Effects of clinically achievable pulmonary antibiotic concentrations on the recovery of bacteria: in vitro comparison of the BioFire FILMARRAY Pneumonia Panel versus conventional culture methods in bronchoalveolar lavage fluid.

Empiric antibiotics may affect bacterial pathogen recovery using conventional culture methods (CCMs), while PCR-based diagnostics are likely less affected. Herein, we conducted an in vitro study of bronchoalveolar lavage fluid (BAL) inoculated with bacteria and clinically relevant antibiotic concentrations to compare the recovery between the BioFire FILMARRAY Pneumonia Panel (Pn Panel) and CCMs. Remnant clinical BAL specimens were inoculated to ~105 cfu/mL using 12 clinical isolates. Isolates consisted of one wild-type (WT) and one or more resistant strains of: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. Piperacillin-tazobactam, cefepime, meropenem, levofloxacin, or vancomycin was added to achieve pulmonary epithelial lining fluid peak and trough concentrations. Post-exposure cfu/mL was quantified by CCMs and simultaneously tested by the PN Panel for identification and semi-quantitative genetic copies/mL. CCM results were categorized as significant growth (SG) (≥1 × 104), no significant growth (NSG) (≥1 × 103, <1 × 104), or no growth (NG) (<1 × 103). The PN Panel accurately identified all isolates, resistance genes, and reported ≥106 genetic copies/mL regardless of antibiotic exposure. The CCM also identified all S. aureus strains exposed to vancomycin. For WT Gram-negative isolates exposed to antibiotics, SG, NSG, and NG were observed in 7/52 (13%), 18/52 (35%), and 27/52 (52%) of CCM experiments, respectively. For resistant Gram-negatives isolates, SG, NSG, and NG were observed in 62/88 (70%), 17/88 (19%), and 9/88 (10%), respectively. These in vitro data demonstrate that the PN Panel is able to identify Gram-negative pathogens in the presence of clinically significant antibiotic concentrations when CCM may not.

The Effect of Commonly Used Local Anesthetic on Bacterial Growth.

INTRODUCTION: Liposomal bupivacaine (LB) has emerged as a superior form of local anesthetic across numerous surgical subspecialties. The purpose of this study is to evaluate the ex-vivo antimicrobial effects of LB in comparison with traditional local anesthetics. METHODS: A standardized inoculum of bacteria commonly associated with surgical site infection was inoculated into a suspension of 1% lidocaine, 0.25% bupivacaine, Exparel (proprietary liposomally packaged 1.3% bupivacaine), and normal saline as a growth control. RESULTS: In all five bacteria tested, the medium inoculated with traditional local anesthetics reduced growth to a greater degree than LB-inoculated plates. Both conventional local anesthetics reduced the growth of all bacteria when compared with the control with the exception of methicillin-susceptible Staphylococcus aureus growth in bupivacaine. LB-inoculated plates had equivalent growth to the control in all plates with the exception of Escherichia coli plates which demonstrated superior growth. CONCLUSIONS: The results of this simple ex-vivo model suggest that the liposomal packaging of bupivacaine may decrease this local anesthetic's innate antibacterial properties.

ANALYSIS OF PARS PLANA VITRECTOMY INCISIONS USING LIVE BACTERIA.

PURPOSE: To analyze small gauge pars plana vitrectomy sclerotomies using live bacteria transformed with green fluorescent protein (GFP). METHODS: Twenty-eight human cadaver eyes were specially harvested for this study. Small gauge vitrectomy was performed on each eye and the wounds were closed with various techniques (sutured, sutureless, and cauterization). Live Staphylococcus epidermidis that has been transformed with a green fluorescent protein was applied to the overlying conjunctival surface. Analysis of all vitreous samples was analyzed with confocal laser microscopy to identify the presence of bacteria. All wounds were analyzed histopathologically. RESULTS: A high concentration of bacteria was noted in 2 of 3 eyes in the sutureless, 23-G perpendicular incision group postinoculation. There were no bacteria detected in any postvitrectomy sample that were closed with cautery or a beveled incision. No bacteria were found in postvitrectomy samples of sutureless 27-G perpendicular incisions and sutureless 27-G beveled incisions. Finally, there were no bacteria detected in both eyes with 23-G perpendicular incisions that had a partial air-fill. CONCLUSION: Live bacteria can be effectively used to analyze wound integrity. Closing sclerotomy sites with cautery proved effective in a model using fresh, human cadaver eyes. 27-G perpendicular incisions may be just as competent as 27-G beveled incisions.

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates.

This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with blaNDM, blaIMP, and blaOXA-48.

Bacterial Contamination in Tips of Electrocautery Devices During Total Hip Arthroplasty.

Surgical equipment can become contaminated during surgery. It is unknown if electrocautery tips can become contaminated in clean orthopedic procedures despite the produced heat. Therefore, we conducted a prospective study to address this concern. The tips from 25 primary and 25 aseptic revision THAs were collected and an additional 5 sterile tips served as negative controls. Aerobic and anaerobic cultures were incubated for a minimum of 3 days. There were 3 positive cultures (6%); one in primary THA (4%) with Lactobacillus and Enterococcus faecalis; two among revisions (8%), one with E. faecalis and another one with alpha hemolytic streptococci and coagulase negative Staphylococcus. The mean exposure time of the contaminated tips was 132.3 minutes. Patients were followed for 90 days postoperatively and none of them developed surgical site infection. This is the first study to demonstrate that electrosurgical devices can become contaminated during THA in laminar flow equipped operating rooms.

Mycobacterium avium complex (MAC) Immune Reconstitution Syndrome (IRIS) With Reduced Susceptibility to Ethambutol in an HIV-Infected Patient.

OBJECTIVE: To describe a case of Mycobacterium avium complex (MAC) lymphadenitis complicated by immune reconstitution syndrome (IRIS) and reduced susceptibility to ethambutol. CASE SUMMARY: A 24-year-old man was diagnosed in October 2012 with advanced HIV infection upon hospitalization for multiple opportunistic infections (OIs). Within 5 months of starting antiretroviral therapy, the patient developed significant cervical lymphadenopathy concerning for MAC/IRIS. Acid-fast bacilli were detected in the primary lymph node biopsy smear, and culture results confirmed the presence of MAC. Susceptibility testing revealed an organism susceptible to azithromycin, with an elevated minimum inhibitory concentration (MIC) to ethambutol (8 µg/mL). Currently, there is no interpretation for an ethambutol MIC of 8 µg/mL for MAC. A review of the primary literature revealed the possibility of decreased ethambutol susceptibility when the MIC is above 1 µg/mL, and therefore, therapy was replaced by rifabutin in combination with azithromycin. DISCUSSION: Current guidelines recommend a 2-drug regimen for the treatment of MAC, specifically a macrolide plus ethambutol. Guidelines also emphasize MAC susceptibility testing for macrolides only. Susceptibility results from this patient's biopsy prompted an evaluation of the effectiveness of his antimycobacterial regimen. CONCLUSIONS: Reduced ethambutol susceptibility in this patient triggered a search of the primary literature that resulted in the decision to replace ethambutol with rifabutin. Additional clinical trials are needed to define susceptibility breakpoints for ethambutol and other antimycobacterial agents used for MAC infection treatment and to direct clinical decisions when elevated MICs to primary agents are identified.

Directed carbapenemase testing is no longer just for Enterobacterales: cost, labor, and workflow assessment of expanding carbapenemase testing to carbapenem-resistant P. aeruginosa.

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5-14) versus 10 min (range 8-22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3-14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus $40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3-14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases.

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage.

BACKGROUND: Group A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy. METHODS: Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis. RESULTS: Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models. CONCLUSION: Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine. TRIAL REGISTRATION: The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

Impact of Direct From Blood Culture Identification of Pathogens Paired With Antimicrobial Stewardship Interventions in a Pediatric Hospital.

OBJECTIVE: Identification of organisms directly from positive blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has the potential for improved clinical outcomes through earlier organism identification and shorter time to appropriate clinical intervention. The uses of this technology in pediatric patients and its impact in this patient population have not been well described. METHODS: Direct from positive blood culture organism identification via MALDI-TOF was implemented in September 2019. A quality improvement project was performed to assess its impact on admissions for contaminant blood cultures and time to effective and optimal antimicrobials and clinical decision-making. A pre- and post-implementation retrospective review for consecutive September through February time periods, was conducted on patients with positive monomicrobial blood cultures. Statistics were evaluated using Mann-Whitney U and χ2 tests. RESULTS: One hundred nineteen patients with 131 unique blood cultures (65 in pre- and 66 in post-implementation) were identified. Time to identification was shorter, median 35.4 hours (IQR, 22.7-54.3) versus 42.3 hours (IQR, 36.5-49) in post- and pre-groups, respectively (p = 0.02). Patients were less likely to be admitted for a contaminated blood culture in the post-implementation, 26% versus 11% in the pre-implementation (p = 0.03) group. In patients treated for bacteremia, there was a shorter time to optimal therapy from Gram stain reporting in the post-implementation (median 42.7 hours [IQR, 27.2-72]) versus pre-implementation (median 60.8 hours [IQR, 42.9-80.6]) (p = 0.03). CONCLUSIONS: Direct from positive blood culture identification by MALDI-TOF decreased time to effective and optimal antimicrobials and decreased unnecessary admission in pediatric patients for contaminated blood cultures.

Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

Potential role of CSF cytokine profiles in discriminating infectious from non-infectious CNS disorders.

Current laboratory testing of cerebrospinal fluid (CSF) does not consistently discriminate between different central nervous system (CNS) disease states. Rapidly distinguishing CNS infections from other brain and spinal cord disorders that share a similar clinical presentation is critical. New approaches focusing on aspects of disease biology, such as immune response profiles that can have stimulus-specific attributes, may be helpful. We undertook this preliminary proof-of-concept study using multiplex ELISA to measure CSF cytokine levels in various CNS disorders (infections, autoimmune/demyelinating diseases, lymphomas, and gliomas) to determine the potential utility of cytokine patterns in differentiating CNS infections from other CNS diseases. Both agglomerative hierarchical clustering and mixture discriminant analyses revealed grouping of CNS disease types based on cytokine expression. To further investigate the ability of CSF cytokine levels to distinguish various CNS disease states, non-parametric statistical analysis was performed. Mann-Whitney test analysis demonstrated that CNS infections are characterized by significantly higher CSF lP-10/CXCL10 levels than the pooled non-infectious CNS disorders (p = 0.0001). Within the infection group, elevated levels of MDC/CCL22 distinguished non-viral from viral infections (p = 0.0048). Each disease group of the non-infectious CNS disorders independently showed IP-10/CXCL10 levels that are significantly lower than the infection group [(autoimmune /demyelinating disorders (p = 0.0005), lymphomas (p = 0.0487), gliomas (p = 0.0294), and controls (p = 0.0001)]. Additionally, of the non-infectious diseases, gliomas can be distinguished from lymphomas by higher levels of GRO/CXCL1 (p = 0.0476), IL-7 (p = 0.0119), and IL-8 (p = 0.0460). Gliomas can also be distinguished from autoimmune/demyelinating disorders by higher levels of GRO/CXCL1 (p = 0.0044), IL-7 (p = 0.0035) and IL-8 (p = 0.0176). Elevated CSF levels of PDGF-AA distinguish lymphomas from autoimmune/demyelinating cases (p = 0.0130). Interrogation of the above comparisons using receiver operator characteristic analysis demonstrated area under the curve (AUC) values (ranging from 0.8636-1.0) that signify good to excellent utility as potential diagnostic discriminators. In conclusion, our work indicates that upon formal validation, measurement of CSF cytokine levels may have clinical utility in both identifying a CNS disorder as infectious in etiology and, furthermore, in distinguishing viral from non-viral CNS infections.

Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan.

Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.

Evaluation of the BacterioScan 216Dx for Standalone Preculture Screen of Preserved Urine Specimens in a Clinical Setting.

BACKGROUND: The BacterioScan 216Dx laser microbial growth monitoring system was evaluated as an option for preurine culture screening of preserved urine specimens at an acute care medical center. METHODS: The BacterioScan 216Dx system performance characteristics and the economic impact (cost effectiveness) for the laboratory were assessed. Urinalysis performance compared to urine culture was assessed if urinalysis was ordered as part of the patient care set. RESULTS: When compared to urine culture, the BacterioScan had an overall performance with corresponding 95% confidence intervals of 76% (68-83) sensitivity, 84% (80-87) specificity, 55% (48-63) positive predictive value, and 93% (90-95) negative predictive value for 610 randomly selected preserved urine specimens. Urinalysis compared to urine culture overall performance was 59% (48-69) sensitivity, 87% (83-90) specificity, 53% (43-63) positive predictive value, 89% (86-92) negative predictive value for 414 urine specimens. CONCLUSIONS: While the system did improve the turnaround time to a negative report, adoption of the BacterioScan system would increase the reagent budget for laboratory urine culture by 2.34 times the current cost, potentially making BacterioScan prohibitive in a budget restricted environment. Additionally, performance when compared to traditional urine culture was less than acceptable for a diagnostic laboratory to use as a stand-alone urinary tract infection screen.

Disseminated tuberculosis with prostatic abscesses in an immunocompromised patient-A case report and review of literature.

We describe a case of disseminated Mycobacterium tuberculosis (mTB) with prostatic abscess in a newly diagnosed HIV patient in the United States. The patient is a 34 year-old male who presented with respiratory symptoms and was diagnosed with HIV/AIDS complicated by disseminated mTB infection of the lungs, liver, and prostate. His prostate showed abscess formation on imaging that required drainage however he did not present with any genitourinary complaints. Our literature review revealed that prostatic involvement in mTB in the form of granulomatous prostatitis is uncommon; however, abscess formation is extremely rare and only few such cases have been published. Nearly 50% of the patients with prostatic abscess formation present without symptoms and therefore a high level of suspicion should be maintained; imaging should be performed early and prophylactic antibiotics for non-specific urinary symptoms should be avoided as this may lead to drug resistance of mTB to flouroquinolones.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to differentiate between Candida albicans and Candida dubliniensis.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis in conjunction with the direct formic acid (FA) sample processing method was evaluated for the ability to differentiate the closely related species of Candida albicans and Candida dubliniensis. The results showed that MALDI-TOF-MS, using the direct FA method, was reliable to differentiate between these species.

Nocardia arthritidis as a cause of disseminated nocardiosis in a patient with chronic lymphocytic leukemia.

A case of disseminated nocardiosis caused by Nocardia arthritidis in an immunocompromised patient with a history of chronic lymphocytic leukemia and rheumatoid arthritis is presented. This report highlights the use for multilocus sequence typing (MLST) in addition to single gene molecular sequencing to identify rare Nocardia species.

Diagnostic Challenges of Cryptococcus neoformans in an Immunocompetent Individual Masquerading as Chronic Hydrocephalus.

Cryptococcus neoformans can cause disseminated meningoencephalitis and evade immunosurveillance with expression of a major virulence factor, the polysaccharide capsule. Direct diagnostic assays often rely on the presence of the cryptococcal glucuronoxylomannan capsular antigen (CrAg) or visualization of the capsule. Strain specific phenotypic traits and environmental conditions influence differences in expression that can thereby compromise detection and timely diagnosis. Immunocompetent hosts may manifest clinical signs and symptoms indolently, often expanding the differential and delaying appropriate treatment and diagnosis. We describe a 63-year-old man who presented with a progressive four-year history of ambulatory dysfunction, headache, and communicating hydrocephalus. Serial lumbar punctures (LPs) revealed elevated protein (153-300 mg/dL), hypoglycorrhachia (19-47 mg/dL), lymphocytic pleocytosis (89-95% lymphocyte, WBC 67-303 mg/dL, and RBC 34-108 mg/dL), and normal opening pressure (13-16 cm H2O). Two different cerebrospinal fluid (CSF) CrAg assays were negative. A large volume CSF fungal culture grew unencapsulated C. neoformans. He was initiated on induction therapy with amphotericin B plus flucytosine and consolidation/maintenance therapy with flucytosine, but he died following discharge due to complications. Elevated levels of CSF Th1 cytokines and decreased IL6 may have affected the virulence and detection of the pathogen.

Diagnostic Options and Challenges for Dengue and Chikungunya Viruses.

Dengue virus (DENV) and Chikungunya virus (CHIKV) are arboviruses that share the same Aedes mosquito vectors and thus overlap in their endemic areas. These two viruses also cause similar clinical presentations, especially in the initial stages of infection, with neither virus possessing any specific distinguishing clinical features. Because the outcomes and management strategies for these two viruses are vastly different, early and accurate diagnosis is imperative. Diagnosis is also important for surveillance, outbreak control, and research related to vaccine and drug development. Available diagnostic tests are aimed at detection of the virus, its antigenic components, or the host immune antibody response. In this review, we describe the recent progress and continued challenges related to the diagnosis of DENV and CHIKV infections.

Bordetella petrii recovered from chronic pansinusitis in an adult with cystic fibrosis.

To date Bordetella petrii has infrequently been identified within the clinical setting likely due to the asaccharolytic nature of this organism. We present a case of B. petrii recovered on two separate events in a patient with adult cystic fibrosis experiencing chronic pansinusitis.

Identification of Staphylococcus epidermidis in the clinical microbiology laboratory by molecular methods.

Biochemical assays for the phenotypic identification of coagulase-negative staphylococci in the clinical microbiology laboratory have been well described in previous publications (Becker and Von Eiff Manual of Clinical Microbiology, ASM Press, Washington, pp. 308-330, 2011; Kloos and Wolfshohl J Clin Microbiol 16:509-516, 1982). This discussion focuses on identification of Staphylococcus epidermidis through molecular and proteomic methods. Molecular assays have been shown to be more discriminatory between the coagulase-negative staphylococcal species than are phenotypic assays (Zadoks and Watts Vet Microbiol 134:20-28, 2009; Sheraba et al. BMC Res Notes 3:278, 2010; Patteet et al. Eur J Clin Microbiol Infect Dis 31:747-751, 2012). The molecular and proteomic methods that have shown the greatest utilization potential within the clinical laboratory are as follows: PCR amplification and sequencing of discriminatory genes, real-time polymerase chain reaction with species-specific probes in conjunction with a melt-curve analysis, fluorescence in situ hybridization, and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.