John J. Farrell

Salvage Microbiology: Detection of Bacteria Directly from Clinical Specimens following Initiation of Antimicrobial Treatment

Background PCR coupled with electrospray ionization mass spectrometry (ESI-MS) is a diagnostic approach that has demonstrated the capacity to detect pathogenic organisms from culture negative clinical samples after antibiotic treatment has been initiated. [1] We describe the application of PCR/ESI-MS for detection of bacteria in original patient specimens that were obtained after administration of antibiotic treatment in an open investigation analysis. Methods We prospectively identified cases of suspected bacterial infection in which cultures were not obtained until after the initiation of antimicrobial treatment. PCR/ESI-MS was performed on 76 clinical specimens that were submitted for conventional microbiology testing from 47 patients receiving antimicrobial treatment. Findings In our series, 72% (55/76) of cultures obtained following initiation of antimicrobial treatment were non-diagnostic (45 negative cultures; and 10 respiratory specimens with normal flora (5), yeast (4), or coagulase-negative staphylococcus (1)). PCR/ESR-MS detected organisms in 83% (39/47) of cases and 76% (58/76) of the specimens. Bacterial pathogens were detected by PCR/ESI-MS in 60% (27/45) of the specimens in which cultures were negative. Notably, in two cases of relapse of prosthetic knee infections in patients on chronic suppressive antibiotics, the previous organism was not recovered in tissue cultures taken during extraction of the infected knee prostheses, but was detected by PCR/ESI-MS. Conclusion Molecular methods that rely on nucleic acid amplification may offer a unique advantage in the detection of pathogens collected after initiation of antimicrobial treatment and may provide an opportunity to target antimicrobial therapy and “salvage” both individual treatment regimens as well as, in select cases, institutional antimicrobial stewardship efforts.

Enterovirus D68-associated acute respiratory distress syndrome in adult, United States, 2014.

To the Editor: Each year, nonpolio enteroviruses cause 10–15 million infections in the United States (1). Enterovirus D68 (EV-D68) is an uncommon strain of nonpolio enterovirus that emerged in Illinois and Missouri in August 2014 in association with severe respiratory infections in children and spread across the United States (2). On August 23, 2014, the infection control department for Comer’s Children’s Hospital at the University of Chicago initially notified the Centers for Disease Control and Prevention of an increased number of children hospitalized with unusually severe respiratory illness (3). From mid-August to December 4, 2014, there were 1,121 laboratory-confirmed cases of EV-D68 in the United States (2). Almost all EV-D68 infections have occurred in children, many of whom had a history of asthma or wheezing (2). One day before the first report (August 22, 2014), a 26-year-old obese woman with an unremarkable medical history was transferred to the medical intensive care unit at Saint Francis Medical Center, a tertiary care medical center in Peoria, Illinois, USA, with severe acute respiratory distress syndrome (ARDS). The transfer was from a nearby community hospital where she had sought care 4 days earlier for influenza-like symptoms consisting of cough, wheezing, progressive shortness of breath, nausea, and vomiting. In the community hospital emergency department, she mentioned that 2 children at home had similar symptoms and that her mother had recently been hospitalized with an acute respiratory illness. Despite treatment with supplemental oxygen, nebulized albuterol, and intravenous antimicrobial drugs for community-acquired pneumonia, her condition deteriorated, and she was intubated on hospital day 2, after which the antimicrobial drug treatment was changed from intravenous ceftriaxone and azithromycin to intravenous vancomycin and piperacillin/tazobactam. Results of bronchoscopy performed on hospital day 4 were unremarkable, and bacterial cultures of alveolar lavage samples were negative. Her transfer to St. Francis Medical Center was prompted by persistent mechanical ventilation requirements of 100% fraction of inspired oxygen; positive end-inspiratory pressure of 12 mm/H2O consistent with classic ARDS (hypoxemia, indicated by a ratio of arterial oxygen partial pressure to fractional inspired oxygen <200 mm Hg); and bilateral infiltrates on chest radiograph (Figure) without evidence of left heart failure (4). On hospital day 8 (cumulative), a nasopharyngeal swab sample was tested by FilmArray Respiratory Panel multiplex PCR (BioFire Diagnostics, Salt Lake City, UT, USA); results were positive for rhinovirus/enterovirus. That day, intravenous methylprednisolone therapy was initiated. Figure Chest radiograph obtained (with portable machine) of semirecumbent adult patient with enterovirus D68–associated acute respiratory distress syndrome on hospital day 3. During a prolonged hospital stay, the patient required mechanical ventilation for 32 days, underwent a second bronchoscopic evaluation, required a percutaneous tracheostomy (and subsequent decannulation), and underwent endoscopic gastrostomy tube placement (and removal). She was discharged from the hospital after 55 days and ultimately recovered completely. To determine the etiology of the clinical syndrome for the patient reported here, molecular diagnostic testing of respiratory tract clinical specimens was required. Institutional review board approval was obtained for molecular diagnostics and sequencing of the patient’s nasopharyngeal swab specimens and bronchoalveolar lavage (BAL) fluid samples. The FilmArray platform is capable of detecting enteroviral infections caused by EV-D68 but cannot differentiate between rhinoviruses and enteroviruses (5). Confirmation of EV-D68 requires EV-D68–specific PCR (6). A novel, research-based diagnostic modality that is capable of rapid identification of viral pathogens directly from clinical specimens is the combination of PCR and electrospray ionization mass spectrometry (ESI-MS) (7), which was instrumental in early recognition of the novel pandemic strain of influenza A(H1N1) virus that emerged in 2009 (8). For a variety of viral pathogens, PCR/ESI-MS sensitivity is 94% and specificity is 98% (9). In this case, PCR/ESI-MS detected a human enterovirus from the right middle lobe and left lingular segment BAL fluid samples. For the assay, 2 primer pairs were used; both confirmed the presence of human enterovirus, but only 1 matched the signatures for EV-D68. For confirmation, we pursued testing with EV-D68–specific PCR, which was performed by the Special Projects Laboratory of the Washington University Department of Pediatrics. This assay amplifies a segment of the viral protein 1 gene, which enables discrimination of EV-D68 from other enteroviruses and rhinoviruses (K.M. Wylie et al., unpub. data). The nasopharyngeal swab sample and the right middle lobe and lingula BAL fluid specimens were positive for EV-D68. PCR/ESI-MS of BAL fluid followed by EV-D68–specific PCR testing of 1 nasopharyngeal swab and 2 BAL fluid samples confirmed our clinical suspicion of ARDS secondary to EV-D68 in an adult. The patient’s history of contact with sick family members and clinical signs (nonproductive cough, nausea, and vomiting) were suggestive of a viral infection. Lessons learned from the emergence of swine-origin influenza A(H1N1)pdm09 virus and recognition (in the midst of the pandemic) that younger age and obesity were risk factors for severe disease were also suggestive of a viral respiratory infection. We are developing a specific rapid molecular assay for EV-D68, which should help clinicians recognize when EV-D68 is present in the community. During those times, EV-D68 infection should be included in the differential diagnosis of severe respiratory infection. Documentation of EV-D68 infection may help with clinical management for individual patients and minimize unnecessary use of antimicrobial drugs within communities.

Identification of Streptococcus intermedius Central Nervous System Infection by Use of PCR and Electrospray Ionization Mass Spectrometry

ABSTRACT We describe the utility of PCR and electrospray ionization with mass spectrometry (PCR/ESI-MS) of culture-negative cerebrospinal fluid (CSF) in order to identify Gram-positive cocci noted on a Gram stain of CSF from a previously healthy 26-year-old man with community-acquired pneumonia (CAP) and multiple brain abscesses. CSF samples were obtained 2 weeks apart, first by lumbar puncture and 2 weeks later from an external ventricular drain that was inserted into the right ventricle. Both CSF cultures were negative. A Gram stain of bronchoalveolar lavage (BAL) fluid was notable for many Gram-positive cocci (GPC), but cultures of BAL fluid and subcarinal lymph node biopsy tissue were negative. PCR/ESI-MS detected Streptococcus intermedius, a common cause of brain abscesses, in both CSF samples as well as in the fixed tissue from the biopsy. This unique case confirms S. intermedius pulmonary infection as the source of metastatic CNS infection and reveals the potential of PCR/ESI-MS to detect a streptococcal pathogen not captured by conventional cultures.

Salvage microbiology: opportunities and challenges in the detection of bacterial pathogens following initiation of antimicrobial treatment

Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field, we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with the implementation of these strategies in clinical practice.

PCR and Electrospray Ionization Mass Spectrometry for Detection of Persistent Enterococcus faecalis in Cerebrospinal Fluid following Treatment of Postoperative Ventriculitis

ABSTRACT 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.

Direct Detection of Indirect Transmission of Streptobacillus moniliformis Rat Bite Fever Infection

ABSTRACT 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.

Rapid Identification of Aspergillus terreus from Bronchoalveolar Lavage Fluid by PCR and Electrospray Ionization with Mass Spectrometry

ABSTRACT We describe the application of PCR and electrospray-ionization with mass spectrometry (PCR/ESI-MS) to culture-negative bronchoalveolar lavage (BAL) fluid in order to identify septate hyphae noted by Gomori methenamine silver (GMS) staining of the fluid that was obtained from an immunocompromised woman with neutropenia following induction chemotherapy for treatment of acute myelogenous leukemia (AML). The patient was treated with empirical antifungal therapy, including intrathecal amphotericin B, while results of fungal cultures were pending. Ultimately, Aspergillus terreus, an amphotericin-resistant mold, was cultured from bilateral brain abscesses. PCR/ESI-MS correctly identified the mold.

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 β-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.

Identification of Occult Fusobacterium nucleatum Central Nervous System Infection by Use of PCR-Electrospray Ionization Mass Spectrometry

ABSTRACT Anaerobic bacteria are often difficult to detect, especially after the initiation of antibiotics. We describe the application of PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) using a sample of cerebrospinal fluid to identify an anaerobic Gram-negative bacillus, Fusobacterium nucleatum, in a patient with “culture-negative” meningitis and cerebral abscesses.

A rare case of aerococcus urinae infective endocarditis

ABSTRACT Introduction: Aerococcus urinae is a rare cause of infective endocarditis. Aerococcus is a gram positive cocci that is easily misidentified as Staphylococci or Streptococci. The true incidence rate of this pathogen is likely underestimated. Recent advances in laboratory diagnostic methods with matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) have lead to increased recognition of this pathogen in the clinical microbiology lab, and awareness as a cause of infective endocarditis in the infectious disease community. Case reports: Aerococcus usually affects males with underlying urinary tract conditions. Herein, we report a case of prosthetic aortic valve endocarditis caused by Aerococcus urinae. Discussion: Our patient was considered high risk for cardiac surgery and was treated successfully with intravenous antibiotics alone for six weeks. Conclusion: Infective endocarditis should be considered in all cases of Aerococcus bacteremia and appropriate diagnostic evaluations pursued. Abbreviations: AV: Aortic valve; IE: Infective endocarditis