Evaluation of the FilmArray™ Meningitis/Encephalitis panel with focus on bacteria and Cryptococcus spp.

PURPOSE: Molecular methods provide fast and accurate detection of both bacteria and viruses in the cerebrospinal fluid (CSF) causing infection in the central nervous system (CNS). In the present study we evaluated the bacterial detection performance of the fully automated FilmArray™ Meningitis/Encephalitis (ME) panel (bioMérieux) by comparing it with culture and multiplexed in-house PCR. METHODS: Three sample types were analysed; Contrived samples with known bacterial/fungal concentration (n = 29), clinical samples from patients with verified cause of CNS infection (n = 17) and external quality assessment (EQA) samples (n = 11). Another six samples were purposely prepared with multiple targets to evaluate multiplex capacity. RESULTS: The FilmArray™ had a slightly higher limit of detection for Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes and Streptococcus agalactiae compared to in-house PCR methods but performed equal or better when compared to culture. The FilmArray™ ME panel detected the expected pathogen in 17 of 17 clinical samples and yielded detection of three additional viruses of which one was confirmed with comparator techniques. All but one of the EQA samples were correctly detected. CONCLUSIONS: The results of this study are promising and the FilmArray™ ME panel could add to the diagnostic algorithm in CNS-infections. However, the limit of detection for the important pathogens N. meningitidis and S. pneumoniae could be improved.

Clinical presentation of invasive disease caused by Neisseria meningitidis serogroup Y in Sweden, 1995 to 2012.

Over the period 1995-2012, the incidence of invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y (NmY) increased significantly in Sweden. This is mainly due to the emergence of a predominant cluster named strain type YI subtype 1, belonging to the ST-23 clonal complex (cc). The aim of this study was to examine the clinical picture of patients with invasive disease caused by NmY and to analyse whether the predominant cluster exhibits certain clinical characteristics that might explain the increased incidence. In this retrospective observational study, the medical records available from patients with IMD caused by Nm serogroup Y in Sweden between 1995 and 2012 were systematically reviewed. Patient characteristics, in-hospital findings and outcome were studied and differences between the dominating cluster and other isolates were analysed. Medical records from 175 of 191 patients were retrieved. The median age was 62 years. The all-cause mortality within 30 days of admission was 9% (15/175) in the whole material; 4% (2/54) in the cohort with strain type YI subtype 1 and 11% (12/121) among patients with other isolates. Thirty-three per cent of the patients were diagnosed with meningitis, 19% with pneumonia, 10% with arthritis and 35% were found to have bacteraemia but no apparent organ manifestation. This survey included cases with an aggressive clinical course as well as cases with a relatively mild clinical presentation. There was a trend towards lower mortality and less-severe disease in the cohort with strain type YI subtype 1 compared with the group with other isolates.

Surveillance of invasive Neisseria meningitidis with a serogroup Y update, Sweden 2010 to 2012.

An increase of invasive meningococcal disease caused by Neisseria meningitidis serogroup Y has been noted in Sweden since 2005, and to a lower extent throughout Europe. The present study describes the epidemiology of invasive N. meningitidis isolates in Sweden in the period between 2010 and 2012, with a focus on serogroup Y. We also aimed to find an optimal molecular typing scheme for both surveillance and outbreak investigations. All invasive N. meningitidis isolates in Sweden during the study period (n=208) were genetically characterised. Serogroup Y predominated with 22/57, 31/61 and 44/90 of all invasive isolates (incidence 0.23, 0.33 and 0.46 per 100,000 population) in 2010, 2011 and 2012 respectively. In each of these years, 15/22, 22/31 and 19/44 of serogroup Y isolates were genetically clonal (Y: P1.5­2,10­1,36­2: F4­1: ST-23(cc23), 'porB allele 3­36, fHbp allele 25 and penA allele 22). Our findings further support those of others that currently recommended FetA typing could be replaced by FHbp. Moreover, in line with a previous study that we conducted, the current results indicate that highly variable multilocus variable-number tandem repeat analysis (HV-MLVA) can be used as a first-hand rapid method for small outbreak investigations.

Genetic characterisation of the emerging invasive Neisseria meningitidis serogroup Y in Sweden, 2000 to 2010.

Neisseria meningitidis serogroups B and C have been responsible for the majority of invasive meningococcal disease in Europe. Recently, an increase of N. meningitidis disease due to serogroup Y has been noted in Sweden (in 2010, the proportion was 39%, with an incidence of 0.23 per 100,000 population), as well as in other northern European countries. We aimed to investigate the clonal pattern of the emerging serogroup Y in Sweden during 2000 to 2010. The serogroup Y isolates identified during this time (n=85) were characterised by multilocus sequence typing and sequencing of the fetA, fHbp, penA, porA and porB genes. The most frequent clone (comprising 28 isolates) with identical allele combinations of the investigated genes, was partly responsible for the observed increased number of N. meningitidis serogroup Y isolates. It was sulfadiazine resistant, with genosubtype P1.5-2,10-1,36-2, sequence type 23, clonal complex 23, porB allele 3-36, fetA allele F4-1, fHbp allele 25 and penA allele 22. The first case with disease due to this clone was identified in 2002: there was a further case in 2004, six during 2006 to 2007, eight during 2008 to 2009, with a peak of 12 cases in 2010. An unusual increase of invasive disease in young adults (aged 20­29 years) caused by this clone was shown, but no increase in mortality rate was observed.

Evaluation of a commercial multiplex PCR test (SeptiFast) in the etiological diagnosis of community-onset bloodstream infections.

The commercial polymerase chain reaction (PCR) test, SeptiFast, is designed to identify the DNA of individual bacterial and fungal pathogens in whole blood. We aimed to evaluate the usefulness of the test for the detection of community-onset bloodstream infections. We prospectively included adult patients who were subjected to blood culture (BC) at an infectious diseases department. For the evaluation, one BC/PCR set (two BC bottles and one PCR tube) per patient was used. When several sets were obtained and analyzed, the first set with any positive result was evaluated. Among 1,093 consecutively included patients, BC was positive in 138 and PCR was positive in 107. Fifty positive PCR results were supported by BC in the same BC/PCR set, ten were supported by other cultures, and, additionally, ten were supported by the clinical presentation. Compared with BC, PCR showed specificities and negative predictive values of >97% for all detectable pathogens. The following sensitivities and positive predictive values (PPVs) were noted: Staphylococcus aureus, 67% and 43%; Streptococcus pneumoniae, 12% and 67%; other Streptococcus species, 43% and 77%; Escherichia coli, 53% and 56%; and Klebsiella species, 43% and 23%. If support from other cultures and the clinical presentation were included in the reference standard, the PPVs for the detection of these bacteria were 57%, 100%, 92%, 75%, and 69%, respectively. Although the specificities were high, the low sensitivities and suboptimal PPVs noted in the present study discourage routine use of the test in its present form for the detection of community-onset bloodstream infections.

Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia.

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.

Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes.

Methicillin-resistant Staphylococcus aureus (MRSA) is an established nosocomial pathogen, but has recently begun to appear in the community. The clones in the community may not have originated in the hospital setting, and are referred to as community-acquired MRSA (CA-MRSA). Resistance to methicillin is mediated by the gene mecA, which is carried by the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec typing (I-IV) of all clinical isolates of MRSA (n = 92) from 1987 to 2004 in Orebro County, Sweden, was performed by real-time LightCycler PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB. Forty-one isolates harboured type IV SCCmec, of which ten could be classified further as subtype IVa, and 27 as subtype IVc. No isolates belonged to subtype IVb, but four isolates could not be subtyped, and may be examples of novel type IV SCCmec subtypes. Thirty-five MRSA isolates, assigned to six different pulsotypes by pulsed-field gel electrophoresis, did not belong to SCCmec types I-IV. The Panton-Valentine leukocidin (PVL) genes were identified in two of these pulsotypes. Only SCCmec type IV has been associated previously with the PVL toxin, but the results suggest that new PVL-positive clones with novel SCCmec types may be arising and disseminating in the community.

Pharyngeal carriage of serogroup W135 Neisseria meningitidis in Hajjees and their family contacts in Morocco, Oman and Sudan.

In 2000 the global outbreak that began in Saudi Arabia was caused by a W135:2a:P1.5,2 strain of Neisseria meningitidis belonging to the ET-37 complex and to ST-11. There was concern that introduction of this epidemic clone (EC) might lead to a wave of outbreaks in the African meningitis belt. The WHO therefore initiated studies of meningococcal carriage among pilgrims and their family contacts in Morocco, Oman and Sudan, 3 to 12 months after the Hajj 2000. In Morocco, 1186 persons were swabbed 3 times. Ninety-five meningococcal strains were isolated from 2.7% of the specimens. Pulsed-field gel electrophoresis showed that 32 (33.6%) were identical with the EC. In Sudan, 5 strains identical with the EC were obtained after sampling 285 persons. In Oman, among 18 meningococcal strains isolated from 399 subjects, 11 (61.1%) belonged to the EC. The important pharyngeal carriage of W135 (EC) and its role in the 2001-2002 outbreaks in Burkina Faso argues for the necessity of reinforcing surveillance, and adapting and planning responses in Africa and the Middle East using the most appropriate vaccine.

Detection of Panton-Valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects.

The prevalence of the Panton-Valentine leukocidin (PVL) gene in Staphylococcus aureus was investigated with a simple, reproducible and rapid real-time LightCycler SYBR Green I PCR assay. The PVL gene was detected in one isolate from 65 patients with S. aureus bacteraemia, in four isolates from 55 patients with respiratory tract infections, and in two isolates from 91 patients with cutaneous infections. In contrast, 15 of 25 cutaneous isolates of methicillin-resistant S. aureus (MRSA) were positive. All PVL-positive cutaneous MRSA isolates were community-acquired and comprised three different clones as determined by pulsed-field gel electrophoresis. The PVL gene was detected in isolates from patients with recurrent primary skin infections and S. aureus bacteraemia, but PVL did not seem to be an important virulence factor in the pathogenesis of staphylococcal bacteraemia.

Analysis of PorA variable region 3 in meningococci: implications for vaccine policy?

Outer membrane protein (OMP) vaccines are being developed against Neisseria meningitidis serogroup B which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited data is available in Europe from genosubtyping meningococci. We therefore undertook a retrospective analysis of the three main variable regions, VR1, VR2 as well as VR3, of the porA gene from N. meningitidis isolated from different countries, mainly from Scotland and Sweden. Analysis of this gene showed that, amongst 226 strains studied, there were a total of 78 different strains. No new VR1 or VR2 alleles were found but five new VR3 alleles are described. Our data indicates the importance of analysing the VR3 region of PorA in addition to VR1 and VR2 and also highlights, in general terms, the need for genosubtyping meningococci. Such analyses have major implications for the design of new meningococcal vaccines.

Comparison of serogroup W-135 meningococci isolated in Sweden during a 23-year period and those associated with a recent hajj pilgrimage.

An outbreak of serogroup W-135 meningococcal disease was reported among pilgrims returning from the annual hajj (pilgrimage to Mecca) in mid-March 2000. Molecular characterization was used to investigate the similarity of the hajj-associated W-135 strains with those isolated in Sweden during a 23-year period (1978 to 2000). The same hajj-associated genosubtype, genosubtype P1.5,2,36b, has been documented in Sweden since 1979, while pulsed-field gel electrophoresis and the sulfadiazine resistance of the W-135 isolates indicated that the outbreak was probably due to a new clone of W-135 meningococci.

Long-term persistence of a discotheque-associated invasive Neisseria meningitidis group C strain as proven by pulsed-field gel electrophoresis and porA gene sequencing.

A cluster of a Neisseria meningitidis serogroup C strain causing invasive disease was investigated. Five out of seven cases were associated with a particular discotheque. The strains were indistinguishable, as revealed by pulsed-field gel electrophoresis and sequencing of variable regions of the porA gene, but caused strikingly different clinical presentations during 5 months.

Genosubtyping by sequencing group A, B and C meningococci; a tool for epidemiological studies of epidemics, clusters and sporadic cases.

Genosubtyping, by sequencing variable regions (VRs) 1, 2 and 3 of the porA gene, was evaluated as a tool to detect clonality of isolates in meningococcal epidemics in Africa and clusters of disease in Sweden. All 63 examined meningococcal isolates were successfully genosubtyped. The isolates belonging to group A type 4 with genosubtype P1.20,9,35a showed little heterogeneity in African epidemics in 1988 and onwards. In Sweden, two meningococcal clones of group B type 15, with genosubtypes P1.7,16,35 and P1.7,16f,35, dominated during two clusters of meningococcal disease in 1995-96 and in sporadic cases thereafter. The characterisation of group C meningococci isolated during 1992 in Sweden indicated a cluster (type 2a with genosubtype P1.5a,10d,36b) connected with a discotheque visit. Two variants of VR2 (10p and 25b), not previously described, were found among the examined isolates. Nucleotide sequence analysis of VRs in the porA gene proved a valuable epidemiological tool since almost all isolates could be genosubtyped, in contrast to the phenotypic methods presently used.

Detection of borreliacidal antibodies by flow cytometry. An accurate, highly specific serodiagnostic test for Lyme disease.

BACKGROUND: Borreliacidal antibodies can be detected in serum samples from patients with early or late Lyme disease symptoms. When these serum samples are incubated with Borrelia burgdorferi and complement, spirochetes are rapidly killed. Detection of these antibodies can be used as a serodiagnostic test. METHODS: Individual serum samples containing IgM or IgG borreliacidal antibodies were Used to develop a method for detection using flow cytometry. An additional 10 case-defined Lyme disease serum samples and 10 normal serum samples were used to confirm appropriate flow cytometric parameters. To determine specificity, 157 normal serum samples and 104 potential cross-reactive serum samples were tested for borreliacidal activity and antibodies to B burgdorferi using indirect fluorescent antibody or enzyme immunoassay. RESULTS: Flow cytometry can be used to detect borreliacidal activity within 16 to 24 hours after incubation of B burgdorferi organisms. Lyme disease serum, and complement. Significant borreliacidal activity was detected in all Lyme disease serum samples. The percentages of positive normal serum samples were comparable (6% to 10%) using all three assays. In addition, the indirect fluorescent antibody and enzyme immunoassay identified 41 (39%) and 47 (45%) potential cross-reactive serum samples as positive, respectively. In contrast, significant borreliacidal activity was not detected in any potential cross-reactive serum samples. CONCLUSION: Detection of borreliacidal antibody, unlike indirect fluorescent antibody and enzyme immunoassay, is an accurate, highly specific serodiagnostic test for detection of Lyme disease.