scholarly journals Comparison of a Commercial DNA Probe Test and Three Cultivation Procedures for Detection of Mycobacterium Paratuberculosis in Bovine Feces

1992 ◽  
Vol 4 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Diana L. Whipple ◽  
Paul A. Kapke ◽  
Phil R. Andersen
Keyword(s):  
Positive Culture ◽  
Culture Method ◽  
Dna Probe ◽  
Fecal Culture ◽  
Mycobacterium Paratuberculosis ◽  
Culture Methods ◽  
Colony Forming Units ◽  
Probe Test ◽  
Centrifugation Method ◽  
Bovine Feces

Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 104 M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.

scholarly journals Comparison of the Lysis Centrifugation Method with the Conventional Blood Culture Method in Cases of Sepsis in a Tertiary Care Hospital

2012 ◽  
Vol 4 (02) ◽  
pp. 089-093 ◽  
Author(s):  
Harshal R Parikh ◽  
Anuradha S De ◽  
Sujata M Baveja
Keyword(s):  
Blood Culture ◽  
Tertiary Care ◽  
Culture Method ◽  
Blood Cultures ◽  
P Value ◽  
Care Hospital ◽  
Culture Methods ◽  
Centrifugation Method ◽  
Considerable Morbidity ◽  
Time Required

ABSTRACT Introduction: Physicians and microbiologists have long recognized that the presence of living microorganisms in the blood of a patient carries with it considerable morbidity and mortality. Hence, blood cultures have become critically important and frequently performed test in clinical microbiology laboratories for diagnosis of sepsis Objectives: To compare the conventional blood culture method with the lysis centrifugation method in cases of sepsis. Materials and Methods: Two hundred nonduplicate blood cultures from cases of sepsis were analyzed using two blood culture methods concurrently for recovery of bacteria from patients diagnosed clinically with sepsis – the conventional blood culture method using trypticase soy broth and the lysis centrifugation method using saponin by centrifuging at 3000 g for 30 minutes. Results: Overall bacteria recovered from 200 blood cultures were 17.5%. The conventional blood culture method had a higher yield of organisms, especially Gram positive cocci. The lysis centrifugation method was comparable with the former method with respect to Gram negative bacilli. The sensitivity of lysis centrifugation method in comparison to conventional blood culture method was 49.75% in this study, specificity was 98.21% and diagnostic accuracy was 89.5%. In almost every instance, the time required for detection of the growth was earlier by lysis centrifugation method, which was statistically significant. Contamination by lysis centrifugation was minimal, while that by conventional method was high. Time to growth by the lysis centrifugation method was highly significant (P value 0.000) as compared to time to growth by the conventional blood culture method. Conclusion: For the diagnosis of sepsis, combination of the lysis centrifugation method and the conventional blood culture method with trypticase soy broth or biphasic media is advocable, in order to achieve faster recovery and a better yield of microorganisms.

A rapid DNA probe test compared to culture methods for identification of subgingival plaque bacteria

2003 ◽  
Vol 30 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Ching Yin Tsai ◽  
Larry F. Wolff ◽  
Greg Germaine ◽  
Jim Hodges
Keyword(s):  
Dna Probe ◽  
Subgingival Plaque ◽  
Culture Methods ◽  
Probe Test

scholarly journals Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 20
Author(s):  
Luigi De Grossi ◽  
Davide Santori ◽  
Antonino Barone ◽  
Silvia Abbruzzese ◽  
Matteo Ricchi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Liquid Culture ◽  
Culture Media ◽  
Small Ruminants ◽  
Culture Method ◽  
Mycobacterium Avium Subsp ◽  
Type I ◽  
Culture Methods ◽  
Specific Pcr ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

scholarly journals Comparing the Yield of Staphylococcus aureus Recovery with Static versus Agitated Broth Incubation

2018 ◽  
Vol 2018 ◽  
pp. 1-3
Author(s):  
Carol E. Muenks ◽  
Patrick G. Hogan ◽  
Carey-Ann D. Burnham ◽  
Stephanie A. Fritz
Keyword(s):  
Staphylococcus Aureus ◽  
Enrichment Culture ◽  
Culture Method ◽  
Ambient Air ◽  
Built Environments ◽  
Culture Methods ◽  
Semiquantitative Assessment ◽  
Direct Plating ◽  
Static Conditions ◽  
Broth Enrichment

Given the lack of standardization of methodologies for microbial recovery from built environments, we sought to compare the yield of Staphylococcus aureus with a broth enrichment method when incubated in agitated versus static conditions. Five unique strains of S. aureus at five different concentrations were cultured to compare direct plating, agitated broth enrichment, and static broth enrichment culture methods. All samples were incubated at 35° in ambient air. The lowest concentration recovered across three replicates and five strains did not differ between culture methods (Fisher’s exact test, p=0.50); notably, recovery of S. aureus was equivalent between static and agitated broth incubation. When broth enrichment was used (both static and agitated), the burden of S. aureus growth was higher (by semiquantitative assessment of 4-quadrant streaking) compared to the direct plating culture method. Optimizing strategies for microbial recovery is essential, particularly in areas of lower biomass, given the paucity of research concerning microbial communities of built environments. The results of this study, in conjunction with other experiments investigating microbiomes of built environments, can help inform protocols for standardizing culturing methods within built environments.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Same-Day Simultaneous Diagnosis of Bacterial and Fungal Infections in Clinical Practice by Nanopore Targeted Sequencing

2020 ◽  
Author(s):  
Ming Wang ◽  
Aisi Fu ◽  
Ben Hu ◽  
Gaigai Shen ◽  
Ran Liu ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Bacterial Infections ◽  
Fungal Infections ◽  
Culture Method ◽  
Targeted Sequencing ◽  
Therapeutic Monitoring ◽  
Rrna Gene ◽  
Culture Methods ◽  
Detection Range ◽  
Sequencing Platform

AbstractBACKGROUNDAs approximately 19% of global deaths are attributable to infectious diseases, early diagnosis of infection is very important to reduce mortality. Traditional infection detection strategies have limited sensitivity, detection range, and turnaround times; a detection technology that can simultaneously detect bacterial and fungal infections within 24 h is urgently need in clinical settings.METHODSWe developed nanopore targeted sequencing (NTS) for same-day simultaneous Diagnosis of fungal and bacterial infections. NTS was developed by amplification of 16s rRNA gene (for bacteria), IST1/2 gene (for fungal), and rpoB (for Mycobacterium spp.) using multiple primers, and sequenced by a real-time nanopore sequencing platform. An in-house bioinformatic analyze pipeline was used to diagnose the infectious pathogens by mapping the sequencing results with the constructed databases.RESULTSComparison of 1312 specimens from 1257 patients using NTS and culture method; NTS detected pathogens in 58.71% of specimens from patients, compared to 22.09% detected using the culture method. NTS showed significantly higher sensitivity than culture methods for many pathogens. Importantly, a turnaround time of <24 h for all specimens, and a pre-report within 6 h in emergency cases was possible in clinical practice. Modification of antibiotic therapy and maintenance of original anti-infection regimens in 51.52% (17/33) and 36.36% (12/33) of patients was in accordance with NTS results, and quantitative monitoring of clinical treatment effects was evaluated in four patients by continuous NTS tests.CONCLUSIONSApplication of NTS in clinically detected pathogens can improve targeted antibiotic treatment and therapeutic monitoring.

Direct detection ofCampylobacter jejuniin human stool samples by real-time PCR

2008 ◽  
Vol 54 (9) ◽  
pp. 742-747 ◽  
Author(s):  
Shiyong Lin ◽  
Xinying Wang ◽  
Haoxuan Zheng ◽  
Zhengguo Mao ◽  
Yong Sun ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Culture Method ◽  
Turnaround Time ◽  
Culture Methods ◽  
Time Saving ◽  
Whole Process ◽  
Pure Cultures ◽  
Stool Samples

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102CFU·mL–1and 103CFU·g–1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5–4 h), so it could be used for clinical diagnostic and epidemiological purposes.

scholarly journals Bovine tuberculosis: Diagnosis by PCR technique in bovine milk samples

2013 ◽  
Vol 37 (2) ◽  
pp. 156-160
Author(s):  
Mohammed J. Alwan
Keyword(s):  
Bovine Milk ◽  
Culture Method ◽  
Bacterial Isolation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Culture Methods ◽  
Milk Samples ◽  
Tuberculosis Diagnosis ◽  
Pcr Method ◽  
Polymerase Chain

     The aim of this study was to determine the percentage of borne tuberculin infection in milk sample by using PCR.  (102) milk samples were collected from  cows , AL-Dejella (39) samples,  AL-Suara (20) samples cow station, AL-Fthalia (20) samples,  AL-Azezia (11) samples and AL-Twarege (12) samples (Iraq) during the period  July 10th   2010 to  Nov.30th   2010. The samples were examined by direct smear stained by Ziehle-Neelson stain, culture methods and confirmed the isolates by Polymerase Chain Reaction (PCR) assay. The results showed that  5, 102 (4.9%) milk samples were M. bovis positive that were detected by direct milk smear method and 10 out 102(9.8%) M.bovis +ve were detected by culture method and PCR assay. The results also showed that high percentage of bacterial isolates from milk samples AL-Dejella city show (12.8%) by culturing and PCR method followed by AL-Suara (10%), AL-Fthelia (10%), Al-Twarege (8.3%) but no bacterial isolation was recorded in AL-Azezia milk samples. This study concluded that M.bovis infection was spreading in dairy cow within the mentioned areas and PCR was more sensitive, rapid, and accurate technique for M.bovis infection diagnosis.

scholarly journals Evaluation of a manual identification system for detection of Mycobacterium tuberculosis in a primary tuberculosis laboratory in China

2019 ◽  
Vol 47 (6) ◽  
pp. 2666-2673 ◽  
Author(s):  
Ping Zhao ◽  
Qin Yu ◽  
Yu Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Performance ◽  
Culture Method ◽  
Cross Sectional Study ◽  
Identification System ◽  
Culture Methods ◽  
Cross Sectional ◽  
Indicator Tube ◽  
Conventional Culture ◽  
Time To Detection

Objective To compare the diagnostic performance of the manual BACTEC™ Mycobacteria Growth Indicator Tube (MGIT™) system (M-MGIT) with the automated BACTEC™ MGIT™ 960 system (A-MGIT) and Löwenstein-Jensen (L-J) culture method in detecting mycobacteria in sputum specimens from patients with suspected pulmonary tuberculosis (TB). Methods For this cross-sectional study, sputum samples were taken from patients aged ≥18 years attending a TB clinic in Beijing, China between July 2015 and October 2016. Processed sputum samples were inoculated into the MGIT systems and L-J medium for up to 6 and 8 weeks, respectively. Results The M-MGIT and A-MGIT methods detected significantly more Mycobacterium tuberculosis complex (MTC) isolates than L-J culture from the 565 sputum samples (39%, 40% and 32%, respectively). Using a positive result from any of the three culture systems as reference, the sensitivity of M-MGIT, A-MGIT and L-J methods were 92%, 94%, and 74%, respectively. The time-to-detection of mycobacteria was 12.9±4.2 days for M-MGIT, 11.8±5.2 days for A-MGIT and 24.2±8.7 days for L-J. Conclusions M-MGIT has a similar diagnostic performance to A-MGIT, and is a fast and reliable alternative to conventional culture methods in the diagnosis of pulmonary TB in a developing country.

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