scholarly journals Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs

1987 ◽  
Vol 98 (3) ◽  
pp. 301-310 ◽  
Author(s):  
Lynne S. Todd ◽  
Diane Roberts ◽  
Barbara A. Bartholomew ◽  
R. J. Gilbert
Keyword(s):  
Enzyme Immunoassay ◽  
Positive Culture ◽  
Advantages And Disadvantages ◽  
Cultural Methods ◽  
Negative Results ◽  
Negative Culture ◽  
Animal Feeding ◽  
Culture Positive ◽  
Initial Results ◽  
Culture Negative

SUMMARYTheSalmonellaBio-EnzaBead Screening Kit, in its modified form with both the MOPC 467 and the 6H4 antibodies, was used for the detection of salmonellas in naturally contaminated foods and animal feeding stuffs in parallel with a traditional cultural procedure.Initial results showed an 82% agreement between the enzyme immunoassay (EIA) and cultural methods when using the criterion recommended by the manufacturer as a cut-off for all types of foods. By adjusting the cut-off for each type of food, the number of EIA positive, culture negative samples was reduced although the number of EIA negative, culture positive samples increased. The EIA may be more sensitive than the cultural methods as in many cases the EIA positive, culture negative results could be real positives which were not detected by the cultural methods.The screening kit provides a simple and convenient method for the detection of salmonella in foods and feeds and a presumptive positive result can be reported within 48 h. The advantages and disadvantages of the method are discussed.

scholarly journals Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Thomas Kellner ◽  
Brendon Parsons ◽  
Linda Chui ◽  
Byron M. Berenger ◽  
Jianling Xie ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Rectal Swab ◽  
Bacterial Pathogen ◽  
Positive Culture ◽  
Content Type ◽  
Negative Culture ◽  
Percent Agreement ◽  
Stool Samples ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTAlthough enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially forSalmonellaspp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen.Salmonellaspp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement forSalmonellaspp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive forSalmonellatested positive by GPP. Specimens GPP positive/culture negative forSalmonellaoriginated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2Campylobacter-positive specimens, 0/4Escherichia coliO157-positive specimens, 0/9Salmonella-positive specimens, and 2/3Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive forSalmonellaspp.

scholarly journals Detection of Antibodies toChlamydia trachomatisWith Peptide-Based Species-Specific Enzyme Immunoassay

1997 ◽  
Vol 5 (5) ◽  
pp. 349-354 ◽  
Author(s):  
Ale Närvänen ◽  
Mirja Puolakkainen ◽  
Wu Hao ◽  
Kohsuke Kino ◽  
Jukka Suni
Keyword(s):  
Enzyme Immunoassay ◽  
Blood Donors ◽  
Positive Culture ◽  
Specific Enzyme ◽  
Antibody Prevalence ◽  
Serum Samples ◽  
Iga Antibodies ◽  
Species Specific ◽  
Culture Positive ◽  
Culture Negative

Objective:We have evaluated the sensitivity and specificity of a new synthetic peptide-based species-specific enzyme immunoassay (EIA) for detection ofChlamydia trachomatisIgG and IgA antibodies.Methods:Synthetic peptides derived from variable domain IV of major outer membrane protein (MOMP) were used as antigen in indirect EIA. IgG and IgA antibodies were measured in parallel with serum samples fromC. trachomatisculture positive, culture negative, and antigen positive patients, and women with suspectedC. trachomatisinfection and blood donors. Sera from children under 15 years of age were used as controls.Results:Culture positive women, culture positive men, and antigen positive women had positive peptide serology in 84.2%, 61.3%, and 93.1% of the cases, respectively. AmongC. trachomatissuspected women, the antibody prevalence was 63.6%. Randomly collected blood donors showed a prevalence of 21.5%. Children withC. pneumoniaeantibodies determined with the microimmuno-fluorescence (MIF) method did not show any reactivity in theC. trachomatispeptide EIA.Conclusions:The results suggest that the new EIA test is highly specific forC. trachomatis, andC. pneumoniaeantibodies do not interfere. Both IgG and IgA antibodies appear within at least 2 weeks in acute phase of infection among both culture positive and culture negative patients.

Prognostic Value Of Blood Cultures as an Illness Severity Marker In Emergency Medical Admissions

2020 ◽  
Vol 19 (2) ◽  
pp. 83-89
Author(s):  
Richard Conway ◽  
◽  
Brian O’Connell ◽  
Declan Byrne ◽  
Deirdre O’Riordan ◽  
...  
Keyword(s):  
Blood Culture ◽  
Prognostic Value ◽  
Positive Culture ◽  
Illness Severity ◽  
Blood Cultures ◽  
Multivariable Logistic Regression Model ◽  
Negative Culture ◽  
Culture Performance ◽  
Culture Positive ◽  
Culture Negative

Background: Positive blood cultures predict mortality. The prognostic value of blood culture performance itself has not been fully defined. Methods: We evaluated medical admissions from 2002-2017. We defined blood culture category as 1) no culture 2) negative culture 3) positive culture. We employed a multivariable logistic regression model to evaluate outcomes. Results: We evaluated 78,568 blood cultures in 106,586 admissions. 30-day in-hospital mortality for no culture was 2.8% (95%CI 2.7, 2.9), culture negative 8.9% (95%CI 8.5, 9.3) and culture positive 16.7% (95%CI 15.5, 17.9). There was significant interaction between blood culture category and illness severity, OR 1.06 (95%CI 1.05, 1.08), and comorbidity, OR 1.09 (95%CI 1.09, 1.10). Conclusion: Performance and results of blood cultures are independently associated with increased mortality.

scholarly journals Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Jie Liu ◽  
Mathieu Almeida ◽  
Furqan Kabir ◽  
Sadia Shakoor ◽  
Shahida Qureshi ◽  
...  
Keyword(s):  
Direct Detection ◽  
Positive Culture ◽  
Accurate Method ◽  
Diagnostic Methods ◽  
Metagenomic Sequencing ◽  
Illumina Hiseq ◽  
Content Type ◽  
Sequence Composition ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe underestimation ofShigellaspecies as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection ofShigellavia a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nineShigellaculture-positive and qPCR-positive (culture+qPCR+) samples, nine culture-negative but qPCR-positive (culture−qPCR+) samples, and nine culture-negative and qPCR-negative (culture−qPCR−) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 108± 5.6 × 107high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to “Shigella” among the three groups. The proportions ofShigella-specific nonhuman sequence reads between culture+qPCR+(0.65 ± 0.42%) and culture−qPCR+(0.55 ± 0.31%) samples were similar (Mann-Whitney U test,P= 0.627) and distinct from the culture−qPCR−group (0.17 ± 0.15%,P< 0.05). The read counts of sequences previously targeted byShigella/enteroinvasiveEscherichia coli(EIEC) qPCR assays, namely,ipaH,virA,virG,ial,ShET2, andipaH3, were also similar between the culture+qPCR+and culture−qPCR+groups and distinct from the culture−qPCR−groups (P< 0.001). Kraken performed well versus other methods: its precision and recall ofShigellawere excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates thatShigella/EIEC qPCR-positive samples are similar to those ofShigellaculture-positive samples inShigellasequence composition, thus supporting qPCR as an accurate method for detectingShigella.

scholarly journals Detection of Ureaplasma urealyticum in Endotracheal Tube Aspirates from Neonates by PCR

1998 ◽  
Vol 36 (5) ◽  
pp. 1236-1239 ◽  
Author(s):  
S. Nelson ◽  
A. Matlow ◽  
G. Johnson ◽  
C. Th’ng ◽  
M. Dunn ◽  
...  
Keyword(s):  
Early Detection ◽  
Endotracheal Tube ◽  
Ureaplasma Urealyticum ◽  
Positive Culture ◽  
Additional Patient ◽  
Sampling Point ◽  
Negative Results ◽  
Amplicon Size ◽  
Culture Positive ◽  
Respiratory Specimens

A PCR-based test was optimized for the detection ofUreaplasma urealyticum from neonatal respiratory specimens, with primers directed against the multiple-banded antigen gene (L. J. Teng, X. Zheng, J. I. Glass, H. Watson, J. Tsai, and G. H. Cassell, J. Clin. Microbiol. 32:1464–1469, 1994). Endotracheal tube aspirates (225) from 103 low-birth-weight neonates (<1,250 g) were taken, when possible, at days 0, 4, and 14 after birth and examined by culture and by PCR. Of 77 specimens positive by either method, 73 were detected by PCR and 60 were detected by culture. Overall, 36% of the neonates were positive for U. urealyticum by either method. Of 16 patients with PCR-positive–culture-negative results, 13 had positive cultures at another sampling point, and one additional patient had a twin with positive cultures. Of 11 patients with day 0 specimens positive by PCR alone, 9 subsequently became culture positive, demonstrating the utility of this test in early detection. Multiple serovars were present in over 50% of positive specimens, with serovars 3 and 14 in combination being most prevalent. The amplicon size generated from the specimen by PCR correctly predicted the biovars isolated in over 85% of positive specimens. Thus, this PCR test was valuable in allowing early detection of U. urealyticum in neonatal respiratory specimens, as well as in providing biovar information.

scholarly journals Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections

2016 ◽  
Vol 54 (9) ◽  
pp. 2262-2266 ◽  
Author(s):  
Nadia Wohlwend ◽  
Sacha Tiermann ◽  
Lorenz Risch ◽  
Martin Risch ◽  
Thomas Bodmer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Positive Culture ◽  
Fecal Calprotectin ◽  
Enteric Pathogens ◽  
Significant Linear Trend ◽  
Content Type ◽  
Culture Positive ◽  
Culture Negative

A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR forCampylobacter jejuni,Campylobacter coli,Salmonellaspp., and shigellosis disease-causing agents (Shigellaspp. and enteroinvasiveEscherichia coli[EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%)Campylobacterspp., 17 (1.6%)Salmonellaspp., and 20 (1.9%)Shigellaspp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle (CT) values of culture-positive specimens were significantly lower than those of culture-negative ones (CTvalues, 24.3 versus 28.7;P< 0.001), indicating that the relative bacterial load per fecal specimen was significantly associated with the culture results. InCampylobacterinfections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative (n =40), PCR-positive/culture-negative (n =14), and PCR-positive/culture-positive (n =15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups (P< 0.001), and a significant linear trend was identified (P< 0.001). Furthermore, the fecal calprotectin concentrations andCTvalues were found to be correlated (r= −0.658). Our results demonstrate that molecular screening ofCampylobacterspp.,Salmonellaspp., andShigellaspp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation.

scholarly journals PCR-Enzyme Immunoassay for Detection ofStreptococcus pneumoniae DNA in Cerebrospinal Fluid Samples from Patients With Culture-Negative Meningitis

1998 ◽  
Vol 36 (12) ◽  
pp. 3605-3608 ◽  
Author(s):  
Thomas Cherian ◽  
M. K. Lalitha ◽  
Anand Manoharan ◽  
Kurien Thomas ◽  
Robert H. Yolken ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Enzyme Immunoassay ◽  
Neurological Disorders ◽  
Latex Agglutination ◽  
Gram Negative Bacteria ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Negative Culture ◽  
Culture Negative ◽  
Prior Antibiotic Treatment

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of Streptococcus pneumoniae and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of S. pneumoniae tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.

scholarly journals False-Positive Xpert MTB/RIF Results in Retested Patients with Previous Tuberculosis: Frequency, Profile, and Prospective Clinical Outcomes

2018 ◽  
Vol 56 (3) ◽  
Author(s):  
Grant Theron ◽  
Rouxjeane Venter ◽  
Liezel Smith ◽  
Aliasgar Esmail ◽  
Philippa Randall ◽  
...  
Keyword(s):  
False Positive ◽  
Positive Culture ◽  
Previous Treatment ◽  
Treatment Completion ◽  
Diagnostic Dilemma ◽  
Negative Results ◽  
Previously Treated Patients ◽  
Positive Results ◽  
Culture Negative

ABSTRACTGlobally, Xpert MTB/RIF (Xpert) is the most widely used PCR test for the diagnosis of tuberculosis (TB). Positive results in previously treated patients, which are due to old DNA or active disease, are a diagnostic dilemma. We prospectively retested sputum from 238 patients, irrespective of current symptoms, who were previously diagnosed to be Xpert positive and treated successfully. Patients who retested as Xpert positive and culture negative were exhaustively investigated (repeat culture, chest radiography, bronchoscopy with bronchoalveolar lavage, long-term clinical follow-up). We evaluated whether the duration since previous treatment completion, mycobacterial burden (the Xpert cycle threshold [CT] value), and reclassification of Xpert-positive results with a very low semiquantitation level to Xpert-negative results reduced the rate of false positivity. A total of 229/238 (96%) of patients were culture negative. Sixteen of 229 (7%) were Xpert positive a median of 11 months (interquartile range, 5 to 19 months) after treatment completion. The specificity was 93% (95% confidence interval [CI], 89 to 96%). Nine of 15 (40%) Xpert-positive, culture-negative patients reverted to Xpert negative after 2 to 3 months (1 patient declined further participation). Patients with false-positive Xpert results had a lower mycobacterial burden than patients with true-positive Xpert results (CT, 28.7 [95% CI, 27.2 to 30.4] versus 17.6 [95% CI, 16.9 to 18.2];P< 0.001), an increased likelihood of a chest radiograph not compatible with active TB (5/15 patients versus 0/5 patients;P= 0.026), and less-viscous sputum (15/16 patients versus 2/5 patients whose sputum was graded as mucoid or less;P= 0.038). All patients who initially retested as Xpert positive and culture negative (“Xpert false positive”) were clinically well without treatment after follow-up. The duration since the previous treatment poorly predicted false-positive results (a duration of ≤2 years identified only 66% of patients with false-positive results). Reclassifying Xpert-positive results with a very low semiquantitation level to Xpert negative improved the specificity (+3% [95% CI, +2 to +5%]) but reduced the sensitivity (−10% [95% CI, −4 to −15%]). Patients with previous TB retested with Xpert can have false-positive results and thus not require treatment. These data inform clinical practice by highlighting the challenges in interpreting Xpert-positive results, underscore the need for culture, and have implications for next-generation ultrasensitive tests.

scholarly journals Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

2020 ◽  
Vol 8 (4) ◽  
pp. 587
Author(s):  
Guerrino Macori ◽  
Siobhán C. McCarthy ◽  
Catherine M. Burgess ◽  
Séamus Fanning ◽  
Geraldine Duffy
Keyword(s):  
Escherichia Coli ◽  
Target Gene ◽  
Positive Culture ◽  
Induction Method ◽  
Negative Results ◽  
Specific Target Gene ◽  
Cultural Isolation ◽  
Two Samples ◽  
Free Particles ◽  
Culture Negative

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

scholarly journals Nested Duplex PCR To Detect Bordetella pertussis and Bordetella parapertussis and Its Application in Diagnosis of Pertussis in Nonmetropolitan Southeast Queensland, Australia

1999 ◽  
Vol 37 (3) ◽  
pp. 606-610 ◽  
Author(s):  
D. J. Farrell ◽  
G. Daggard ◽  
T. K. S. Mukkur
Keyword(s):  
Bordetella Pertussis ◽  
Positive Culture ◽  
Case Definition ◽  
Immunoglobulin M ◽  
Contact Tracing ◽  
Duplex Pcr ◽  
Bordetella Parapertussis ◽  
Negative Results ◽  
Specimen Collection ◽  
Culture Positive

A duplex PCR to detect Bordetella pertussis andBordetella parapertussis was developed with the insertion sequences IS481 (B. pertussis) and IS1001 (B. parapertussis) and evaluated with specimens from 520 consecutive patients presenting with possible pertussis. No culture-positive–PCR-negative results occurred, giving the method a sensitivity of 100%. For B. pertussis, 58 of 520 patients (11.2%) were positive by PCR compared to 17 of 520 patients positive (3.3%) by culture. For B. parapertussis, 7 of 520 patients (1.3%) were positive by PCR compared to 2 of 520 patients positive (0.4%) by culture. Two patients were positive for both B. pertussis and B. parapertussis. Patient records were reviewed to determine the validity of PCR-positive–culture-negative results. Forty-two of 49 patients who could be evaluated fulfilled the criteria for a case definition of pertussis, with 32 patients being <1 year of age and having classical pertussis symptoms. The seven patients who did not fulfil the criteria were aged 7 to 55 years and had a persistent cough for >2 weeks. The method was also used to investigate a classroom outbreak in whichB. pertussis culture was positive for 5 of 28 patients. All five culture-positive specimens were confirmed by PCR, and an additional eight were positive by PCR. Of 25 patients from a suspected pertussis outbreak in a girls’ dormitory, seven of seven specimens were negative for B. pertussis, although 13 of 25 patients were positive for B. pertussis immunoglobulin M (IgM) (2 of which produced equivocal IgA results, with 23 of 25 patients being negative). Five symptomatic patients were subsequently found to be positive (by IgM and particle agglutination assays) forMycoplasma pneumoniae, demonstrating the value of PCR in rapidly excluding B. pertussis infection in an outbreak situation. Twenty-two of 71 (30.1%) throat swabs were positive by PCR compared to 2 of 71 (2.8%) throat swabs positive by culture, indicating that a reassessment of the use of throat swabs should be considered, particularly for older patients, in contact tracing, and in situations in which specimen collection is difficult.

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