Evaluation of the BioFire® FilmArray® Meningitis/Encephalitis panel in an adult and pediatric Ugandan population

BackgroundMeningitis causes significant mortality in sub-Saharan Africa and limited diagnostics exist. We evaluated the utility of the BioFire® FilmArray® Meningitis/Encephalitis multiplex PCR panel (BioFire ME) in HIV-infected adults and HIV-infected and uninfected children presenting with suspected meningitis in Uganda.MethodsWe tested cerebrospinal fluid (CSF) using a stepwise meningitis diagnostic algorithm including BioFire ME. We determined the diagnostic performance of BioFire ME for cryptococcal meningitis, using cryptococcal antigen (CrAg) and CSF culture as reference standards, and assessed other central nervous system (CNS) pathogens identified by the panel.ResultsWe evaluated 328 adult and 42 pediatric CSF specimens using BioFire ME. Of the adult CSF samples tested, 258 were obtained at baseline, and 70 were obtained from repeat lumbar punctures in cryptococcal meningitis. For Cryptococcus, sensitivity was 82%, specificity was 98%, PPV was 98%, and NPV was 79% in baseline specimens using CSF CrAg as the reference standard. Among follow-up specimens, a negative BioFire ME for Cryptococcus predicted CSF culture sterility with 84% NPV. Overall sensitivity was decreased at low fungal burdens: 29% for 0–99 Cryptococcus CFU/mL compared to 94% for ≥100 CFU/mL in baseline specimens. Other pathogens detected included E. Coli, H. influenzae, S. pneumoniae, CMV, enterovirus, HSV, HHV-6, and VZV. Two specimens tested positive for S. pneumoniae and one for Cryptococcus in the pediatric population.ConclusionsMultiplex PCR is a promising rapid diagnostic test for meningitis in adults and children in resource-limited settings. Cryptococcus at low fungal burdens in CSF may be missed by BioFire ME.     

Diagnostic test accuracy of the BioFire® FilmArray® meningitis/encephalitis panel: a systematic review and meta-analysis

BackgroundThe FilmArray® meningitis/encephalitis (ME) panel is a multiplex PCR assay which can detect the most commonly identified pathogens in central nervous system infections. It significantly decreases the time to diagnosis of ME and data has yielded several positive outcomes. However, in part, reports of both false positive and false negative detections have resulted in concerns about adoption.ObjectivesThe aim was to evaluate the ME panel in a diagnostic test accuracy review.Data sourcesThe PubMed and EMBASE databases were systematically searched through May 2019.Study eligibility criteriaEligible studies were those providing sensitivity and specificity data for the ME panel compared with a reference standard. Studies providing details on false positive and false negative results of the panel as well as further investigation (adjudication) of the discordant results between the panel and comparator assays were included and assessed separately.ParticipantsPatients with suspected ME for whom a panel was ordered were included.MethodsThe ME panel was compared to reference standard methods for diagnosing community-acquired ME. We performed a meta-analysis and calculated the summary sensitivity and specificity of the ME panel. Moreover, we evaluated the false positive and false negative results of the panel.ResultsThirteen studies (3764 patients) were included in the review and 8 of them (3059 patients) were pooled in a meta-analysis. The summary estimates of sensitivity and specificity with 95% confidence intervals (CI) was 90% (95% CI 86–93%) and 97% (95% CI 94–99%), respectively. When we looked specifically at studies that assessed further the false positive and false negative results, false positive detections were 11.4% and 4% before and after adjudication, respectively. The highest proportion of false positive was observed for Streptococcus pneumoniae followed by Streptococcus agalactiae. False negative isolates were 2.2% and 1.5% before and after adjudication, respectively. Herpes simplex virus 1 and 2, enterovirus and Cryptococcus neoformans/gattii had the highest proportions of false negative determinations. False negative C. neoformans/gattii were mostly patients with positive antigen titres, on treatment or cleared disease.ConclusionsThe currently available literature suggests that the ME panel has high diagnostic accuracy. However, the decision for implementation should be individualized based on the needs of the patient population, the capabilities of the laboratory, and the knowledge of the healthcare providers that will utilize the test.     

Usefulness of the FilmArray meningitis/encephalitis (M/E) panel for the diagnosis of infectious meningitis and encephalitis in Taiwan

Background/purposeEarly recognition of causative pathogens is critical for the appropriate management of central nervous system infection and improved outcomes. The BioFire^® FilmArray^® Meningitis/Encephalitis Panel (BioFire^® ME Panel, BioFire Diagnostics) is the first U.S. Food and Drug Administration (FDA)-approved multiplex PCR assay that allows the rapid detection of 14 pathogens, including bacteria (n = 6), viruses (n = 7), and fungi (n = 1), from cerebrospinal fluid (CSF). The performance of the panel is expected to be dependent on the epidemiology of M/E in different geographical regions.MethodsIn this preliminary study, we used the BioFire^® ME Panel in 42 subjects who presented to the emergency department with symptoms of M/E in our hospital. The results were compared to conventional culture, antigen detection, PCR, and various laboratory findings.ResultsThe panel detected six positive samples, of which five were viral and one bacterial. We observed an overall agreement rate of 88% between the BioFire^® ME Panel results and the conventional methods. There were no false-positive findings, but five discordant results were observed for enterovirus, herpes simplex virus type 1, Escherichia coli, and Cryptococcus species.ConclusionsThe BioFire^® ME Panel performed equivalently to the traditional PCR methods for virus detection, and better than bacterial cultures. This revolutionary system represents a paradigm shift in the diagnosis of M/E and may aid in the rapid identification of community-acquired M/E. However, the usefulness of this tool is limited in regions with a high prevalence of infectious M/E caused by microorganisms not included in the panel.     

Reactivation of herpes simplex type 1 in pneumococcal meningitis

BackgroundAcute bacterial meningitis (ABM) and herpes simplex type 1 (HSV-1) encephalitis are two rare but serious infections affecting the central nervous system (CNS). Concurrent bacterial and viral CNS infection has occasionally been reported.ObjectivesTo illustrate the possibility of intrathecal infection with both Streptococcus pneumonia and HSV-1 by presenting a case and to examine whether herpesvirus reactivation is common in ABM.Study designWe report a case diagnosed with HSV-1 reactivation in the cerebrospinal fluid (CSF) during treatment for pneumococcal ABM. A retrospective analysis of CSF samples from 21 patients with ABM was performed, with analysis of DNA from HSV-1 and four other neurotropic herpesviruses.ResultsAll 21CSF samples were negative for HSV-1, HSV-2, varicella zoster-virus, Epstein–Barr virus and human herpesvirus 6 DNA by PCR.ConclusionsAlthough herpesvirus infection does not seem to be a common phenomenon in ABM we suggest that HSV-1 reactivation could be kept in mind if patients with ABM show symptoms or signs compatible with encephalitis.     

Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis

In a two-centre study, the routine DNA preparation and PCR amplification protocols were compared for herpes simplex virus (HSV) detection in cerebrospinal fluids (CSFs) of 43 patients with suspected herpes simplex encephalitis (HSE). The combined clinical, radiological and laboratory results indicated HSE in 6/43 (14%) patients. Discrepant PCR results between the two centres were obtained in 8 (18%) cases consisting of 5 false-positive and 3 false-negative results. Seven out of 8 (88%) discrepant results were associated with the method of CSF preparation using protease K digestion followed by heat inactivation. In contrast, CSF digestion with proteinase K followed by DNA purification on silica spin columns was better yielding discrepant PCR results in only 1 of 78 analyses (1.3%). The results point to the need for standardization and inter-laboratory quality control for routine clinical work. J. Med. Virol. 57:31–35, 1999. © 1999 Wiley-Liss, Inc.     

Rapid identification of bloodstream bacterial and fungal pathogens and their antibiotic resistance determinants from positively flagged blood cultures using the BioFire FilmArray blood culture identification panel

Background/purposeRapid and accurate identification of pathogens and their antibiotic resistance directly from flagged blood cultures can aid clinicians in optimizing early antibiotic treatment and improve the clinical outcomes, especially in settings associated with high rates of bloodstream infection caused by vancomycin-resistant Enterococci (VRE) and carbapenem-resistant Enterobacteriaceae (CRE). We compared the results of the BioFire FilmArray Blood Culture Identification (BCID) panel with those of conventional methods for identifying the pathogens and their antibiotic susceptibility status.MethodsIn total, 100 randomly selected positive blood cultures (BACTEC Plus Aerobic/F bottles or BACTEC Anaerobic Lytic/10 bottles) were analyzed. The pathogen detection efficiency of FilmArray BCID panel was compared with that of conventional method using MALDI-TOF MS system (Bruker MALDI Biotyper) and susceptibility testing by the Vitek 2 system. The sequencing analysis of antibiotic resistance genes was performed for discrepant results obtained from MALDI Biotyper and Vitek 2.ResultsAmong the 100 positively flagged blood cultures, 94% of FilmArray BCID panel results were consistent with the MALDI Biotyper results. All five VRE isolates positive for vanA/vanB genes, 10 of 12 Staphylococcus species positive for mecA gene, and only one Klebsiella pneumoniae isolate positive for K. pneumoniae carbapenemase gene (bla_KPC) detected in the FilmArray BCID panel were also concordant with results by the results by conventional susceptibility testing/molecular confirmation.ConclusionsThe FilmArray BCID panel results not only demonstrated good correlation with conventional blood culture identification and susceptibility results but also provided results rapidly, especially for the early detection of MRSA, VRE and bla_KPC–mediated CRE.     

Diagnostic accuracy of rapid one-step PCR assays for detection of herpes simplex virus-1 and -2 in cerebrospinal fluid: a systematic review and meta-analysis

BackgroundRapid and accurate diagnosis of herpes simplex virus (HSV)-1 and -2 (HSV1/2) in cerebrospinal fluid (CSF) is important for patient management.ObjectivesSummarize the diagnostic accuracy of commercial rapid sample-to-answer PCR assays (results in <90 minutes, without a separate nucleic acid extraction step) for HSV1/2 detection in CSF.Data sourcesFour databases (MEDLINE, EMBASE, Scopus, and CENTRAL) and five conference abstract datasets from January 2012 to March 2022.Study eligibility criteriaEligible diagnostic accuracy studies provided sufficient data for the construction of a standard diagnostic accuracy two-by-two table.ParticipantsPatients with suspected meningitis and/or encephalitis.TestsFilmArray Meningitis-Encephalitis Panel and Simplexa HSV 1&2 Direct Kit PCR.Reference StandardReal-time PCR assay.Assessment of risk of biasTwo investigators independently extracted data, rated risk of bias, and assessed quality using QUADAS-2.Methods of data synthesisAccuracy estimates were pooled using Bayesian random effects models.ResultsThirty-one studies were included (27 FilmArray; 4 Simplexa), comprising 9924 samples, with 95 HSV-1 and 247 HSV-2 infections. Pooled FilmArray sensitivities were 84.3% (95% credible interval, 72.3–93.0) and 92.9% (95% credible interval (CrI), 82.0–98.5) for HSV-1 and HSV-2, respectively; specificities were 99.8% (95% CrI, 99.6–99.9) and 99.9% (95% CrI, 99.9–100). Pooled Simplexa sensitivities were 97.1% (95% CrI, 88.1–99.6) and 97.9% (95% CrI, 89.6–99.9), respectively; specificities were 98.9% (95% CrI, 96.8–99.7) and 98.9% (95% CrI, 97.1–99.7). Pooled FilmArray sensitivities favoured industry-sponsored studies by 10.0 and 13.0 percentage points for HSV-1 and HSV-2, respectively. Incomplete reporting frequently led to unclear risk of bias. Several FilmArray studies did not fully report true negative data leading to their exclusion.ConclusionsOur results suggest Simplexa is accurate for HSV1/2 detection in CSF. Moderate FilmArray sensitivity for HSV-1 suggests additional testing and/or repeat CSF sampling is required for suspected HSV encephalitis when the HSV-1 result is negative. Low prevalence of HSV-1 infections limited summary estimates' precision. Underreporting of covariates limited exploration of heterogeneity.     

Evaluation of the Biofire FilmArray meningitis/encephalitis assay for the detection of Cryptococcus neoformans/gattii

ObjectivesCryptococcal meningitis (CM) remains an important cause of morbidity and mortality among immunocompromised patients. Laboratory diagnostics for CM includes antigen detection, staining and culture. Data on the performance of the Biofire® FilmArray® meningitis/encephalitis (ME) panel for detecting Cryptococcus neoformans/gattii is limited, with several reports describing false negativity for this target.MethodsA retrospective analysis of 1384 physician-ordered ME panel tests ordered between January 2017 to October 2018 was performed. ME panel results were compared to cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) and CSF culture testing and clinical significance of cryptococcal detection was determined.ResultsThere were 34 patients positive for cryptococcal detection by either ME panel, CSF CrAg or CSF culture in 2.7% of CSF specimens tested (38/1384). Of the 34 patients positive for cryptococcal detection, 85.3% were human immunodeficiency virus positive (29/34). The ME panel detected 32/38 (84.2%) cryptococcal-positive specimens, culture detected 28/38 (73.7%) and CSF CrAg was positive in 37/38 specimens (97.4%). The ME panel had a sensitivity and specificity of 96.4% (95% CI 81.7–99.9%) and 99.6% (95% CI 99.2–99.9%) compared with culture, and 83.8% (95% CI 68.0–93.8%) and 99.9% (95% CI 99.6–100.0%) compared to CSF CrAg testing, respectively. CrAg titres were lower among ME panel-negative, culture-negative specimens compared with ME panel-positive, culture-negative specimens (reciprocal median end-point titres of 128 ± 60 vs. 1920 ± 1730, p 0.04). All five CrAg-positive, ME panel- and culture-negative specimens were obtained from previously treated CM patients.DiscussionThe ME panel had high correlation with CSF culture and a somewhat lower correlation with CSF CrAg testing. The potential utility of using negative ME panel test results to predict culture sterility among patients undergoing treatment for CM warrants further study.     

An international external quality assessment of nucleic acid amplification of herpes simplex virus.

BACKGROUND: There is an increasing awareness of the need for external quality control of diagnostic virology. OBJECTIVES: To assess the quality of nucleic acid amplification tests (NAT) of herpes simplex within Europe. STUDY DESIGN: Herpes simplex virus (HSV) proficiency panels were produced at the Swedish Institute for Infectious Disease Control on behalf of the European Union Concerted Action for Quality Control of Nucleic Acid Amplification in 1999 and 2000. Nine reference laboratories evaluated the production process. Each panel consisted of 12 coded samples with various concentrations of inactivated, freeze-dried HSV type 1 (HSV-1), and HSV type 2 (HSV-2), or negative controls. Positive samples included HSV-1 and HSV-2 in a range of concentrations (2 x 10(2) to 2 x 10(7) genome copies per ml) similar to those found in cerebrospinal fluids from patients with HSV encephalitis. RESULTS: Sixty-six participants reported a total of 76 data sets for panel 1, and 71 reported 78 data sets for panel 2. The majority of the participants employed qualitative 'in-house' polymerase chain reaction (PCR) methods, either in a single, nested or semi-nested format. For panel 2, 9 laboratories reported use of 'real-time' PCR in contrast to 3 for panel 1. Three laboratories submitted quantitative results on both panels. Thirty percent of the data sets had correct results for the entire panel 1. In 6 data sets (8%) a total of 11 false positive results were reported. For panel 2, 28% of the data sets had correct result. Nineteen false positive results were reported in 14 data sets (18%), but most of the incorrect results reflected a lack of test sensitivity. CONCLUSIONS: The relatively high frequency of false positive results and the large number of false-negative results, albeit at low copy number, stress the need for improvement in the quality of HSV NAT and for external quality control programmes.     

A multicenter prospective trial to asses a new real-time polymerase chain reaction for detection of Treponema pallidum,herpes simplex-1/2 and Haemophilus ducreyi in genital,anal and oropharyngeal ulcers

Treponema pallidum, herpes simplex virus types 1 or 2 (HSV-1/2) and Haemophilus ducreyi are sexually transmitted pathogens that can cause genital, anal and oropharyngeal ulcers. Laboratory evaluation of these pathogens in ulcers requires different types of specimens and tests, increasing the risk of improper specimen handling and time lapse until analysis. We sought to develop a new real-time PCR (TP-HD-HSV1/2 PCR) to facilitate the detection of T. pallidum, HSV-1/2 and H. ducreyi in ulcers. The TP-HD-HSV1/2 PCR was tested (i) in a retrospective study on 193 specimens of various clinical origin and (ii) in a prospective study on 36 patients with genital, anal or oropharyngeal ulcers (ClinicalTrials.gov # NCT01688258). The results of the TP-HD-HSV1/2 PCR were compared with standard diagnostic methods (T. pallidum: serology, dark field microscopy; HSV-1/2: PCR; H. ducreyi: cultivation). Sensitivity and specificity of the TP-HD-HSV1/2 PCR for T. pallidum were both 100%, for HSV-1 100% and 98%, and for HSV-2 100% and 98%, respectively. T. pallidum and HSV-1/2 were detected in 53% and 22% of patients in the prospective study; H. ducreyi was not detected. In the prospective study, 5/19 (26%) specimens were true positive for T. pallidum in the TP-HD-HSV1/2 PCR but non-reactive in the VDRL. The TP-HD-HSV1/2 PCR is sensitive and specific for the detection of T. pallidum and HSV-1/2 in routine clinical practice and it appears superior to serology in early T. pallidum infections.     

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of pneumococcal and meningococcal meningitis in a tertiary care hospital

This study evaluated the performance of a real-time polymerase chain reaction (PCR) assay in comparison with Gram staining and culture of cerebrospinal fluid for the diagnosis of meningococcal and pneumococcal meningitis in patients with suspected bacterial meningitis. The sensitivity for detection of Neisseria meningitidis in cerebrospinal fluid was 87% (20/23) for the PCR assay, 27% (6/22) for Gram staining, and 17% (4/23) for culture. The sensitivity for detection of Streptococcus pneumoniae in cerebrospinal fluid was 100% (14/14) for the PCR assay, 62% (8/13) for Gram staining, and 36% (5/14) for culture. Therefore, we recommend that real-time PCR of cerebrospinal fluid for detection of N. meningitidis and S. pneumoniae become a part of the routine diagnostic procedure for patients with suspected bacterial meningitis.     

Investigation of the presence of carbapenemases in carbapenem-resistant Klebsiella pneumoniae strains by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and comparison with real-time PCR method

PurposeWe aimed to develop a new procedure for rapid detection of the carbapenemase activity using MALDI-TOF MS, and to determine the sensitivity and specificity of the method. Also, we aimed to determine the distribution of carbapenemase genes among the K.pneumoniae strains isolated in our hospital using real-time PCR.MethodBetween January 2017–February 2019; K. pneumoniae strains(n = 74) isolated from blood culture samples were included. Klebsiella pneumoniae NCTC 13438 was used as a positive control and Escherichia coli ATCC 25922 as a negative control. First, Imipenem, meropenem, and ertapenem MIC values of strains were determined. Then bla_KPC, bla_OXA-48, and/or bla_NDM genes were investigated with PCR. Carbapenemase activity was investigated in strains with the newly developed method using MALDI-TOF MS. The performance of the new method was evaluated for both the second and fourth hours of the incubation period.ResultsWhile 65 strains were found resistant to tested carbapenems, nine of them were susceptible. Of the 65 resistant strains, 57 had bla_OXA-48, 15 had bla_NDM, and four had bla_KPC genes. Bla_OXA-48 and bla_NDM genes were detected together in 11 strains. Bla_OXA-48, bla_NDM, and bla_KPC genes were not detected in any of the susceptible strains. The sensitivity and specificity of MALDI-TOF MS at the second hour were 83.1% and 100%, respectively. At the fourth hour, the sensitivity and specificity of MALDI-TOF MS were 100%. No false-positive results were observed.ConclusionThe sensitivity of the method at the fourth hour was better than the second hour. The false-negative results observed in the second hour disappeared when the incubation period was extended to 4 h. MALDI-TOF MS which is still under development is a fast, cost-effective, promising method for the detection of carbapenemase activity.     

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

BackgroundThe rapid molecular diagnosis of enteroviral meningitis has been shown important for an adequate management of the patients.ObjectivesA new CE-marked real-time RT-PCR assay (ENTEROVIRUS R-gene?, Argene) was evaluated in two university hospital virology laboratories.Study designReactivity, analytical sensitivity and specificity were evaluated using 54 prototype and 173 clinical human enterovirus (HEV) strains, a 12-sample HEV proficiency panel, and 30 non-HEV microorganisms. The clinical performance of the ENTEROVIRUS R-gene? assay was evaluated by testing 197 cerebrospinal fluid (CSF) and 103 respiratory specimens, comparatively to the routinely used diagnostic techniques.ResultsSixty-four out of the 65 HEV serotypes tested were detected. The analytical sensitivity ranged between 10^?2.64 and 10^2.39 TCID₅₀/50 μl. Cross-reactivity was observed with four human rhinoviruses. On 59 CSF specimens analyzed prospectively, the results of the ENTEROVIRUS R-gene? assay showed a 94.8% concordance with those of the Smart enterovirus (EV) assay (Cepheid). On 138 CSF specimens tested retrospectively, the results of the ENTEROVIRUS R-gene? assay showed a 97.1% concordance with those of either the GeneXpert EV assay (Cepheid) or the in-house RT-PCR HEV assays used at the time of specimen collection. On 103 respiratory specimens, the concordance between the results of the ENTEROVIRUS R-gene? assay and those of the routine RT-PCRs or viral culture was 90.2% and 96.1% before and after retest, respectively.ConclusionsThe new test was found able to detect a large panel of enterovirus serotypes; it was sensitive when used on clinical specimens; and, easy and rapid to perform on a routine basis.     

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR

BackgroundSusceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out.ObjectivesThe goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting.Study designA DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates. The DRA consisted of pre-culture of a clinical sample for 48 h and subsequent culture in the presence of antivirals for 24 h. Viral DNA concentration in the culture lysates was measured by an internally controlled quantitative real-time HSV-1 PCR and corrected for cell count and lysis by beta-globin PCR. DRA results were compared to results from PRA and sequence analysis.ResultsDRA results were in accordance with PRA results for both aciclovir and foscarnet susceptibility and appeared to have good discriminative value for low-level resistance due to UL30 gene mutations. Although the direct application of DRA in clinical samples appeared not possible, short pre-culture of 48 h was sufficient and ensured results within a clinically relevant time frame of 5 days.ConclusionsDRA is an accurate, rapid and easy to perform phenotypical susceptibility test for HSV-1.     

Role of <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> in pediatric encephalitis

A retrospective study investigating all the infectious encephalitis cases hospitalized at the pediatric intensive care unit of Edouard Herriot University Hospital in Lyon, France, was carried out in order to estimate the prevalence of Mycoplasma pneumoniae in acute childhood encephalitis. From January 2001 to December 2005, the cases of 29 children were selected and reviewed. M. pneumoniae related encephalitis was considered as probable in five cases (17%) on the basis of positive serological tests or positive polymerase chain reaction (PCR) tests in throat or nasopharyngeal swab while the PCR tests performed from the cerebrospinal fluid were negative. This study suggests that M. pneumoniae may be a major cause of infectious encephalitis in children as well as enteroviruses or Epstein-Barr virus detected in five and three cases, respectively.     

Urinary detection of toscana virus nucleic acids in neuroinvasive infections

BackgroundToscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion.ObjectivesTo evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen.Study designA total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing.ResultsPhlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample.ConclusionsThese preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available.     

Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility

We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of ≤0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.     

Broad-Range PCR-Electrospray Ionization Mass Spectrometry for Detection and Typing of Adenovirus and Other Opportunistic Viruses in Stem Cell Transplant Patients

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log₁₀ copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.     

The use of broad-range bacterial PCR in the diagnosis of infectious diseases: a prospective cohort study

ObjectivesBroad-range PCR has the potential to detect virtually any bacterial species via amplification and nucleotide sequencing of a DNA region common to all bacteria. We aimed to evaluate its usefulness and clinical relevance when applied to a wide variety of primary sterile materials.MethodsA prospective study including 1370 samples (75 heart valves, 151 joint tissue samples, 230 joint aspirates, 848 whole blood samples and 66 culture-negative cerebrospinal fluid samples) were studied by using a commercial PCR system for detecting 16S rDNA (Molzym). The PCR results were compared with culture and were considered to provide added diagnostic value only if the PCR approach revealed new pathogens that were missed by culture.ResultsThe added value of PCR was evident in 173 of 555 PCR-positive samples (0.126; 0.109–0.144 (proportion from all tested samples; 95% confidence interval)), most frequently in examinations of heart valves (0.56; 0.448–0.672) and joint tissue samples (0.219; 0.153–0.284). In contrast, the lowest rate of PCR with added value was noted for blood samples, regardless of the patient cohort they had been drawn from (nononcologic patients from intensive care: 0.065; 0.043–0.087, haematooncologic children: 0.048; 0.027–0.070). Moreover, PCR missed up to 7.1% of blood culture findings (0.071; 0.048–0.095) regarded as clinically relevant, which was the second highest failure rate after joint tissue samples (0.099; 0.052–0.147).ConclusionsBroad-range PCR substantially increases detection rate of pathogens, especially from heart valves and joint samples. However, a concurrent risk of false-negative PCR results justifies the need for parallel culture.     

Respiratory Virus Detection in Immunocompromised Patients with FilmArray Respiratory Panel Compared to Conventional Methods

Respiratory virus infections cause significant morbidity and mortality in immunocompromised patients. Timely diagnosis is needed to provide optimal clinical care. Diagnostic tests routinely available at most institutions are limited by poor sensitivity and a slow turnaround time. We collected 90 respiratory samples from 87 immunocompromised patients (56 bronchoalveolar lavage and 34 nasopharyngeal aspirate samples) in order to compare the performance of routine respiratory virus testing available at our institution to the FilmArray respiratory panel assay, a novel diagnostic tool which utilizes multiplex PCR to test for 21 respiratory pathogens with a 1-h turnaround time. Samples with discordant results and 13 samples with concordant results underwent further verification testing by laboratory-developed real-time PCR. The FilmArray assay identified viral pathogens in more samples than did clinical testing (30/90 versus 16/90; McNemar P = 0.001). Most of the additional viral pathogens identified by the FilmArray respiratory panel assay that were confirmed by verification testing were pathogens not assessed by routine clinical tests, including rhinovirus/enterovirus, human metapneumovirus, and coronavirus. The FilmArray respiratory panel assay allowed for increased identification of respiratory viral pathogens in this cohort of immunocompromised patients.