Ann Croft

Evaluation of Partial 16S Ribosomal DNA Sequencing for Identification of Nocardia Species by Using the MicroSeq 500 System with an Expanded Database

ABSTRACT Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i) conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii) molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii) when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Optimization of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis for Bacterial Identification

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.

Application of SmartGene IDNS Software to Partial 16S rRNA Gene Sequences for a Diverse Group of Bacteria in a Clinical Laboratory

ABSTRACT Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories.

Performance of the Phoenix bacterial identification system compared with disc diffusion methods for identifying extended-spectrum β-lactamase, AmpC and KPC producers

Phenotypic identification of AmpC, KPC and extended-spectrum beta-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam +/- clavulanic acid (CA) and cefpodoxime +/- CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC(+), but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC(+) isolates, all of which tested as ESBL(+) or ESBL(+) AmpC(+) by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.

Spotting the Spirochete: Rapid Diagnosis of Leptospirosis in Two Returned Travelers

Leptospirosis is a zoonosis caused by a ubiquitous spirochete of the genus Leptospira and is endemic to the tropics. Human infection occurs through contact with soil or water that has been contaminated with infected animal urine. Fresh water recreational activities (swimming, rafting, and kayaking) in contaminated lakes and rivers have been associated with infection, especially during flood stage. 1‐4 Prompt diagnosis and treatment are important in reducing the morbidity and mortality that can be associated with this illness. The most common method of diagnosis is a 4-fold rise in antibody titers; 5 however, this does not allow for a rapid diagnosis in the acute phase. Two cases were recently diagnosed at our facility with the aid of dark-field microscopy and subsequently confirmed by culture.We propose that this technique may be used effectively in the prompt detection of spirochetes, leading to timely management decisions. Case 1: A 35-year-old white female spent 3 weeks traveling in Ecuador. She spent 2 weeks kayaking in mountain streams at flood stage and the following week kayaking on the coast. There she developed diarrhea that resolved with a short course of ciprofloxacin. Three weeks after returning to the United States, she presented with a 3-day history of fever, chills, nausea,vomiting,myalgias,and headache associated with new onset diarrhea and abdominal cramps. Her initial examination was remarkable only for right upper and lower abdominal tenderness. Her laboratory studies are summarized in the Table. She was empirically treated with oral ciprofloxacin and sent home. She returned to the clinic the following day with persistent symptoms,intense headache, fever, facial flushing, bilateral conjunctival suffusion, and a tense abdomen. A lumbar puncture revealed normal cerebrospinal fluid (CSF). As she now appeared ill, she was admitted to the hospital and treated empirically with intravenous (IV) ampicillin and levofloxacin. Blood cultures that were obtained at the clinic visit were processed for dark-field microscopy by the centrifugation procedure described in the 7th edition of the Manual of Clinical Microbiology. 6 Three of the four blood cultures had visible spirochetes. The residual pellet was inoculated to Fletcher’s medium for culturing leptospira. Meanwhile, the patient was continued on IV ampicillin and supportive care. Her symptoms resolved within 2 days and she was discharged to home on day 3 with amoxicillin. Case 2: A 36-year-old white female had had a neardrowning experience with extensive ingestion of fresh water while she was white-water rafting in Costa Rica. Tw elve days after returning to the United States, she presented as an outpatient with high fevers, chills, myalgias, nausea, and right upper quadrant abdominal pain. After obtaining blood for testing (including cultures for Leptospira spp), she was treated with oral doxycycline. Two days later, her symptoms persisted and she presented to the emergency room. She appeared ill and was unable to tolerate oral antibiotics. She was subsequently admitted and treated with IV doxycycline. Her laboratory results are also summarized in the Table. Her blood was also processed for dark-field microscopy and was found to be positive for spirochetes. These results were reported to the clinicians within 5 hours of the request. She was continued on IV doxycycline and discharged on the third day with a 3-week course of oral doxycycline.