Usefulness of the FilmArray blood culture identification panel for identifying causative pathogens of bone and joint infections

As bone and joint infections (BJIs) require long-term treatment, identifying their causative pathogens is vital. However, the detection rate of conventional culturing remains inadequate. This study aimed to evaluate the usefulness of the FilmArray blood culture identification (BCID) panel for identifying causative pathogens in patients with BJIs. We tested a BCID panel using collected samples, in addition to conventional cultures. The primary outcome was to evaluate the diagnostic performance of the BCID panel, calculated using conventional culturing methods. A total of 44 patients who underwent BJI-related specimen collection were enrolled. Of the 44 patients, 22 were diagnosed with a BJI. Conventional culture identified 15 of 22 organisms (68.2%), whereas the BCID panel identified 14 of 22 organisms (63.4%). The overall sensitivity and specificity of the BCID panel were 73.3% and 57.1%, respectively, compared to those of the conventional culture. However, the sensitivity reached 100% when only pathogens included in the BCID panel were considered. In seven culture-negative cases, the BCID panel identified three organisms (42.9%). The BCID panel also indicated the appropriate therapy against a BJI caused by methicillin-resistant Staphylococcus aureus by detecting the mecA gene. This study demonstrated that the BCID panel has the potential for early and accurate diagnosis of the causative organism of BJI using specimens such as joint fluid and bone tissue. Our results suggest that BCID panels, in addition to routine culture, may improve our ability to diagnose the causative microorganisms of BJI in clinical practice, thereby contributing to the selection of appropriate antimicrobial agents.     

Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.     

Spectrum of enteropathogens in cases of traveler's diarrhea that were detected using the FilmArray GI panel: New epidemiology in Japan

IntroductionTraveler's diarrhea (TD) is the most common illness among people traveling from resource-rich regions to resource-limited regions, although the precise microbial etiology is unclear in many cases.MethodsStool specimens were prospectively collected from 106 consecutive patients with TD and 16 healthy controls without TD, and were tested using both the FilmArray gastrointestinal panel (BioFire Diagnostics) and conventional stool cultures.ResultsThe 106 patients had traveled to Southeast Asia (55 cases), South Asia (22 cases), Africa (11 cases), and East Asia (7 cases). Among the 106 specimens, 95 specimens (89.6%) were positive for pathogens during the FilmArray testing. The FilmArray testing also identified multiple pathogens in 75.8% of the specimens from positive cases. Conventional stool cultures only detected pathogens in 23.6% of the specimens.ConclusionThe FilmArray gastrointestinal panel significantly improved the detection of enteropathogens and allowed for a rapid assessment of the TD's etiology. In addition, conventional stool cultures are likely to underestimate co-infections with multiple infectious pathogens.     

FilmArray® respiratory panel performance in respiratory samples from neonatal care units

FilmArray Respiratory Panel (RP) (Idaho Technology, Inc., Salt Lake City, UT, USA) performance was retrospectively evaluated in respiratory samples collected from neonates in 2 reference neonatology units. Using the FilmArray RP assay, 121/152 (79.6%) samples were positive for at least 1 respiratory virus, while 31/152 (20.4%) were negative. FilmArray RP results were concordant in 68/72 (94.4%) respiratory samples tested with laboratory-developed real-time PCR assays, while in 4/72 (5.6%) samples, the FilmArray RP assay detected an additional virus (2 human rhinovirus/enterovirus and 2 bocavirus). In addition, FilmArray RP results for 70 of 80 (87.5%) respiratory samples tested were concordant with the Seegene Seeplex RV15® detection assay (Seegene, Inc., Seoul, South Korea), while 10/80 (12.5%) were discordant. The advantages of the FilmArray RP are the rapid detection of respiratory viruses (1 hour), the wide number of pathogens detectable in a single assay, and the reduced hands-on time.     

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect bla_OXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for bla_OXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.     

Multiplex real-time polymerase chain reaction for rapid detection of β-lactamase–negative,ampicillin-resistant Haemophilus influenzae

We evaluated a multiplex real-time quantitative polymerase chain reaction (PCR) method for quantification of Haemophilus influenzae and rapid detection of β-lactam–resistant strains. We designed 5 PCR primer sets to simultaneously detect the β-lactam–resistant genes and quantify the pathogen. To demonstrate the validity of this assay, we used 191 clinical isolates, including 141 H. influenzae strains, and 100 purulent sputum samples, including 30 samples from which H. influenzae had been isolated. This assay showed 92.9% sensitivity and 91.8% specificity for detecting β-lactam–resistant genes, relative to the conventional phenotypic method, and this assay correlated well with conventional quantitative culture counts. By using this assay, we could quantify H. influenzae and identify β-lactam susceptibility in only 3 h and with only one tube. This method will be helpful for the rapid detection of H. influenzae infections and the selection of appropriate antibiotics.     

Influence of GeneXpert MRSA/SA test implementation on clinical outcomes of Staphylococcus aureus bacteremia — a before–after retrospective study

Use of GeneXpert MRSA/SA in diagnostic algorithms of Staphylococcus aureus bacteremia may influence both patients' clinical outcomes and antibiotic stewardship. We evaluated these outcomes in a retrospective cohort before (1/6/2015–31/5/2016) and after (1/6/2016–31/8/2017) the introduction of the test in adult patients with Gram-positive cocci in clusters in blood cultures. We included 254 patients (125 preintervention, 129 postintervention). No significant difference in 30-day mortality or clinical success was demonstrated between periods. Appropriate antibiotic therapy rates were significantly higher in the postintervention group, and vancomycin use was significantly reduced (80.6% vs 53.6%, P?<?0.01; 2.3±0.38 vs 2.98±1.02 defined daily doses/100 patient days, P?=?0.026, respectively). Appropriate beta-lactam use was also significantly higher (56.7% postintervention vs 23.1% preintervention, P?<?0.01). Use of GeneXpert MRSA/SA test has a positive effect on antibiotic stewardship measures, though it has no significant effect on clinical outcomes including mortality in this fatal infection.     

Clinical utility of pan-microbial PCR assays in the routine diagnosis of infectious diseases

The goals of the study were to examine the analytical properties and the clinical utility of pan-microbial PCR (PM-PCR) assays in a retrospective study conducted in 2014–2015 at the Tel-Aviv Sourasky Medical Center. PM-PCR included in-house assays for pan-bacterial, pan-fungal, and pan-mycobacterial PCR followed by sequencing. The clinical utility of the assays was decided based on defined criteria/categories. There were 585?PM-PCR tests performed on samples from 306 patients. The positivity rates of PM-PCR for bacterial, fungal, and mycobacterial infections were 72/316 (22.7%), 16/186 (8.6%), and 6/83 (7.2%), and the sensitivity values were 65%, 76%, and 85%, respectively. PCR results had influenced the management in 14/82 (17%) of PCR-positive cases and in 13/222 (5.8%) of PCR-negative cases (P?=?0.005). The causes for the low clinical utility were related to lack of effect on the initial treatment in PCR-negative cases and concurrent positive cultures or presumed contamination in PCR-positive cases.     

Plasma cell-free metagenomic next generation sequencing in the clinical setting for the diagnosis of infectious diseases: a systematic review and meta-analysis

Plasma cell-free metagenomic next-generation sequencing (cf-mNGS) is a non-invasive method that may be able to identify thousands of pathogens through a hypothesis-free approach. There is a lack of consensus on how this test compares to conventional microbiologic testing. We conducted a systematic review and meta-analysis of published studies evaluating the accuracy of plasma cf-mNGS in hospitalized patients and present pooled estimates of the positive (PPA) and negative percent agreement (NPA) compared to a composite reference standard that included all conventional microbiological testing and clinical history as assessed by an adjudication panel or clinical treatment team. Five retrospective studies (n = 552) were included. The majority of the patients (56%?88%) were immunocompromised. The pooled PPA was 67% (95% CI, 54%?81%) and the pooled NPA was 70% (95% CI, 63%?77%). The pooled diagnostic performance characteristics suggest that cf-mNGS provides limited evidence for ruling in or out the presence of infection as commonly used.     

Direct identification of bacteria in positive blood cultures: comparison of two rapid methods,FilmArray and mass spectrometry

We evaluated the accuracy and performance of the FilmArray Direct from Positive Blood Culture system (BCID) (BioFire Diagnostics, Salt Lake City, UT, USA) and the VITEK Mass Spectrometry System (Vitek MS; bioMerieux, Durham, NC, USA) to identify bacterial isolates from 161 positive blood culture bottles. The BCID uses multiplex PCR to identify 90–95% of common isolates to the genus or species/complex level as well as mecA, Van A/B, and bla_KPC genes in approximately 1 hour. Of 151 monomicrobic isolates, the FilmArray correctly identified 48/49 (98%) to the genus and 84/84 (100%) to the species/complex level, while 18/151 (12%) gave no identification, as expected from the database. Mass spectrometry correctly identified 142/151 (94%) monomicrobic cultures to the genus level, 137/151 (91%) to the species level, with only 8/151(5%) giving no identification. Although mass spectrometry has a much larger database, the filtration system was cumbersome in contrast to the 3–5 minutes hands-on-time for the BCID.     

Detection of “Xisco” gene for identification of Streptococcus pneumoniae isolates

We describe a PCR-assay differentiating Streptococcus pneumoniae from closely-related species of the Mitis group of the genus Streptococcus and identification of pneumococcus clinical isolates, based on the “Xisco” gene discriminatory marker. The complete “Xisco” gene sequence was observed in all S. pneumoniae genomes analyzed and absent in all non-pneumococcus genomes.     

An enhanced method for the identification of Leishmania spp. using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region

The accurate identification of Leishmania spp. is important for the treatment of infected patients. Molecular methods offer an alternative to time-consuming traditional laboratory techniques for species determination. We redesigned a 7SL RNA gene-based polymerase chain reaction and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania guyanensis, and the previously indistinguishable Leishmania braziliensis and Leishmania panamensis. The Leishmania donovani complex members remain indistinguishable by this method, as are the representatives of Leishmania amazonensis/Leishmania garnhami and Leishmania mexicana/Leishmania pifanoi.     

YEAST PANEL multiplex PCR for identification of clinically important yeast species: stepwise diagnostic strategy,useful for developing countries

Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assays, which are time-consuming and prone to errors. Wrong species identification may result in suboptimal treatment and inaccurate epidemiological data. To improve rapidity and accuracy of species identification, a diagnostic strategy using a stepwise “YEAST PANEL multiplex PCR assays” targeting 21 clinically important yeast species of Candida, Trichosporon, Rhodotorula, Cryptococcus, and Geotrichum was designed. Four hundred CBS reference strains were used for optimization and specificity testing. Eight hundred clinical species were prepared in blinded sets for multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption time of flight mass spectrophotometry (MALDI-TOF MS) investigation. Results obtained from YEAST PANEL multiplex PCR assay were 100% consistent with those of MALDI-TOF MS. Utilization of pure colony testing showed distinct amplicons for each species, thus eliminating the need for DNA extraction. The targeted yeast species of this assay are responsible for 95% of the yeast infections. In conclusion, due to the high accuracy and coverage of a broad range of yeasts, this assay could be useful for identification in routine laboratories and epidemiological studies.     

Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles

We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10⁵ CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.     

Real-time polymerase chain reaction method for detection of toxigenic Clostridium difficile from stools and presumptive identification of NAP1 clone

This study describes the development of a cost-effective, multiplex real-time polymerase chain reaction (RTPCR) method for detection of toxigenic Clostridium difficile from stools and presumptive identification of the NAP-1 strain. The diagnostic value of the new method is for the detection of toxigenic C. difficile which has the following performance characteristics: 99.8% specificity, 95.1% sensitivity, 97.5% positive predictive value, and 99.5% negative predictive value. Examination of 24 specimens presumptively identified as NAP1 strain by RTPCR with Pulsed-field gel electrophoresis performed on C. difficile isolated from those specimens showed 100% agreement. This RTPCR showed equivalent test performance characteristics as the 2 commercially available assays which were evaluated. The estimated cost per test is CAD$9.50 and which is significantly less than the commercial assays. The average turnaround time from setup to detection is 3.5 h. The RTPCR method described here is a cost-effective and highly sensitive test which can be implemented in a clinical laboratory to assist clinicians in establishing the diagnosis of C. difficile infection and indirectly determine the presence of the hypervirulent epidemic binary toxin (BI)/NAP 1 strain for prompt infection control interventions.     

Prevalence of antimicrobial-resistant Cutibacterium isolates and development of multiplex PCR method for Cutibacterium species identification

IntroductionCutibacterium species such as C. acnes, C. avidum, and C. granulosum are known anaerobic skin inhabitants and often cause surgical site infections. These species are genetically similar and are difficult to identify rapidly. In addition, their pathogenicity and antimicrobial resistance remain unknown. In this study, antimicrobial resistance in Cutibacterium isolates was studied and a multiplex PCR method for their identification was developed.MethodsA total of 497 C. acnes, 71 C. avidum, and 25 C. granulosum strains which were isolated from the acne pustule and infectious regions, were used.ResultsThe antimicrobial resistance rates of C. acnes, C. avidum, and C. granulosum strains isolated from patients with acne vulgaris were higher than those of strains isolated from patients with infectious diseases. In particular, macrolide-clindamycin-resistant strains were isolated most frequently from all species. Among the resistant strains, strains with 23S rRNA mutations were the most common in C. acnes (24.3%, 71/292), whereas C. avidum and C. granulosum strains were most frequently found with erm(X). For the first time, a C. granulosum strain carrying pTZC1, which codes erm(50) and tet(W), was isolated from patients with acne vulgaris. Regarding the rapid identification of causative pathogens from infectious regions, three Cutibacterium species were identified with 100% sensitivity and specificity using multiplex PCR method.ConclusionsOur data showed that antimicrobial resistance differed among Cutibacterium species. The multiplex PCR method may contribute to the rapid detection of Cutibacterium species and selection of appropriate antimicrobial agents.     

Reliable identification of clinically prevalent species and subspecies of staphylococci by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis

We identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 10 species and 5 subspecies of Staphylococcus among 139 clinical isolates and compared it with conventional tests. All isolates showed species-specific whole-cell protein profiles, even atypical strains, with up to 60% and at least 73.5% of interspecies and intraspecies similarity, respectively. Except for 5 isolates that presented biochemical profiles of Staphylococcus hominis subsp. hominis, but were identified as S. hominis subsp. novobiosepticus by SDS-PAGE, there was 100% accordance between the methods used. Comparison with the partial 16S-rDNA sequences confirmed by SDS-PAGE showed the high accuracy of this method to identify staphylococci subspecies and species.     

Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.