PCR-electrospray Ionization Mass Spectrometry Research Articles

MP25-02 THE NORMAL HEALTHY URINARY MICROBIOME IN THE MAPP NETWORK STUDY: RELATION TO GENDER, AGE AND MENOPAUSAL STATUS

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I (MP25)1 Sep 2021MP25-02 THE NORMAL HEALTHY URINARY MICROBIOME IN THE MAPP NETWORK STUDY: RELATION TO GENDER, AGE AND MENOPAUSAL STATUS J. Curtis Nickel, Alisa Stephens, A. Lenore Ackerman, Jennifer Anger, Henry LaI, and Garth D Ehrlich J. Curtis NickelJ. Curtis Nickel More articles by this author , Alisa StephensAlisa Stephens More articles by this author , A. Lenore AckermanA. Lenore Ackerman More articles by this author , Jennifer AngerJennifer Anger More articles by this author , Henry LaIHenry LaI More articles by this author , and Garth D EhrlichGarth D Ehrlich More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002022.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To understand the role of the urinary microbiome in disease states and interpret non-culture-based diagnostic urine testing, we must have a better understanding of the microbiome in asymptomatic healthy individuals. We examined the impact of gender, age, and hormonal status on the healthy human urinary microbiome in asymptomatic control subjects enrolled in the multi-institution NIDDK Multidisciplinary Approach to the Study of Chronic Pelvic Pain Network (MAPP) study. METHODS: Asymptomatic healthy controls, recruited to be age- and sex-matched to patients in the Trans-MAPP Epidemiology and Phenotyping Study, provided midstream urine collection for PCR-electrospray ionization mass spectrometry identification of urinary microbiota. The microbiomes of male and female participants were described and analyzed for differences in composition and diversity at the species and genus level by sex, age, and, in females, by hormonal status. RESULTS: The total number of species detected was 66 with mean of 1.2 species (sd 1.1) per male (n=97; mean age =43) and 2.3 (sd 1.3) per female (n=110, mean age = 38) in asymptomatic healthy controls. Species and genera diversity analyses showed significantly greater richness and diversity in females. In regard to species (Figure), Bifidobacterium subtile, Lactobacillus crispatus, and Lactobacillus johnsonii were more predominant in females. The genera Bifidobacterium, Staphylococcus, Lactobacillus, and Corynebacterium were more predominant in females. While for males the most prevalent organisms included Staphylococcus and Propionibacterium, only Propionibacterium approached a significant difference between genders. No significant difference in the presence and/or diversity of micro-organisms with hormonal status could be observed. Sex-specific age trends, particularly diversity, were larger for females than males (Figure). CONCLUSIONS: The urinary microbiome of healthy asymptomatic subjects differed between genders and age in females, but not hormonal status. Gender differences may be attributable to the detection of urethral/vaginal organisms in females and prostate organisms in males. These findings will better allow us to interpret the results of microbiome reports in the midstream urine specimens of patients with urinary symptoms. Source of Funding: NIH/NIDDK © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e453-e454 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information J. Curtis Nickel More articles by this author Alisa Stephens More articles by this author A. Lenore Ackerman More articles by this author Jennifer Anger More articles by this author Henry LaI More articles by this author Garth D Ehrlich More articles by this author Expand All Advertisement Loading ...

Performance of PCR/Electrospray Ionization-Mass Spectrometry on Whole Blood for Detection of Bloodstream Microorganisms in Patients with Suspected Sepsis.

Blood culture (BC) often fails to detect bloodstream microorganisms in sepsis. However, molecular diagnostics hold great potential. The molecular method PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect DNA from hundreds of different microorganisms in whole blood. The aim of the present study was to evaluate the performance of this method in a multicenter study including 16 teaching hospitals in the United States (n = 13) and Europe (n = 3). First, on testing of 2,754 contrived whole blood samples, with or without spiked microorganisms, PCR/ESI-MS produced 99.1% true-positive and 97.2% true-negative results. Second, among 1,460 patients with suspected sepsis (sepsis-2 definition), BC and PCR/ESI-MS on whole blood were positive in 14.6% and 25.6% of cases, respectively, with the following result combinations: BC positive and PCR/ESI-MS negative, 4.3%; BC positive and PCR/ESI-MS positive, 10.3%; BC negative and PCR/ESI-MS positive, 15.3%; and BC negative and PCR/ESI-MS negative, 70.1%. Compared with BC, PCR/ESI-MS showed the following sensitivities (coagulase-negative staphylococci not included): Gram-positive bacteria, 58%; Gram-negative bacteria, 78%; and Candida species, 83%. The specificities were >94% for all individual species. Patients who had received prior antimicrobial medications (n = 603) had significantly higher PCR/ESI-MS positivity rates than patients without prior antimicrobial treatment-31% versus 22% (P < 0.0001)-with pronounced differences for Gram-negative bacteria and Candida species. In conclusion, PCR/ESI-MS showed excellent performance on contrived samples. On clinical samples, it showed high specificities, moderately high sensitivities for Gram-negative bacteria and Candida species, and elevated positivity rates during antimicrobial treatment. These promising results encourage further development of molecular diagnostics to be used with whole blood for detection of bloodstream microorganisms in sepsis.

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS) is not suitable for rapid bacterial identification in peritoneal dialysis effluent.

Peritoneal dialysis (PD)-related peritonitis is a serious complication of PD, but routine microbiological culture is slow and could not identify the organism in 15% cases. We examine the accuracy of polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS), a PCR-based method developed for the direct detection of bacteria in blood, for rapid identification of microorganisms from PD effluent. We recruited 73 consecutive patients with PD-related peritonitis. Dialysis effluent was collected for routine bacterial culture, PCR/ESI-MS, and bacterial DNA quantification before initiation of antibiotic therapy. By digital PCR with universal bacterial primers, bacterial DNA was detectable in all PD effluent specimens. For the entire cohort, taking standard bacterial culture as the gold standard, the PCR/ESI-MS assay correctly identified 34.3% of the causative organisms, failed to identify any organism in 52.1% cases, and identified a different organism in 8.2% cases. For the 14 episodes of peritonitis that were culture negative by conventional bacterial culture, the PCR/ESI-MS assay identified an organism in only four cases. The detection rate of the IRIDICA BAC BSI assay was not affected by the use of biocompatible PD solution or concomitant exit-site infection. The PCR/ESI-MS assay could not identify the causative organism in over 50% of the PD effluent samples in patients with PD-related peritonitis and should be not used for such purpose. The reason for the poor performance needs further investigation.

Evaluation of a PCR-electrospray ionization mass spectrometry platform for detection and identification of fungal pathogens directly from prospectively collected bronchoalveolar lavage specimens

The incidence of invasive fungal infections is on the rise worldwide due to the growth of the immunocompromised population. We report here the use of a diagnostic assay that utilizes a universal extraction method, broad spectrum PCR amplification and analysis via electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify more than 200 pathogenic fungi directly from bronchoalveolar lavage (BAL) specimens in less than 8 hours. In this study, we describe both analytical and clinical performance of the assay, when run with prospectively collected clinical BAL specimens. In 146 patients with probable and possible fungal infections defined by EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria, the PCR/ESI-MS assay demonstrated a sensitivity of 90.9% (95% CI: 76.4–96.9%) and a specificity of 82.3% (95% CI: 74.2–88.2%). This data demonstrates the utility of a non-culture based broad fungal targets molecular diagnostic tool for rapid and accurate diagnosis of invasive fungal infections in patients at risk of developing fungal diseases.

2267. The Effect of Opportunistic Infection (OI) Prophylaxis on the Gastrointestinal Microbiome (GIM) and Immune Reconstitution (IR) in Veterans With HIV and AIDS

BackgroundDespite antiretroviral therapy (ART), some patients do not achieve IR. Moreover, GI bacterial translocation may lead to a decrease in CD4 counts with an increase in IL-6 in blood. However, the effect of OI prophylaxis on the GIM, bacterial translocation and IR has not been studied in HIV+ veterans. Here we studied the gut microbiome and bacterial translocation in VA patients with (i) stable HIV on ART (controls), (ii) newly diagnosed HIV starting on ART (new dx) and OI prophylaxis, and (iii) resuming ART (resumers).MethodsBlood and stool specimens from 16 controls, 4 new dx and 3 resumers were obtained at 3 visits, as well as clinical and virological data. PCR electrospray ionization mass spectrometry (ESI-MS) was performed on blood samples to detect bacteria. Quantitative cultures and gut microbiome (deep sequencing bacterial 16S rRNA) was done on stool.ResultsThere was no relation between CD4 count, log CFU TMP-SMX-resistant Gram-negative bacteria (GNRs) or total anaerobes. Except for 2 control patients with a decrease in CD4 count <200, none took TMP-SMX. One of these control patients started TMP-SMX, while the other took atovaquone. Neither had TMP-SMX R GNRs in stool, despite low CD4 /TMP-SMX. Major stool phyla in controls were Bacteroidetes (37 ± 19%), Firmicutes (37 ± 14%), Proteobacteria (15 ± 14%); while resumers had 54 ± 15% Bacteroidetes, 33 ± 12% Firmicutes and 7 ± 1% Proteobacteria. Only one new dx individual had CD4 count <200 at dx and took doxycycline initially for hidradenitis supparativa. Dapsone was initiated due to sulfa allergy. He was also diagnosed with lung cancer, treated with resection/XRT and received cefazolin. His VL became undetectable but CD4 <200. He had persistence of TMP-SMX-resistant GNRs despite dapsone and a shift in his GIM was observed over the first 6 months of care, i.e., Bacteroidetes decreased from 61.5% to 29.5% and Firmicutes increased from 30.6% to 53.3%.ConclusionOI prophylaxis does not affect the GIM of stable HIV VA patients on ART. TMP-SMX-resistant GNRs in stool are unrelated to TMP-SMX exposure or CD4 count. Other antibiotics such as doxycycline can alter GI microbiota and may affect immune reconstitution.Disclosures All authors: No reported disclosures.

Broad-range survey of vector-borne pathogens and tick host identification of Ixodes ricinus from Southern Czech Republic.

Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host–vector–pathogen interactions that results in spatial distribution of infection risk. Broad-range polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens. Borrelia burgdorferi sensu lato spirochetes were the most common pathogen detected in the ticks; 21% of ticks were positive for a single genospecies and 2% were co-infected with two genospecies. Other tick-borne pathogens detected included Rickettsia helvetica (3.9%), R. monacensis (0.2%), Anaplasma phagocytophilum (2.8%), Babesia venatorum (0.9%), and Ba. microti (0.5%). The vertebrate host of the ticks was determined using PCR followed by reverse line blot hybridization from the tick's blood-meal remnants. The host was identified for 61% of ticks. DNA of two hosts was detected in 16% of samples with successful host identification. The majority of ticks had fed on artiodactyls (50.7%) followed by rodents (28.6%) and birds (7.8%). Other host species were wild boar, deer, squirrels, field mice and voles.

Demise of Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry as an Infectious Diseases Diagnostic Tool.

Although there are several US Food and Drug Administration (FDA)-approved/cleared molecular microbiology diagnostics for direct analysis of patient samples, all are single target or panel-based tests. There is no FDA-approved/cleared diagnostic for broad microbial detection. Polymerase chain reaction (PCR)/electrospray ionization-mass spectrometry (PCR/ESI-MS), commercialized as the IRIDICA system (Abbott) and formerly PLEX-ID, had been under development for over a decade and had become CE-marked and commercially available in Europe in 2014. Capable of detecting a large number of microorganisms, it was under review at the FDA when, in April 2017, Abbott discontinued it. This turn of events represents not only the loss of a potential diagnostic tool for infectious diseases but may be a harbinger of similar situations with other emerging and expensive microbial diagnostics, especially genomic tests.

Occurrence and Diversity of Clinically Important Vibrio Species in the Aquatic Environment of Georgia.

Among the more than 70 different Vibrio species inhabiting marine, estuarine, and freshwater ecosystems, 12 are recognized as human pathogens. The warm subtropical climate of the Black Sea coastal area and inland regions of Georgia likely provides a favorable environment for various Vibrio species. From 2006 to 2009, the abundance, ecology, and diversity of clinically important Vibrio species were studied in different locations in Georgia and across seasons. Over a 33-month period, 1,595 presumptive Vibrio isolates were collected from the Black Sea (n = 657) and freshwater lakes around Tbilisi (n = 938). Screening of a subset of 440 concentrated and enriched water samples by PCR-electrospray ionization/mass spectrometry (PCR-ESI/MS) detected the presence of DNA from eight clinically important Vibrio species: V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus, V. harveyi, V. metschnikovii, and V. cincinnatiensis. Almost 90% of PCR/ESI-MS samples positive for Vibrio species were collected from June through November. Three important human-pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, and V. vulnificus) were detected in 62.8, 37.8, and 21.4% of samples testing positive for Vibrios, respectively. The results of these activities suggest that natural reservoirs for human-pathogenic Vibrios exist in Georgian aquatic environments. Water temperature at all sampling sites was positively correlated with the abundance of clinically important Vibrio spp. (except V. metschnikovii), and salinity was correlated with species composition at particular Black Sea sites as well as inland reservoirs.

Viral Respiratory Tract Infections in Adult Patients Attending Outpatient and Emergency Departments, Taiwan, 2012-2013: A PCR/Electrospray Ionization Mass Spectrometry Study.

Viral etiologies of respiratory tract infections (RTIs) have been less studied in adult than in pediatric populations. Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinoviruses in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for rhinoviruses and enteroviruses.Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples.Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation, PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and rhinoviruses (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (<5% of cases for each type of virus). Coinfection was observed in 4.1% of cases. In the control group, only 1 (2.4%) sample tested positive for a respiratory virus by PCR/ESI-MS. Patients who were undergoing steroid treatment, had an active malignancy, or suffered from chronic obstructive pulmonary disease (COPD) were at risk for rhinovirus, hMPV, or parainfluenza infections, respectively. Overall, immunocompromised patients, patients with COPD, and patients receiving dialysis were at risk for noninfluenza respiratory virus infection. Rhinoviruses (12.7%), influenza A virus (10.9%), and parainfluenza viruses (7.3%) were the most common viruses involved in the 55 cases of lower RTIs. The factors of parainfluenza infection, old age, and immunosuppression were independently associated with lower RTIs.In conclusion, PCR/ESI-MS improved the diagnostic yield for viral RTIs. Non-influenza respiratory virus infections were associated with patients with comorbidities and with lower RTIs. Additional studies that delineate the clinical need for including non-influenza respiratory viruses in the diagnostic work-up in these populations are warranted.

Utilizing Rapid Diagnostics for Detection of Candida Species

Rapid diagnostic tests are now available to aid in the detection of invasive candidiasis (IC) and promise significant advantages to conventional detection and identification methods. The most progress has been made on rapid species identification when blood culture bottles turn positive and/or when isolated pure colonies are available, while the diagnosis of IC directly from a clinical specimen (e.g., the patient’s blood) remains problematic. For the latter, nucleic acid-based tests (PCR, PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS), and T2 magnetic resonance) and immunologic tests [1,3-β-D-glucan (BG) and Candida antigen and antibody tests] are promising but each contains certain limitations which have restricted their impact on clinical practice. In-house PCR assays are not standardized, and commercially available PCR tests are limited by minimal clinical data and a long turnaround time. The PCR/ESI-MS and T2 magnetic resonance platforms both require substantial up-front resources, and further clinical experience is required to understand their role in the clinical laboratory. Also, the BG and Candida antigen and antibody tests may be useful in certain scenarios (e.g., a negative BG may help rule out IC) but require significant clinical correlation and interpretation of results. On the other hand, PNA FISH and FilmArray Blood Culture ID Panel can substantially reduce the time-to-identification from a positive blood culture, impact clinical practice, and are quite reasonable to implement in the clinical laboratory. While the clear advantages for MALDI-TOF MS are speed, versatility, and excellent accuracy for identification of organisms from culture plates (which is its primary use in the clinical laboratory), further optimization is required before it can reliably be used for organism identification directly from blood culture bottles and its cost is currently prohibitively expensive for a small clinical laboratory. MALDI-TOF MS is very appealing, however, for rapid identification of organisms in a large clinical laboratory. All of these rapid diagnostic methods complement but do not replace the culture, however, as having the organism itself is still critical for susceptibility testing and strain typing

Molecular identification of Mucorales in human tissues: contribution of PCR electrospray-ionization mass spectrometry

Molecular methods are crucial for mucormycosis diagnosis because cultures are frequently negative, even if microscopy suggests the presence of hyphae in tissues. We assessed PCR/electrospray-ionization mass spectrometry (PCR/ESI-MS) for Mucorales identification in 19 unfixed tissue samples from 13 patients with proven or probable mucormycosis and compared the results with culture, quantitative real-time PCR, 16S–23S rRNA gene internal transcribed spacer region (ITS PCR) and 18S PCR sequencing. Concordance with culture identification to both genus and species levels was higher for PCR/ESI-MS than for the other techniques. Thus, PCR/ESI-MS is suitable for Mucorales identification, within 6 hours, for tissue samples for which microscopy results suggest the presence of hyphae.

Correction: Taliaferro, L. et al. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection. Viruses 2014, 6, 1876-1896

We have noted some inaccuracies in Table 1 and Table 2 of our article “Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection” (Viruses 2014, 6, 1876–1896) [1].[...]

Detection of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry Applied to Synovial Fluid

PCR coupled with electrospray ionization mass spectrometry applied to synovial fluid specimens had an 81% sensitivity and a 95% specificity for the diagnosis of prosthetic joint infection.

Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection

BackgroundDiverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. ObjectivesTo develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. Study designWe developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6–8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. ResultsThe assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). ConclusionsThe assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.

Diagnosis of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry

We compared PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) to culture using sonicate fluid from 431 subjects with explanted knee (n = 270) or hip (n = 161) prostheses. Of these, 152 and 279 subjects had prosthetic joint infection (PJI) and aseptic failure, respectively. The sensitivities for detecting PJI were 77.6% for PCR-ESI/MS and 69.7% for culture (P = 0.0105). The specificities were 93.5 and 99.3%, respectively (P = 0.0002).

Broad-Range PCR-Electrospray Ionization Mass Spectrometry for Detection and Typing of Adenovirus and Other Opportunistic Viruses in Stem Cell Transplant Patients

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.

Detection and Identification of Yeasts from Formalin-Fixed, Paraffin-Embedded Tissue by Use of PCR-Electrospray Ionization Mass Spectrometry

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

Identification of Mycobacterium Species and Mycobacterium tuberculosis Complex Resistance Determinants by Use of PCR-Electrospray Ionization Mass Spectrometry

PCR coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) is a novel technology that has recently been used to identify pathogens from clinical specimens or after culture within about 6 h. We evaluated the MDR-TB (multidrug-resistant tuberculosis) assay, which uses PCR-ESI-MS for detection and identification of Mycobacterium spp. and Mycobacterium tuberculosis complex (MTBC) resistance determinants from solid and broth Middlebrook culture media. The performance of the MDR-TB assay was compared to identification using nucleic acid hybridization probes and 16S rRNA gene sequencing for 68 MTBC and 97 nontuberculous mycobacterial (NTM) isolates grown on agar and 107 cultures grown in Bactec MGIT broth. MTBC resistance profiles from the MDR-TB assay were compared to results with the agar proportion method. The PCR-ESI-MS system correctly identified all MTBC isolates and 97.9% and 95.8% of the NTM isolates from characterized agar cultures and MGIT broth cultures to the species level, respectively. In comparison to the agar proportion method, the sensitivity and specificity for the detection of drug resistance using the MDR-TB assay were 100% and 92.3% for rifampin, 100% and 93.8% for isoniazid, 91.6% and 94.4% for ethambutol, and 100% and 100% for fluoroquinolones, respectively. The MDR-TB assay appears to be a rapid and accurate method for the simultaneous detection and identification of mycobacterial species and resistance determinants of MTBC from culture.

Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry

Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.

PCR-Electrospray Ionization Mass Spectrometry for Direct Detection of Pathogens and Antimicrobial Resistance from Heart Valves in Patients with Infective Endocarditis

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.