Efficient SARS-CoV-2 variant detection and monitoring with Spike Screen next-generation sequencing.

The emergence and rapid spread of SARS-CoV-2 prompted the global community to identify innovative approaches to diagnose infection and sequence the viral genome because at several points in the pandemic positive case numbers exceeded the laboratory capacity to characterize sufficient samples to adequately respond to the spread of emerging variants. From week 10, 2020, to week 13, 2023, Slovenian routine complete genome sequencing (CGS) surveillance network yielded 41 537 complete genomes and revealed a typical molecular epidemiology with early lineages gradually being replaced by Alpha, Delta, and finally Omicron. We developed a targeted next-generation sequencing based variant surveillance strategy dubbed Spike Screen through sample pooling and selective SARS-CoV-2 spike gene amplification in conjunction with CGS of individual cases to increase throughput and cost-effectiveness. Spike Screen identifies variant of concern (VOC) and variant of interest (VOI) signature mutations, analyses their frequencies in sample pools, and calculates the number of VOCs/VOIs at the population level. The strategy was successfully applied for detection of specific VOC/VOI mutations prior to their confirmation by CGS. Spike Screen complemented CGS efforts with an additional 22 897 samples sequenced in two time periods: between week 42, 2020, and week 24, 2021, and between week 37, 2021, and week 2, 2022. The results showed that Spike Screen can be applied to monitor VOC/VOI mutations among large volumes of samples in settings with limited sequencing capacity through reliable and rapid detection of novel variants at the population level and can serve as a basis for public health policy planning.

Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era.

BACKGROUND: The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting. METHODS: Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2. RESULTS: The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022. CONCLUSION: The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

Performance of nasopharyngeal swab and saliva in detecting Delta and Omicron SARS-CoV-2 variants.

A prospective cohort study was conducted during the Delta and Omicron severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) epidemic waves from paired nasopharyngeal swab (NPS or NP swab) and saliva samples taken from 624 participants. The study aimed to assess if any differences among participants from both waves could be observed and if any difference in molecular diagnostic performance could be observed among the two sample types. Samples were transported immediately to the laboratory to ensure the highest possible sample quality without any freezing and thawing steps before processing. Nucleic acids from saliva and NPS were prospectively extracted and SARS-CoV-2 was detected using a real-time reverse-transcription polymerase chain reaction. All observed results were statistically analyzed. Although the results obtained with NP and saliva agreed overall, higher viral loads were observed in NP swabs regardless of the day of specimen collection in both SARS-CoV-2 epidemic waves. No significant difference could be observed between the two epidemic waves characterized by Delta or Omicron SARS-CoV-2. To note, Delta infection resulted in higher viral loads both in NP and saliva and more symptoms, including rhinorrhea, cough, and dyspnea, whereas Omicron wave patients more frequently reported sore throat. An increase in the mean log RNA of SARS-CoV-2 was observed with the number of expressed symptoms in both waves, however, the difference was not significant. Data confirmed that results from saliva were concordant with those from NP swabs, although saliva proved to be a challenging sample with frequent inhibitions that required substantial retesting.

Mycoplasma pneumoniae P1 Genotype Indicates Severity of Lower Respiratory Tract Infections in Children.

Mycoplasma pneumoniae strains can be classified into two major genetic groups, P1 type 1 (P1-1) and P1 type 2 (P1-2). It remains unknown if clinical manifestations of lower respiratory tract infections (LRTI) in children differ between the two genotypes. We aimed to determine if the M. pneumoniae P1 genotype is associated with severity of LRTI in children. Medical charts of 420 children (≤15 years old) with signs of acute LRTI who were PCR positive for M. pneumoniae from pharyngeal swabs in a recent M. pneumoniae epidemic were analyzed. We used a culture and pyrosequencing approach for genotyping PCR-positive samples. We compared epidemiological and clinical data of children with either P1-1 or P1-2 LRTI. P1-2-infected children presented with a significantly higher median baseline C-reactive protein level and were admitted to the hospital more often. The P1 genotype had a significant predictive value in a multiple linear regression model predicting C-reactive protein levels in our study sample. Moreover, the P1 genotype significantly affected the likelihood of hospital admission in a logistic regression model. Our modeling results were also confirmed on an additional independent sample of children with M. pneumoniae LRTI. Results from our large patient group indicate that the two M. pneumoniae P1 genotypes may have different pathogenic potential and that LRTI with P1-2 strains may have a more severe disease course than those with P1-1 strains in children. P1 genotyping is not routinely performed but could be used as a predictor of M. pneumoniae LRTI severity, enabling patient-tailored treatments.

Clinical characteristics of infections caused by Mycoplasma pneumoniae P1 genotypes in children.

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.

Confirmed inguinal lymphogranuloma venereum genovar L2c in a man who had sex with men, Slovenia, 2015.

A laboratory-confirmed lymphogranuloma venereum (LGV) case in Slovenia was reported in 2015, in a human immunodeficiency virus (HIV)-negative man presenting with inguinal lymphadenopathy. He reported unprotected insertive anal intercourse with two male partners in Croatia. Variant L2c of Chlamydia trachomatis was detected in clinical samples. Although the patient was eventually cured, the recommended treatment regimen with doxycycline had to be prolonged.

Prevalence, genotyping and macrolide resistance of Mycoplasma pneumoniae among isolates of patients with respiratory tract infections, Central Slovenia, 2006 to 2014.

In this retrospective study we employed real-time polymerase chain reaction (PCR) to analyse the occurrence of Mycoplasma pneumoniae among upper and lower respiratory tract infections (RTI) in the Central Region of Slovenia between January 2006 and December 2014. We also used a culture and pyrosequencing approach to genotype strains and infer their potential macrolide resistance. Of a total 9,431 tested samples from in- and out-patient with RTI, 1,255 (13%) were found to be positive by M. pneumoniae PCR. The proportion of positive samples was 19% (947/5,092)among children (≤16 years-old) and 7% (308/4,339) among adults (>16 years-old). Overall, among those PCR tested, the highest proportions of M. pneumonia infections during the study period were observed in 2010 and 2014. In these two years, 18% (218/1,237) and 25% (721/2,844) of samples were positive respectively,indicating epidemic periods. From the 1,255 M. pneumoniae PCR-positive samples, 783 (614 from paediatric and 169 from adult patients) were successfully cultured. Of these, 40% (312/783) were constituted of strains belonging to the P1 type II genomic group, while 60% (469/783) contained strains of the P1 type I group. Two isolates comprised both P1 type Iand II strains. Results of a genotype analysis by year,showed that the dominant M. pneumoniae P1 type during the 2010 epidemic was P1 type II (82% of isolates;81/99), which was replaced by P1 type I in the 2014 epidemic (75%; 384/510). This observation could indicate that the two epidemics may have been driven by a type shift phenomenon, although both types remained present in the studied population during the assessed period of time. Only 1% of strains (7/783) were found to harbour an A2063G mutation in the 23S rRNA gene,which confers macrolide resistance, suggesting that the occurrence of M. pneumoniae macrolide resistance still seems to be sporadic in our geographic area.

Sequencing analysis of SARS-CoV-2 cases in Slovenian long-term care facilities to support outbreak control.

Introduction: Residents of long-term care facilities (LTCFs) are at high risk of morbidity and mortality due to COVID-19, especially when new variants of concern (VOC) emerge. To provide intradisciplinary data in order to tailor public health interventions during future epidemics, available epidemiologic and genomic data from Slovenian LTCFs during the initial phases of the COVID-19 pandemic was analyzed. Methods: The first part of the study included SARS-CoV-2 reverse-transcription Real-Time PCR (rtRT-PCR) positive LTCF residents, from 21 facilities with COVID-19 outbreaks occurring in October 2020. The second part of the study included SARS-CoV-2 rtRT-PCR positive LTCF residents and staff between January and April 2021, when VOC Alpha emerged in Slovenia. Next-generation sequencing (NGS) was used to acquire SARS-CoV-2 genomes, and lineage determination. In-depth phylogenetic and mutational profile analysis were performed and coupled with available field epidemiological data to assess the dynamics of SARS-CoV-2 introduction and transmission. Results: 370/498 SARS-CoV-2 positive residents as well as 558/699 SARS-CoV-2 positive residents and 301/358 staff were successfully sequenced in the first and second part of the study, respectively. In October 2020, COVID-19 outbreaks in the 21 LTCFs were caused by intra-facility transmission as well as multiple independent SARS-CoV-2 introductions. The Alpha variant was confirmed in the first LTCF resident approximately 1.5 months after the first Alpha case was identified in Slovenia. The data also showed a slower replacement of existing variants by Alpha in residents compared to staff and the general population. Discussion: Multiple SARS CoV-2 introductions as well as intra-facility spreading impacted disease transmission in Slovenian LTCFs. Timely implementation of control measures aimed at limiting new introductions while controlling in-facility transmission are of paramount importance, especially as new VOCs emerge. Sequencing, in conjunction with epidemiological data, can facilitate the determination of the need for future improvements in control measures to protect LTCF residents from COVID-19 or other respiratory infections.

Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

Improvement of pneumococcal pneumonia diagnostics by the use of rt-PCR on plasma and respiratory samples.

BACKGROUND: The aim of the study was to assess the performance of a real-time polymerase chain reaction (rt-PCR) assay on plasma and respiratory samples for the diagnosis of pneumococcal pneumonia. METHODS: Three hundred and forty patients (160 children and 180 adults) with community-acquired pneumonia were included prospectively from January 2011 to May 2012. Blood samples were obtained simultaneously for culture and rt-PCR targeting the lytA gene. Respiratory samples were also obtained: nasopharyngeal swab in nearly all patients and sputum or tracheal aspirate when available. RESULTS: Streptococcus pneumoniae was detected in 222 (65%) of 340 patients: 143 (89%) children and 79 (44%) adults. Pneumonia was assigned as definite pneumococcal in 96 (28.2%) of 340 patients, according to S. pneumoniae detected in blood: in 54 (33.8%) children - by rt-PCR in 51 (31.9%) and by culture in 5 (3.1%); and in 42 (23.3%) adults - by rt-PCR in 41 (22.8%) and by culture in 12 (6.7%). Pneumonia was considered as probably pneumococcal in 19 (10.6%) adults according to S. pneumoniae detected in sputum/tracheal aspirate, by rt-PCR in 19 and by culture in 5. In 18 adults and 89 children with S. pneumoniae detected only in the nasopharynx, pneumonia was considered as possibly pneumococcal; however it should be noted that nasopharyngeal colonization with S. pneumoniae is also common in children with other aetiologies of pneumonia. CONCLUSIONS: rt-PCR on plasma and other samples performed significantly better than culture for the detection of pneumococcal pneumonia (p < 0.0005) in children and adults.

A simplified nasopharyngeal swab collection procedure for minimizing patient discomfort while retaining sample quality.

A nasopharyngeal swab (NPS) is the most frequently collected sample type when molecular diagnosis of respiratory viruses, including SARS CoV-2, is required. An optimal collection technique would provide sufficient sample quality for the diagnostic process and would minimize the discomfort felt by the patient. This study compares a simplified NPS collection procedure with only one rotation of the swab to a more standard procedure with five rotations. Swabs were collected from 76 healthy volunteers by the same healthcare professional on 2 consecutive days at a similar hour to minimize variability. The number of Ubiquitin C copy number per sample was measured by real-time quantitative PCR and patient discomfort was assessed by questionnaire. No statistically significant difference (p = 0.15) was observed in the Ubiquitin C copy number per sample between a NPS collected with one rotation (5.2 ± 0.6 log UBC number copies/sample) or five rotations (5.3 ± 0.5 log UBC number copies/sample). However, a statistically significant difference was observed in discomfort between these two procedures, the second being much more uncomfortable. Additional analysis of the results showed a weak correlation between discomfort and the number of human cells recovered (Spearman's rho = 0.202) and greater discomfort in younger people. The results of this study show that a NPS collected with one slow rotation has the same quality as a NPS collected with five rotations. However, the collection time is shorter and, most importantly, less unpleasant for patients.

Changes in HRSV Epidemiology but Not Circulating Variants in Hospitalized Children due to the Emergence of SARS-CoV-2.

This study assesses the circulation of human respiratory syncytial virus (HRSV) genotypes before, during, and toward the end of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in children and determines the influence of the pandemic on HRSV circulation patterns and evolution. Phylogenetic analysis of the hypervariable glycoprotein G gene was performed on 221/261 (84.7%) HRSV-positive samples and shows two separated clusters, one belonging to HRSV-A (129/221) and another to HRSV-B (92/221). All Slovenian HRSV-A strains contained the 72-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GA2.3.5. All Slovenian HRSV-B strains similarly contained a 60-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GB5.0.5a. During the 3-year period (2018-2021) covered by the study, no significant differences were observed within strains detected before the SARS-CoV-2 pandemic, during it, and after the implementation of nonpharmaceutical preventive measures. Slovenian HRSV-A strains seem to be more diverse than HRSV-B strains. Therefore, further whole-genome investigations would be required for better monitoring of the long-term impact of SARS-CoV-2 endemic circulation and the formation of new HRSV lineages and epidemiological patterns.

Interspecies transmission of porcine-originated G4P[6] rotavirus A between pigs and humans: a synchronized spatiotemporal approach.

As a leading viral cause of acute gastroenteritis in both humans and pigs, rotavirus A (RVA) poses a potential public health concern. Although zoonotic spillover of porcine RVA strains to humans is sporadic, it has been detected worldwide. The origin of chimeric human-animal strains of RVA is closely linked to the crucial role of mixed genotypes in driving reassortment and homologous recombination, which play a major role in shaping the genetic diversity of RVA. To better understand how genetically intertwined porcine and zoonotic human-derived G4P[6] RVA strains are, the present study employed a spatiotemporal approach to whole-genome characterization of RVA strains collected during three consecutive RVA seasons in Croatia (2018-2021). Notably, sampled children under 2 years of age and weanling piglets with diarrhea were included in the study. In addition to samples tested by real-time RT-PCR, genotyping of VP7 and VP4 gene segments was conducted. The unusual genotype combinations detected in the initial screening, including three human and three porcine G4P[6] strains, were subjected to next-generation sequencing, followed by phylogenetic analysis of all gene segments, and intragenic recombination analysis. Results showed a porcine or porcine-like origin for each of the eleven gene segments in all six RVA strains. The G4P[6] RVA strains detected in children most likely resulted from porcine-to-human interspecies transmission. Furthermore, the genetic diversity of Croatian porcine and porcine-like human G4P[6] strains was propelled by reassortment events between porcine and porcine-like human G4P[6] RVA strains, along with homologous intragenotype and intergenotype recombinations in VP4, NSP1, and NSP3 segments. Described concurrent spatiotemporal approach in investigating autochthonous human and animal RVA strains is essential in drawing relevant conclusions about their phylogeographical relationship. Therefore, continuous surveillance of RVA, following the One Health principles, may provide relevant data for assessing the impact on the protectiveness of currently available vaccines.

Comparative Evaluation of Six SARS-CoV-2 Real-Time RT-PCR Diagnostic Approaches Shows Substantial Genomic Variant-Dependent Intra- and Inter-Test Variability, Poor Interchangeability of Cycle Threshold and Complementary Turn-Around Times.

Several professional societies advise against using real-time Reverse-Transcription PCR (rtRT-PCR) cycle threshold (Ct) values to guide clinical decisions. We comparatively assessed the variability of Ct values generated by six diagnostic approaches by testing serial dilutions of well-characterized isolates of 10 clinically most relevant SARS-CoV-2 genomic variants: Alpha, Beta, Gamma, Delta, Eta, Iota, Omicron, A.27, B.1.258.17, and B.1 with D614G mutation. Comparison of three fully automated rtRT-PCR analyzers and a reference manual rtRT-PCR assay using RNA isolated with three different nucleic acid isolation instruments showed substantial inter-variant intra-test and intra-variant inter-test variability. Ct value differences were dependent on both the rtRT-PCR platform and SARS-CoV-2 genomic variant. Differences ranging from 2.0 to 8.4 Ct values were observed when testing equal concentrations of different SARS-CoV-2 variants. Results confirm that Ct values are an unreliable surrogate for viral load and should not be used as a proxy of infectivity and transmissibility, especially when different rtRT-PCR assays are used in parallel and multiple SARS-CoV-2 variants are circulating. A detailed turn-around time (TAT) comparative assessment showed substantially different TATs, but parallel use of different diagnostic approaches was beneficial and complementary, allowing release of results for more than 81% of non-priority samples within 8 h after admission.

Evaluation of Two Broadly Used Commercial Methods for Detection of Respiratory Viruses with a Recently Added New Target for Detection of SARS-CoV-2.

The clinical symptoms caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nonspecific and can be associated with most other respiratory viruses that cause acute respiratory tract infections (ARI). Because the clinical differentiation of COVID-19 patients from those with other respiratory viruses is difficult, the evaluation of automated methods to detect important respiratory viruses together with SARS-CoV-2 seems necessary. Therefore, this study compares two molecular assays for the detection of respiratory viruses, including SARS-CoV-2: the Respiratory Viruses 16-Well Assay (AusDiagnostics, Pty Ltd., Mascot, Australia) and the Allplex™ RV Essential Assay coupled with the Allplex™-nCoV Assay (Seegene Inc., Seoul, Korea). The two methods (AusDiagnostics and AlplexTM-nCoV Assay SARS-CoV-2) had 98.6% agreement with the reference method, cobas 6800, for the detection of SARS-CoV-2. Agreement between the AusDiagnostics assay and the AlplexTM RV Essential Assay for the detection of seven respiratory viruses was 99%. In our experience, the Respiratory Viruses 16-Well Assay proved to be the most valuable and useful medium-throughput method for simultaneous detection of important respiratory viruses and SARS-CoV-2. The main advantages of the method are high specificity for all targets included and their simultaneous detection and medium throughput with the option of having multiple instruments provide a constant run.

Decreasing Pasteurization Treatment Efficiency against Amoeba-Grown Legionella pneumophila-Recognized Public Health Risk Factor.

Legionellae are gram-negative bacteria most commonly found in freshwater ecosystems and purpose-built water systems. In humans, the bacterium causes Legionnaires' disease (LD) or a Pontiac fever. In this study, the different waters (drinking water, pool water, cooling towers) in which Legionella pneumophila has been isolated were studied to assess the possible risk of bacterial spreading in the population. The influence of physical and chemical parameters, and interactions with Acanthamoeba castellanii on L. pneumophila, were analyzed by Heterotrophic Plate Count, the Colony-forming units (CFU) methods, transmission electron microscopy (TEM), and Sequence-Based Typing (SBT) analysis. During the study period (2013-2019), a total of 1932 water samples were analyzed, with the average annual rate of Legionella-positive water samples of 8.9%, showing an increasing trend. The largest proportion of Legionella-positive samples was found in cooling towers and rehabilitation centers (33.9% and 33.3%, respectively). Among the isolates, L. pneumophila SGs 2-14 was the most commonly identified strain (76%). The survival of Legionella was enhanced in the samples with higher pH values, while higher electrical conductivity, nitrate, and free residual chlorine concentration significantly reduced the survival of Legionella. Our results show that growth in amoeba does not affect the allelic profile, phenotype, and morphology of the bacterium but environmental L. pneumophila becomes more resistant to pasteurization treatment.

Real-Life Head-to-Head Comparison of Performance of Two High-Throughput Automated Assays for the Detection of SARS-CoV-2 RNA in Nasopharyngeal Swabs: The Alinity m and cobas 6800 SARS-CoV-2 Assays.

The Alinity m (Abbott Molecular, Des Plaines, IL) automated molecular analyzer allows continuous loading of samples and sample-to-result molecular detection of several microorganisms. The detection of SARS-CoV-2 by the Alinity m was compared with that of the cobas 6800 (Roche Molecular Systems, Branchburg, NJ; standard comparator) in a manufacturer-independent clinical evaluation on 2157 consecutive nasopharyngeal swab samples. Valid initial results on Alinity m and cobas 6800 were obtained from 2129 (98.7%) and 2157 (100%) samples, respectively. The overall percent agreement (95% CI) was 98.3% (2092/2129 [97.6%-98.7%]); positive percent agreement, 100% (961/961 [99.6%-100%]); negative percent agreement, 96.8% (1131/1168 [95.7%-97.7%]); and high κ value, 0.965 (0.954-0.976). There were 37 discordant results on Alinity m and, based on discordant analyses, including previous and/or follow-up PCR results, 22 could be considered analytically true positive with high probability. Due to a lack of additional information and an inability to perform repeated/further testing, the status of the remaining 15 discordant results remained unresolved. The throughput of the two analyzers was compared using testing on 564 samples in parallel across two 8-hour shifts in clinical practice. The turnaround times were compared using processing of 94 routine samples in parallel on each working day for 5 consecutive days. The two analyzers showed similar performance, with certain differences that have potential importance in some laboratory settings.

Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis.

Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the ISFtu2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica. We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals (n = 532) and Ixodes ricinus ticks (n = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

Detection of point mutations in the gyrA gene of Helicobacter pylori isolates in Slovenia.

BACKGROUND/AIMS: The prevalence of antibiotic-resistant H. pylori clinical isolates is a growing concern. So far, fluoroquinolones have not been used to treat H. pylori on a large scale, but recent studies have reported a high rate of quinolones-resistance in H. pylori too. The aim of our study was to asses the mechanism of resistance to ciprofloxacin in H. pylori clinical isolates from patients living in Slovenia. METHODOLOGY: Out of 397 H. pylori clinical isolates, obtained in the period 1997 to 2004, 33 (8.3%) ciprofloxacin-resistant H. pylori isolates were recognized. DNA sequences of the gyrA gene were determined and translated into amino acid sequences. RESULTS: Based on the results of this analysis, various point mutations in the ciprofloxacin-resistant clinical isolates were revealed. The most common mutations in H. pylori gyrA gene were found at codons corresponding to Asp91 (57.6%) and Asn87 (36.4%). Sequence analysis revealed amino acid substitutions also at codons Ala97 to Val, Ala129 to Thr and a double substitution at Asn87 to Lys and Val107 to Ile. CONCLUSIONS: These results suggest that H. pylori resistance to ciprofloxacin is already present in the Slovenian population and that it seems to be mediated through amino acid substitutions in the gyrA gene. Furthermore, the results obtained from the study also demonstrate no significant association between the type of gyrA mutation and the ciprofloxacin MIC level.

Genotyping of Chlamydia trachomatis directly from urogenital and conjunctiva samples using an ompA gene pyrosequencing-based assay.

The aim of the present study was to develop and validate a multitarget pyrosequencing-based protocol for basic Chlamydia trachomatis genotyping directly from clinical samples and to characterize the distribution of genotypes among Slovenian sexually active population. The newly developed combination of assays that targets the variable domains VD-I and VD-IV of the C. trachomatis ompA gene, was optimized and validated with 11 reference C. trachomatis strains and by comparison to complete ompA conventional sequencing. In addition, 183 clinical specimens which were previously diagnosed as C. trachomatis positive were evaluated by pyrosequencing. The pyrosequencing products showed a 100% match to corresponding sections of the respective conventional ompA sequences. Based on our results the most frequent genotype in urogenital samples was E (51.1%) followed by F (21.4%), G and K (6.9%), D (6.1%), H (3.8%), J (2.3%) and Ia and Ja (0.8%). In conjunctiva samples the genotype distribution was E (63.3%), D and F (13.3%), K (6.7%) and G (3.3%). Pyrosequencing thus proved itself to be a rapid method for C. trachomatis typing, which is important for better understanding the pathogenesis and epidemiology of this pathogen.