Differential Identification of Mycobacterial Species Using High-Resolution Melting Analysis.

Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

Pathogen Identification in Suspected Cases of Pyogenic Spondylodiscitis.

Pyogenic spinal infection continues to represent a worldwide problem. In approximately one-third of patients with pyogenic spondylodiscitis, the infectious agent is never identified. Of the cases that lead to organismal identification, bacteria are more commonly isolated from the spine rather than fungi and parasites. This study applied universal prokaryotic 16S rRNA PCR as a rapid diagnostic tool for the detection of bacterial agents in specimens from patients suspected of pyogenic spondylodiscitis. Gram and Ziehl-Neelsen staining were used as a preliminary screening measure for microbiologic evaluation of patient samples. PCR amplification targeting 16S rRNA gene was performed on DNA extracted from 57 cases including specimens from epidural abscesses, vertebral, and disc biopsies. Positive samples were directly sequenced. MRI findings demonstrated that disc destruction and inflammation were the major imaging features of suspected pyogenic spondylodiscitis cases, as 44 cases showed such features. The most common site of infection was the lumbar spine (66.7%), followed by thoracic spine (19%), the sacroiliac joint (9.5%), and lumbar-thoracic spine (4.8%) regions. A total of 21 samples amplified the 16S rRNA-PCR product. Sanger sequencing of the PCR products identified the following bacteriological agents: Mycobacterium tuberculosis (n = 9; 42.9%), Staphylococcus aureus (n = 6; 28.5%), Mycobacterium abscessus (n = 5; 23.8%), and Mycobacterium chelonae (n = 1; 4.8%). 36 samples displayed no visible 16S rRNA PCR signal, which suggested that non-bacterial infectious agents (e.g., fungi) or non-infectious processes (e.g., inflammatory, or neoplastic) may be responsible for some of these cases. The L3-L4 site (23.8%) was the most frequent site of infection. Single disc/vertebral infection were observed in 9 patients (42.85%), while 12 patients (57.15%) had 2 infected adjacent vertebrae. Elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) inflammatory markers were noted in majority of the patients. In conclusion, microbiological methods and MRI findings are vital components for the proper diagnosis of pyogenic spondylodiscitis. Our findings suggest that molecular methods such as clinical application of 16S rRNA PCR and sequencing may be useful as adjunctive diagnostic tools for pyogenic spondylodiscitis. The rapid turnaround time of 16S rRNA PCR and sequencing submission and results can potentially decrease the time to diagnosis and improve the therapeutic management and outcome of these infections. Although S. aureus and M. tuberculosis were the most common causes of pyogenic spinal infections in this study, other infectious agents and non-infectious etiologies should be considered. Based on study results, we advise that antibiotic therapy should be initiated after a definitive etiological diagnosis.

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes.

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64, and mtp40. On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110- and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110, among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard.

Isolation of Streptococcus pyogenes from children with pharyngitis and emm type analysis.

BACKGROUND: The group A streptococcus (GAS) M protein, encoded by the emm gene, acts as a major virulence factor. Emm-typing is the GAS gold standard molecular typing and is based on the DNA sequence of the nucleotides of the emm gene. The aim of the present study was to isolate GAS from patients and to detect the emm types of the isolates using emm typing. METHODS: A total of 1000 throat samples were collected from patients with pharyngitis referred to Aboozar Children's Hospital in Ahvaz, Iran. We performed antimicrobial susceptibility testing on all isolates using the Kirby-Bauer disk diffusion method. Additionally, amplification of the emm gene was performed using polymerase chain reaction using the standard primers and described protocol. RESULTS: From all throat samples screened, 25 isolates (2.5%) were identified as GAS. Antibiotic susceptibility testing revealed that all the GAS isolates were susceptible to penicillin and erythromycin, but 44% showed resistance to vancomycin. Based on polymerase chain reaction for the emm gene, the obtained emm types were: emm-3, observed in 20 isolates (80%); emm-1 observed in four isolates (16%); and emm-75 observed in one isolate (4%). CONCLUSION: The result of the present study showed that penicillin and erythromycin are still the most effective antibiotics against the organism. The emm typing revealed that emm type-3 was detected in most of the isolates from patients with purulent pharyngitis. On the basis of the findings of this study, we may conclude that emm typing provides new insights on the genetic diversity of the M proteins, and is of demonstrable value for molecular studies of GAS.

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15-100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

Detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients in Ahwaz, Iran.

The antimicrobial activities of 6 common antimicrobial agents including carbapenems were tested against 100 clinical Pseudomonas aeruginosa isolates using a disk diffusion method. All imipenem (IPM)-resistant isolates were screened for metallo-beta-lactamase (MBL) production by Etest assay and were subsequently subjected to polymerase chain reaction (PCR) analysis with the bla(IMP) and bla(VIM) genes. Of 41 IPM-nonsusceptible isolates detected, 8 (19.51%) appeared to produce MBL, as determined by Etest. Using PCR assay, these isolates were positive for bla(VIM) genes, whereas none were positive for bla(IMP) genes.