Rapid Detection of Pathogenic Fungi from Clinical Specimens Using LightCycler Real-Time Fluorescence PCR

In the study presented here a LightCycler real-time PCR system was used for the diagnosis of fungal infections from clinical tissue samples. Nine specimens were investigated from six patients with suspected or proven invasive fungal infections. Seven of nine samples were positive in a broad-range fungal PCR assay. In four samples, Aspergillus fumigatus was detected both by a species-specific hybridization assay as well as by sequencing of amplification products. In addition, the broad-range fungal PCR assay and PCR sequencing detected and identified, respectively, the following organisms in the specimens noted: Candida albicans in a culture-negative liver biopsy, Histoplasma capsulatum in a bone marrow sample, and Conidiobolus coronatus in a facial soft tissue specimen. Real-time PCR is a promising tool for the diagnosis of invasive fungal infections in human tissue samples and offers some advantages over culture methods, such as rapid analysis and increased sensitivity.     

Comparison of Sample Preparation Methods for Detection of Legionella pneumophila in Culture-Positive Bronchoalveolar Lavage Fluids by PCR

A prospective study was conducted on 25 Legionella pneumophila culture-positive and 98 culture-negative bronchoalveolar lavage fluid samples to compare two DNA preparation methods: a rapid modified Chelex-based protocol and a proteinase K method. PCR was found to be more sensitive with the Chelex-based method (P = 0.03). No difference was found concerning the inhibition rate.     

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.     

Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10² Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.     

Identification of Mycobacterium avium subsp. paratuberculosis in Biopsy Specimens from Patients with Crohn's Disease Identified by In Situ Hybridization

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient Mycobacterium avium subsp. paratuberculosis in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS900 as a probe.     

Detection,Identification, and Distribution of Fungi in Bronchoalveolar Lavage Specimens by Use of Multilocus PCR Coupled with Electrospray Ionization/Mass Spectrometry

As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.     

Detection of non-jejuni and -coli Campylobacter Species from Stool Specimens with an Immunochromatographic Antigen Detection Assay

The STAT! Campy immunochromatographic assay for Campylobacter antigen was compared to culture for 500 clinical stool specimens. Antigen was detected in six culture-negative, PCR-positive specimens. C. upsaliensis, a pathogenic species that is traditionally difficult to recover in routine stool cultures, was detected in two of these culture-negative specimens. This study provides evidence that antigen testing may cross-react with at least one additional non-jejuni and -coli Campylobacter species that may be missed by routine culture for campylobacteriosis.     

Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of Bordetella pertussis

We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis.     

Comparison of BD GeneOhm Cdiff and Seegene Seeplex ACE PCR Assays Using Toxigenic Clostridium difficile Culture for Direct Detection of tcdB from Stool Specimens

We evaluated the performances of 2 PCR assays (BD GeneOhm and Seegene ACE) for direct detection of tcdB from stool specimens. The concordance rate between BD and Seegene was 96.3%. The sensitivities, specificities, positive predictive values (PPVs), and negative predictive values (NPVs) of BD and Seegene were 95.7%, 96.5%, 91.8%, and 98.2% and 90.0%, 97.1%, 92.6%, and 96.0%, respectively.     

Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.     

Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species,Legionella pneumophila,and Legionella pneumophila Serogroup 1

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.     

Comparison of PCR, Nested PCR, and Random Amplified Polymorphic DNA PCR for Detection and Typing of Ureaplasma urealyticum in Specimens from Pregnant Women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5′ end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P = 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.     

Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures

A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques.Bloodstream infections (BSI) caused by Gram-negative bacilli (GNB) are associated with significant mortality representing roughly 22% of all BSI (1, 4). In recent years, increased multidrug resistance in these pathogens has highlighted the importance of rapid identification and appropriate therapy (2, 3, 6). Conventional phenotypic and biochemical methods for identification of GNB bacteremia routinely involve Gram staining and subculturing from positive blood cultures followed by testing with biochemical and/or automated devices and may require 1 to 3 days to complete. The peptide nucleic acid fluorescence in situ hybridization (PNA FISH) platform provides identification of select GNB within hours. Three FDA-cleared GNB PNA FISH assays (AdvanDx, Woburn, MA) are currently available: Escherichia coli PNA FISH (single-color assay), E. coli/Pseudomonas aeruginosa PNA FISH (dual-color assay distinguishing E. coli and P. aeruginosa), and EK/P. aeruginosa PNA FISH (dual-color assay distinguishing P. aeruginosa from E. coli and/or Klebsiella pneumoniae).The purpose of this study was to compare the performances of shortened procedures for E. coli/P. aeruginosa PNA FISH and EK/P. aeruginosa PNA FISH to the original procedures and to conventional laboratory identification techniques.A total of 368 GNB-positive blood cultures bottles from four U.S. sites were evaluated. All samples were deidentified remnant clinical specimens; routine identification results were left blinded until PNA FISH testing was completed. Two automated microbial blood culture detection systems were used: BacT/Alert 3D (bioMérieux, Durham, NC) and Bactec 9240 (BD, Cockeysville, MD). The GNB-positive BacT/Alert bottles included 49 standard aerobic (SA) and 51 standard anaerobic (SN). GNB-positive Bactec bottles included 61 standard/10 aerobic, 56 standard anaerobic, 62 plus aerobic, 35 plus anaerobic, 41 lytic/10 anaerobic, and 13 Peds Plus. The study protocol was approved by the institutional review board at each institution.Samples were analyzed with both E. coli/P. aeruginosa and EK/P. aeruginosa PNA FISH assays using both the standard and shortened procedures (four tests per sample). The two assays share the same procedure and components, with the exception of the specific PNA FISH probe solution. Results of the PNA FISH assays were read independently by test operators blinded to results obtained by standard methods: Vitek (bioMérieux), MicroScan (Siemens), and/or API (bioMérieux). The shortened procedure eliminated the 10-min ethanol immersion step and shortened the hybridization step from 90 to 30 min. Slides were examined under a fluorescence microscope (60× or 100× oil objective) equipped with a dual-bandpass fluorescein isothiocyanate/Texas Red (FITC/TXR) filter. For both assays, positive samples were determined as multiple bright fluorescent rods in multiple fields of view: green for E. coli and red for P. aeruginosa. In the EK/P. aeruginosa PNA FISH assay, K. pneumoniae and E. coli were identical in morphology and degree of green fluorescence.Sensitivity, specificity, and total agreement were calculated. Exact 95% confidence intervals (CIs) were determined using the online calculator provided at http://www.measuringusability.com/.A total of 383 GNB (from 368 blood cultures; 15 mixed cultures), including 151 E. coli, 36 P. aeruginosa, and 65 K. pneumoniae isolates and 131 organisms from 35 other species, were identified by the routine techniques of the four laboratories. There was 100% agreement between the new and standard procedures for both E. coli/P. aeruginosa PNA FISH and EK/P. aeruginosa PNA FISH (368 of 368 and 370 of 370 results, respectively). In two mixed cultures of P. aeruginosa and K. pneumoniae, EK/P. aeruginosa PNA FISH produced both red and green results, which accounts for the greater number of positive results obtained by EK/P. aeruginosa PNA FISH (370) than the number of culture bottles sampled (368).Compared to routine identification methods, all 151 (100% sensitivity) E. coli isolates were correctly identified by both the standard and shortened procedures of E. coli/P. aeruginosa PNA FISH. Likewise, all 215 (100% sensitivity) E. coli and/or K. pneumoniae isolates were correctly identified by both of the EK/P. aeruginosa PNA FISH procedures. The sensitivity for P. aeruginosa for both assays with both procedures was 97.2% (35 of 36 results). One sample which contained a mixed culture of P. aeruginosa and vancomycin-resistant Enterococcus (VRE) was negative for P. aeruginosa by both PNA FISH methods and assays. The specificity of E. coli/P. aeruginosa PNA FISH for both procedures was 100% (181 of 181 results) and 100% (119 of 119 results) for EK/P. aeruginosa PNA FISH compared to routine methods (Tables ​(Tables11 and ​and22).

TABLE 1.

Performance of shortened E. coli/P. aeruginosa PNA FISH versus routine laboratory identification methods and standard PNA FISH

Sequential Flow Cytometry and Fluorescence In Situ Hybridization for the Study of Formalin-Fixed, Paraffin-Embedded Breast Cancer Cells

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.     

The Efficacy of In Situ PCR,CARD and Nanogold Systems for Gene Detection

Ovarian endometrial cysts, one of the typical manifestations of endometriosis, are generated by the retention of cyclic hemorrhages and are classified as tumor-like lesions rather than neoplasms. Clonality analysis provides important information about the histogenesis and progression of neoplastic diseases. As it is generally accepted that most neoplasms are monoclonal in origin, however, the clonality of endometrial cysts remains uncertain. Using the human androgen receptor gene (HUMARA) as an X-linked polymorphic marker, we examined the clonal status of epithelial cells in endometrial cysts. We separated 21 fresh epithelial cell samples from 11 endometrial cysts and found that all were monoclonal in the methylation pattern of the HUMARA alleles. Moreover, in each of the five cysts from which epithelial cells were sampled from multiple and distant areas, the methylation patterns of all samples from a single cyst were identical. These data indicate that endometrial cysts are monoclonal in origin and suggest their neoplastic potentiality.