susceptibility patterns of three other 5-nitroiminazoles (secnidazole, tinidazole, and ornidazole), which are active against T. vaginalis with metronidazole in vitro. Metronidazole, the drug of choice for the treatment of trichomoniasis, showed the highest level of resistance, while the three regimens showed very low levels of resistance. These data indicate that all three are viable options as a replacement for metronidazole if these in vitro findings are found to correlate with clinical outcomes.

Stability of a monovalent rotavirus vaccine after exposure to different temperatures observed in KwaZulu-Natal, South Africa.

BACKGROUND: Rotavirus infection and its associated hospitalization of children less than 5 years old in middle- and low-income countries remains a public health challenge. We hypothesized that the Rotarix®potency is affected by non-optimal temperatures which translates into reduced vaccine effectiveness in these settings. OBJECTIVE: To assess the effect of non-optimal temperatures on the potency of the Rotarix® vaccine in South Africa. METHODS: Rotarix® vaccine was exposed to temperatures reflecting breaches in the cold chain. Vero cells (ATCC CCL-81) grown in a 24-well tissue culture plates were infected with Rotarix® vaccine viruses after exposure to non-optimal temperatures and the potency of the vaccine was determined using the plaque assay. RESULTS: Exposure of the Rotarix® vaccine to seasonal temperatures in KwaZulu-Natal for 6 hours and to extreme temperatures of 40oC for 72 hours as well as to -20°C and -80°C for 12 hours did not affect the potency of the vaccine beyond its expected standard of >7 x 105 PFU/ml. CONCLUSION: This study revealed that the Rotarix® vaccine remains potent even after exposure to non-optimal temperatures. However, this study only explored the effect of a constant 'adverse' temperature on vaccine potency and not the effect of temperature fluctuations.

Resistance Profile of Neisseria gonorrhoeae in KwaZulu-Natal, South Africa Questioning the Effect of the Currently Advocated Dual Therapy.

BACKGROUND: We report on the antimicrobial resistance profile of Neisseria gonorrhoeae isolates and the distribution of tetM genes in isolates with high-level tetracycline resistance in KwaZulu-Natal, South Africa. METHODS: Male and female patients presenting with urethral and/or vaginal discharge were recruited into the study. Urethral and cervical secretions were cultured on New York City agar. Confirmatory tests for N. gonorrhoeae included Gram stain, catalase, oxidase, and carbohydrate utilization tests. Beta-lactamase was tested by means of the chromogenic cephalosporin test. Minimum inhibitory concentrations were determined using agar dilution with multipoint inoculation. Polymerase chain reaction with gel electrophoresis was used to detect the presence and type of the tetM gene. RESULTS: N. gonorrhoeae was isolated from the specimens of 319 (26%) of the 1220 recruited patients. Of these 319 isolates, 71% were resistant to 3 or more drugs. Resistance to azithromycin was found in 68% of the isolates. All isolates showed high-level tetracycline resistance with minimum inhibitory concentration values of 16 and 32 mg/mL. The tetM gene was present in 293 (92%). The American type was found in 264 (90%) and the Dutch type in 29 (10%). Twenty-six (8%) did not carry a tetM gene. CONCLUSIONS: The current syndromic management with dual ceftriaxone and azithromycin is due to the high level of azithromycin resistance factually single-drug therapy. High-level tetracycline resistance based on a resistance mechanism other than ribosome protection by the tetM gene product is present in N. gonorrhoeae infecting South African patients.

An evaluation of infection control in private and public sector intensive care units in South Africa.

BACKGROUND: Appropriate infection control policies and practices are key to reducing the risk of healthcare-associated infections in patients in intensive care units (ICUs). OBJECTIVE: To evaluate infection control in ICUs using the Infection Control Assessment Tool (ICAT). METHODS: Six public and five private adult ICUs were included. Seven modules from the ICAT were administered including ICU, hand hygiene, and isolation and standard precautions. Modules were scored on a quantitative scale as per the tool guidelines and trained independent nurses observed practices. RESULTS: All ICUs reported to have a 1:1 nurse-to-patient ratio. One public ICU did not have the required 1:2 hand wash basin-to-bed ratio. We observed 100% adherence to handwashing or alcohol rub at each of the five moments of hand hygiene; however, the correct amount of alcohol rub was used in only 2% (n = 2) of the 117 observations. The median score for isolation and standard precautions was 79%. DISCUSSION: There was good infection control practice in ICUs. However, ICUs did not have isolation policies for all the infections listed in the ICAT and did not screen visitors to the ICU. We identified shortcomings in the ICAT and a more suitable tool is required for our healthcare setting.

Challenges with Surveillance of Healthcare-Associated Infections in Intensive Care Units in South Africa.

BACKGROUND: The incidence of healthcare-associated infections (HAIs) in the public health sector in South Africa is not known due to the lack of a surveillance system. We report on the challenges experienced in the implementation of a surveillance system for HAIs in intensive care units (ICUs). METHODS: A passive, paper-based surveillance system was piloted in eight ICUs to measure the incidence of ventilator-associated pneumonia, catheter-associated urinary tract infection, and central line-associated bloodstream infection. Extensive consultation with the ICU clinical and nursing managers informed the development of the surveillance system. The Plan-Do-Study-Act method was utilized to guide the implementation of the surveillance. RESULTS: The intended outputs of the surveillance system were not fully realized due to incomplete data. The organizational culture did not promote the collection of surveillance data. Nurses felt that the surveillance form added to their workload, and the infection control practitioners were unable to adequately supervise the process due to competing work demands. CONCLUSIONS: A manual system that adds to the administrative workload of nurses is not an effective method of measuring the burden of HAIs. Change management is required to promote an organizational culture that supports accurate data collection for HAIs.

Cellular Architecture of Spinal Granulomas and the Immunological Response in Tuberculosis Patients Coinfected with HIV.

Mycobacterium tuberculosis (M.tb) and HIV are individually responsible for the most deaths worldwide among all infectious agents, and coinfection with M.tb and HIV is a significant public health challenge in the developing world. Although the lung is the primary target organ for tuberculosis (TB), M.tb can also cause extrapulmonary tuberculosis (EPTB) such as in the bones and joints. Treatment of EPTB is much more challenging than treatment of pulmonary TB. The hallmark of the host immune response against TB is the formation of organized structures called granulomas that are infiltrated with immune cells and are rich in cytokines and chemokines. Inside granulomas, the host confines the M.tb bacteria to a particular region of the organ and avoids dispersion. In this study, we analyzed immune cells in bone granulomas of patients with EPTB that are also coinfected with HIV. We found that HIV-infected TB patients have dispersed bone granulomas, with reduced T cell numbers and a concomitant increase in plasma cells. Additionally, HIV-infected patients exhibited dramatically increased serum levels of IgM and IgG1 antibodies, which is indicative of T-cell-independent B-cell activation and mucosal T-cell activation, respectively. Interestingly, we also observed that CD29+ stem cells are increased in HIV-TB coinfection, suggesting a link with HIV infection. Therefore, our work provides new insights into the architecture of spinal TB granulomas and the role of B-cells and humoral immunity against a highly infectious intracellular pathogen. We propose that our findings will inform biomarker identification for EPTB and possibly the development of related therapeutics and/or vaccines to protect HIV-infected patients against disseminated TB.

Molecular Characterization of Corynebacterium diphtheriae Outbreak Isolates, South Africa, March-June 2015.

In 2015, a cluster of respiratory diphtheria cases was reported from KwaZulu-Natal Province in South Africa. By using whole-genome analysis, we characterized 21 Corynebacterium diphtheriae isolates collected from 20 patients and contacts during the outbreak (1 patient was infected with 2 variants of C. diphtheriae). In addition, we included 1 cutaneous isolate, 2 endocarditis isolates, and 2 archived clinical isolates (ca. 1980) for comparison. Two novel lineages were identified, namely, toxigenic sequence type (ST) ST-378 (n = 17) and nontoxigenic ST-395 (n = 3). One archived isolate and the cutaneous isolate were ST-395, suggesting ongoing circulation of this lineage for >30 years. The absence of preexisting molecular sequence data limits drawing conclusions pertaining to the origin of these strains; however, these findings provide baseline genotypic data for future cases and outbreaks. Neither ST has been reported in any other country; this ST appears to be endemic only in South Africa.

Genomic diversity in autopsy samples reveals within-host dissemination of HIV-associated Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of death worldwide, especially among individuals infected with HIV. Whereas phylogenetic analysis has revealed M. tuberculosis spread throughout history and in local outbreaks, much less is understood about its dissemination within the body. Here we report genomic analysis of 2,693 samples collected post mortem from lung and extrapulmonary biopsies of 44 subjects in KwaZulu-Natal, South Africa, who received minimal antitubercular treatment and most of whom were HIV seropositive. We found that purifying selection occurred within individual patients, without the need for patient-to-patient transmission. Despite negative selection, mycobacteria diversified within individuals to form sublineages that co-existed for years. These sublineages, as well as distinct strains from mixed infections, were differentially distributed throughout the lung, suggesting temporary barriers to pathogen migration. As a consequence, samples taken from the upper airway often captured only a fraction of the population diversity, challenging current methods of outbreak tracing and resistance diagnostics. Phylogenetic analysis indicated that dissemination from the lungs to extrapulmonary sites was as frequent as between lung sites, supporting the idea of similar migration routes within and between organs, at least in subjects with HIV. Genomic diversity therefore provides a record of pathogen diversification and repeated dissemination across the body.

Mechanisms of first-line antimicrobial resistance in multi-drug and extensively drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa.

BACKGROUND: In South Africa, drug resistant tuberculosis is a major public health crisis in the face of the colossal HIV pandemic. METHODS: In an attempt to understand the distribution of drug resistance in our setting, we analysed the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of tuberculosis in clinical isolates of Mycobacterium tuberculosis in the KwaZulu-Natal province. RESULTS: Classical mutations were detected in the katG, inhA and embB genes associated with resistance to isoniazid and ethambutol. Diverse mutations were recorded in the multidrug resistant (MDR) and extensively drug resistant (XDR) isolates for the rpoB and pncA gene associated with resistance to rifampicin and pyrazinamide. CONCLUSIONS: M.tuberculosis strains circulating in our setting display a combination of previously observed mutations, each mediating resistance to a different drug. The MDR and XDR TB isolates analysed in this study displayed classical mutations linked to INH and EMB resistance, whilst diverse mutations were linked to RIF and PZA resistance. The similarity of the XDR strains confirms reports of the clonality of the XDR epidemic. The successful dissemination of the drug resistant strains in the province underscores the need for rapid diagnostics to effectively diagnose drug resistance and guide treatment.

Strains of Mycobacterium tuberculosis differ in affinity for human osteoblasts and alveolar cells in vitro.

Although the lung is the primary site of infection of tuberculosis, Mycobacterium tuberculosis is capable of causing infection at other sites. In 5-10 % such extra-pulmonary tuberculosis is located in bone tissue of the spine. It is unknown whether host or microbial factors are responsible for the site where extra-pulmonary tuberculosis manifests itself. One MDR isolate belonging to strain F28, one susceptible F11 and one isolate each of susceptible, MDR and XDR F15/LAM4/KZN were cultured in Middlebrook 7H9 media. Human osteoblasts (SaOS-2) and human alveolar epithelial cells (A549) were exposed to these different isolates of M. tuberculosis and invasion capacity and intra-cellular multiplication rates were established. Mouse macrophage (MHS) cells exposed to M. tuberculosis H37Rv served as control. The invasion capacity of F15/LAM4/KZN representatives increased with the level of resistance. The F28 MDR strain showed similar invasion capacity as the XDR F15/LAM4/KZN for pulmonary epthelial cells, whilst the fully susceptible F11 strain displayed a propensity for osteoblasts. The differences observed may in part explain why certain strains are able to cause infection at specific extra-pulmonary sites. We postulated that the development of extra-pulmonary tuberculosis depends on the ability of the microbe to pass effectively through the alveolar epithelial lining and its affinity for cells other than those in pulmonary tissue.

ClassTR: Classifying Within-Host Heterogeneity Based on Tandem Repeats with Application to Mycobacterium tuberculosis Infections.

Genomic tools have revealed genetically diverse pathogens within some hosts. Within-host pathogen diversity, which we refer to as "complex infection", is increasingly recognized as a determinant of treatment outcome for infections like tuberculosis. Complex infection arises through two mechanisms: within-host mutation (which results in clonal heterogeneity) and reinfection (which results in mixed infections). Estimates of the frequency of within-host mutation and reinfection in populations are critical for understanding the natural history of disease. These estimates influence projections of disease trends and effects of interventions. The genotyping technique MLVA (multiple loci variable-number tandem repeats analysis) can identify complex infections, but the current method to distinguish clonal heterogeneity from mixed infections is based on a rather simple rule. Here we describe ClassTR, a method which leverages MLVA information from isolates collected in a population to distinguish mixed infections from clonal heterogeneity. We formulate the resolution of complex infections into their constituent strains as an optimization problem, and show its NP-completeness. We solve it efficiently by using mixed integer linear programming and graph decomposition. Once the complex infections are resolved into their constituent strains, ClassTR probabilistically classifies isolates as clonally heterogeneous or mixed by using a model of tandem repeat evolution. We first compare ClassTR with the standard rule-based classification on 100 simulated datasets. ClassTR outperforms the standard method, improving classification accuracy from 48% to 80%. We then apply ClassTR to a sample of 436 strains collected from tuberculosis patients in a South African community, of which 92 had complex infections. We find that ClassTR assigns an alternate classification to 18 of the 92 complex infections, suggesting important differences in practice. By explicitly modeling tandem repeat evolution, ClassTR helps to improve our understanding of the mechanisms driving within-host diversity of pathogens like Mycobacterium tuberculosis.

Within-Host Heterogeneity of Mycobacterium tuberculosis Infection Is Associated With Poor Early Treatment Response: A Prospective Cohort Study.

The clinical management of tuberculosis is a major challenge in southern Africa. The prevalence of within-host genetically heterogeneous Mycobacterium tuberculosis infection and its effect on treatment response are not well understood. We enrolled 500 patients with tuberculosis in KwaZulu-Natal and followed them through 2 months of treatment. Using mycobacterial interspersed repetitive units-variable number of tandem repeats genotyping to identify mycobacterial heterogeneity, we report the prevalence and evaluate the association of heterogeneity with treatment response. Upon initiation of treatment, 21.1% of participants harbored a heterogeneous M. tuberculosis infection; such heterogeneity was independently associated with a nearly 2-fold higher odds of persistent culture positivity after 2 months of treatment (adjusted odds ratio, 1.90; 95% confidence interval, 1.03-3.50).

Anti-dormant mycobacterial activity and target analysis of nybomycin produced by a marine-derived Streptomyces sp.

In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0µg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death.

The pros, cons, and unknowns of search and destroy for carbapenem-resistant enterobacteriaceae.

Antibiotic drug discovery has not kept pace with the development of microbial resistance to these agents. There are ever increasing reports where the causative agents of serious infections are multi-drug resistant and in some cases resistant to all known antibiotics. The emergence and spread of carbapenemase-producing Enterobacteriaceae has heightened awareness regarding antibiotic stewardship programs and infection prevention and control measures. There has been much controversy regarding the utility of the "search and destroy" strategy to prevent the spread of carbapenem-resistant Enterobacteriaceae. These controversies center on screening and management of carriers, including decontamination and isolation. It is however clear that a functional infection prevention and control program is fundamental to any strategy that serves to address the spread of microbes within a healthcare facility.

Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1ß and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence.

Cervical ectopy: associations with sexually transmitted infections and HIV. A cross-sectional study of high school students in rural South Africa.

OBJECTIVES: It has been hypothesised that ectopy may be associated with increased susceptibility to sexually transmitted infections (STIs). In this cross-sectional study, we wanted to explore the association between STIs (including HIV) and cervical ectopy. METHODS: We included 700 sexually active young women attending randomly selected high schools in a rural district in KwaZulu-Natal, South Africa. The district is endemic of HIV and has a high prevalence of STIs. We did computer-assisted measurements of the ectocervical area covered by columnar epithelium (ectopy) in colposcopic images and STI analyses on cervicovaginal lavage and serum samples. All participating women answered a questionnaire about sexual behaviour and use of contraceptives. RESULTS: The mean age was 19.1 years. Ectopy was found in 27.2%, HIV in 27.8%, chlamydia in 25.3% and gonorrhoea in 15.6%. We found that age, parity, chlamydia and gonorrhoea, years since menarche, years since sexual debut and number of sexual partners were associated with ectopy. In multivariate analysis with chlamydia infection as the dependent variable, women with ectopy had increased odds of having chlamydia infection (adjusted OR 1.78, p=0.033). In women under 19 years of age, we found twofold higher odds of being HIV-positive for those with ectopy (OR 2.19, p=0.014). CONCLUSIONS: In conclusion, cervical ectopy is associated with Chlamydia trachomatis infection and HIV in the youngest women.

High prevalence and incidence of asymptomatic sexually transmitted infections during pregnancy and postdelivery in KwaZulu Natal, South Africa.

BACKGROUND: We report the prevalence and incidence of 3 treatable sexually transmitted pathogens (Neiserria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis) in women who were HIV infected or at high risk for HIV infection, in pregnancy and postpartum, respectively. METHOD: Vulvovaginal specimens collected at the first antenatal visit and again at 14 weeks postpartum were tested for N. gonorrhoeae, C. trachomatis, and T. vaginalis in the laboratory. Women were routinely tested for HIV-1 with a point-of-care test. RESULTS: Among 1480 women, 32.3% (95% confidence interval, 29.9-34.7) tested positive for any of the sexually transmitted infections (STIs) in pregnancy and 19.2% (95% confidence interval, 16.9-21.5) were positive when retested 14 weeks postpartum (incidence rate, 79.2 per 100 person-years). The prevalence of N. gonorrhoeae and T. vaginalis infections in pregnancy and the incidence rate of any STI at 14 weeks postpartum were significantly higher in HIV-1-infected women (P < 0.0001 amd P = 0.0079). More than 50% of N. gonorrhoeae, T. vaginalis, and C. trachomatis infections in pregnancy were asymptomatic. CONCLUSIONS: The high prevalence of asymptomatic STIs in pregnancy is compelling evidence that demands the development and validation of point-of-care tests for STIs be expedited. In addition, the high incidence of STIs 3 months postpartum suggests that women in this study setting resume unprotected sexual intercourse soon after delivery.

Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis.

Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.

Isoniazid induces apoptosis of activated CD4+ T cells: implications for post-therapy tuberculosis reactivation and reinfection.

Tuberculosis (TB) remains the second highest killer from a single infectious disease worldwide. Current therapy of TB is lengthy and consists of multiple expensive antibiotics, in a strategy referred to as Directly Observed Treatment, Short Course (DOTS). Although this therapy is effective, it has serious disadvantages. These therapeutic agents are toxic and are associated with the development of a variety of drug-resistant TB strains. Furthermore, patients treated with DOTS exhibit enhanced post-treatment susceptibility to TB reactivation and reinfection, suggesting therapy-related immune impairment. Here we show that Isoniazid (INH) treatment dramatically reduces Mycobacterium tuberculosis antigen-specific immune responses, induces apoptosis in activated CD4(+) T cells, and renders treated animals vulnerable to TB reactivation and reinfection. Consequently, our findings suggest that TB treatment is associated with immune impairment.