Evaluation of clinical specimens for influenza A virus positivity using various diagnostic methods

The diagnostic method for Influenza A virus, utilizing the SERION ELISA Antigen kit (SERION EIA), if results were evaluated according to the manufacturer's instructions, has repeatedly failed to detect a great number of clinical samples positive by virus isolation and RT-PCR. Therefore we compared the SERION EIA with the one-step 44/107L-Px immunocapture enzyme immunoassay (44/107L-Px EIA), developed in our laboratory (Tkácová and Varecková, J. Virol. Methods 60, 65-71, 1996). Seventy-three clinical specimens, of which 65 were positive by virus isolation (used as reference method), were tested by both EIAs. By the SERION EIA, out of the 65 reference-positive samples only 8 (12%) were positive, 5 (8%) were ambiguous, and 52 (80%) were negative, which corresponded to the sensitivity of 12%. On the contrary, the sensitivity of the 44/107L-Px EIA was 74%. However, the calculation of cut-off values for the evaluation of positivity of clinical specimens in these two assays were not the same. If the evaluation procedure used for the 44/107L-Px EIA was applied to the SERION EIA, the sensitivity and the specificity of both EIAs became comparable, namely 71% and 100% for the SERION EIA and 74% and 100% for the 44/107L-Px EIA, respectively. From these results it follows that not the detection ability of the SERION EIA, but the evaluation procedure recommended by its manufacturer led to a loss of large number of positive specimens.     

Automated methods for identification of bacteria from clinical specimens.

Automated methods for measuring enzyme activities of bacterial suspensions in saline are described. The methods were applied to bacteria cultured from urine specimens, and specific enzyme profiles characteristic for Escherichia coli, Klebsiella sp, Proteus sp, and Pseudomonas sp were established. Identification of 294 freshly isolated strains by automated and conventional methods were compared. Results from automated identification based on eight enzyme tests and assay of protein content, all performed on a bacterial suspension made from one colony in 1 ml of saline, agreed 100% with those obtained by conventional methods. Identification was achieved in 6 hours.     

Evaluation of various rapid agglutination methods for the identification of Staphylococcus aureus

A latex agglutination test (SeroSTAT Staph; Scott Laboratories, Fiskeville, R.I.) and two hemagglutination tests (Staphyloslide; BBL Microbiology Systems, Cockeysville, Md.; and Hemastaph; Remel, Lenexa, Kans.) were compared with the slide coagulase (SC) and tube coagulase (TC) tests at room temperature (22 to 25 degrees C) and at 37 degrees C for the rapid identification of Staphylococcus aureus. A total of 380 clinical strains of staphylococci were tested. The TC test performed at room temperature yielded the largest number of TC-positive results (n = 239), and based on this observation 239 organisms were classified as S. aureus and 141 were classified as non-S. aureus. The SC, TC (37 degrees C), SeroSTAT Staph, Staphyloslide, and Hemastaph tests correctly identified 210 (87.9%), 221 (92.5%), 238 (99.6%), 239 (100%), and 236 (98.7%) of the S. aureus isolates, respectively. Of the S. aureus isolates that were TC positive at room temperature 68% required 24 h of incubation before coagulase production was detected. There was one false-negative SeroSTAT Staph result and one false-negative Hemastaph result. The Staphyloslide test yielded two noninterpretable results (both organisms were later confirmed as non-S. aureus), whereas there were six noninterpretable results recorded with the Hemastaph test (four organisms were classified as non-S. aureus, and two were classified as S. aureus). The SeroSTAT Staph, Staphyloslide, and Hemastaph tests were all more sensitive than the conventional SC and TC (37 degrees C) tests and were considerably more rapid than the TC test at either temperature.     

Comparison of three commercial media for direct identification and discrimination ofCandida species in clinical specimens

One hundred and ninety-two clinical specimens were used to compare the three chromogenic media Albicans ID, Candiselect, and CHROMagar Candida to a standard method using a Sabouraudch-loramphenicol agar medium and standard methods for identification of yeasts. The detection rates were 83.79, 83.24, 86.59 and 84.91 % respectively. For the chromogenic media, the rates of direct identification (growth plus specific pigmentation) forCandida albicans were 56.50, 37.68 and 11.59% after 24 hours' incubation and 92.75, 91.30 and 88.57% after 72 hours' incubation respectively, with 100% specificity. Furthermore, CHROMagar Candida identified the fiveCandida tropicalis and the twoCandida krusei strains detected after 48 hours' incubation.     

Comparative study using phenotypic, genotypic, and proteomics methods for identification of coagulase-negative staphylococci

Five methods were compared to determine the most accurate method for identification of coagulase-negative staphylococci (CoNS) (n = 142 strains). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed the best results for rapid and accurate CoNS differentiation (99.3% of strains correctly identified). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing (100% of strains correctly identified when both methods are performed simultaneously).     

Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens

In 1972 there were only 11 genera and 26 species in the family Enterobacteriaceae. Today there are 22 genera, 69 species, and 29 biogroups or Enteric Groups. This paper is a review of all of the new organisms. It has a series of differential charts to assist in identification and a large chart with the reactions of 98 different organisms for 47 tests often used in identification. A simplified version of this chart gives the most common species and tests most often used for identification. The sources of the new organisms are listed, and their role in human disease is discussed. Fourteen new groups of Enterobacteriaceae are described for the first time. These new groups are biochemically distinct from previously described species, biogroups, and Enteric Groups of Enterobacteriaceae. The new groups are Citrobacter amalonaticus biogroup 1, Klebsiella group 47 (indole positive, ornithine positive), Serratia marcescens biogroup 1, and unclassified Enteric Groups 17, 45, 57, 58, 59, 60, 63, 64, 68, and 69.     

Comparison of the Bactec and lysis concentration methods for recovery ofBrucella species from clinical specimens

In a comparison of the Bactec system and the lysis concentration procedure in the isolation ofBrucella species in 54 patients the recovery rate was similar (60 % and 55 %, respectively). However, the recovery time was significantly shorter with the lysis concentration method than with the Bactec system (3.5 days versus 14 days). The lysis concentration procedure for the culture ofBrucella is simple, inexpensive and reliable, and produces results for the clinician relatively quickly.     

Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis.

A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha-demethylase (L1A1) gene fragment that encodes a highly conserved region was used to detect yeast DNA in clinical specimens. Positive PCR products were obtained from genomic DNAs of Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) glabrata, and C. kefyr. No human, bacterial, or parasitic DNA was amplified. The sensitivity was evaluated for C. albicans genomic DNA by using various DNA concentrations (200 pg to 2 fg). The amplified DNAs of Candida species with unknown P-450 L1A1 gene sequences were subcloned and sequenced. Identification at the species level was achieved by digestion of the PCR products with different restriction enzymes. A specific restriction enzyme analysis pattern was determined for each species investigated. Subsequently, we used PCR to detect specific yeast DNA directly with clinical specimens such as blood and bronchoalveolar lavage specimens. After appropriate treatment, the specimens were processed by PCR and the results were compared with those obtained by traditional diagnostic procedures such as cultures and serology. Although preliminary, the PCR results seem to correlate well, at least for blood, with those of antigen detection assays and traditional blood cultures, with a better and earlier detection of candidemia.     

Comparison of four methods for identification of gram-negative non-fermenters: Organisms less commonly encountered in clinical specimens

Four commercial kits - Oxi/Ferm (OF), API 20E (AP), Minitek (MT), Flow N/F (NF) were evaluated, without additional tests, for identification of 105 opportunistic Gram-negative non-fermentative rods. OF correctly identified 42% of strains, with 35% as part (but not first) of a spectrum of identifications (SI) and 23% incorrect identification. MT yielded 75% correct identification, with 12% SI and 13% incorrect. AP correctly identified 64% of strains, with 26% SI, 10% incorrect. NF correctly speciated 70% of strains, with 24% SI, 6% incorrect. All 4 methods show deficiencies in identification of these rare but increasingly clinically encountered organisms. Addition of new tests/modification of existing ones would render these systems more capable of identifying this organisms group.     

Four methods for identification of gram-negative nonfermenting rods: organisms more commonly encountered in clinical specimens.

Four commercial kits, Oxi/Ferm (OF), API 20E (AP), Minitek (MT; BBL Microbiology Systems), and Flow N/F (NF), were evaluated, without additional tests, for identification of 258 gram-negative nonfermentative rods. OF and MT were read after 48 h of incubation, and AP and NF were read after both 24 and 48 h of incubation, respectively. Overall, OF correctly identified 51% of strains, with 46% as part (but not first) of a spectrum of identifications (SI), and 3% incorrect species identification. MT yielded 85% correct identification, with 15% SI. Of 126 glucose-positive strains, or those with greater than or equal to 3 positive AP reactions after 24 h, 60% were correctly identified, with 40% SI; incubation for an additional 24 h raised the rate of correct identification to 99%, with 1% SI. A total of 132 strains yield less than 3 positive AP reactions after 24 h and were identified after 48 h only; of these, 82% were correctly identified, with 17% SI and 1% incorrect species identification. NF correctly identified 79% of cultures after 24 h, with 21% SI; corresponding figures after an additional 24 h of incubation were 80% and 20%, respectively. All four commercial methods show promise; OF is easiest to inoculate, but requires extra tests for optimal identification. AP reliably identifies the majority of clinically important nonfermenters, with fairly good species identification of saccharolytic strains after 24 h. MT yields reliable identification of most nonfermenters and has the advantage of flexibility. NF is easy to inoculate, yields satisfactory identification rates, and may be read after 24 h of incubation.     

Availability of immunostaining methods for identification of mixed-up tissue specimens

The immunostaining method of ABO blood determinants in tissue sections is useful when surgical tissue specimens, submitted for diagnosis, are improperly identified. This article reports two cases in which the surgical specimens became mixed-up and the ABO immunostaining method was used to identify the patients' tissues correctly and prevent unnecessary surgery.     

Comparative study of various API galeries for the identification of gram negative bacteria

116 strains of Gram negative bacteria were identified with the use of API ATB 32 GN and Rapid 20 E galeries, in order to evaluate their performance as compared with API 20 E or API NE galeries used as reference. The identification concur (bacterial genus and species) in approximately 80 per cent of the cases. There are only 2 major discrepancies with ABT 32 GN galleries and only one with Rapid 20 E. In other cases, the profile that is obtained only permits identification of the genus but without a sufficient differentiation, requiring a study of additional characteristics or repetition of the test. These galeries enable to solve most routine diagnosis problems due to Gram negative bacteria, with the advantage of Rapid (Rapid 20 E) or automatized reading (ATB 32 GN).     

rpoB gene sequence-based identification of Staphylococcus species

The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.     

Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with special reference to urinary tract infection in catheterized patients

Various clinical specimens were processed to find the prevalence rate of enterococci and to identify the species of clinical isolates of enterococci. Screening of various clinical specimens revealed that enterococci were prevalent in 22.19% of the total specimens, with Foley's catheters and burn wounds to be the major site of isolation. High rate of colonization was noted as opposed to infection. Conventional test scheme proposed by Facklam and Collins were successfully used to speciate enterococcal strains. Seven species of enterococci were identified in the study from a set of 202 cultures, with E.faecalis (49.50%) and E. faecium (35.64%) predominating. E. avium (9.40%), E. hirae (2.47%), E. raffinosus (1.98%) and one isolate each of E.gallinarum and E. casseliflavus were the other members of Enterococcus species identified. Urinary tract infection (UTI) by enterococci due to catherisation was found in 8.92% of the patients and is probably the result of high rate of colonization of Foley's catheters and use of broad-spectrum antibiotics.     

Comparative study of three methods of identification of Enterobacteriaceae.

Three separate hospital clinical microbiology laboratories using three different identification systems participated in the identification of Enterobactericeae from a central pool of 'unknown" clinical isolates. With conventional tubed media, API-20E (Anlytab Products Inc.) and R/B tube (Corning Diagnostics) systems, there was a 91.1% agreement in the species designation. No significant differences at the 95% confidence level were found among the systems. Evaluation of individual tests within the systems used revealed lysine decarboxylase of the conventional and citrate of the API-20E system to be significantly different from the same test within the other two systems. The lysine decarboxylase of the conventional system had species relatedness, whereas the differences in citrate of the API-20E system were not related to a particular species. These individual test variations did not affect final organism identification. Reproducibility, evaluated as the system's ability to designate the same identification on two separate occasions, was 92 to 94% for each system. Exact duplication of selected sets of reactions was 60% for conventional, 45% for API-20E, and 61% for R/B. The variations in sets of reactions differed with the system and with the organism involved. The findings suggest equivalency among the three systems in ability to identify common clinical isolates of Enterobacteriaceae and point out the limited usefulness of these systems for biochemical biotyping.     

Simplified scheme for routine identification of human Staphylococcus species.

From a total of 40 characters that were previously used to differentiate species of staphylococci, 13 key characters were selected to make a simplified scheme that could be easily used by the routine clinical laboratory for identifying human staphylococci. These key characters included coagulase activity, hemolysis, nitrate reduction, and aerobic acid production from fructose, xylose, arabinose, ribose, maltose, lactose, sucrose, trehalose, mannitol, and xylitol. In the simplified scheme, 924 strains of staphylococci were placed into 11 positions, each of which contained the major portion (greater than or equal to 80%) of strains of one of the recognized species. Several positions contained a rare or few uncommon strains of one or more additional species and these could be resolved on the basis of other key characters.     

Evaluation of API An-IDENT and RapID ANA II systems for identification of Actinomyces species from clinical specimens.

We compared the accuracy of the An-IDENT system (bioMerieux Vitek, Inc., Hazelwood, Mo.) and the RapID ANA II system (Innovative Diagnostic Systems, Norcross, Ga.) with that of conventional biochemical tests for the identification of 85 strains of Actinomyces species. In our hands, the overall accuracy of the An-IDENT was 59% and that of the RapID ANA II was 24%. The error rate for the An-IDENT was 18% and that for the RapID ANA II was 38%. The results of this study suggest that although the An-IDENT was more accurate than the RapID ANA II (P < 0.005), neither system, in our hands, was able to identify Actinomyces species with an acceptable degree of accuracy. It is recommended that suspected Actinomyces isolates be identified by conventional testing.