ID Test Research Articles

Identification of coagulase-negative staphylococci isolated from caprine milk samples by PCR-RFLP of groEL gene

Coagulase-negative staphylococci (CNS) are the most common cause of intramammary infection (IMI) in small ruminants. In this study, 143 CNS isolates were collected from 1385 sarda goats belonging to 29 herds with high somatic cell count scores. In order to make light of the role of CNS species in goat mastitis, all isolates were subjected to identification with the API Staph ID test, and then to amplification of staphylococcal groEL gene by PCR assay. Amplicons of groEL gene were submitted to restriction fragment length polymorphism analysis with the enzyme AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, groEL gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (53, corresponding to 37%), followed by S. caprae (45, corresponding to 31.4%), S. chromogenes (19, corresponding to 13.2%) and S. simulans (7 corresponding to 4.8%). In conclusion, the PCR-RFLP assay of groEL gene is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing goats mastitis.

P3-S2.04 Clinical evaluation of the BD HSV2 Qx assay for the direct qualitative testing of HSV2 as compared to viral culture and a laboratory-based PCR assay using male and female external anogenital lesions

BackgroundTo compare the performance characteristics of the BD ProbeTecTM HSV2 Qx Assay* on the BD ViperTM System in Extracted Mode to viral culture and a well-characterised molecular assay for the...

P3-S2.03 Clinical evaluation of the BD HSV1 Qx assay for the direct qualitative testing of HSV1 as compared to viral culture and a laboratory-based PCR assay using male and female external anogenital lesions

BackgroundTo compare the performance characteristics of the BD ProbeTecTM HSV1 Qx Assay* on the BD ViperTM System in Extracted Mode to viral culture and a well-characterised molecular assay for the...

Evaluation of the Bactec MGIT 960 System in Combination with the MGIT TBc Identification Test for Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

The sensitivity and specificity of the MGIT TBc identification (TBc ID) test for Mycobacterium tuberculosis complex (MTC) detection in positive Bactec MGIT cultures were 95.2% and 99.2%, respectively. When MTC-positive results obtained from two additional molecular methods were included, the sensitivity of the MGIT TBc ID test was 85.4%, while that of culture was 95.7%.

Evaluation of TBc Identification Immunochromatographic Assay for Rapid Identification of Mycobacterium tuberculosis Complex in Samples from Broth Cultures

Tuberculosis (TB) is a disease of major public health concern worldwide, especially in developing countries. In addition, the human immunodeficiency virus (HIV) epidemic has increased the incidence of infection with nontuberculous mycobacteria (NTM). Rapid, accurate, and simple methods for differentiation of Mycobacterium tuberculosis complex (MTBC) isolates from NTM is greatly needed for successful control of the TB epidemic. This study was done to evaluate the clinical performance of the BD MGIT TBc identification test (TBc ID) for rapid identification of MTBC in samples from broth cultures. A total of 229 Ziehl-Neelsen (ZN) stain-positive MGIT cultures were tested using the TBc ID test, and the results were compared with those of the AccuProbe MTBC identification test (GenProbe, San Diego, CA). The agreement between the TBc ID test and the AccuProbe assay was 96% (kappa = 0.92; confidence interval [CI] = 0.869 to 0.971). The sensitivity, specificity, and negative and positive predictive values of the TBc ID test compared to the AccuProbe assay were 100%, 92.4%, 100%, and 92.2%, respectively. After additional molecular testing, the agreement between the two methods increased to 97.8% (kappa = 0.96; CI = 0.917 to 0.994), and the specificity and positive predictive value increased to 95.6% and 95.7%, respectively. The TBc ID test is a simple, sensitive, and specific test for identification of MTBC in samples from acid-fast bacillus (AFB) smear-positive cultures. The TBc ID test could be a good alternative to the AccuProbe test in TB diagnostic laboratories.

Evaluation of the Rapid MGIT TBc Identification Test for Culture Confirmation of Mycobacterium tuberculosis Complex Strain Detection

A culture confirmation test for the detection of Mycobacterium tuberculosis complex strains that uses a lateral-flow immunochromatographic assay to detect the MPB64 antigen, the MGIT TBc identification (TBc ID) test, has been developed. We evaluated the performance of the TBc ID test in the detection of the M. tuberculosis complex in 222 primary-positive liquid cultures. We compared these results to those of nucleic acid-based identification and conventional biochemical tests. The validity of the TBc ID test was determined, and all of the nontuberculous mycobacteria (NTM) and Nocardia species tested were found to be negative. The detection limit of the TBc ID test was 5 × 10(5) CFU/ml, and for IS6110 real-time PCR it was 5 CFU/ml. All of the M. tuberculosis and M. africanum cultures were found to be positive, while M. bovis and M. bovis BCG cultures were negative. With the exception of 1 contaminated culture, the 221 culture-positive isolates contained 171 (77.5%) M. tuberculosis isolates, 39 (17.6%) NTM species, and 11 (5.0%) unidentified species. Two culture-positive isolates harbored a 63-bp deletion at position 196 of the mpb64 gene. The sensitivity, specificity, positive predictive values, and negative predictive values of the TBc ID test were 98.8, 100, 100, and 95.1%, respectively. Furthermore, the approximate turnaround time for real-time PCR was 4 h (including buffer and sample preparation), while for the TBc ID test it was less than 1 h. We suggest an algorithm for the primary identification of M. tuberculosis in liquid culture using the TBc ID test as an alternative to conventional subculture followed by identification using biochemical methods.

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB). Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene. The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.

Blood stream infections caused by Acinetobacter ursingii in an obstetrics ward

The genus Acinetobacter is an important causative pathogen of nosocomial infections in the healthcare setting. The objectives of this study were to determine the species of causative pathogens and the sources of Acinetobacter blood stream infections that occurred in 2 immunocompetent pregnant women admitted to an obstetrics ward within a 2-month period. Phenotypic identification of the two isolates from blood stream infections was inconsistent among the ID test, the MicroScan WalkAway and the Vitek2 systems. In addition to the growth profile and detailed biochemical analysis, genotypic identification and phylogenetic tree analysis based on the almost complete 16S rRNA sequence and the partial rpoB gene sequence confirmed the identification of these isolates as A. ursingii. Environmental investigation of the obstetrics ward revealed A. ursingii and different strains of Acinetobacter junii in specimens obtained from the ward shower bath, although the source and route of transmission for the A. ursingii infections were not clarified. Our findings show that A. ursingii can inhabit the hospital environment.

Respuesta serológica y protección en terneras revacunadas con cepa 19 en dosis reducida

From 8 dairy farms having more than 20% of brucellosis prevalence, 247 heifer-calves were selected after 2 to 6 months of the traditional immunization withBrucella abortus Strain 19 (S19). Two groups were established in each farm and a total number of 157 animals received a revaccination with 4.5 x 109 CFU of S19 through the conjunctival route, while 90 animals remained unvaccinated.All the animals were bled at days 45, 90, 180, 360, 540 and 720 after revaccination and the sera were tested by SAT and RBT. The reactors to one or both of these tests were retested by a double immunodiffusion test (ID) using aB. abortus polysaccharide antigen to discard the vaccine response and detect a possible natural infecction. No significative differences (p > 0.05) between the control and revaccinated groups were found respect to SAT and/or RBT reactors. It is noticeable that the number of reactors decreased until the day 360 and increased slowly in the 540 and 720 sampling days, concurrent with the appearence of ID reactors. It is concluded that the effect of revaccination proposed did not enhanced the protection of animals during the period of study. At the same time is was showed that the traditional use of S19 did not protect efficiently because some animals became reactors to ID test after 2 to 2 1/2 years of being vaccinated.

Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR–RFLP of 16S rRNA and gap genes

The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR–RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR–RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR–RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis.

Effect of Physico-Chemical Factors on the Disintegration Behavior of Calcareous Shale

Calcareous shale, which is often encountered in construction projects, has a variety of physical and chemical properties that influence disintegration behavior of the rock mass. The diverse behavior of calcareous minerals in shale adds to the complexity of geotechnical investigations. Abundance of calcareous minerals is known to improve shale rock strength, although, when exposed to seasonal wetting and drying cycles, disintegration is rapid. The intent of this study is to statistically evaluate the disintegration behavior of calcareous shales in relation to their physico-chemical properties. Shale samples from fresh rock cuts and talus were tested using a multi-cycle slake durability index (Id), X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) elemental analysis, bulk density, natural water content, and porosity. A relationship was found between the degree of disintegration and the abundance of calcareous minerals, especially calcite and gypsum. Higher porosity and elevated water content were observed in highly weathered shales. Additionally, the efficiency of the Id test as an indicator of the degree of disintegration was evaluated. Grain-size distribution (GSD) analysis of disintegrated talus was compared with fragmental material of two- and five-cycle Id (Id2 and Id5) tests. The Coefficient of Uniformity (Cu) and Coefficient of Curvature (Cc) of Id5 materials showed satisfactory correlation with Cu and Cc of talus material. It is demonstrated that when compared to the conventional Id2 test, multi-cycle Id tests more precisely predict the disintegration pattern of shale and can be used to evaluate shale in terms of degree of disintegration.

Multiplexed SNP detection panels for human identification

SNP profiling is a very powerful tool for human identification. Two panels of 49 and 41 SNPs were selected based on high average heterozygosity (>0.4) and low global Fst values (<0.06) as a set of highly discriminative SNPs suitable for human identification in all geographic populations. SNPs were selected based on testing samples from 44 populations across the globe using TaqMan ® allelic discrimination formats. Coding SNPs, and SNPs with known functional or phenotypic manifestations were excluded. To test the performance characteristics of the two panels of SNPs and to compare their utility to STR analysis for human identification and paternity testing, we genotyped a panel of 41 individuals from three different CEPH families spanning three generations. The test samples were genotyped using the two SNP panels and a 15-loci STR kit. Further, utility of the SNP panels in human ID testing and related applications was demonstrated using degraded DNA and DNA from blood, semen and saliva samples. The development of highly multiplexed SNP detection systems enabling lower cost and higher throughput via automation will result in increased use of SNP profiling in applications like human identification, cell line authentication, species identification and bacterial strain typing.

Characteristics of Environmental Paracoccidioides brasiliensis Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.

A Prospective Study of the Case-Notes of MRSA-Positive Patients: A Vehicle of MRSA Spread

Methicillin-resistant Staphylococcus aureus (MRSA) has received much publicity and remains a major problem for the health service. The aim of this study was to determine whether case-notes of MRSA-positive patients can act as a vehicle for MRSA transmission. A prospective study was performed of patients with active MRSA infection identified from a microbiology department database. Two swabs were taken from the cover of the case-notes and the page with the most recent entry for identification of MRSA using MRSA ID (Chromogenic agar medium), Pastorex and DNase tests. Positive case-notes were re-swabbed 96 h later. A total of 50 MRSA-positive patients were identified from medical, surgical, high dependency and intensive care wards. Three sets of notes were MRSA positive (3/50; 6%). None of the positive case-notes was positive on re-swabbing at 96 h. Case-notes of MRSA-positive patients can act as carriers of MRSA with the potential to spread MRSA to both healthcare and non-healthcare workers without direct contact with infected patients. We recommend extending the universal precautions to the handling of case-notes belonging to MRSA-infected patients.

Initial experience of a commercial donor egg bank

Traditional egg donation, although well-established and effective, is still limited by donor availability, scheduling challenges, high cost and frequent need for travel. The use of frozen donor eggs alleviates many of these problems and additionally allows for egg quarantine and shipment. This report outlines the initial experience of a commercial donor egg bank. Retrospective chart review. Anonymous donors between the age of 21–32 underwent controlled ovarian hyperstimulation followed by egg retrieval and cryopreservation of all MII oocytes using a slow-freeze protocol with sodium-depleted, choline-substituted HTF medium (SAGE IVF). All donors were screened according to FDA regulations for donor eligibility testing and returned for repeat ID testing 6 months after egg retrieval. Recipients of donor eggs underwent endometrial preparation with oral estradiol and vaginal micronized progesterone. A minimum of 8 eggs were thawed per transfer attempt using a rapid-thaw process in the same choline-substituted media, and all surviving eggs inseminated with ICSI. Embryos were transferred on day 3 or day 5 after assisted hatching. A shared-risk model was utilized whereby recipients were granted a second batch of eggs at no cost if a viable pregnancy (26 weeks GA) was not achieved. All subjects signed an IRB-approved informed consent prior to participation in the program. Nineteen donors age 24.9 ± 3.4 years (mean ± SD) underwent 21 egg donation cycles yielding 385 mature eggs (18.3 ± 9.4) for cryopreservation. Fourteen recipients age 44.1 ± 4.0 years underwent 19 embryo transfers. For these transfers, 214 eggs (11.3 ± 1.8 per cycle) were thawed of which 162 (75.7%) survived, 123 (75.9%) fertilized and 91.4% cleaved. An average of 3.7 ± 0.9 embryos were transferred per cycle yielding a clinical pregnancy rate per transfer of 6/19 (31.6%) and an implantation rate of 9/71 (12.7%). Of the 8 recipients who have completed the program, 6 (75%) had a clinical pregnancy. 1) Commercial donor egg banking is a potentially viable alternative to traditional egg donation. 2) Cumulative pregnancy rates comparable to traditional egg donation are possible when a program incorporates a shared-risk model. 3) Until cryopreservation technology ceases to be experimental, commercial donor egg banks should continue to operate under IRB oversight.

Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction–restriction fragment length polymorphism analysis of a partial groEL gene sequence

A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.

Response of healthy dogs to infusions of human serum albumin

To evaluate the clinical and immunologic response in healthy dogs to infusions of human serum albumin (HSA). 9 healthy purpose-bred mixed-breed dogs. Each dog was administered a 25% HSA solution once or twice. Various physical examination and laboratory variables were serially evaluated. Antibody against HSA was assayed before and after infusion by use of an ELISA. Intradermal testing was also conducted. A repeated-measures ANOVA or Friedman repeated-measures ANOVA on ranks was used to compare results for the variables. Adverse clinical reactions were observed after the first or second infusion in 3 dogs. Anaphylactoid reactions were observed in 1 of 9 dogs during the first infusion and in 2 of 2 dogs administered a second infusion. Two dogs developed severe edema and urticaria 6 or 7 days after an initial infusion. All dogs developed anti-HSA antibodies. Positive responses for ID tests were observed in 8 of 9 dogs. Short-term increases were detected in blood protein, total bilirubin, and calcium concentrations after HSA infusion. Serum cholesterol concentrations and platelet counts decreased after HSA infusion. Administration of HSA resulted in profound reactions in 2 of 9 dogs administered a single infusion and in 2 of 2 dogs administered a second infusion. This indicates that there is risk of life-threatening adverse reactions to HSA infusion in healthy dogs.

Comparison of Genotypic and Phenotypic Methods for Species-Level Identification of Clinical Isolates of Coagulase-Negative Staphylococci

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.

Identification Procedure for Pasteurella Pneumotropica in Microbiologic Monitoring of Laboratory Animals

Discrepancies have been recognized in the identification of Pasteurella pneumotropica between testing laboratories. To determine the causes of the differences and to propose a reliable identification procedure for P. pneumotropica, a working group was organized and 69 isolates identified or suspected as P. pneumotropica were collected from 8 laboratories in Japan. These isolates were examined by colony morphology, Gram-staining, the slide agglutination test using two antisera (ATCC35149 and MaR), two commercially available biochemical test kits (ID test, API20NE) and two primer sets of PCR tests (Wang PCR, CIEA PCR). The 69 isolates and two reference strains were divided into 10 groups by test results. No single procedure for P. pneumotropica identification was found. Among tested isolates, large differences were not observed by colony morphology and Gram-straining except for colony colors that depended on their biotypes. Sixty-eight out of 69 isolates were positive by the slide agglutination test using two antisera except for one isolate that tested with one antiserum. The ID test identified 61 out of 69 isolates as P. pneumotropica and there was no large difference from the results of CIEA PCR. From these results, we recommend the combination of colony observation, Gram-straining, the slide agglutination tests with two antisera and biochemical test using the ID test for practical and reliable identification of this organism.

Investigation of gas–solid flow structure after a 90° vertical-to-horizontal elbow for low conveying gas velocities

Abstract The characteristics of gas–solid flow after a 90° vertical-to-horizontal elbow, where the flow in the vertical pipe is upward, were investigated both numerically and experimentally for low conveying gas velocities. The experiments were conducted in a 0.154-m ID test section using pulverized coal particles having a mean particle diameter of 50 μ. The time-averaged local particle concentration and velocity were measured using a fiber optic probe at various locations along the length of the horizontal pipe after the elbow. The effects of gas flow velocity, gas to solids mass flow rate ratio and elbow radius to pipe diameter ratio on particle concentration and velocity profiles, and deposit characteristics were investigated. Computational fluid dynamics simulations were performed to study the particle deposition characteristics. Experiments show that saltation velocity is affected by the gas to solids mass flow rate ratio and elbow radius to pipe diameter ratio. The maximum deposit thickness typically occurred near the leading edge of the deposit. Average deposit thickness decreased with increasing gas to solids mass flow rate ratio. The numerical simulations indicate that the coarse particles move into the rope created in the elbow and secondary flows carry the small particles around the pipe circumference through the particle-free regions.