Genexpert rapid detection of rifampicin resistance among patients with pulmonary tuberculosis

The routine conventional phenotypic bacteriological tests available for pulmonary tuberculosis (PTB) diagnosis, such as microscopy by Ziehl Neelsen staining, and Lowenstein Jensen method of solid culture, requires a long time for revealing positivity, resulting in a delayed diagnosis. After Mycobacterium tuberculosis (MTB) genome was discovered, nucleic acid amplification techniques were developed with more rapid detection and identification of rifampicin resistance in respiratory and extra-pulmonary specimens. GeneXpert is a DNA-polymerase chain reaction technique based on NAA, which allows an accurate genotypic method for a quick diagnosis of PTB diagnosis. The aim of the study was to assess retrospectively the yield of genotypic assay GeneXpert in revealing multidrug resistant TB compare to phenotypic confirmation by solid culture. 512 patients with positive and negative smears of suspected PTB were investigated by conventional phenotypic assays. PTB diagnosis assessed by positive genotypic assay and confirmed by phenotypic conventional solid culture method was 78.12%. The yield of GeneXpert in revealing rifampicin resistance was high (n=79/512; 15.43%) with 12.5% additional resistance against isoniazid revealed by positive solid cultures and drug susceptibility test. GeneXpert assay is a very useful method for rapid detection of MTB and drug resistance, which facilitates timely diagnosis and appropriate management of pulmonary tuberculosis.

Genetic Diversity of Local Greek and Bulgarian Grapevine (Vitis Vinifera L.) Varieties

The aim of this study was to estimate the genetic diversity of Greek and Bulgarian grapevine varieties with the use of microsatellite markers. The studied samples were collected from various productive vineyards, consisting of eight Greek and nine Bulgarian native varieties. In order to create a genetic profile for each sample, a multiplex PCR reaction method was used amplifying simultaneously seven microsatellite loci. Statistical analysis of data showed that there was a high degree of genetic heterogeneity among most of the varieties studied, highlighting the discriminative power of the chosen set of markers. Moreover, the synonymy of (I) Greek Pamid and Bulgarian Pamid and (II) Greek Zoumiatiko and Bulgarian Dimyat was suggested, as each variety pair had identical allele profiles in all loci examined. Regarding the Greek Mavrud and Bulgarian Mavrud varieties, there was a close genetic relationship between them, however, they did not share common alleles in all microsatellite loci and, therefore, should not be characterized as synonyms. On the other hand, Greek and Bulgarian Keratsouda, which were supposed to be common varieties, were found to be genetically different, supporting that these two varieties should be considered as homonyms. Despite the genotypic assay performed herein, we believe that additional molecular work is needed for the efficient management of Greek and Bulgarian grapevine genepools, as well as to safely suggest any synonym or homonym annotation.

Analysis of Ganciclovir-Resistant Cytomegalovirus Infection Caused by the UL97 Gene Mutation in Codons 460 and 520 in Pediatric Patients: A Case Series

Background Drug-resistant cytomegalovirus (CMV) infection has been increasingly recognized. However, there are limited data in pediatric patients. In this study, the prevalence and factors associated with CMV infection with UL97 mutations in pediatric patients treated with ganciclovir but not responding to treatment were evaluated. Methods This retrospective study was conducted from January 2013 to December 2017. All patients who were suspected of having ganciclovir-resistant CMV infection and had never had ganciclovir prophylaxis were included. Genotypic assay for UL97 mutations in codons 460 and 520 conferring ganciclovir resistance was performed. Factors associated with the presence of UL97 mutations were analyzed. Results Of 34 patients included, 10 patients (29.4%) had a genotypically confirmed UL97 mutation. The median age (interquartile range [IQR]) was 3 (0.85–8.68) years. Ganciclovir resistance was tested at a median time (IQR) of 22.5 (14.3–31) days after initiation of ganciclovir. All resistant isolates harbored a UL97 mutation in codon 460. Compared with patients infected with CMV without UL97 mutation, those infected with UL97 mutation strains were younger (median age [IQR], 3.02 [0.85–8.68] vs 10.45 [2.7–16.4] years) and had a higher maximum viral load (median [IQR], 5.06 [4.74–6.05] vs 4.42 [4.03–4.87] copies/mL). Six of 10 (60%) patients were successfully treated with high-dose ganciclovir (7.5 mg/kg twice daily). Conclusions UL97 mutation ganciclovir-resistant CMV infection was not uncommon in the pediatric population. Screening for this mutation should be considered in patients experiencing virological worsening while ganciclovir is given, even if patients have not previously received ganciclovir prophylaxis.

A7 Co-receptor tropism determined by genotypic assay in HIV-1 non-B subtypes circulating in Cuba: Implications for pathogenesis and Maraviroc resistance

The V3 loop of the HIV-1 envelope (env) gene is involved in binding to the chemokine receptors CCR5 and CXCR4, thus determining viral tropism. With the aim of genetically characterizing the C2V3 env region of HIV-1 samples from Cuban patients, naive to Maraviroc (MVC) therapy, 115 plasma samples were taken in the period of 2014–6 and analyzed by sequencing of the C2V3 region. HIV-1 subtyping was performed using COMET V.2 and Rega subtyping toolV.3 software. Subtypes were confirmed by phylogenetic analyses using Mega-6. Prediction of co-receptor tropism was performed using the geno2pheno algorithm. The viral mutations associated to MVC resistance were analyzed, as well as the association of the subtype with clinical, epidemiological, virological, and immunological variables. The subtypes detected using the C2V3 region were CRF20, 23, 24_BG (35 patients, 30.4%); Subtype B (33 patients, 28.7%); CRF19_cpx (30 patients, 26.1%); CRF18_cpx (10 patients, 8.7%); and others (7 patients, 6.1%). Overall, 60 per cent of the viruses exhibited R5 phenotype, 14.8 per cent were R5X4 and 25.2 per cent were X4. Interestingly, CRF19_cpx virus was associated with having phenotype X4 [46.7%, P = 0.0047, odds ratio (OR): 3.96, 95% confidence interval (95% CI): 1.59–9.84], with infection in young individuals (39.1%, P = 0.025, OR: 3,548; 95% CI: 1,136–11,077) and with higher values of viral load (P ≤ 0.05). The comparison of the amino acid sequences of the V3 loop showed differences between the B and non-B subtypes (P = 0.0001). Mutations reported to be associated with MVC resistance, were detected in 75.7 per cent of the samples, in positions 11 (6.1%), 13 (49.6%), 25 (6.1%), 316 (7.0%), 323 (11.3%), and 319 (3.5%) of Gp120, particularly in the recombinant forms CRF19_cpx and CRF_BGs. HIV variants that use the CXCR4 co-receptor were associated with more than 10 years of diagnosis, with older individuals, in the AIDS stage, with low CD4 counts and higher viral load levels (P < 0.05). The results support the hypothesis previously stated that CRF19_cpx viruses could be more pathogenic and would have limitations for the use of MVC. The high rate of mutations associated to MVC among non-B Cuban subtypes should be further studied.

Association between biofilm formation and susceptibility to antibiotics in Staphylococcuslentus isolated from urinary catheterized patients.

Staphylococcuslentus is a coagulase negative gram positive cocci recognized as opportunistic pathogens and rarely biofilm forming and have many virulence factors,but recently causes nosocomial and community infections. Biofilm formation S. lentus may be associated with ability to resistant antibiotics lead to increase in mortality rate due to difficult in eradicate infections To evaluate the biofilm forming capacity in S.lentus and its susceptibility to antibiotics by using phenotypic and genotypic assay. Twenty eight biofilm bacteria among of them S. lentus were isolated and identified from urine catheterized patients who were hospitalized in different department of four Iraqi hospitals (Al-Diwaniyah teaching,Al- Hilla teaching,Al Qassim and Al Hashimiyah hospitals) S. lentus were examined for detection biofilm formation by detecting icaA gene intercellular adhesion gene which express adhesion factor to form biofilm in staphylococci by using PCR method and tested for antimicrobial susceptibility testing by disc diffusion method and VITEK2 system CLSI guidelines. Three isolates of S. lentus revealed the ability 100% to form biofilm phenotypicallywhich contained icaA gene with clearly antibiotics resistance (100% ) to each penicillin,Carbencillin,Gentamicin,Tobramycin,Oxacillin,Vancomycin,Clindamycin and Ciprofloxacin and (0%) to Azithromycin.icaA genes present in Staphylococcuslentus and responsible for biofilm formation which consider as indicator and the biofilm formation is a strong cause of multidrug resistance in bacteria.