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.

Differential expression of groEL-1, incB, pyk-F, tal, hctA and omcB genes during Chlamydia trachomatis developmental cycle.

Chlamydia trachomatis infects squamous and columnar epithelia at the mucosal surface. Research on gene expression patterns of C. trachomatis has predominantly focused on non-native host cells, with limited data on growth kinetics and gene expression of chlamydia in keratinocytes. Here, we investigated whether early, mid, and late chlamydial genes observed in HeLa cell line studies were co-ordinately regulated at the transcriptional level even in the keratinized cell line model and whether the expression was stage-specific during the developmental cycle. HaCaT cell lines were infected with chlamydia clinical isolates (US151and serovar E) and reference strain (L2 434). Expression of groEL-1, incB, pyk-F, tal, hctA, and omcB genes was conducted with comparative real-time PCR and transcriptional events during the chlamydial developmental cycle using transmission electron microscopy. The relative expression level of each gene and fold difference were calculated using the 2-ΔΔCT method. The expression of groEL-1 and pyk-F genes was highest at 2 hours post-infection (hpi) in the L2 434 and serovar E. The expression of incB gene increased at 2 hpi in L2 434 and serovar E but peaked at 12 hpi in serovar E. L2 434 and US151 had similar tal expression profiles. Increased expression of hctA and omcB genes were found at 2 and 36 hpi in L2 434. Both clinical isolates and reference strains presented the normal chlamydial replication cycle comprising elementary bodies and reticulate bodies within 36 hpi. We show different gene expression patterns between clinical isolates and reference strain during in vitro infection of keratinocytes, with reference strain-inducing consistent expression of genes. These findings confirm that keratinocytes are appropriate cell lines to interrogate cell differentiation, growth kinetics, and gene expression of C. trachomatis infection. Furthermore, more studies with different clinical isolates and genes are needed to better understand the Chlamydial pathogenesis in keratinocytes.

Differences in Chlamydia trachomatis growth rates in human keratinocytes among lymphogranuloma venereum reference strains and clinical isolates.

The pathogenesis of the primary stage of lymphogranuloma venereum (LGV) is poorly understood. There is no skin cell model and LGV pathogenesis studies are therefore carried out on cells of different origin. Moreover, such studies usually use reference strains, which may have evolved over the years in culture. In this study, a model was developed in which Chlamydia trachomatis enters and grows in human keratinocytes at 37 and 33 °C. Keratinocytes were infected with fresh clinical isolates and the three LGV reference strains L1, L2 and L3. Growth was monitored for 5 days post-infection using fluorescence microscopy and image analysis software. Chlamydial replication was quicker at 37 than at 33 °C, despite 33 °C being the temperature of human skin. The serovar L2 reference strain grew significantly faster than the other strains, although the fresh clinical isolates were also serovar L2. When grown in keratinocytes at 33 °C, the L2 and L3 reference strains produced much larger inclusions than the other strains tested. This model, which utilizes keratinocytes, better simulates the conditions present at the initial site of infection in LGV than previously published literature, making it a useful tool for future LGV pathogenesis studies. In addition, the results indicate that fresh clinical isolates should be included in LGV pathogenesis studies.