Characterization and drug resistance of Trichomonas vaginalis clinical isolates

Sexually transmitted infections (STIs) are a major cause of acute illnesses, infertility, long term disability and death with far reaching health, social and economic consequences. Trichomonas vaginalis is the causative organism of trichomoniasis which classically presents in women as a malodorous green-yellowish discharge accompanied by itching and burning. In men infection can cause non-gonococcocal urethritis and chronic prostatitis. Complications of T. vaginalis include preterm delivery, low birth weight, predisposition to HIV infection and cervical cancer. Previous studies in South Africa have focused mostly on T. vaginalis detection with reported rates of prevalence of T. vaginalis infections ranging between 20% to 49%. Despite estimates showing T. vaginalis being the most prevalent sexually transmitted disease worldwide, very little is known about the genetic diversity of T. vaginalis clinical isolates. Furthermore, the degree of metronidazole resistance in a particular setting needs to be investigated, since this has implications on the treatment regimen of trichomoniasis. The purpose of this study was: i) To detect T. vaginalis in HIV positive female patients from the Anti-Retroviral clinic of the Tshwane District Hospital, Pretoria using three methods, namely microscopy, culture and PCR; ii) To characterize T. vaginalis isolates using both the random amplified polymorphic DNA (RAPD) assay and the intergenic spacer regionpolymerase chain reaction-restriction fragment length polymorphism (IGS-PCR RFLP) assay and iii) To phenotypically determine metronidazole resistance of the T. vaginalis isolates and to compare the results with random amplified polymorphic DNA (RAPD) assay results. Self-collected vaginal swabs from 380 women were included in the first part of the study. Trichomonas vaginalis was detected using: wet mount microscopy, culture (modified Diamond’s medium) and molecular detection using a commercial kit, Trichomonas vaginalis 240/250 IC (Sacace Biotechnology, Italy). The genetic relatedness of 92 culture positive T. vaginalis isolates was determined. Five primers (TV1, TV2, TV3, TV5 and TV6) were used for the RAPD assay. The PCR-IGS-RFLP products were digested with five enzymes, namely: AluI, HinfI, RsaI, Sau3AI and Tsp509. A 24 h and 48 h interval microtiter assay was used to detemine the metronidazole antimicrobial susceptibility of 30 T. vaginalis isolates. A total of 8% (30/380) of specimens were positive for T. vaginalis using microscopy, 24% (92/380) of specimens were positive using culture and 31% (118/380) of the specimens were positive using the commercial PCR kit Trichomonas vaginalis 240/250 IC (Sacace Biotechnology, Italy). RAPD assay analysis showed a high level of genetic diversity between the different T. vaginalis isolates. The dendrogrammes obtained from the RAPD markers grouped the 92 T.vaginalis isolates into between nine to 24 clusters with a 70% similarity, while the PCR-IGS RFLP assay results for the isolates were genetically indistinguishable. The minimal inhibitory concentration (MIC) for metronidazole was between 0.06 to 25 μg/ml. Only 6% (2/30) of the T. vaginalis isolates were resistant. The dendrogrammes constructed in the second part of the study did not group the metronidazole resistant isolates together in one cluster. No link between resistance and a specific T. vaginalis genotype could be indicated. This study proved that PCR is a more sensitive diagnostic tool for the detection of T. vaginalis to improve the diagnosis of trichomoniasis. A high prevalence of T. vaginalis in HIV positive women in South Africa was observed. The RAPD assay proved to be useful in discriminating between the different T. vaginalis isolates, while the IGS-PCR RFLP assay was not a suitable marker. In future, other T. vaginalis genes, such as the ferredoxin and beta-tubulin genes could be investigated to determine the genetic variability of T. vaginalis isolates. Although metronidazole is the only antimicrobial drug used for treatment of trichomoniasis in South Africa, a low prevalence of in vitro resistance was found. This study emphasized the importance of in vitro antimicrobial drug susceptibility testing to ensure continunous screening for possible cases of metronidazole resistance and to monitor MIC changes.Dissertation (MSc)--University of Pretoria, 2013.Medical MicrobiologyMScUnrestricte

A novel molecular strategy for surveillance of multidrug resistant tuberculosis in high burden settings

BACKGROUND In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. METHODS A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods. RESULTS Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases. CONCLUSION We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wildtype strains as needed.Supporting Information. S1 File. (XLSX)http://www.plosone.orgam2016Medical Microbiolog

High Cryptococcal Antigen Titers in Blood Are Predictive of Subclinical Cryptococcal Meningitis Among Human Immunodeficiency Virus-Infected Patients

Background High mortality rates among asymptomatic cryptococcal antigen (CrAg)–positive patients identified through CrAg screening, despite preemptive fluconazole treatment, may be due to undiagnosed cryptococcal meningitis. Methods Symptoms were reviewed in CrAg-positive patients identified by screening 19233 individuals with human immunodeficiency virus infection and CD4 cell counts <100/µL at 17 clinics and 3 hospitals in Johannesburg from September 2012 until September 2015, and at 2 hospitals until June 2016. Cerebrospinal fluid samples from 90 of 254 asymptomatic patients (35%) and 78 of 173 (45%) with headache only were analyzed for cryptococcal meningitis, considered present if Cryptococcus was identified by means of India ink microscopy, culture, or CrAg test. CrAg titers were determined with stored blood samples from 62 of these patients. The associations between blood CrAg titer, concurrent cryptococcal meningitis, and mortality rate were assessed. Results Cryptococcal meningitis was confirmed in 34% (95% confidence interval, 25%–43%; 31 of 90) of asymptomatic CrAg-positive patients and 90% (81%–96%; 70 of 78) with headache only. Blood CrAg titer was significantly associated with concurrent cryptococcal meningitis in asymptomatic patients (P 160 (sensitivity, 88.2%; specificity, 82.1%); the odds ratio for concurrent cryptococcal meningitis was 34.5 (95% confidence interval, 8.3–143.1; P < .001). Conclusions About a third of asymptomatic CrAg-positive patients have concurrent cryptococcal meningitis. More effective clinical assessment strategies and antifungal regimens are required for CrAg-positive patients, including investigation for cryptococcal meningitis irrespective of symptoms. Where it is not possible to perform lumbar punctures in all CrAg-positive patients, blood CrAg titers should be used to target those most at risk of cryptococcal meningitis

Evaluation of a Novel Semiquantitative Cryptococcal Antigen Lateral Flow Assay in Patients with Advanced HIV Disease.

Higher cryptococcal antigen (CrAg) titers are strongly associated with mortality risk in individuals with HIV-associated cryptococcal disease. Rapid tests to quantify CrAg levels may provide important prognostic information and enable treatment stratification. We performed a laboratory-based validation of the IMMY semiquantitative cryptococcal antigen (CrAgSQ) lateral flow assay (LFA) against the current gold standard CrAg tests. We assessed the diagnostic accuracy of the CrAgSQ in HIV-positive individuals undergoing CrAg screening, determined the relationship between CrAgSQ scores and dilutional CrAg titers, assessed interrater reliability, and determined the clinical correlates of CrAgSQ scores. A total of 872 plasma samples were tested using both the CrAgSQ LFA and the conventional IMMY CrAg LFA, of which 692 were sequential samples from HIV-positive individuals undergoing CrAg screening and an additional 180 were known CrAg-positive plasma samples archived from prior studies. Interrater agreement in CrAgSQ reading was excellent (98.17% agreement, Cohen's kappa 0.962, P?<?0.001). Using the IMMY CrAg LFA as a reference standard, CrAgSQ was 93.0% sensitive (95% confidence interval [CI] 80.9% to 98.5%) and 93.8% specific (95% CI, 91.7% to 95.6%). After reclassification of discordant results using CrAg enzyme immunoassay testing, the sensitivity was 98.1% (95% CI, 90.1% to 100%) and specificity 95.8% (95% CI, 93.9% to 97.2%). The median CrAg titers for semiquantitative score categories (1+ to 4+) were 1:10 (interquartile range [IQR], 1:5 to 1:20) in the CrAgSQ 1+ category, 1:40 (IQR, 1:20 to 1:80) in the CrAgSQ 2+ category, 1:640 (IQR, 1:160 to 1:2,560) in the CrAgSQ 3+ category, and 1:5,120 (IQR, 1:2,560 to 1:30,720) in the CrAgSQ 4+ category. Increasing CrAgSQ scores were strongly associated with 10-week mortality. The IMMY CrAgSQ test had high sensitivity and specificity compared to the results for the IMMY CrAg LFA and provided CrAg scores that were associated with both conventional CrAg titers and clinical outcomes

Correlation of rpoB Mutations with Minimal Inhibitory Concentration of Rifampin and Rifabutin in Mycobacterium tuberculosis in an HIV/AIDS Endemic Setting, South Africa

Abstract Treatment of tuberculosis (TB) and HIV co-infection is often complicated by drug-drug interactions between anti-mycobacterial and anti-retroviral (ARV) agents. Rifabutin (RFB) is an alternative to rifampin (RIF) for TB regimens and is recommended for HIV patients concurrently receiving protease inhibitors because of reduced induction of CYP3A4. This study sought to determine the proportion of RFB susceptible isolates among RIF resistant strains in a high HIV prevalence setting in South Africa. In addition, the study explored the association between rpoB mutations and minimum inhibitory concentration (MIC) levels of RIF and RFB. A total of 189 multidrug resistant (MDR) M. tuberculosis isolates from the Centre for Tuberculosis (CTB) repository were analyzed. The MICs were determined using a Sensititre MYCOTB system and the rpoB gene was sequenced. Of the 189 MDR isolates, 138 (73%) showed resistance to both RIF and RFB, while 51 (27%) isolates were resistant to RIF but RFB susceptibility. S531L was the most frequent rpoB mutation in 105 89 (56%) isolates, followed by H526Y in 27 89 (14%) isolates. Resistance to both RIF and RFB was found predominantly in association with mutations S531L (91105, 87%), H526Y (2027, 74%), and H526D (1519, 79%), while D516V (1517, 88%) and L533P (34, 75%) were found in RFB susceptible isolates. This study has shown that up to 27% of MDR-TB patients in South Africa may benefit from a treatment regimen that includes RFB

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed

Correlation of rpoB mutations with minimal inhibitory concentration of rifampin and rifabutin in Mycobacterium tuberculosis in an HIV/AIDS endemic setting, South Africa

CITATION: Rukasha, I. et al. 2016. Correlation of rpoB mutations with minimal inhibitory concentration of rifampin and rifabutin in Mycobacterium tuberculosis in an HIV/AIDS endemic setting, South Africa. Frontiers in Microbiology, 7:1947, doi: 10.3389/fmicb.2016.01947.The original publication is available at http://journal.frontiersin.org/journal/microbiologyENGLISH SUMMARY : Treatment of tuberculosis (TB) and HIV co-infections is often complicated by drug-to-drug interactions between anti-mycobacterial and anti-retroviral agents. Rifabutin (RFB) is an alternative to rifampin (RIF) for TB regimens and is recommended for HIV patients concurrently receiving protease inhibitors because of reduced induction of CYP3A4. This study sought to determine the proportion of RFB susceptible isolates among RIF-resistant strains in a high HIV prevalence setting in South Africa. In addition, the study explored the association between rpoB mutations and minimum inhibitory concentrations (MIC) of RIF and RFB. A total of 189 multidrug resistant (MDR) Mycobacterium tuberculosis isolates from the Centre for Tuberculosis repository were analyzed. The MICs were determined using a MYCOTB Sensititre plate method and the rpoB gene was sequenced. Of the 189 MDR isolates, 138 (73%) showed resistance to both RIF and RFB, while 51 (27%) isolates were resistant to RIF but retained susceptibility to RFB. The S531L was the most frequent rpoB point mutation in 105/189 (56%) isolates, followed by H526Y in 27/189 (14%) isolates. Resistance to both RIF and RFB was found predominantly in association with mutations S531L (91/105, 87%), H526Y (20/27, 74%), and H526D (15/19, 79%), while D516V (15/17, 88%), and L533P (3/4, 75%) were found in RIF-resistant, RFB-susceptible isolates. This study has shown that up to 27% of MDR-TB patients in South Africa may benefit from a treatment regimen that includes RFB.http://journal.frontiersin.org/article/10.3389/fmicb.2016.01947/fullPublisher's versio

High Cryptococcal Antigen Titers in Blood Are Predictive of Subclinical Cryptococcal Meningitis Among Human Immunodeficiency Virus-Infected Patients.

Background: High mortality rates among asymptomatic cryptococcal antigen (CrAg)-positive patients identified through CrAg screening, despite preemptive fluconazole treatment, may be due to undiagnosed cryptococcal meningitis. Methods: Symptoms were reviewed in CrAg-positive patients identified by screening 19233 individuals with human immunodeficiency virus infection and CD4 cell counts 160 (sensitivity, 88.2%; specificity, 82.1%); the odds ratio for concurrent cryptococcal meningitis was 34.5 (95% confidence interval, 8.3-143.1; P < .001). Conclusions: About a third of asymptomatic CrAg-positive patients have concurrent cryptococcal meningitis. More effective clinical assessment strategies and antifungal regimens are required for CrAg-positive patients, including investigation for cryptococcal meningitis irrespective of symptoms. Where it is not possible to perform lumbar punctures in all CrAg-positive patients, blood CrAg titers should be used to target those most at risk of cryptococcal meningitis