AccuProbe Test Research Articles

Identification of Mycobacterium avium and Mycobacterium intracellulare using three DNA probe tests, and their distributions in Japan

Identification of Mycobacterium avium complex (MAC) was made using three DNA probe tests for MAC: Gen-Probe Rapid Diagnostic System for the MAC (Gen-Probe Inc., San Diego, U.S.A.), AccuProbe MAC Culture Identification or Confirmation Test (Gen-Probe Inc.); and SNAP Culture Identification Diagnostic Kit (MAC) (Syngene Inc., San Diego, U.S.A.). Various strains of MAC belonging to serovars 21 to 28 were identified by the DNA probe tests and showed the following. First, Serovar 21 and 25 belonged to M. avium and M. intracellulare, respectively. Each of them reacted with species-specific probes used in the three DNA probe tests [i.e., either M. avium-probe (in SNAP test; Probe A) or M. intracellulare-probe (in SNAP test; Probe I)]. Second, serovars 22-24 and 26-28 consisted of M. intracellulare, MAC strains that reacted with Probe X of SNAP test but lacked the reactivity with M. avium- and M. intracellulare probes of all the DNA probe tests, M. scrofulaceum that showed no reactivity with M. avium- or M. intracellulare-probe or Probe X, and M. scrofulaceum that had only the reactivity with Probe X. When the disease-associated MAC strains (35 strains), isolated in the Kanto to Kyushu areas in Japan, were identified using AccuProbe test, both the M. avium and M. intracellulare strains identified by the Gen-Probe test reacted with the MAC-probe but not with the M. tuberculosis complex (MTC)-probe.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Coccidioidomycosis in India: Report of a third imported case

We describe the third fatal case of imported coccidioidomycosis in India in a 31-year-old mechanical engineer originally from Andhra Pradesh, India, who lived in Gwinner, North Dakota. He had traveled to Arizona in summer of 2006, where he drove tractors in a dusty environment at a tractor production facility near Phoenix, Arizona. He was human immunodeficiency virus (HIV) positive. Initially, he was treated in Fargo, North Dakota, in 2006, with liposomal amphotericin B followed by oral fluconazole. Antiretroviral treatment for HIV infection was started. He moved back to India and was admitted to the intensive care unit of St. John's Medical College and Hospital, Bangalore, India. His blood cultures yielded Coccidioides sp. The identity of the isolate was confirmed using the Gen Probe Accuprobe test at the Centers for Disease Control and Prevention, Atlanta, Georgia. In spite of initiation of treatment with antifungal agents (amphotericin B and fluconazole), his condition deteriorated and he expired three days following his admission to the hospital.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

Frequency of tuberculous cervical lymphadenitis detection at a single laboratory in islamabad.

To determine the frequency of tuberculous cervical lymphadenitis and to evaluate the diagnostic efficacy of microscopy and conventional Lowenstein Jensen (LJ) culture technique in the diagnosis of cervical lymphadenitis caused by M. tuberculosis (MTB) Study Design: A descriptive, cross-sectional study. Place and Duration of the Study: Department of Mycobacteriology, Public Health Laboratories Division, National Institute of Health, Islamabad, from January 2003 to December 2004. A total of 220 patients from Pakistan Institute of Medical Sciences (PIMS), Islamabad, Federal Government Services Hospital (FGSH), Islamabad and Rawalpindi General Hospital (RGH), Rawalpindi, presenting with enlarged cervical lymph nodes (for at least six months), pain/ weight loss and low grade fever were studied for the presence of MTB from 142 lymph node biopsies, 60 FNA samples and 18 discharge fluids/swabs. All the samples were examined at NIH by ZN staining smear and culture on conventional LJ medium as well as on Bactec 12B medium using Bactec 460 TB system. The drug susceptibility testing of the isolates was performed on Bactec 460 TB system. NAP test on Bactec 460-TB system, Accuprobe and biochemical tests were employed to identify the mycobacterial isolates. M. tuberculosis accounted for 173 out of 220 cases of cervical lymphadenopathy. Maximum incidence was found to be in the age group 10-30 years with male to female ratio of 1:1.7. Discharge sinuses and abscess formation were uncommon. Biopsy tissue samples gave the maximum yield of positive mycobacterial cultures as all 142 biopsy samples being positive while only 50% (30/60) of FNA and 5.5% (1/18) of discharge fluids/swabs were positive. All the isolates were identified as M. tuberculosis. No atypical mycobacteria were recovered from the samples examined. All isolates were found to be susceptible to first line anti-tuberculous drugs i.e. Streptomycin, Isoniazid, Rifampicin and Ethambutol (SIRE). Tuberculosis was the major cause of cervical lymphadenopathy in the referral area. Culture of the biopsy tissue from the affected lymph nodes is a method of choice for laboratory diagnosis of tuberculous cervical lymphadenopathy.

Isolation of Nontuberculous Mycobacteria Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

The isolation rate of nontuberculous mycobacteria (NTM) in clinical laboratories and the incidence of NTM infections are on the increase recently in Korea, but there have been only a few studies that reveal the general aspect of NTM isolation or species distribution. Therefore, this study was performed to examine the isolation rate of NTM, species identification, and the clinical significance of mycobacterial cultures. From August 2004 to May 2005, we examined mycobacterial isolates by AccuProbe test to differentiate NTM from Mycobacterium tuberculosis complex. NTM was then identified by polymerase chain reaction-restriction fragment length analysis (PCR-RFLP). A total of 6,742 specimens from 2,784 patients were requested for mycobacterial culture. Mycobacteria were isolated from 776 specimens (11.5%). The isolation rates of NTMs among the total culture positive specimens and culture positive sputum specimens were 24.4% (189/776) and 25.3% (169/667), respectively. Fourteen species of NTM identified in 172 of the 175 specimens tested included M. avium (39.0%), M. intracellulare (22.7%), and M. abscessus (19.8%). Using AccuProbe tests and PCR-RFLP method for mycobacterial cultures processed in a clinical laboratory, we were able to identify NTMs to the species level. The isolation rate of NTM in this study was similar to that reported in past studies in Korea. In addition, we found that some of the NTMs isolated in this study could cause pulmonary diseases.

Direct Identification of Gram-Positive Cocci from Routine Blood Cultures by Using AccuProbe Tests

Rapid and reliable identification of bacteria directly from blood cultures is important in clinical practice to guide appropriate antibiotic therapy. In this study, the performance of the AccuProbe (Gen-Probe, Inc., San Diego, Calif.) in direct identification of Staphylococcus aureus, Streptococcus pneumoniae, enterococci, and group A and B streptococci from positive blood culture bottles was evaluated by using 6-year routine clinical laboratory blood culture material from Paijat-Hame Central Hospital, Lahti, Finland. With the enterococcal and group A and B streptococcal probes, the diagnostic performance of the test was excellent at a cutoff value of 50,000 relative light units (RLU) as recommended by the manufacturer. However, with the S. aureus probe, although the specificity was very high (99.8%), the sensitivity was low (72.4%). To improve the clinical usability of the direct AccuProbe identification, optimal cutoff values for the individual AccuProbe tests were defined by using receiver-operating characteristic analysis. Consequently, cutoff values for S. aureus and S. pneumoniae tests were adjusted to 30,000 RLU and for enterococci and to 55,000 RLU for group A and B streptococci. With these adjustments, the performance of the AccuProbe tests, especially that for S. aureus, was significantly improved.

Confirmation of nontypeable Streptococcus pneumoniae-like organisms isolated from outbreaks of epidemic conjunctivitis as Streptococcus pneumoniae.

Eleven isolates representing five distinct outbreaks of pneumococcal conjunctivitis were examined for phenotypic and genetic characteristics. None of the strains possessed capsules, and all strains were susceptible to optochin, bile soluble, and Gen-Probe AccuProbe test positive. All 11 isolates were confirmed as Streptococcus pneumoniae by DNA-DNA reassociation experiments.

Comparison of Gen-Probe AccuProbe Group B streptococcus culture identification test with conventional culture for the detection of Group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women☆

The performance of the AccuProbe Group B Streptococcus Culture Identification Test (Gen-Probe Incorporated, San Diego, CA, USA) for the detection of group B streptococci (GBS) directly from LIM broth cultures of vaginal-anorectal swab specimens from pregnant women (two swabs per patient in most cases) was evaluated by comparing results to those of conventional GBS culture. Of 411 specimens analyzed, 82 were positive and 312 were negative for GBS by both methods. After initial testing, the percent agreement was 95.9%. The initial sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 90.1%, 97.5%, 91.1%, and 97.2%, respectively. Results were discrepant for 17 specimens: eight were GBS positive by probe and negative by culture; nine were negative by probe and positive by culture. To resolve discrepancies, culture plates were re-examined for GBS colonies, AccuProbe testing was repeated on the initial LIM broth cultures, and the second swab (if received) was inoculated to LIM broth for AccuProbe testing after overnight incubation. After discrepant resolution testing, the percent agreement between the two test methods was 97.8%. The final sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively. These data suggest that the AccuProbe test is a reliable method for detecting GBS in vaginal-anorectal specimens, providing results more rapidly than conventional culture. However, strict adherence to the manufacturer’s test protocol is necessary to limit technical errors.

Advances in bacteriological diagnosis

Microbiological diagnosis of urogenital tuberculosis, traditionally based on the results from bacterioscopy and cultures, may sometimes be insufficient apart from requiring a particularly long time (4–8 weeks). In an attempt to reduce the times of response and to improve sensitivity, new methods have been put forward using DNA probes in culture media (fast methods) and nucleic acid amplification systems (ultra-fast methods). The authors describe the various methods and report their experience using the fast (AccuProbe) and the ultra-fast methods (MTBDA). They still consider the culture examination as the gold standard, but times of response may be reduced (10–15 days) using the AccuProbe test. The ultra-fast methods MTBDA may be put to good use when there is a clinical and/or radiological suspicion of tuberculosis with a negative culture or in cases where a rapid differential diagnosis is necessary.

Detection of Mycobacterium tuberculosis in mixed broth cultures using DNA probes.

OBJECTIVE: To investigate the ability of a commercial acridinium ester-labeled DNA probe (AccuProbe, Gen-Probe Inc., USA) to detect the presence of Mycobacterium tuberculosis complex in a mixed culture with Mycobacterium avium complex. METHODS: The density of organisms required to produce a positive result for Mycobacterium tuberculosis complex alone in broth culture was compared with the density required to produce a positive result in the presence of Mycobacterium avium complex. RESULTS: A threshold density of 1.5 x 106 CFU/mL was required for detection of Mycobacterium tuberculosis and this threshold remained unaltered in the presence of Mycobacterium avium complex. The presence of Mycobacterium avium complex had no effect on detection of Mycobacterium tuberculosis in a mixed broth culture incubated and probed over a 21-day period. CONCLUSIONS: The findings of the study suggest that the presence of Mycobacterium avium complex has no effect on the detection of Mycobacterium tuberculosis and that the Accuprobe test is potentially capable of detecting a dual infection with organisms of both complexes.

Pooling of Noncollaborative Multilaboratory Data for Evaluation of the Use of DNA Probe Test Kits in Identifying Listeria monocytogenes Strains

The Accuprobe test kit, a DNA probe culture confirmation test kit for identifying bacterial isolates as Listeria monocytogenes was evaluated with 148 food-borne Listeria isolates. The identities of the isolates were confirmed by conventional tests. A subset of the Listeria isolates was also used to evaluate the Gene-Trak Listeria monocytogenes isolation kit. The data obtained with each kit were pooled with data for that kit from independent published studies. Interlaboratory performances were then estimated statistically, to achieve the main features of orthodox interlaboratory collaborative studies. The large number of strains required in this noncollaborative evaluation method advantageously provided a more representative sampling of the potential isolates that may be found in practice. Exact comparability between the two kit studies was not possible nor is it a feature of orthodox collaborative identification kit studies. However, the kits apparently performed equivalently given the different statistical confidences dictated by the study designs.

Genotypic characterization of five subspecies of Mycobacterium kansasii.

Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.

Direct identification of Staphylococcus aureus in blood cultures by detection of the gene encoding the thermostable nuclease or the gene product.

This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bacterium or the nuc gene encoding it. The protein was detected by an enzyme diffusion test in o-toluidine blue DNA agar with a test time of at least 4 h, by a monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay (sELISA) with a test time of approximately 4 h, and by a MAb-based sandwich enzyme-linked immunofiltration assay (sELIFA) with a test time of 25-30 min (sample preparation included). The nuc gene was amplified by a polymerase chain reaction (PCR) with a test time (amplification plus detection) of approximately 3.5 h. The tests were optimized for direct examination of blood-containing cultures. All tests were positive with 67/67 blood cultures which grew S. aureus, negative with 35/35 cultures which grew coagulase-negative staphylococci, and negative with 37/37 cultures with various other bacteria. These results showed positive agreement with those of the commercial AccuProbe test but not with the StaphAurex agglutination kit. With an artificially seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc-PCR, 9.5 h; sELIFA, 12.5 h; enzymatic test, 16-36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mecA gene in all the coagulase-negative staphylococci (48.6%) which showed oxacillin resistance. The sELIFA had the particular advantage of its short test time, the PCR its high sensitivity and the possibility of simultaneous detection of the species-specific nuc gene and genes encoding other clinically important characters of the bacteria. These tests offer the prospect of direct application to a variety of clinical specimens for rapid diagnosis of S. aureus infection.

Inhibitory effects of enrichment media on the Accuprobe test for Listeria monocytogenes.

During an evaluation of the Accuprobe kit for the detection of Listeria monocytogenes, some of the enrichment media used were found to interfere with the test. Microscopic examination during the lysis step of the test revealed that media containing high salt greatly reduced or prevented cell lysis. This prevented the probe from binding to the cellular RNA, resulting in false-negative results.

Use of acridinium-ester-labeled DNA probes for identification of mycobacteria in Bactec 13A blood cultures

AccuProbe tests for mycobacteria (Gen-Probe) cannot be performed directly on Bactec 13A cultures because of interfering substances. This problem can be circumvented by subculturing to Bactec 12B media. Artifactual chemiluminescence greater than the positive cutoff was seen in 15 of 19 13A cultures containing Mycobacterium avium complex compared with 0 of 19 in 12B subcultures. Of the subcultures, 89% were tested after overnight incubation.

Strains of Mycobacterium terrae complex which react with DNA probes for M. tuberculosis complex

Following a recent report that two isolates of Mycobacterium terrae complex had given positive reactions with M. tuberculosis complex DNA probes, a joint study was undertaken to determine the extent of these findings in the clinical culture collection holdings of two state health laboratories. A total of 117 M. terrae complex strains (identified by standard biochemical methods) were subjected to M. tuberculosis complex probe testing with the two then-available kits (from Syngene, Inc., and Gen-Probe, Inc.). In addition to the two original isolates first reported, two further M. terrae complex isolates were found to react with the M. tuberculosis complex probes. Two modifications of the Accuprobe (Gen-Probe, Inc.) test method were evaluated. Extension of the selection time to 8 min was the most convenient modification and rendered the M. terrae complex isolates negative when tested with the Accuprobe M. tuberculosis complex probe. However, the effects of increased selection time on the overall sensitivity of the M. tuberculosis complex probe require further study.

Comparison of monoclonal antibody methods and a ribosomal ribonucleic acid probe test forNeisseria gonorrhoeae culture confirmation

Recently, a chemiluminescent nucleic acid probe test that specifically detects the ribosomal ribonucleic acid of Neisseria gonorrhoeae has been released for clinical laboratory use (AccuProbe Neisseria gonorrhoeae). In this study, three coagglutination tests (GonoGen I, Meritec GC, and GC Omni), the GonoGen II immunofiltration method and the Micro Trak Neisseria gonorrhoeae fluorescent monoclonal antibody test were compared with AccuProbe for identification of gonococci. Strains tested (n = 376) included 194 Neisseria gonorrhoeae, 82 Neisseria meningitidis, 32 Neisseria lactamica, 32 Neisseria species, 32 Moraxella catarrhalis, 2 Moraxella spp. and 2 Kingella denitrificans. The GonoGen I, Meritec GC and GC Omni coagglutination tests produced clearly positive results for 93.8%, 92.3% and 95.9% of the gonococci, respectively. The GonoGen II unequivocally identified 91.8% and the MicroTrak fluorescent antibody test identified 90.7% with 2+ or greater fluorescence. AccuProbe identified 100% of the gonococci tested. GonoGen I and GonoGen II were 98% specific, Meritec GC was 99% specific and the specificity of the GC Omni, MicroTrak fluorescent antibody and AccuProbe tests was 100%. While antibody-based tests were reliable when results were clearly interpretable, the AccuProbe was the only confirmatory test that was 100% accurate. Serotyping studies indicate that an array of beta-lactamase positive and negative gonococcal serotypes fail to react with the monoclonal antibody-based tests in general and with the fluorescent antibody test in particular.

Use of chemiluminescent DNA probes in the rapid detection of oxacillin resistance in clinically isolated strains of Staphylococcus aureus

Chemiluminescent DNA probes (AccuProbe, species specific; and FlashTrack, bacterial generic) were used to determine oxacillin resistance in Staphylococcus aureus. Ribosomal RNA was measured at designated intervals in the presence and absence of antibiotic. A total of 48 (AccuProbe assay) and 24 (FlashTrack) S. aureus isolates with known oxacillin susceptibility patterns were inoculated into Bactec 6A bottles both with and without 4 μg/ml oxacillin and incubated at 35°C for 4 h. Aliquots were removed at 0 and 4 h, and pellets of bacteria were obtained via selective centrifugation. Probe assay counts (relative light units, RLUs) were performed. Of 21 oxacillin-resistant S. aureus (ORSA) strains, 20 showed a >5-fold RLU increase during the incubation period (AccuProbe assay): 25 of 27 oxacillin-susceptible strains demonstrated a ⩽4-fold increase. AccuProbe test sensitivity and specificity were 95% and 92%, respectively. With the generic FlashTrack probe assay, all nine ORSA isolates showed a ⩾4-to 10-fold increase in RLUs, and all 15 oxacillin-susceptible strains showed a ⩽4-fold increase in RLUs during the 4-h incubation. The FlashTrack test sensitivity and specificity were both 100%. Probe assays were completed within 5 h. This study suggests that rapid and reliable determination of oxacillin resistance in S. aureus clinical isolates can be accomplished using commercially available DNA probes.

Identification of Streptococcus pneumoniae with a DNA probe

The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae.