Evaluation of Endothelial Cells Differentiated from Amniotic Fluid-Derived Stem Cells

Amniotic fluid holds great promise as a stem cell source, especially in neonatal applications where autologous cells can be isolated and used. This study examined chemical-mediated differentiation of amniotic fluid-derived stem cells (AFSC) into endothelial cells and verified the function of AFSC-derived endothelial cells (AFSC-EC). AFSC were isolated from amniotic fluid obtained from second trimester amnioreduction as part of therapeutic intervention from pregnancies affected with twin-twin transfusion syndrome. Undifferentiated AFSC were of normal karyotype with a subpopulation of cells positive for the embryonic stem cell marker SSEA4, hematopoietic stem cell marker c-kit, and mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105. Additionally, these cells were negative for the endothelial marker CD31 and hematopoietic differentiation marker CD45. AFSC were cultured in endothelial growth media with concentrations of vascular endothelial growth factor (VEGF) ranging from 1 to 100 ng/mL. After 2 weeks, AFSC-EC expressed von Willebrand factor, endothelial nitric oxide synthase, CD31, VE-cadherin, and VEGF receptor 2. Additionally, the percentage of cells expressing CD31 was positively correlated with VEGF concentration up to 50 ng/mL, with no increase at higher concentrations. AFSC-EC showed a decrease in stem cells markers c-kit and SSEA4 and were morphologically similar to human umbilical vein endothelial cells (HUVEC). In functional assays, AFSC-EC formed networks and metabolized acetylated low-density lipoprotein, also characteristic of HUVEC. Nitrate levels for AFSC-EC, an indirect measure of nitric oxide synthesis, were significantly higher than undifferentiated controls and significantly lower than HUVEC. These results indicate that AFSC can differentiate into functional endothelial-like cells and may have the potential to provide vascularization for constructs used in regenerative medicine strategies

Comparison of three commercial molecular assays for detection of rifampin and isoniazid resistance among Mycobacterium tuberculosis isolates in a high-HIV-prevalence setting

In a head to head comparison of the MTBDRplus Version 2.0 (Hain Lifescience), Xpert® MTB/RIF (Cepheid) and the Anyplex™ MTB/NTM (Seegene) assays we demonstrate equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampicin resistance with further analysis of discordances. The Xpert does not detect Isoniazid resistance while Anyplex showed high false positivity.National Health Laboratory Serviceshttp://jcm.asm.org2016-03-31hb201

Evaluation of the GenoType MTBDRsl Version 2.0 Assay for second-line drug resistance detection of Mycobacterium tuberculosis isolates in South Africa

Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDRsl version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (gyrA and gyrB genes) and second-line injectable drugs (SLID) (rrs and eis genes). The study evaluated the diagnostic performance of the GenoType MTBDRsl VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on Mycobacterium tuberculosis complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDRsl VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDRsl VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDRsl VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings.CTB NICD/NHLShttp://jcm.asm.org2017-09-30Medical Microbiolog

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

BACKGROUND : Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium(PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. METHODS : Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. RESULTS : Complete inactivation ofM. tuberculosis occurredwithin 30 min of exposure at a ratio of 1:3 for sputumto PS-MTM. Sputum specimen in PS-MTMshowed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ctvalues (b5% on a mean starting value of 22.47). Of the 256 clinical sputumspecimens, 10.2%were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. CONCLUSIONS : Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.http://www.elsevier.com/locate/jmicmeth2016-10-31hb201

Next-generation sequencing for identifying pyrazinamide resistance in Mycobacterium tuberculosis

No abstract available.http://cid.oxfordjournals.orgam201

Field evaluation of a novel preservation medium to transport sputum specimens for molecular detection of Mycobacterium tuberculosis in a rural African setting

Molecular tests are revolutionizing diagnosis of tuberculosis (TB). Disease burden is concentrated in resource-poor countries with inadequate infrastructure and capacity resulting in delays for specimens to reach the laboratory. We assessed the performance of an innovative method using a swab to inoculate sputum in a transport medium, PrimeStore® - Molecular Transport Medium (PS-MTM) for subsequent molecular detection of Mycobacterium tuberculosis at a centralized facility. A sputum specimen was obtained from suspected TB patients at rural healthcare facilities in South Africa and a swab taken and placed into PS-MTM from this specimen, prior to it being processed by either liquid culture or Xpert MTB/Rif assay (Xpert). A subset from a larger cohort study of a 141 patients was included for analysis, which included 47 laboratory-confirmed TB patients. M. tuberculosis was detected at 29% by culture, 29% by Xpert and 31% and 36% by real-time PCR of PS-MTM for the culture and Xpert specimen respectively. Concordance between the method under evaluation with culture was 82% (McNemar, p=0.55) and 84% (McNemar, p=0.05) for Xpert. Stratified by culture result, detection rate by real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (p=0.32), but significantly higher if culture was negative (p=0.008). These results suggest that swab collection of sputum into PS-MTM provides a promising application for diagnosis of TB in rural healthcare settings thereby potentially improving the options available for the diagnosis of TB in countries incapable of applying decentralized high-tech molecular testing.Department of Medical Microbiology, University of Pretoria and Anova Health Institute.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-31562017-06-30hb2016Medical Microbiolog

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings

SETTING : Mopani District, South Africa. OBJECTIVE : To explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStorew Molecular Transport Medium (PSMTM) compared to settings where microscopy or Xpertw MTB/RIF is used as the baseline test. DESIGN : Two sputum specimens were collected from patients with cough of72 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMixw polymerase chain reaction (PM-PCR). RESULTS : Of 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PSMTM/PSMTM/ PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/ PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P , 0.001), but similar to Xpert (P ¼ 0.33). CONCLUSION: PCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.http://www.ingentaconnect.comcontent/iuatld/ijtld2015-11-30hb201

Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing

To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of Mycobacterium tuberculosis on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of Rv0678 mutations. In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected Rv0678 mutants) and clinical pan-susceptible isolates were determined (n = 70). In phase II, a tentative CC for CFZ (n = 55) was proposed. In phase III, the proposed CC was validated using clinical drug-resistant tuberculosis (DR-TB) isolates stratified by Rv0678 mutation (n = 85). The MIC distribution of CFZ for laboratory and clinical pan-susceptible strains ranged between 0.125 μg/ml and 0.5 μg/ml. As the MIC values of DR-TB isolates used for phase II ranged between 0.25 μg/ml and 1 μg/ml, a CC of 1 μg/ml was proposed. Validation of the CC in phase III showed that probably susceptible and probably resistant Rv0678 mutants overlapped at 1 μg/ml. We therefore recommend a CC of 1 μg/ml, with additional testing at 0.5 μg/ml to define an intermediate category. This was the first comprehensive study to establish a CC for routine phenotypic DST of CFZ using the MGIT960 system to guide therapeutic decisions.https://www.ingentaconnect.com/content/iuatld/ijtld2019-11-01hj2019Medical Microbiolog

Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains

A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin- resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug- resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.http://jcm.asm.org/am201

Systematic rifampicin resistance errors with Xpert® MTB/RIF Ultra : implications for regulation of genotypic assays

No abstract available.https://www.ingentaconnect.com/content/iuatld/ijtldhj2021Medical Microbiolog