Evaluation of methods for the rapid identification of Neisseria gonorrhoeae in a routine clinical laboratory.

Of 78 isolates of Neisseria gonorrhoeae, 21 failed to grow and produce acid in unsupplemented cystine-Trypticase agar (CTA); whereas positive reactions were obtained by using serum-supplemented CTA and fluorescent antibody (FA). An additional 290 strains of Neisseria were evaluated by FA and by a rapid carbohydrate degradation technique (RF). There was agreement between the two methods 92% of the time on the initial trial and 99% of the time with repeats on discrepancies. The RF and FA tests provided rapid and reliable identification of N. gonorrhoeae, alleviating the problems of CTA due to lack of growth and need for overnight incubation.     

Yeast identification in routine clinical microbiology laboratory and its clinical relevance

Rapid identification of yeast infections is helpful in prompt appropriate antifungal therapy. In the present study, the usefulness of chromogenic medium, slide culture technique and Vitek2 Compact (V2C) has been analysed. A total of 173 clinical isolates of yeast species were included in the study. An algorithm to identify such isolates in routine clinical microbiology laboratory was prepared and followed. Chromogenic medium was able to identify Candida albicans, C. tropicalis, C. krusei, C. parapsilosis and Trichosporon asahii. Chromogenic medium was also helpful in identifying "multi-species" yeast infections. The medium was unable to provide presumptive identification of C. pelliculosa, C. utilis, C. rugosa, C. glabrata and C. hemulonii. Vitek 2 compact (V2C) differentiated all pseudohypae non-producing yeast species. The algorithm followed was helpful in timely presumptive identification and final diagnosis of yeast infections, including multi-species yeast infections.     

Distribution and incidence of viridans streptococcal species in routine clinical specimens

Five hundred consecutive isolates of viridans streptococci were identified to the species level in an effort to determine their distribution and incidence in routine clinical specimens. Viridans streptococci accounted for significant percentages of streptococcal isolates from urine, wounds, body fluids, and blood. The most commonly isolated strains belonged to the Streptococcus milleri, Streptococcus mitis, Streptococcus sanguis I, and Streptococcus sanguis II species. Patient charts were reviewed in order to investigate the possible role as a urinary pathogen of strains belonging to a subgroup of S. milleri. Although these strains frequently are isolated from urine, they appear to play no pathogenic role in urinary tract infections.     

Molecular method for Bartonella species identification in clinical and environmental samples

A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.     

Molecular method for identification of Rickettsia species in clinical and environmental samples

We present a PCR method targeting the 23S-5S internal transcribed spacer coupled with reverse line blotting that allows Rickettsia species detection and identification in a single step. The method is highly sensitive and specific in identifying Rickettsia species from both patient and environmental samples. The generic approach used allowed us to identify new pathogens.     

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory

European Journal of Clinical Microbiology & Infectious Diseases - The study evaluates the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)...     

Proposed tests for the routine identification ofRochalimaea species

A study was conducted to establish tests for the routine identification ofRochalimaea species. Strains used were reference strains ofRochalimaea vinsonii andRochalimaea quintana, and a type strain and six human isolates ofRochalimaea henselae. Rochalimaea species were confirmed to be gram-negative, oxidase-negative, non-motile, urease-negative, indole-negative, catalase-negative, glucose-nonfermenting organisms which failed to grow on MacConkey agar. Further testing of the organisms in a commercial identification system with the addition of hemin (100 µg/ml) to the medium revealed biochemical reactivity of the organisms not previously observed. The Voges-Proskauer reaction, tests for hydrolysis of hippurate and esculin, leucine arylamidase activity and the lactose test allowed identification and differentiation of the three species.Rochalimaea henselae was the only species with a positive lactose test andRochalimaea quintana was the only species with a positive Voges-Proskauer reaction. Further studies are needed to confirm the validity of these tests for identification ofRochalimaea species.     

Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples

The aim of this study is to assess the usefulness of a simple, low-cost method for the detection and species identification of Leishmania isolated by in vitro culture or detected directly from clinical samples. A total of 110 samples were used in this study. Among these, 21 were human and canine peripheral bloods, 63 skin lesion material samples, eight reference strains and 18 Leishmania culture. Detection of Leishmania DNA with PCR using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently sensitive at the level of 0.1 parasites per PCR reaction. Furthermore, followed by single-strand conformational polymorphism (SSCP), the PCR-ITS1 allowed the species identification of Leishmania. The inter-specific polymorphism of Leishmania was first validated on reference strains, and then this method was applied on clinical samples and culture. Typing identified all human and canine visceral leishmaniasis samples (21 samples) as L. infantum, 95.23% of the cutaneous leishmaniasis samples as L. major and 3.17% as L. killicki and 1.58% as L. infantum. A scheme of the PCR diagnosis procedure for the detection and identification of Leishmania parasites is proposed in this study.     

Re-utilization of clinical laboratory patient-derived samples

Our hospital has traditionally placed a portion of Clinical Laboratories patient-derived samples into temporary storage for purposes of re-examination. Occasionally, following a brief review, a part of these samples were provided not only for research but also for quality control. However, recent trends to protect confidential personal information have made it difficult to offer patient-derived samples to those who perform research and quality control even after a strict and complicated review of their requests. Because of recent progresses in the various "-omics" such as genomics, proteomics, and/or metabolomics, a strong need of patient-derived samples for research exists. Therefore, we have developed and, since 2002, provided a "Bio Sample Bank" to smoothly and efficiently expedite provision of patient-derived samples for research and quality control purposes. So far we have collected more than 130,000 samples. We wish to disclose our experiences concerning this bank and to develop interest in the importance of our system and its significant benefits to clinical procedures and patients.     

Evaluation of API NH, a new 2-hour system for identification of Neisseria and Haemophilus species and Moraxella catarrhalis in a routine clinical laboratory.

API NH is a new 2-h system (bioMérieux, La Balme-les-Grottes, France) for the identification of most Neisseria and Haemophilus spp. of clinical significance and of Moraxella catarrhalis and for the detection of penicillinase production. Furthermore, this system allows the biotyping of Haemophilus influenzae and Haemophilus parainfluenzae. Three hundred eighteen strains belonging to these species, previously identified by conventional methods, were tested. Among the 305 strains belonging to species included in the data base, 225 (73.8%) were identified without additional tests, 79 (25.9%) were correctly identified after extra tests, and 1 strain (0.3%) was misidentified. For 131 (90.3%) of the 145 H. influenzae and H. parainfluenzae strains, results of biotyping were in agreement with results of standard methods. API NH is an accurate and reliable method for the routine identification of these bacteria in a clinical laboratory, for biotyping of Haemophilus spp., and for the detection of penicillinase-producing strains. The system is ready to use and time saving; inoculation of the system and reading of results are easy.     

Detection of extended spectrum beta-lactamases in the routine clinical microbiology laboratory

AIMS: To compare three methods of confirming the presence of an extended spectrum beta-lactamase (ESBL) enzyme with the initial detection (i.e., screening) by the Vitek AMS. METHODS: Gram-negative bacteria which flagged as ESBL-positive in the Vitek GNS card, or were suspected of harbouring an enzyme, were further tested by each of the following methods: (a) combination disc test using cefpodoxime, ceftazidime and cefotaxime with and without clavulanate; (b) cefotaxime ESBL Etest; and (c) Jarlier keyhole method with cefpodoxime (10 microg), cefotaxime (5 microg) and aztreonam (30 microg) placed 15mm away from an augmentin (30 microg) disc. RESULTS: A total of 52 isolates were investigated, representing an 18-month time period. Fifty of these were positive by Vitek. Twenty-eight (56%) were confirmed by other methods (true positives). Of the 44% Vitek-positive/confirmatory test-negative (false positives), eight were Escherichia coli which was 53% of all E. coli tested. The majority of other false-positive isolates were Klebsiella oxytoca (24% overall) which were all Vitek- and Etest-positive but negative by the combination disc test. CONCLUSIONS: All ESBL-positive strains by Vitek should be confirmed by the combination disc test using all three antibiotics. This will enable differentiation of 'true' ESBLs from false-positive organisms, including K1 hyperbetalactamase-producing Klebsiella oxytoca and AmpC-producing organisms. The cefpodoxime combination discs gave the best differentiation in this study with only one ESBL organism being missed.     

Application of adrenocorticotropin assays in a routine clinical laboratory.

The radioimmunoassay of ACTH was used in a routine laboratory to localize the site of the lesion in 20 patients with Cushing's syndrome. Eight of the patients had no detectable circulating ACTH and had adrenal tumors removed, 12 had high levels and were diagnosed as having pituitary Cushing's syndrome. Very high levels of plasma ACTH were found in eight patients who had primary adrenal insufficiency, while ACTH was undetectable in ten patients with secondary hypoadrenalism. The routine use of this assay in endocrinology should reduce the hospitalization of patients under investigation for disorders of the pituitary--adrenal axis. Eight patients who had the ectopic ACTH syndrome and carcinoma of the lung were found to have very high levels of ACTH with no diurnal variation. Forty-seven patients with oat-cell carcinoma but without evidence of the ectopic ACTH syndrome had normal ACTH levels. A possible role of ACTH and other peptide hormones as tumor markers is mentioned.     

Performance of the new VITEK 2 GP card for identification of medically relevant gram-positive cocci in a routine clinical laboratory

The VITEK 2 gram-positive (GP) identification card (bioMerieux, Marcy l'Etoile, France) has been redesigned to achieve greater accuracy in the identification of gram-positive cocci. A total of 43 biochemical tests, including 17 enzymatic tests, are present in the card and interpreted in a kinetic mode, for up to 8 h. The VITEK 2 database, used in conjunction with the GP identification card, allows the identification of 115 different taxa. A total of 364 strains of GP cocci (217 Streptococcaceae strains and 147 Micrococcaceae strains) belonging to 31 taxa were tested with the new VITEK 2 GP identification card. Of the 364 strains, 105 were taken from routine primary plating media. A total of 344 strains (94.5%) were correctly identified to the species level and 17 strains (4.7%) were identified with low discrimination, requiring additional tests, whereas 1 strain (0.3%) was incorrectly identified and 2 strains (0.5%) remained unidentified. Within 7 h of the start of incubation, more than 90% of all strains were identified. Of the 105 primary cultures, 97% were correctly identified to the species level, 2% were identified with low discrimination, and 1% remained unidentified. Identification performance data were independent of each of the three plating media used. It is concluded that the new VITEK 2 GP identification card provides reliable results for the identification of GP cocci under routine laboratory conditions.     

Biochemical identification of citrobacteria in the clinical laboratory.

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).     

Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.     

Evaluation of the LOGIC system for the rapid identification of members of the family Enterobacteriaceae in the clinical microbiology laboratory.

The LOGIC identification system (J.D. Perry, M. Ford, N. Hjersing, and F.K. Gould, J. Clin. Pathol. 41: 1010-1012, 1988) for gram-negative rods, performed with microdilution plates instead of tubes, was evaluated for strains isolated from all tested specimens except those obtained from stools (for which a different strategy was followed). Systematically extended with the oxidase test and lactose fermentation, it proved to be very reliable and extremely simple and economical.     

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.