A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals ; Bacteria ; genetics ; isolation & purification ; Bacteriological Techniques ; methods ; Endometritis ; microbiology ; veterinary ; Female ; Multiplex Polymerase Chain Reaction ; standards ; Polymerase Chain Reaction ; veterinary ; Sensitivity and Specificity ; Sheep ; Sheep Diseases ; microbiology ; Tibet

OBJECTIVELower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes.

METHODSThree multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens.

RESULTSThe detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens.

CONCLUSIONThis study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.

Bacteria ; classification ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; genetics ; Electrophoresis, Capillary ; methods ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Respiratory Tract Infections ; microbiology ; Sensitivity and Specificity

OBJECTIVEThe influence of anti-tuberculosis (TB) treatment history on tuberculous lymphadenitis (TBLN) diagnosis is unclear. Therefore, this study aims to evaluate the diagnostic methods, including histology, microbiology, and molecular tests, used for TBLN.

METHODSIn this study, suspected patients with TBLN and having different anti-TB treatment background were enrolled. All the samples were tested simultaneously by histology, Ziehl-Neelsen (ZN) staining, mycobacterial culture (culture), Xpert MTB/RIF (xpert), real-time PCR, and high-resolution melting curve PCR (HRM). Thereafter, the performance of these methods on samples with different anti-TB treatment background was assessed.

RESULTSIn our study, 89 patients were prospectively included 82 patients with TBLN and 7 with other diseases. The overall sensitivities of Xpert, real-time PCR, histology, ZN staining, and culture were 86.6%, 69.5%, 58.5%, 43.9%, and 22.0%, respectively. The anti-TB treatment history revealed dramatic influences on the sensitivity of culture (P < 0.0001). In fact, the treatment that lasted over 3 months also influenced the sensitivity of Xpert (P < 0.05). However, the treatment history did not affect the performance of remaining tests (P > 0.05). For rifampicin drug susceptibility test (DST), the anti-TB treatment showed only significant influence on the success rate of culture DST (P = 0.001), but not on those of Xpert and HRM tests (P > 0.05).

CONCLUSIONOther tests as well as culture should be considered for patients with TBLN having retreatment history or over 1-month treatment to avoid false negative results.

Adolescent ; Adult ; Aged ; Antitubercular Agents ; therapeutic use ; Bacteriological Techniques ; Drug Resistance, Bacterial ; Female ; Humans ; Male ; Middle Aged ; Tuberculosis, Lymph Node ; diagnosis ; drug therapy ; microbiology ; Young Adult

BACKGROUND/AIMS: Surgical site infection (SSI) is a major complication after radical neck dissection (RND) in patients with head and neck cancer (HNC). We investigated the incidence, risk factors, and etiology of SSI among patients who underwent RND. METHODS: A retrospective cohort study was performed on HNC patients, excluding those with thyroid cancer, who underwent first RND at a teaching hospital between January 2006 and June 2010. Medical records were collected and analyzed to evaluate the risk factors and microbiological etiologies. RESULTS: A total of 370 patients underwent first RND. The overall incidence of SSI was 19.7% (73/370). Multivariate analysis showed that male sex (odds ratio [OR], 4.281; p = 0.004), cardiovascular diseases (OR, 1.941; p = 0.020), large amount of blood loss during surgery (OR, 4.213; p = 0.001), and surgery lasting longer than 6 hours (OR, 4.213; p = 0.002) were significantly associated with SSI. The most common causative pathogen was Staphylococcus aureus (32.6%), and 93.2% of S. aureus isolates were methicillin-resistant. Klebsiella pneumoniae (13/92, 14.1%), Pseudomonas aeruginosa (11/92, 12.0%), and Enterococcus species (11/92, 12.0%) were also frequently detected. CONCLUSIONS: Based on our results, we predict that certain groups of patients are at high risk for SSIs after major HNC surgery. Preventive measures or close monitoring in these patients may be required to reduce the likelihood of postoperative SSIs. Furthermore, even though additional research is required, we would consider changing the prophylactic antibiotic regimens according to the causative organisms.
Aged ; Bacteriological Techniques ; Blood Loss, Surgical ; Cardiovascular Diseases/complications ; Chi-Square Distribution ; Female ; Head and Neck Neoplasms/*surgery ; Humans ; Incidence ; Logistic Models ; Male ; Medical Records ; Middle Aged ; Multivariate Analysis ; Neck Dissection/*adverse effects ; Odds Ratio ; Operative Time ; Republic of Korea ; Retrospective Studies ; Risk Factors ; Sex Factors ; Surgical Wound Infection/diagnosis/epidemiology/*microbiology ; Treatment Outcome

OBJECTIVETo compare the performance of MTBDRplus V2 and Xpert MTB/RIF for detecting smear negative pulmonary tuberculosis (PTB).

METHODSClinical PTB suspects were enrolled consecutively in Anhui Chest Hospital and Xi'an Chest Hospital from January to December in 2014. The sputum samples of smear negative PTB suspects were collected and decontaminated. The sediment was used to conduct MTBDRplus V2, Xpert MTB/RIF and drug susceptibility test (DST). All the samples with discrepant drug susceptibility result between molecular methods and phenotypic method were confirmed by DNA sequencing.

RESULTSA total of 1973 cases were enrolled in this study. The detection rates of Mycobacterium tuberculosis complex (MTBC) by MTBDRplus V2 and Xpert MTB/RIF were 27.67% and 27.98%, respectively. When setting MGIT culture result as a gold standard, the sensitivity and specificity of MTBDRplus V2 were 86.74% and 93.84%, and the sensitivity and specificity of Xpert MTB/RIF were 86.55% and 93.43%, respectively. For the detection of the resistance to rifampin, the sensitivity and specificity of MTBDRplus V2 were 94.34% and 96.62%, and the sensitivity and specificity of Xpert MTB/RIF were 88.68% and 95.96%, respectively. For the detection of the resistance to isoniazid, the sensitivity and specificity of MTBDRplus V2 were 77.38% and 98.02%, respectively.

CONCLUSIONMTBDRplus V2 and Xpert MTB/RIF can be used to detect MTBC in smear negative samples with satisfactory performance.

Antitubercular Agents ; pharmacology ; Bacteriological Techniques ; methods ; Drug Resistance, Bacterial ; Humans ; Isoniazid ; pharmacology ; Mycobacterium tuberculosis ; drug effects ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.
Bacteriological Techniques/methods ; DNA Transposable Elements/genetics ; DNA, Bacterial/analysis/genetics ; Early Diagnosis ; Female ; Gene Amplification ; Humans ; Male ; Multiplex Polymerase Chain Reaction/*methods ; Mycobacterium tuberculosis/genetics/*isolation & purification ; Polymerase Chain Reaction/*methods/standards ; Sensitivity and Specificity ; Tuberculosis/*diagnosis

The evaluation of the quality of a sputum specimen prior to bacterial culture has been an accepted practice. However, optimal sputum criteria for pulmonary tuberculosis (TB) are not well established. We investigated indicators for sputum acceptability in tuberculosis cultures and acid-fast bacilli (AFB) smear. A post-hoc analysis of a randomized trial with 228 sputum specimens from 77 patients was conducted. In the trial, pulmonary TB suspects were requested for collecting three sputum specimens. We performed both TB study (AFB smear and M. tuberculosis culture) and Gram staining in each specimen. By using generalized estimating equations, the association between sputum characteristics and positive TB testings were analyzed. Although acceptable specimens for bacterial pneumonia showed higher TB-culture positive rates than unacceptable specimens (adjusted odds ratio [aOR]=1.66; 95% confidence interval [CI]=1.11-2.49), a specimen with > or =25 white blood cells/low-power field was the better predictor for positive M. tuberculosis cultures (aOR=2.30; 95% CI=1.48-3.58) and acid-fast bacilli smears (aOR=1.85; 95% CI=1.05-3.25). Sputum leukocytosis could be an indicator of sputum acceptability for diagnosing pulmonary tuberculosis.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bacteriological Techniques/*methods ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis/*isolation & purification ; Reproducibility of Results ; Sensitivity and Specificity ; Sputum/cytology/*microbiology ; Tuberculosis, Pulmonary/*diagnosis/*microbiology/pathology ; Young Adult

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.
Acid Etching, Dental ; methods ; Alloys ; chemistry ; Bacterial Adhesion ; physiology ; Bacteriological Techniques ; Biofilms ; Cell Adhesion ; physiology ; Cell Culture Techniques ; Cell Line, Tumor ; Cell Movement ; physiology ; Ceramics ; chemistry ; Coculture Techniques ; Dental Alloys ; chemistry ; Dental Etching ; methods ; Dental Implants ; microbiology ; Dental Materials ; chemistry ; Dental Polishing ; methods ; Humans ; Osseointegration ; physiology ; Osteoblasts ; physiology ; Porphyromonas gingivalis ; physiology ; Prevotella intermedia ; physiology ; Surface Properties ; Titanium ; chemistry ; Yttrium ; chemistry ; Zirconium ; chemistry

BACKGROUND: This study was designed as a quasi-experiment to evaluate automatic inoculation of fecal specimens, using the automated specimen inoculator Previ Isola (bioMerieux, France). METHODS: We evaluated the quality of cultures, recovery rates of enteropathogenic bacteria (Salmonella, Shigella, Campylobacter, and Yersinia species), and cost-effectiveness in terms of technical time. The Previ Isola recovery rates for the two-year period from August 2009 to July 2011 were compared with historical manual inoculation data of the previous two years (August 2007 to July 2009). The regional (Baden-Wurttemberg) and nationwide (Germany) trends of recovery rates for this four-year period were referred. RESULTS: A total of 5,884 fecal specimens were collected over the study period. Most positive cultures were for Salmonella, followed by Campylobacter. Compared with the historical data, the numbers of Campylobacter-positive specimens for a year between August and July were increased significantly, from 19 in 2007-2008 and 10 in 2008-2009 to 32 in 2009-2010 (P=0.002) and 32 in 2010-2011 (P=0.003), respectively. During the study period, the official data for our region and nationwide did not show this increase in the recovery rate of Campylobacter. For Salmonella, Shigella, and Yersinia, no significant changes were observed. Compared with manual inoculation, the mean hands-on time with Previ Isola inoculation was significantly shortened, from 37:30 min to 8:42 min per 15 fecal specimens. CONCLUSIONS: Inoculation by Previ Isola improves the quality of routine culture of fecal specimens, with better sensitivity for Campylobacter and less hands-on time.
Automation ; Bacteria/*isolation & purification ; Bacteriological Techniques/*methods/standards ; Campylobacter/isolation & purification ; Feces/*microbiology ; Humans ; Quality Control ; Salmonella/isolation & purification ; Shigella/isolation & purification ; Yersinia/isolation & purification