Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda.

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.

Identification of histoplasma-specific peptides in human urine.

Histoplasmosis is a severe dimorphic fungus infection, which is often difficult to diagnose due to similarity in symptoms to other diseases and lack of specific diagnostic tests. Urine samples from histoplasma-antigen-positive patients and appropriate controls were prepared using various sample preparation strategies including immunoenrichment, ultrafiltration, high-abundant protein depletion, deglycosylation, reverse-phase fractions, and digest using various enzymes. Samples were then analyzed by nanospray tandem mass spectrometry. Accurate mass TOF scans underwent molecular feature extraction and statistical analysis for unique disease makers, and acquired MS/MS data were searched against known human and histoplasma proteins. In human urine, some 52 peptides from 37 Histoplasma proteins were identified with high confidence. This is the first report of identification of a large number of Histoplasma-specific peptides from immunoassay-positive patient samples using tandem mass spectrometry and bioinformatics techniques. These findings may lead to novel diagnostic markers for histoplasmosis in human urine.

Exploratory study of proteins in urine of patients with histoplasma antigenuria.

Disseminated histoplasmosis is an invasive fungal infection that can be fatal in patients with weak immune system. The goal of our exploratory study was to evaluate differences in urinary protein profiles among samples of healthy individuals, patients with proteinuria (PRU), and histoplasma antigenuria (HIS), and to identify physiological pathways associated with the excreted proteins. Urine samples were depleted of abundant proteins, deglycosylated, digested with trypsin, fractionated and analyzed by nano-LC-QTOF. The total number of human proteins identified in the samples was 117, of which 20 and 23 were unique to the samples from patients with PRU and HIS, respectively. Pathway analysis of proteins identified in samples of PRU and HIS patients suggested increased levels of proteins associated with acute response signaling, coagulation system, prothrombin activation, glucocorticoid regulation and the lipid antigen presentation signaling pathway networks. The obtained data provide information on protein expression associated with HIS, and suggest that further more rigorous studies aimed at the identification of proteins associated with proteinuria of different causes are feasible.

Comparison of traditional phenotypic identification methods with partial 5' 16S rRNA gene sequencing for species-level identification of nonfermenting Gram-negative bacilli.

Correct identification of nonfermenting Gram-negative bacilli (NFB) is crucial for patient management. We compared phenotypic identifications of 96 clinical NFB isolates with identifications obtained by 5' 16S rRNA gene sequencing. Sequencing identified 88 isolates (91.7%) with >99% similarity to a sequence from the assigned species; 61.5% of sequencing results were concordant with phenotypic results, indicating the usability of sequencing to identify NFB.

Performance characteristics of a polyclonal enzyme immunoassay for the quantitation of Histoplasma antigen in human urine samples.

An antigen detection assay was prepared with rabbit anti- Histoplasma antibodies to detect and quantitate Histoplasma capsulatum antigen in urine samples. By using a 4-parameter curve fit, the assay calibration ranges from 2 to 1,000 enzyme immunoassay (EIA) units. We compared results for 99 urine samples with those of a reference laboratory, half of which tested positive or equivocal by that reference laboratory. Performance characteristics were further defined by studying the assay linearity, precision, percentage of positive agreement, and percentage of negative agreement. An acceptable correlation with the reference laboratory (R2=0.7174) was obtained with results ranging from less than 2 (negative samples) to 132 EIA units by the new method. Compared with the reference laboratory, the percentage of agreement for positive samples, excluding equivocal samples, was 92% and for negative samples was 98%. Crossreactivity occurred with culture filtrates of Paracoccidioides brasiliensis, Coccidioides immitis, and Blastomyces dermatiditis. No cross-reactivity was observed with Candida albicans or Aspergillus fumigatus culture filtrates. The current EIA for the detection and quantitation of H capsulatum antigen in urine specimens is a valid assay that agrees well with results from an established reference laboratory.

Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens.

Several Mycobacterium-like organisms related to the Mycobacterium terrae complex have been isolated from clinical samples. In the clinical microbiology laboratory, partial 16S rRNA gene sequencing (approximately the first 500 bp) rather than full 16S rRNA gene sequencing is often used to identify Mycobacterium species. Partial 16S rRNA gene sequence analysis revealed 100 % similarity between 65 clinical isolates and Mycobacterium sp. MCRO 6 (GenBank accession no. X93032). Even after sequencing the nearly full-length 16S rRNA gene, closest similarity was only 99.6 % to Mycobacterium nonchromogenicum ATCC 19530(T). Sequencing of the nearly full-length 16S rRNA gene, the 16S-23S internal transcribed spacer region and the hsp65 gene did not reveal genotypic identity with the type strains of M. nonchromogenicum, M. terrae or Mycobacterium triviale. Although sequence analysis suggested that these clinical isolates represented a novel species, mycolic acid analysis by HPLC failed to distinguish them from M. nonchromogenicum. Therefore, phenotypic analysis including growth characterization, antibiotic susceptibility testing and biochemical testing was performed. These strains from clinical samples should be recognized as representing a novel species of the genus Mycobacterium, for which the name Mycobacterium arupense sp. nov. is proposed. The type strain is AR30097(T) (=ATCC BAA-1242(T) = DSM 44942(T)).

Use of the MGB Eclipse system and SmartCycler PCR for differentiation of Mycobacterium chelonae and M. abscessus.

Although accurate in the identification of Mycobacterium species, partial 16S rRNA gene sequencing does not distinguish Mycobacterium chelonae from M. abscessus. Thus, we designed a SmartCycler PCR assay targeting the 16S-to-23S internal transcribed spacer (ITS) region with use of MGB Eclipse probes to distinguish each species. Comparison with PCR-restriction enzyme analysis of a 441-bp fragment of the hsp65 gene resulted in 100% correlation with 25 isolates of M. chelonae and 25 isolates of M. abscessus. ITS PCR performed on 90 consecutive isolates identified by partial 16S rRNA gene sequencing (26 isolates of the M. chelonae-M. abscessus complex and 64 remaining isolates, including Mycobacterium species, Nocardia species, and other aerobic actinomycetes) showed 100% specificity and sensitivity. The ITS PCR assay is accurate and specific, easy to perform, and a good supplemental test when using partial 16S rRNA gene sequencing to identify M. chelonae and M. abscessus.

Interpretive criteria for use of AccuProbe for identification of Mycobacterium avium complex directly from 7H9 broth cultures.

Rapid identification of Mycobacterium avium complex (MAC) is possible by use of AccuProbe (Gen-Probe, San Diego, Calif.). To evaluate the reliability of the MAC AccuProbe for testing 7H9 cultures inoculated with broth from MGIT cultures positive for acid-fast bacilli or growth on a solid medium, we compared probe results to results obtained by sequencing a portion of the 16S rRNA gene. Isolates were sequenced if the MAC probe result was <100,000 relative light units (RLU) or if it was > or =100,000 RLU and the colony morphology was not classic or there were two colony types. For the 1,389 cultures tested in phase 1, conducted to evaluate cutoff values for the MAC probe in testing of 7H9 cultures inoculated with broth from MGIT cultures, the sensitivity and specificity of the MAC AccuProbe were 97.7% and 88.8%, respectively, according to the manufacturer's interpretive criteria (> or =30,000 RLU is positive). If the cutoff for a positive result were 80,000 RLU, the specificity would be 100% and the sensitivity 92.3%. Of the 344 isolates in phase 2, which was conducted to confirm the 80,000-RLU cutoff for a positive result and therefore included only isolates with a MAC probe result of < or =100,000 RLU, 13 of 16 with results of > or =30,000 but <80,000 RLU were identified as mycobacteria other than MAC, including five Mycobacterium tuberculosis complex isolates. These data support the use of 80,000 RLU as the cutoff for a positive result in testing of 7H9 broth cultures with the MAC AccuProbe.

Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction.

Rapid species identification within the Mycobacterium chelonae-abscessus group by high-resolution melting analysis of hsp65 PCR products.

Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonae-abscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.

Evaluation of a modified gen-probe amplified direct test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid.

Laboratory evidence for tuberculous meningitis is difficult to acquire due to the low numbers of organisms present in cerebrospinal fluid (CSF) and the presence of nucleic acid amplification inhibitors. The Amplified Mycobacterium tuberculosis Direct Test (MTD) is sensitive and specific for the direct detection of M. tuberculosis complex in respiratory samples but has not been approved for CSF. We evaluated a modified version of the current MTD, optimized for use with CSF samples. Samples were prepared by spiking CSF with various numbers of M. tuberculosis complex organisms. The modified MTD performance was compared with results obtained using a purified RNA sample extracted using the Qiagen RNeasy Protect Bacteria Mini Kit. By use of CSF artificially spiked with M. tuberculosis complex, the sensitivity of the modified MTD was 100% (six of six) for CSF samples containing approximately 600 CFU/ml, 78% (seven of nine) for approximately 60 CFU/ml, 50% (three of six) for 6 CFU/ml, and 17% (one of six) for samples with <1 CFU/ml. The specificity of the modified MTD method was 100% (22 of 22). The sensitivity of the Qiagen MTD method was 100% for CSF samples containing approximately 600 CFU/ml (six of six) and approximately 60 CFU/ml (nine of nine), 50% for samples with approximately 6 CFU/ml (three of six), and 50% for samples with <1 CFU/ml (three of six). The specificity of the Qiagen MTD method was 86% (19 of 22). With the Qiagen MTD method, however, initial results were equivocal for 14 of the 27 (52%) positive samples, requiring repeat analysis, whereas with the modified MTD, only 1 of 27 (4%) was equivocal. The modified MTD for CSF samples was less time-consuming and less expensive and resulted in considerably fewer equivocal results than the Qiagen MTD method did.

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

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.

Evaluation of RIDOM, MicroSeq, and Genbank services in the molecular identification of Nocardia species.

The molecular identification of Nocardia species, when compared to phenotypic identification, has two primary advantages: rapid turn-around time and improved accuracy. The information content in the 5'-end of the 16S ribosomal RNA gene is sufficient for identification of most bacterial species. An evaluation was performed to demonstrate the quality of results provided by two specialized databases (RIDOM and MicroSeq 500 versions 1.1 and 1.4.3, library version 500-0125, respectively) and the more general GenBank database. In addition, these results were compared with phenotypic identifications. Partial 5'-16S rDNA sequences from 64 culture collection strains (DSM, CIP, JCM, and ATCC) were derived, in duplicate, independently in two laboratories. Furthermore, the sequences and the conventional identification results of 91 clinical Nocardia isolates were determined. With the exception of N. soli and N. cummidelens, all Nocardia type strains were distinguishable using 5'-16S rDNA sequencing. Assuming a normal distribution for the pairwise distances of all unique Nocardia sequences and choosing a reporting criterion of > or = 99.12% similarity for a "distinct species", a statistical error probability of 1.0% can be calculated. When the various databases were searched with the clinical isolate sequences RIDOM gave a perfect match in 71.4% of cases whereas MicroSeq yielded a perfect match in only 26.4%. The GenBank service gave a 100% similarity in 59.3% but in 70.4% of these cases the results obtained were not exclusive for a single Nocardia species. Conventional methods gave a correct identification in 59 cases, although most recent taxonomic changes were not taken into account. The RIDOM service (http://www.ridom-rdna.de/) is in the process of making available a comprehensive and high-quality database for bacterial identification purposes and provides excellent results for the majority of Nocardia isolates.

Description of a multiplex Bordetella pertussis and Bordetella parapertussis LightCycler PCR assay with inhibition control.

While culture for Bordetella species is highly specific, sensitivity is extremely variable due to patient age, immunization status, antibiotic treatment, and specimen transport conditions. We evaluated a real-time multiplex PCR assay as an alternative to culture for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis. The PCR conditions allowed the simultaneous detection of one B. pertussis organism and five B. parapertussis organisms per reaction. An inhibition control was incorporated into the assay. Of 163 total samples evaluated, 37 of 38 samples positive by either culture or direct fluorescent antibody testing (DFA) were also positive by PCR (97% sensitivity). Of 125 culture- or DFA-negative samples, 101 were also negative by PCR (81% specificity). The described multiplex assay is a rapid, sensitive, contamination-limiting, real-time PCR assay that controls for inhibition. The assay performs well using liquid or swab samples and from dried material on slides.

Impact of nasopharyngeal swab types on detection of Bordetella pertussis by PCR and culture.

A study was designed to assess the performance of various swabs and transport media routinely used to collect specimens submitted for Bordetella culture and PCR. Calcium-alginate swabs inhibited the PCR. No inhibition was detected in any PCRs with dacron or rayon swabs. All swab materials performed similarly for recovery of Bordetella pertussis in culture. The Amies with charcoal transport system performed poorly for culture. Calcium-alginate swabs are not recommended for PCR-based detection of B. pertussis. Dacron and rayon swabs are an excellent choice for both PCR and culture.