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.
View Article and Find Full Text PDFBackground: Disseminated fungal infections are a known serious complication in individuals with cystic fibrosis (CF) following orthotopic lung transplantation. Aspergillus fumigatus and Scedosporium species are among the more common causes of invasive fungal infection in this population. However, it is also important for clinicians to be aware of other emerging fungal species which may require markedly different antifungal therapies.
View Article and Find Full Text PDFUnlabelled: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics.
View Article and Find Full Text PDFThe rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens.
View Article and Find Full Text PDFBackground: Diverse 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.
Objectives: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens.
Background: A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS).
View Article and Find Full Text PDFA prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.
View Article and Find Full Text PDFCultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea.
View Article and Find Full Text PDFWe describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter.
View Article and Find Full Text PDFAchieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood.
View Article and Find Full Text PDFInvasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens.
View Article and Find Full Text PDFDetection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies.
View Article and Find Full Text PDFTechnology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short.
View Article and Find Full Text PDFBackground: The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts.
Objectives: To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity.
We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.
View Article and Find Full Text PDFPDA J Pharm Sci Technol
April 2016
Adventitious contaminations with bacterial, viral, or fungal infectious agents represent a major risk associated with the manufacture and release of pharmaceutical products for human use, including vaccines, protein-based therapeutics, and antibodies. Early detection of contaminants in the biologicals production process might allow immediate action to correct such events without a significant interruption in the rate of production. Among the methods currently used for testing are cell culture, animal inoculation, electron microscopy, and in vitro molecular and antibody assays.
View Article and Find Full Text PDFDiagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus.
View Article and Find Full Text PDFDiagnosis of respiratory viruses traditionally relies on culture or antigen detection. We aimed to demonstrate capacity of the reverse transcription polymerase chain reaction/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to identify clinical relevant respiratory viruses in nasopharyngeal aspirate (NPA) samples and compare the diagnostic performance characteristics relative to conventional culture- and antigen-based methods. An RT-PCR/ESI-MS respiratory virus surveillance kit designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, Adenoviridae types A-F, Coronaviridae, human bocavirus, and human metapneumovirus was evaluated using both mock-ups and frozen archived NPA (N = 280), 95 of which were positive by clinical virology methods.
View Article and Find Full Text PDFMycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs.
View Article and Find Full Text PDFBackground: Pyoderma gangrenosum-like ulcers and cellulitis of the lower extremities associated with recurrent fevers in patients with X-linked (Bruton) agammaglobulinemia have been reported to be caused by Helicobacter bilis (formerly classified as Flexispira rappini and then Helicobacter strain flexispira taxon 8). Consistent themes in these reports are the difficulty in recovering this organism in blood and wound cultures and in maintaining isolates in vitro. We confirmed the presence of this organism in a patient's culture by using a novel application of gene amplification polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry.
View Article and Find Full Text PDFTechnologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs.
View Article and Find Full Text PDFFlaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose.
View Article and Find Full Text PDFMonkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units.
View Article and Find Full Text PDFThe Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V.
View Article and Find Full Text PDFRapid detection and identification of Ehrlichia species improves clinical outcome for patients suspected of ehrlichiosis. We describe an assay that employs multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify Ehrlichia species directly from blood specimens. The results were compared to those of a colorimetric microtiter PCR enzyme immunoassay (PCR-EIA) used as a diagnostic assay.
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