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.

The effect of empiric antimicrobial treatment duration on detection of bacterial DNA in sterile surgical specimens.

Initial antimicrobial treatment of patients with deep seated or invasive infections is typically empiric. Usually, cultures of specimens obtained from the suspected source of infection are performed to identify pathogens and guide continued antimicrobial treatment. When patients present with signs and symptoms of infection, but sterile body fluid or tissue specimens cannot be obtained in a timely fashion, growth of bacterial pathogens in culture may be inhibited following initiation of empiric antibiotic treatment. To address this clinical dilemma, we performed a prospective evaluation of conventional culture vs. PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS) on sterile body fluids and tissues submitted to the diagnostic microbiology lab following initiation of empiric antibiotic treatment for patients with suspected infection. In this series of surgical samples, PCR/ESI-MS identified bacterial pathogen(s) in 56% (49/87) of patients with non-diagnostic cultures. Examination of patients stratified by antibiotic treatment duration demonstrated that PCR/ESI-MS sustains high rates of bacterial DNA detection over time by generalized estimating equation models (p<0.0001).

Culture negative empyema in a critically ill child: an opportunity for rapid molecular diagnostics.

BACKGROUND: Nucleic acid amplification technologies (NAAT) are advancing our ability to make rapid molecular diagnoses in patients with serious culture negative infections. This is the first report of PCR coupled to electrospray ionization mass spectrometry use in the evaluation of complicated community acquired pneumonia in a pediatric patient. CASE PRESENTATION: We present a case of culture negative empyema in a critically ill, Caucasian, 2-year-old girl who was treated with broad-spectrum empiric antibiotics, in which the length of stay was prolonged by adverse effects of the empiric antibiotic treatment. PCR coupled to electrospray ionization mass spectrometry was applied to culture negative fluid and tissue samples from the patient in order to determine the etiology of the empyema. CONCLUSIONS: Using this method, Streptococcus mitis/viridans was identified as the pathogen. A retrospective review of cases of empyema in children at our institution found that 87.5% of cases were negative for identification of a pathogen and antibiotics were administered to 100% of cases prior to collecting pleural fluid for culture. Understanding the role of Streptococcus mitis/viridans group in the etiology of empyema using an advanced NAAT coupled with mass spectrometry can enlighten clinicians as to the impact of this pathogen in community acquired pneumonia and help assist with antibiotic stewardship.

Acute respiratory distress caused by Neosartorya udagawae.

We describe the first reported case of acute respiratory distress syndrome (ARDS) attributed to Neosartorya udagawae infection. This mold grew rapidly in cultures of multiple respiratory specimens from a previously healthy 43-year-old woman. Neosartorya spp. are a recently recognized cause of invasive disease in immunocompromised patients that can be mistaken for their sexual teleomorph, Aspergillus fumigatus. Because the cultures were sterile, phenotypic identification was not possible. DNA sequencing of ITS, calmodulin and ß-tubulin genes supported identification of Neosartorya udagawae. Our case is the first report of ARDS associated with Neosartorya sp. infection and defines a new clinical entity.

Direct detection of indirect transmission of Streptobacillus moniliformis rat bite fever infection.

We describe the evaluation of culture-negative synovial fluid from a 3-year-old boy by PCR and electrospray ionization followed by mass spectrometry (PCR/ESI-MS). Our patient developed a diffuse rash and fever with systemic signs and symptoms of sepsis, but four sets of blood cultures obtained prior to initiation of antibiotics were negative. After 1 week of illness, he developed right-knee swelling. Analysis of synovial fluid was consistent with infection, but cultures of specimens obtained following initiation of antimicrobial treatment were negative for growth. PCR/ESI-MS detected Streptobacillus moniliformis in the synovial fluid sample. Our patient completed an appropriate course of antibiotic treatment and remained completely asymptomatic in follow-up evaluation. This unique case suggests that PCR/ESI-MS may be a useful diagnostic tool for direct detection of unusual or unexpected pathogens directly from clinical specimens, particularly when samples have been obtained from patients following initiation of antibiotic therapy.

Ureaplasma parvum prosthetic joint infection detected by PCR.

We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection.

PCR and electrospray ionization mass spectrometry for detection of persistent enterococcus faecalis in cerebrospinal fluid following treatment of postoperative ventriculitis.

We describe the use of PCR and electrospray ionization followed by mass spectrometry (PCR/ESI-MS) to evaluate "culture-negative" cerebrospinal fluid (CSF) from a 67-year-old man who developed postoperative bacterial ventriculitis following a suboccipital craniotomy for resection of an ependymoma in the 4th ventricle. CSF samples were obtained on seven occasions, beginning in the operating room at the time of insertion of a right ventriculoperitoneal shunt (VPS) and continuing until his death, 6 weeks later. During the course of the illness, two initial CSF specimens taken before the initiation of antimicrobial treatment were notable for growth of Enterococcus faecalis. Once antimicrobial treatment was initiated, all CSF cultures were negative. PCR/ESI-MS detected genetic evidence of E. faecalis in all CSF samples, but the level of detection (LOD) decreased once antimicrobial treatment was initiated. When our patient returned with symptoms of meningitis 3 days after the completion of antibiotic treatment, CSF cultures remained negative, but PCR/ESI-MS again found genetic evidence for E. faecalis at levels comparable to the pretreatment levels seen initially. This unique case and these findings suggest that determination of CSF LOD by PCR/ESI-MS may be a very sensitive indicator of persistent infection in patients on antibiotic therapy for complex CNS infections and may have relevance for treatment duration and assessment of persistent or recurrent infection at the completion of therapy.

Differentiating microbial forensic qPCR target and control products by electrospray ionization mass spectrometry.

Molecular bioforensic research is dependent on rapid and sensitive methods such as real-time PCR (qPCR) for the identification of microorganisms. The use of synthetic positive control templates containing small modifications outside the primer and probe regions is essential to ensure all aspects of the assay are functioning properly, including the primers and probes. However, a typical qPCR or reverse transcriptase qPCR (qRT-PCR) assay is limited in differentiating products generated from positive controls and biological samples because the fluorescent probe signals generated from each type of amplicon are indistinguishable. Additional methods used to differentiate amplicons, including melt curves, secondary probes, and amplicon sequencing, require significant time to implement and validate and present technical challenges that limit their use for microbial forensic applications. To solve this problem, we have developed a novel application of electrospray ionization mass spectrometry (ESI-MS) to rapidly differentiate qPCR amplicons generated with positive biological samples from those generated with synthetic positive controls. The method has sensitivity equivalent to qPCR and supports the confident and timely determination of the presence of a biothreat agent that is crucial for policymakers and law enforcement. Additionally, it eliminates the need for time-consuming methods to confirm qPCR results, including development and validation of secondary probes or sequencing of small amplicons. In this study, we demonstrate the effectiveness of this approach with microbial forensic qPCR assays targeting multiple biodefense agents (bacterial, viral, and toxin) for the ability to rapidly discriminate between a positive control and a positive sample.

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis.

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Microbiota evaluation of patients with a Boston type I keratoprosthesis treated with topical 0.5% moxifloxacin and 5% povidone-iodine.

PURPOSE: To evaluate the efficacy of a prophylactic regimen of daily topical 0.5% moxifloxacin and 5% povidone-iodine (PI) in patients with Boston type I keratoprosthesis (KPro) and to assess the applicability of a novel molecular diagnostic technique to analyze the ocular surface microbiota in these patients. METHODS: Ten patients had their inferior conjunctival fornix sampled for standard culture methods before the addition of topical 5% PI to the prophylactic regimen and were considered the control group (group 1). The inferior conjunctival fornix and the KPro-donor cornea interface of 10 patients treated with the mentioned prophylactic regimen were sampled and analyzed by standard culture methods and using a polymerase chain reaction/electrospray ionization mass spectrometry assay (group 2). RESULTS: Samples from the inferior conjunctival fornix were positive for coagulase-negative staphylococcus in 3 patients and for Aerobasidium pullulans in 1 patient in group 1. The inferior conjunctival fornix and the KPro-donor cornea interface scrapings were positive for coagulase-negative staphylococcus in 2 patients and 1 patient, respectively, in group 2. No bacteria and fungi growth were detected in any patient from group 2 with the molecular diagnostic approach. None of the patients with culture-positive results developed keratitis or endophthalmitis during the study. CONCLUSIONS: Topical 0.5% moxifloxacin associated with topical 5% PI is an effective prophylactic regimen in patients with Boston type I KPro. The molecular diagnostic approach using serial polymerase chain reaction and mass spectrometry was comparable with standard microbiologic techniques as a surveillance tool in these patients.

Clinical accuracy of a PLEX-ID flu device for simultaneous detection and identification of influenza viruses A and B.

Respiratory tract infections caused by influenza A and B viruses often present nonspecifically, and a rapid, high-throughput laboratory technique that can identify influenza viruses is clinically and epidemiologically desirable. The PLEX-ID Flu assay (Abbott Molecular Inc., Des Plaines, IL) incorporates multilocus PCR and electrospray ionization-mass spectrometry to detect and differentiate influenza A 2009 H1N1 (H1N1-p), seasonal H1N1 (H1N1-s), influenza A H3N2, and influenza B viruses in nasopharyngeal swab (NPS) specimens. The clinical performance characteristics of the PLEX-ID Flu assay in symptomatic patients were determined in this multicenter trial. A total of 2,617 prospectively and retrospectively collected NPS specimens from patients with influenza-like illness between February 2008 and 28 May 2010 were eligible for inclusion in the study. Each specimen was tested in parallel by the PLEX-ID Flu assay and by the Prodesse ProFLU+ assay (Prodesse Inc., Madison, WI), to detect influenza A and B viruses. Specimens testing positive for influenza A virus by ProFLU+ were subtyped as H1N1-p, H1N1-s, or H3N2 by using the ProFAST+ assay (Gen-Probe Prodesse Inc.). The reproducibility of the PLEX-ID Flu assay ranged from 98.3 to 100.0%, as determined by testing a nine-specimen panel at three clinical sites on each of 5 days. Positive percent agreements (PPAs) and negative percent agreements (NPAs) of the PLEX-ID Flu assay were 94.5% and 99.0% for influenza A virus and 96.0% and 99.9% for influenza B virus, respectively. For the influenza A virus subtyping characterization, the PLEX-ID Flu assay had PPAs and NPAs of 98.3% and 97.5% for H1N1-p, 88.6% and 100.0% for H1N1-s, and 98.0% and 99.9% for H3N2, respectively. The overall agreements between the PLEX-ID and Prodesse ProFLU+/ProFAST+ assays were 97.1 to 100.0%. Bidirectional Sanger sequencing analysis revealed that 87.5% of 96 discrepant results between the PLEX-ID Flu and ProFLU+/ProFAST+ assays were found upon influenza A virus detection and H1N1-p subtyping. The PLEX-ID Flu assay demonstrated a high level of accuracy for the simultaneous detection and identification of influenza A and B viruses in patient specimens, providing a new laboratory tool for the rapid diagnosis and management of influenza A and B virus infections.

Rapid and high-throughput detection of highly pathogenic bacteria by Ibis PLEX-ID technology.

In this manuscript, we describe the identification of highly pathogenic bacteria using an assay coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS) run on an Ibis PLEX-ID high-throughput platform. The biothreat cluster assay identifies most of the potential bioterrorism-relevant microorganisms including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia mallei and pseudomallei, Brucella species, and Coxiella burnetii. DNA from 45 different reference materials with different formulations and different concentrations were chosen and sent to a service screening laboratory that uses the PCR/ESI-MS platform to provide a microbial identification service. The standard reference materials were produced out of a repository built up in the framework of the EU funded project "Establishment of Quality Assurances for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk" (EQADeBa). All samples were correctly identified at least to the genus level.

Exhaled aerosol transmission of pandemic and seasonal H1N1 influenza viruses in the ferret.

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected 'donor' ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.

Transmission of aerosolized seasonal H1N1 influenza A to ferrets.

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID(50)) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID(50) for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.

Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry.

We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.

Single nucleotide polymorphism typing of Bacillus anthracis from Sverdlovsk tissue.

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis.

Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes.

For many years, analytical mass spectrometry has had numerous supporting roles in the drug development process, including the assessment of compound purity; quantitation of absorption, distribution, metabolism and excretion; and compound-specific pharmacokinetic analyses. More recently, mass spectrometry has emerged as an effective technique for identifying lead compounds on the basis of the characterization of noncovalent ligand-macromolecular target interactions. This approach offers several attractive properties for screening applications in drug discovery compared with other strategies, including the small quantities of target and ligands required, and the capacity to study ligands or targets without having to label them. Here, we review the application of electrospray ionization mass spectrometry to the interrogation of noncovalent complexes, highlighting examples from drug discovery efforts aimed at a range of target classes.

SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.

A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct. The optimized benzimidazoles demonstrated activity in a cellular replicon assay at concentrations comparable to their KD for the RNA target.

The synthesis and 16S A-site rRNA recognition of carbohydrate-free aminoglycosides.

The first carbohydrate-free aminoglycoside analogs bearing the 2-deoxystreptamine moiety were synthesized from asymmetrically protected 2-deoxystrepamine and subsequently demonstrated to have significant binding to the 16S A-site rRNA target and moderate functional activity.