Antimicrobial resistance in Staphylococcus pseudintermedius and the molecular epidemiology of methicillin-resistant S. pseudintermedius in small animals in Finland.

Objectives: To investigate antimicrobial susceptibility in Staphylococcus pseudintermedius and the occurrence of methicillin-resistant S. pseudintermedius (MRSP), to explore the molecular structure of the MRSP population and to analyse risk factors for MRSP. Methods: Susceptibility data for clinical S. pseudintermedius isolates in 2011-15 were analysed using WHONET. All MRSP isolates in 2010-14 ( n = 362) were typed using PFGE. Representative isolates ( n = 87) of clusters were analysed using MLST and staphylococcal cassette chromosome mec (SCC mec ) typing. Risk factors were analysed using logistic regression. Results: Of the clinical S. pseudintermedius ( n = 1958; 98% from dogs), 14% were MRSP. Resistance to other antimicrobials varied between 12% and 39%. No trends were observed over time. Among clinical specimens (from infection sites) and screening specimens (from potential carriers), respectively, 2.5% (267/10 813) and 9% (211/2434) revealed MRSP. MLST revealed 42 different STs, including 19 new ones. Clonal complexes 71, 45 and 258 were the most common, but the MRSP population diversified over the years. A clinical S. pseudintermedius isolate was more likely to be MRSP if the patient was on antimicrobials at the time of sampling or was male. The presence of MRSP in screening specimens was more likely if the patient was on multiple antimicrobials at the time of sampling. Specimens from private clinics (versus the Veterinary Teaching Hospital of the University of Helsinki) had a higher likelihood of MRSP in both analyses. Conclusions: Resistance to antimicrobials among S. pseudintermedius in Finland is high, emphasizing the importance of infection control measures and susceptibility testing prior to therapy. The diverse MRSP population indicates non-clonal spread.

Epidemiology of methicillin resistant Staphylococcus pseudintermedius in guide dogs in Finland.

BACKGROUND: Methicillin resistant Staphylococcus pseudintermedius (MRSP) and Staphylococcus aureus (MRSA) are common multi-drug resistant (MDR) bacteria in dogs. In 2012-2013 three dogs of the Guide Dog School of the Finnish Federation of the Visually Impaired were found to be MRSP positive. Guide dogs have regular contact with each other during their first year of life and prolonged contact when in training. Since dogs are placed in different parts of Finland after training, there is a risk for national spread of MDR bacteria. In this study the prevalence of MRSP and MRSA, as well as the risk factors for MRSP were determined in the Finnish guide dog population. MRSP isolates were investigated using molecular methods and compared to the earlier isolates. RESULTS: Out of 132 tested dogs 4 were MRSP positive thus giving the prevalence estimate of 3% (95% CI: 1-8%) for MRSP in the target population. MRSA was not detected (prevalence estimate 0%, 95% CI: 0-3%). Risk factors associated with MRSP were being a breeding bitch (OR = 8.4; 95% CI: 1.1-64.1, P = 0.012), the number of veterinary visits (OR = 1.23; 95% CI: 1.0-1.5, P = 0.025) and number of antimicrobial courses (OR = 1.63; 95% CI: 1.0-2.55; P = 0.035). Identified MRSP isolates belonged to five different sequence types (ST45, 71, 402, 403 and 404). All ST71 isolates carried SCCmec II-III, while the SCCmec type of the ST45 and ST402 (a single locus variant of ST45) isolates were non-typeable with the method used. CONCLUSIONS: MRSP and MRSA had low prevalence in the studied dog population despite the close contact between dogs, and the MRSP population was heterogenic. Antimicrobial therapy and veterinary visits are risk factors for MRSP even among a small case group.

Performance of the Luminex xTAG Respiratory Viral Panel Fast in a clinical laboratory setting.

The aim of the study was to develop a real-time RT-PCR for the detection of enteroviruses (EVs) and rhinoviruses (RVs) and to assess the performance of the xTAG RVP Fast assay in comparison to a direct fluorescent assay (DFA), a real-time RT-PCR assay for the detection of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), and the EV/RV RT-PCR assay developed in this study. The performance of the RVP Fast assay was assessed in the analysis of 373 nasopharyngeal samples. For the viruses of the DFA panel, detection rates of 27.6% and 23.8% were obtained by RVP and DFA, respectively, in analysis of a set of 297 samples collected in 2009-2010. These results show statistically significant superiority of the RVP Fast assay (P=0.049). For RSV, hMPV, EV, and RV, detection rates of 48.0% and 45.2% were achieved by RVP and RT-PCR, respectively. For individual targets, increased detection of EV/RV (P=0.043) and decreased detection of influenza A virus (P=0.004) by RVP in comparison to real-time RT-PCR was observed. The results of the present study imply the need to adjust the InfA component of the RVP Fast assay to also cover the InfA(H1N1) 2009 virus.

Evaluation of multiplex polymerase chain reaction and microarray-based assay for rapid herpesvirus diagnostics.

Rapid diagnosis is critical to minimize morbidity and mortality associated with infections of the central nervous system (CNS). In this study, we evaluated the performance of a multiplex polymerase chain reaction (PCR) and microarray-based method, Prove-it™ Herpes, in a routine clinical laboratory setting for the diagnostics of 7 herpesviruses in viral CNS infections. Cerebrospinal fluid samples (n = 495), which had arrived for diagnostics in the 5 participating laboratories, were analyzed for herpesvirus DNA both by the current PCR-based method of the laboratory and by the microarray assay. The sensitivity and specificity for the microarray assay were 93% and 99%, respectively. The microarray assay was considered as a rapid and robust diagnostic platform that was easily implemented into the laboratory workflow. The broad herpesvirus coverage and the small sample volume required by the assay could benefit the diagnostics and thus the treatment of life-threatening infections of the CNS, especially among immunocompromised patients.

A novel screening method detects herpesviral DNA in the idiopathic pulmonary fibrosis lung.

BACKGROUND: Herpesviruses could contribute to the lung epithelial injury that initiates profibrotic responses in idiopathic pulmonary fibrosis (IPF). METHODS: We identified herpesviral DNA from IPF and control lung tissue using a multiplex PCR-and microarray-based method. Active herpesviral infection was detected by standard methods, and inflammatory cell subtypes were identified with specific antibodies. Patients that underwent lung transplantation were monitored for signs of herpesviral infection. RESULTS: A total of 11/12 IPF samples were positive for Epstein-Barr virus (EBV) and 10/12 for human herpesvirus 6B (HHV-6B) DNA. Control lung samples (n = 10) were negative for EBV DNA, whereas three samples were positive for HHV-6B. EBV-encoded RNA (EBER) was identified in nine IPF samples and localized mainly to lymphocytic aggregates. HHV-6B antigens were detected in mononuclear cells in IPF lung tissue. CD20+ B lymphocytic aggregates that were surrounded by CD3+ T cells were abundant in IPF lungs. CD23+ cells (activated B cells, EBV-transformed lymphoblasts, and dendritic cells) were observed in the aggregates. IPF patients had no signs of increased herpesviral activation after lung transplantation. CONCLUSIONS: Inflammatory cells are the main source of herpesviral DNA in the human IPF lung. Diagnostic tools should be actively used to elucidate whether herpesviral infection affects the pathogenesis, progression, and/or exacerbation of IPF.

A selective broth enrichment combined with real-time nuc-mecA-PCR in the exclusion of MRSA.

We analyzed the performance of a selective enrichment broth combined with Taqman-based real-time duplex nuc-mecA-PCR to expedite the screening of methicillin-resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4-256 microg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc-mecA-PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24-48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.

Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology.

BACKGROUND: The increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking. OBJECTIVES: To compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers. STUDY DESIGN: A panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation. RESULTS: The novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected. CONCLUSIONS: The diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum.

Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study.

BACKGROUND: New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround time of a new molecular sepsis assay. METHODS: 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE, and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. FINDINGS: 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94.7% (95% CI 93.6-95.7) and a specificity of 98.8% (98.1-99.2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1-2 working days. 34 of 3284 (1.0%) samples were excluded because of technical and operator errors. INTERPRETATION: Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis.

Rapid identification of bacterial pathogens using a PCR- and microarray-based assay.

BACKGROUND: During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples. RESULTS: Comparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence. CONCLUSION: The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

Serological microarray for detection of HSV-1, HSV-2, VZV, and CMV antibodies.

The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray was designed and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) and IgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelated in-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection of antibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteins of herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, and cytomegalovirus (CMV) pp150 phosphoprotein. The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested for herpesviruses by the serological microarray, and the results were compared to those of individual EIAs used for standard diagnostic purposes. The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. However, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarray showed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1, HSV-2, VZV, and CMV antibodies could be detected on the same microarray.

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time PCR.

We developed a real-time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real-time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 microl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections.

Human herpesvirus-6 and -7 DNA in cerebrospinal fluid of facial palsy patients.

CONCLUSIONS: Finding human herpesvirus (HHV)-7 and dual HHV-6A and -6B DNA in cerebrospinal fluid (CSF) of two facial palsy (FP) patients is intriguing but does not allow etiologic conclusions as such. HHV-6 or -7 DNA was revealed in 10% of the CSF samples tested from 70 immunocompetent adolescents and adults; a highly unusual result. How these findings are associated with the diseases they accompany remains to be defined. OBJECTIVE: To determine whether herpes simplex virus (HSV)-1 and -2, varicella-zoster virus (VZV), HHV-6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) DNA could be found in CSF of FP patients or controls. SUBJECTS AND METHODS: In all, 33 peripheral FP patients (26 idiopathic, 5 with herpesvirus infection, 1 puerperal, 1 Melkersson-Rosenthal syndrome) (34 CSF samples) and 36 controls (16 nonidiopathic FP, 7 hearing loss, 6 vertigo, 5 headache, 2 other) previously tested for HSV-1, VZV, and HHV-6 DNA by polymerase chain reaction (PCR) were tested with highly sensitive multiplex-PCR and an oligonucleotide microarray method. RESULTS: One FP patient had HHV-7 DNA and another had HHV-6A and -6B DNA simultaneously. In the control group, one HHV-7, one HHV-6A, and three HHV-6B DNA-positive specimens were found.

Molecular epidemiology of dengue virus strains from Finnish travelers.

We characterized 11 dengue virus (DENV) isolates obtained from Finnish travelers during 2000-2005 using monoclonal antibodies and phylogenetic analysis. The analysis of DENV isolated from travelers contributes to the global picture of strain distribution and circulation. The isolates included all serotypes, including a DENV-2 isolate from Ghana.

Improved multiplex-PCR and microarray for herpesvirus detection from CSF.

BACKGROUND: A multiplex-PCR and microarray-based method was designed for detection of eight herpesviruses from clinical specimens. OBJECTIVES: To improve the method, especially for detection of herpes simplex (HSV) and varicella-zoster viruses (VZV), and to update and validate the method using positive cerebrospinal fluid (CSF) samples. STUDY DESIGN: A new primer pair for HSV-PCR and four detection oligonucleotides for HSVs were designed. Two new detection oligonucleotides for VZV and additional oligonucleotides for CMV, EBV, HHV-6 and -7 were designed. The new and previous multiplex-PCR and microarray method were tested in parallel with dilution series of commercial herpesvirus DNAs and 20 CSF specimens positive for HSV-1, HSV-2, or VZV. RESULTS: New method was more sensitive for detection of HSVs and both two new detection oligonucleotides for VZV functioned well at low levels of viral DNA. CONCLUSIONS: The revised HSV-PCR and new HSV- and VZV-oligonucleotides were found to function well and be more sensitive, thereby increasing reliability of the method.

Search for Herpesviruses in cerebrospinal fluid of facial palsy patients by PCR.

CONCLUSIONS: Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) DNA were not detected in the cerebrospinal fluid (CSF) of patients with acute idiopathic peripheral facial palsy (Bell's palsy). Our results indicate either the absence of these viruses or the presence of technical shortcomings. The role of human herpesvirus 6 (HHV-6) in this disorder and the significance of a positive HHV-6 DNA finding in the central nervous system need further investigation. OBJECTIVE: Our goal was to determine whether DNA of HSV-1, VZV, or HHV-6 can be found by polymerase chain reaction (PCR) in the CSF of peripheral facial palsy patients. MATERIALS AND METHODS: We used PCR to detect the presence of HSV-1, VZV, and HHV-6 DNA in CSF. This was a retrospective case control study with 33 peripheral facial palsy patients (34 CSF samples) in the study group (26 with Bell's palsy, 5 with simultaneously diagnosed herpesvirus infection, 1 with puerperal facial palsy, 1 with Melkersson-Rosenthal syndrome). The control group included 36 patients, most with diagnosed or suspected Borreliosis and facial palsy or sudden deafness. RESULTS: One patient with Bell's palsy had HHV-6 DNA in CSF. Neither HSV-1 nor VZV DNA was detected in patients or controls.

Monitoring of EBV-DNAemia by quantitative real-time PCR after adult liver transplantation.

BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) causes significant morbidity and mortality in transplantation. The clinical significance of Epstein-Barr virus (EBV) in the development of PTLD is clear, but not all EBV-reactivations cause PTLD. OBJECTIVES: We retrospectively analyzed EBV-DNAemia in liver transplant patients by a quantitative TaqMan-based real-time plasma PCR. STUDY DESIGN: Altogether 1284 specimens, obtained from 105 patients for frequent monitoring of cytomegalovirus (CMV) and human herpesvirus-6 and -7 (HHV-6, HHV-7) during the post-transplant year, were retrospectively tested for EBV-DNA. RESULTS: Altogether, 14/105 (13%) patients showed EBV-DNAemia, which usually occurred within 3 months after transplantation and subsided within a few weeks. EBV-DNAemia occurred concurrently with CMV in 10/14, with HHV-6 in 11/14, and with all three betaherpesviruses in 4/14 cases. The peak viral loads were relatively low (median 2100 EBV-DNA copies/ml, range 568-6600), except in one patient who first had low-level EBV-DNA (562-3022 copies/ml) in the early post-transplant period, but on day 175 after transplantation developed high-level DNAemia (9851-86,975copies/ml) which continued for 6 months and developed into PTLD at 6 months after transplantation. CONCLUSION: Low-level EBV-DNAemia is common after liver transplantation, often occurring together with betaherpesviruses, but seldom leads to high viral loads or PTLD. However, monitoring of EBV-DNA levels in the patients can be useful.

Multiplex-PCR and oligonucleotide microarray for detection of eight different herpesviruses from clinical specimens.

BACKGROUND: Human herpesviruses cause clinically important diseases, e.g. infections of the central nervous system. New diagnostic tools are required for rapid and reliable detection of these viruses. OBJECTIVES: A microarray-based method was designed for detection of eight human herpesviruses in cerebrospinal fluid (CSF), whole blood, plasma, serum and proficiency-testing specimens. STUDY DESIGN: Herpes simplex type 1 and 2, varicella-zoster, cytomegalo-, Epstein-Barr and human herpes viruses 6A, 6B and 7 were amplified from clinical specimens by two multiplex-PCRs and transcribed to single-stranded RNAs which were hybridized to oligonucleotides on microarray. The results were compared to those from conventional PCR. In total, 227 specimens were tested including 23 CSF, 10 whole blood, 73 plasma, 10 proficiency-testing samples and 111 negative control samples. RESULTS: Concordant results were obtained in 214/227 (94%). Microarray detected 10 possible double and one triple infection. Negative control samples (70 serum, 30 CSF and 11 proficiency-testing samples) were all negative. CONCLUSIONS: Microarray is suitable for detection of multiple herpesviruses in clinical samples.

Quantitative TaqMan assay for the detection and monitoring of cytomegalovirus infection in organ transplant patients.

Quantitative polymerase chain reaction assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. In recent years, the development of automated nucleic acid extraction devices together with the introduction of real-time technology have been important elements for improvements of these assays. This chapter describes a method for the quantitation of CMV DNA viral load in the plasma samples of organ transplant patients. The method is based on MagNA Pure LC nucleic acid extraction system (Roche) and the TaqMan real-time technology. MagNA Pure LC is highly automated procedure with which 32 plasma samples could be processed within 1.5 h. TaqMan chemistry and Sequence Detector System 7900HT device (Applied Biosystems) are used for the quantitative amplification of the CMV genome. The chapter also describes preparation of the plasmid, which is needed to achieve a quantitative standard curve for quantitation.