Comprehensive real-time epidemiological data from respiratory infections in Finland between 2010 and 2014 obtained from an automated and multianalyte mariPOC® respiratory pathogen test.

Respiratory viruses cause seasonal epidemics every year. Several respiratory pathogens are circulating simultaneously and typical symptoms of different respiratory infections are alike, meaning it is challenging to identify and diagnose different respiratory pathogens based on symptoms alone. mariPOC® is an automated, multianalyte antigen test which allows the rapid detection of nine respiratory infection pathogens [influenza A and B viruses, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, parainfluenza 1-3 viruses and pneumococci] from a single nasopharyngeal swab or aspirate samples, and, in addition, can be linked to laboratory information systems. During the study period from November 2010 to June 2014, a total of 22,485 multianalyte respi tests were performed in the 14 participating laboratories in Finland and, in total, 6897 positive analyte results were recorded. Of the tested samples, 25 % were positive for one respiratory pathogen, with RSV (9.8 %) and influenza A virus (7.2 %) being the most common findings, and 0.65 % of the samples were multivirus-positive. Only small geographical variations in seasonal epidemics occurred. Our results show that the mariPOC® multianalyte respi test allows simultaneous detection of several respiratory pathogens in real time. The results are reliable and give the clinician a picture of the current epidemiological situation, thus minimising guesswork.

Ready to use dry-reagent PCR assays for the four common bacterial pathogens using switchable lanthanide luminescence probe system.

Ready to use dry-reagent PCR assays for Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas spp. and for broad-range bacteria detection were developed. The assays were based on novel switchable lanthanide probes that provide sensitive target DNA detection with exceptionally high signal-to-background ratio, thus enabling clear discrimination between positive and negative results. For example, sensitivity of three S. aureus and two S. pneumonia bacteria (colony forming units) per PCR assay was measured with fluorescence signal more than 30 times over the background signal level. The rapid and easy-to-use assays are suitable for routine clinical diagnostics without molecular biology expertise and facilities.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of viridans group streptococci.

In this study, the performances of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, MALDI Biotyper (Bruker Daltonics) and VITEK MS (bioMérieux), were evaluated in the identification of viridans group streptococci. Two collections of isolates were tested with both methods. From a panel of type collection strains (n = 54), MALDI Biotyper gave correct species-level identification for 51/54 (94 %) strains and 37/54 (69 %) strains for the VITEK MS in vitro diagnostic (IVD) method. Additionally, a collection of blood cultures isolates which had been characterized earlier with partial sequencing of 16S rRNA (n = 97) was analyzed. MALDI Biotyper classified 89 % and VITEK MS 93 % of these correctly to the group level. Comparison of species-level identification from the blood culture collection was possible for 36 strains. MALDI Biotyper identified 75 % and VITEK MS 97 % of these strains consistently. Among the clinical isolates, MALDI Biotyper misidentified 36 strains as Streptococcus pneumoniae. Nevertheless, our results suggest that the current MALDI-TOF methods are a good alternative for the identification of viridans streptococci and do perform as well as or better than commercial phenotypical methods.

One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system.

A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l'Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters (n = 419) and other Gram stain forms (n = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10(4) CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.

Rapid confirmation of suspected methicillin-resistant Staphylococcus aureus colonies on chromogenic agars by a new commercial PCR assay, the GenomEra MRSA/SA Diagnose.

A new automated closed tube PCR assay, the GenomEra(™) MRSA/SA Diagnose (Abacus Diagnostica Oy, Finland) was evaluated for rapid confirmation of methicillin-resistant Staphylococcus aureus (MRSA) from cultured screening specimens. The ability of the assay to detect genotypically different MRSA strains was studied with a collection of 304 MRSA isolates covering 68 spa types. The specificity was investigated with a collection of 146 non-MRSA staphylococcus isolates. The usefulness of the assay for clinical purposes was assessed by a sequential combination of MRSA screening culture and confirmation of the colonies with the GenomEra MRSA/SA Diagnose assay. A total of 145 suspected MRSA colonies on chromogenic plates were analyzed this way. All MRSA isolates from the culture collection and from the clinical screening specimens were confirmed as MRSA with the GenomEra MRSA/SA Diagnose assay and none of the non-MRSA staphylococci caused false-positive results, which indicates both sensitivity and specificity of 100%. The combination of GenomEra MRSA/SA Diagnose with preceding culture on selective MRSA agar permitted MRSA confirmation within 24 h. This practice offers a reliable and quick detection of MRSA that is also suitable in areas where several strain types cause epidemics.

Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis.

Rapid detection is essential for timely initiation of medical post-exposure prophylactic measures in the event of intentional release of biological threat agents. We compared real-time PCR assay performance between the Applied Biosystems 7300/7500 and the RAZOR instruments for specific detection of the causative agents of anthrax, brucellosis, tularemia and plague. Furthermore, an assay detecting Bacillus thuringiensis, a Bacillus anthracis surrogate, was developed for field-training purposes. Assay sensitivities for B. anthracis, Brucella spp., Francisella tularensis and Yersinia pestis were 10-100 fg of target DNA per reaction, and no significant difference in assay performance was observed between the instrument platforms. Specificity testing of the diagnostic panels with both instrument platforms did not reveal any cross-reactivity with other closely related bacteria. The duration of thermocycling with the RAZOR instrument was shorter, i.e. 40 min as compared with 100 min for the Applied Biosystems 7300/7500 instruments. These assays provide rapid tools for the specific detection of four biological threat agents. The detection assays, as well as the training assay for B. thuringiensis powder preparation analysis, may be utilized under field conditions and for field training, respectively.

Induced sputum in the diagnosis of childhood community-acquired pneumonia.

BACKGROUND: The usefulness of induced sputum in searching for causative agents of pneumonia in children has not been studied. METHODS: The study involved 101 children, aged 6 months to 15 years, treated for community-acquired pneumonia at Turku University Hospital (Turku, Finland) from January 2006 to April 2007. Nasopharyngeal aspirate samples were first collected through both nostrils. Sputum production was then induced by inhalation of 5.0% hypertonic saline for 5-10 min and a sputum sample was either aspirated or expectorated. The presence and amount of bacteria and viruses in paired nasopharyngeal aspirate and sputum specimens was analysed and compared using semiquantitative bacterial culture and quantitative PCR techniques. RESULTS: A good quality sputum specimen was obtained from 76 children. The possible causative agent was found in 90% of cases. Streptococcus pneumoniae (46%) and rhinovirus (29%) were the most common microbes detected. Newly discovered viruses human bocavirus and human metapneumovirus were detected in 18% and 13% of the children, respectively. One-quarter of all bacterial findings were only detected in sputum, and the amount of bacteria in the remainder of the sputum specimens compared with nasopharyngeal aspirate was higher in 14% and equal in 70%. The amount of rhinovirus in sputum was higher than in nasopharyngeal aspirate in 82%. CONCLUSIONS: Sputum induction provides good quality sputum specimens with high microbiological yield in children with community-acquired pneumonia. Induced sputum analysis can be useful in the microbiological diagnosis of childhood community-acquired pneumonia.

Evaluation of Vitek 2 for identification of yeasts in the clinical laboratory.

The Vitek 2 system was compared with conventional assimilation, fermentation and morphological methods for its ability to identify yeast isolates from among 151 clinical specimens and 16 known type culture or quality control strains. An unequivocal identification was obtained for 155 (92.8%) isolates, with low discrimination for nine (5.4%) and false identification for three (1.8%) isolates. All isolates of Candida albicans, Candida glabrata and Candida krusei were identified correctly. It was concluded that the Vitek 2 system offers an excellent alternative for the identification of yeasts in a clinical laboratory.

Bronchoalveolar lavage in immunocompromised patients with haematological malignancy--value of new microbiological methods.

PURPOSE: To assess the usefulness of new culture-independent microbiological methods to analyse bronchoalveolar lavage (BAL) fluid from haematological patients with clinical pneumonia. PATIENTS AND METHODS: Results of 135 BALs from 122 disease episodes in 99 patients treated between 1996 and 2002 were retrospectively analysed. Forty-three patients had undergone haematopoietic stem cell transplantation and 56 patients had been treated with conventional chemotherapy for haematological malignancy. In addition to conventional microbiological methods, polymerase chain reaction (PCR) tests for Pneumocystis carinii, cytomegalovirus (CMV), Legionella sp., mycobacterium, Mycoplasma pneumoniae, and Chlamydia pneumoniae and the Aspergillus antigen test were performed. RESULTS: Three (2.2%) quantitative and four (3.0%) special bacterial cultures gave an aetiological diagnosis. A respiratory virus was isolated in 10 episodes (8.2%). The diagnostic yield increased to 35.6% (48 of 135) by other methods. The P. carinii PCR test was positive in 21 of 24 patients with P. carinii pneumonia, being the only microbiological indication of P. carinii in four cases. The CMV PCR test was positive in 18 patients, but in 14 patients the clinical significance of the finding remained unproven. The Aspergillus antigen test was positive in seven of nine patients with aspergillosis, being the only microbiological indication of Aspergillus in three cases. The result of BAL indicated commencement of specific antimicrobial treatment in 27 episodes (22.1%). CONCLUSION: The contribution of new culture-independent methods to the total diagnostic yield was of note. Among these methods, the P. carinii PCR and Aspergillus antigen tests proved the most valuable, while the CMV PCR test was not clinically useful.

Application of Bacillus anthracis PCR to simulated clinical samples.

We evaluated PCR for the detection of Bacillus anthracis DNA from simulated clinical specimens relevant for the microbiological diagnosis of anthrax or exposure to B. anthracis spores. In simulated blood specimens, the lowest limit of detection was 400 CFU per mL of blood, which may be sufficient for samples from patients with septic anthrax. Screening nasal swabs by PCR may not be sensitive enough to rule out dangerous exposure to anthrax spores, as a minimum of 2000 spores per sample was required for detectable amplification. As spores survived some standard DNA purification methods, special attention should be paid to laboratory safety when preparing samples possibly containing live spores.

Aetiological diagnosis of brain abscesses and spinal infections: application of broad range bacterial polymerase chain reaction analysis.

OBJECTIVE: To evaluate the usefulness of the broad range bacterial rDNA polymerase chain reaction (PCR) method combined with DNA sequencing in the aetiological diagnosis of intracranial or spinal infections in neurosurgical patients. METHODS: In addition to conventional methods, the broad range bacterial PCR approach was applied to examine pus or tissue specimens from cerebral or spinal lesions in patients treated in a neurosurgical unit for a clinical or neuroradiological suspicion of bacterial brain abscess or spondylitis. RESULTS: Among the 44 patients with intracranial or spinal lesions, the final diagnosis suggested bacterial disease in 25 patients, among whom the aetiological agent was identified in 17. A causative bacterial species was identified only by the rDNA PCR method in six cases, by both the PCR methodology and bacterial culture in six cases, and by bacterial culture alone in five. All samples in which a bacterial aetiology was identified only by the PCR approach were taken during antimicrobial treatment, and in three patients the method yielded the diagnosis even after >/= 12 days of parenteral treatment. One case also identified by the PCR approach alone involved a brain abscess caused by Mycoplasma hominis, which is not readily cultured by routine methods. CONCLUSIONS: In patients with brain abscesses and spinal infections, the broad range bacterial rDNA PCR approach may be the only method to provide an aetiological diagnosis when the patient is receiving antimicrobial treatment, or when the causative agent is fastidious.

Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens.

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect Legionella DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73 Legionella pneumophila strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other Legionella strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species L. micdadei, L. bozemaniae, and L. dumoffii. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with Legionella culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of Legionella DNA.

LCx Mycobacterium tuberculosis assay is valuable with respiratory specimens, but provides little help in the diagnosis of extrapulmonary tuberculosis.

BACKGROUND: Commercial nucleic acid amplification tests, designed for the detection of Mycobacterium tuberculosis DNA/RNA in respiratory samples, are often applied also in nonrespiratory specimens in order to verify the diagnosis of extrapulmonary tuberculosis. AIM. To evaluate the value of the Abbott LCx Mycobacterium tuberculosis assay for the diagnosis of pulmonary and extrapulmonary tuberculosis based on routine clinical laboratory results. METHODS: The assay was used to analyse 350 respiratory and 826 nonrespiratory specimens from 961 patients, of whom 3.6% had culture-proven tuberculosis. The results obtained by the LCx assay were compared with the records on mycobacterial isolates of the national reference laboratory and, in the case of positive findings, with clinical data. RESULTS: In comparison with culture, the sensitivity, specificity and positive/negative predictive value of the assay on respiratory specimens were 87.5%, 99.7%, 93.3% and 99.4%, respectively. With nonrespiratory specimens, the overall sensitivity, specificity and positive/negative predictive value of the LCx assay were 73.3%, 98.0%, 40.7% and 99.5%, respectively. When clinical and histological data were also included, the positive predictive value of LCx with nonrespiratory specimens was 45.8%. CONCLUSION: Critical interpretation of the nucleic acid amplification results obtained from nonrespiratory specimens is necessary in both laboratory and clinical settings.

Direct amplification of rRNA genes in diagnosis of bacterial infections.

A broad-range bacterial PCR targeting rRNA genes (rDNAs) was used to directly analyze 536 clinical samples obtained from 459 hospitalized patients during a 4-year study period. The molecular diagnosis based on DNA sequencing of the PCR product was compared to that obtained by bacterial culture. The bacteriological diagnosis was concordant for 447 (83%) specimens. Broad-range rDNA PCR was the only method that yielded an etiologic diagnosis for 11 (2.4%) of 459 patients. Compared to culture and clinical assessment, the sensitivity of the PCR method combined with sequencing was 74.2%, and the specificity was between 98.7 and 99.6%. At present, the described molecular approach proved superior to bacterial culture in two clinical situations: infections caused by bacteria with unusual growth requirements and specimens taken during antimicrobial treatment of the patient.