Diagnosis of external ventricular drainage related infections with real-time 16S PCR and third-generation 16S sequencing.

OBJECTIVE: Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). METHODS: Subjects with suspected EVDRI were prospectively included at Uppsala University Hospital. Subjects were included into three groups: subjects with negative CSF culture with and without antibiotic treatment and subjects with positive CSF culture, respectively. CSF was analysed with real-time 16S PCR and third-generation 16S sequencing. Real-time 16S PCR positivity/negativity and number of 16S sequence reads were compared between groups. For culture positive subjects, species identification in third-generation sequencing and routine culture was compared. RESULTS: 84 subjects were included. There were 18, 44 and 22 subjects in the three groups. Real-time PCR was positive in 17 of 22 subjects in the culture positive group and negative in 61 of the 62 subjects in the two culture negative groups. The sensitivity and specificity for real-time 16S PCR compared to culture was estimated to 77% and 98%, respectively. Species identification in 16S sequencing and culture was concordant in 20 of 22 subjects. The number of 16S sequence reads were significantly higher in the culture positive group than in both culture negative groups (p < 0.001). There was no significant difference in number of 16S sequences between the two culture negative groups. CONCLUSIONS: Real-time 16S PCR predict culture results with sufficient reliability. Third-generation 16S sequencing could enhance sensitivity and species identification in diagnostics of EVD-related infections. False negative culture results appear to be uncommon in patients with suspected EVDRI.

The use and usefulness of point-of-care tests in patients with pharyngotonsillitis - an observational study in primary health care.

BACKGROUND: Rapid antigen detection tests (RADT) for Group A streptococci (GAS) and point-of-care tests for C-reactive protein (CRP) are commonly used in patients with pharyngotonsillitis in Sweden and Denmark although CRP testing is not supported by guidelines. We aimed to describe (1) the proportion of patients tested with RADT and/or CRP, (2) the relation between test results and antibiotic prescribing, and (3) the association between CRP level and microbial aetiology. METHODS: We used a post-hoc-analysis of data collected in primary health care in a prospective aetiological study of 220 patients 15-45 years old diagnosed with pharyngotonsillitis. The outcomes of RADTs and CRP tests were related to antibiotic prescribing and microbial aetiology. RESULTS: A RADT was used in 94% of the patients. A CRP test was used in 50% of the patients but more commonly in those with a negative RADT (59%) than in those with a positive RADT (38%) (p = 0.005). Most (74%) CRP tests were used in patients with a negative RADT. Antibiotic prescribing differed greatly between patients with a positive RADT (96%) and patients with a negative RADT (17%) (p < 0.001). In patients with a negative RADT, there was a positive association between CRP value and antibiotic prescribing (OR 1.05; 95% CI 1.02-1.07; p < 0.001). Patients with CRP values ≤ 30 mg/l were seldomly prescribed antibiotics. Patients with GAS in culture had the highest median CRP (46 mg/l), which was higher than in patients without GAS (8 mg/l; p < 0.001). However, the positive predictive value for GAS never exceeded 0.60 (95% CI 0.31-0.83) at the investigated CRP levels. CONCLUSIONS: The widespread use of tests is a major deviation from national guidelines. Most CRP tests were used in patients with a negative RADT, suggesting a belief in the added value of a CRP test, and the CRP result seemed to influence antibiotic prescribing. However, as an aetiological test, CRP is not useful for predicting GAS.

Prevalence of SARS-CoV-2 Omicron Sublineages and Spike Protein Mutations Conferring Resistance against Monoclonal Antibodies in a Swedish Cohort during 2022-2023.

Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study, 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden, collected between March 2022 and May 2023, were whole-genome sequenced using amplicon-based sequencing methods on Oxford Nanopore GridION, Illumina MiSeq, Illumina HiSeq, or MGI DNBSEQ-G400 instruments. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. We found that the dominant sublineages changed over time, and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.

Short incubation of disc diffusion for Streptococcus pneumoniae and Haemophilus influenzae to reduce time to susceptibility report.

BACKGROUND: Rapidly instituted antimicrobial therapy is important in severe infections, and reduced time to the antimicrobial susceptibility testing (AST) report is thus of importance. Disc diffusion (DD) is a cheap, rapidly adaptable, flexible and comprehensive method for phenotypic AST. Previous studies have shown that early reading of inhibition zones for non-fastidious species is possible. OBJECTIVES: To evaluate zone reading after short incubation of DD in Haemophilus influenzae (n = 73) and Streptococcus pneumoniae (n = 112). METHODS: The readability was evaluated and susceptibility interpretation (SIR) was performed, using the EUCAST 18 ±â€Š2 h incubation breakpoint table (version 12.0), after 6 and 8 h of incubation. Categorical agreement (CA) and error rates were calculated using standard DD and broth microdilution as reference. RESULTS: The proportion of readable zones in H. influenzae was 19% (6 h) and 89% (8 h). The CA was 98% after 8 h. The corresponding readability in S. pneumoniae was 63%/98% and CA was 95%/97% after 6 and 8 h, respectively. Early reading of the screening discs (benzylpenicillin 1 unit in H. influenzae and oxacillin 1 µg in S. pneumoniae) correctly identified 18/22 of the H. influenzae isolates and all the readable S. pneumoniae isolates with reduced ß-lactam susceptibility. For non-ß-lactam agents, very major errors were most common for quinolones in S. pneumoniae. Introduction of areas of technical uncertainty (ATUs) reduced the error rate to ≤1.1%. CONCLUSIONS: We conclude that shortened incubation is feasible for H. influenzae and S. pneumoniae. To reduce the risk of false categorization a buffer zone (i.e. ATU) near the breakpoints must be used.

Helicobacter pylori and Pro-Inflammatory Protein Biomarkers in Myocardial Infarction with and without Obstructive Coronary Artery Disease.

Myocardial infarction (MI) with obstructive coronary artery disease (MI-CAD) and MI in the absence of obstructive coronary artery disease (MINOCA) affect different populations and may have separate pathophysiological mechanisms, with greater inflammatory activity in MINOCA compared to MI-CAD. Helicobacter pylori (Hp) can cause systemic inflammation and has been associated with cardiovascular disease (CVD). We aimed to investigate whether Hp infection is associated with concentrations of protein biomarkers of inflammation and CVD. In a case-control study, patients with MINOCA (n = 99) in Sweden were included, complemented by matched subjects with MI-CAD (n = 99) and controls (n = 100). Protein biomarkers were measured with a proximity extension assay in plasma samples collected 3 months after MI. The seroprevalence of Hp and cytotoxin-associated gene A (CagA) was determined using ELISA. The associations between protein levels and Hp status were studied with linear regression. The prevalence of Hp was 20.2%, 19.2%, and 16.0% for MINOCA, MI-CAD, and controls, respectively (p = 0.73). Seven proteins were associated with Hp in an adjusted model: tissue plasminogen activator (tPA), interleukin-6 (IL-6), myeloperoxidase (MPO), TNF-related activation-induced cytokine (TRANCE), pappalysin-1 (PAPPA), soluble urokinase plasminogen activator receptor (suPAR), and P-selectin glycoprotein ligand 1 (PSGL-1). Hp infection was present in one in five patients with MI, irrespective of the presence of obstructive CAD. Inflammatory proteins were elevated in Hp-positive subjects, thus not ruling out that Hp may promote an inflammatory response and potentially contribute to the development of CVD.

Comparison of SARS-CoV-2 whole genome sequencing using tiled amplicon enrichment and bait hybridization.

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) pandemic has led to extensive virological monitoring by whole genome sequencing (WGS). Investigating the advantages and limitations of different protocols is key when conducting population-level WGS. SARS-CoV-2 positive samples with Ct values of 14-30 were run using three different protocols: the Twist Bioscience SARS­CoV­2 protocol with bait hybridization enrichment sequenced with Illumina, and two tiled amplicon enrichment protocols, ARTIC V3 and Midnight, sequenced with Illumina and Oxford Nanopore Technologies, respectively. Twist resulted in better coverage uniformity and coverage of the entire genome, but has several drawbacks: high human contamination, laborious workflow, high cost, and variation between batches. The ARTIC and Midnight protocol produced an even coverage across samples, and almost all reads were mapped to the SARS-CoV-2 reference. ARTIC and Midnight represent robust, cost-effective, and highly scalable methods that are appropriate in a clinical environment. Lineage designations were uniform across methods, representing the dominant lineages in Sweden during the period of collection. This study provides insights into methodological differences in SARS­CoV­2 sequencing and guidance in selecting suitable methods for various purposes.

Helicobacter Pylori Virulence Factor Cytotoxin-Associated Gene A (CagA) Induces Vascular Calcification in Coronary Artery Smooth Muscle Cells.

Helicobacter pylori (H. pylori) has been associated with cardiovascular diseases. The pro-inflammatory H. pylori virulence factor cytotoxin-associated gene A (CagA) has been detected in serum exosomes of H. pylori-infected subjects and may exert systemic effects throughout the cardiovascular system. The role of H. pylori and CagA in vascular calcification was hitherto unknown. The aim of this study was to determine the vascular effects of CagA through human coronary artery smooth muscle cell (CASMC) osteogenic and pro-inflammatory effector gene expression as well as interleukin 1ß secretion and cellular calcification. CagA upregulated bone morphogenic protein 2 (BMP-2) associated with an osteogenic CASMC phenotype switch and induced increased cellular calcification. Furthermore, a pro-inflammatory response was observed. These results support that H. pylori may contribute to vascular calcification through CagA rendering CASMCs osteogenic and inducing calcification.

[Four blood culture bottles from one venipuncture instead of divided into two: new Swedish national recommendation for blood culture]. / Ta fyra blododlingsflaskor i en och samma venpunktion.

Blood culture is the standard method for microbiological diagnostics in sepsis. In many countries, the traditional recommendation for blood culture has been collection of four blood culture bottles with two bottles from each of two venipunctures. New studies show that four blood culture bottles from the same venipuncture provides similar culture results and higher number of obtained blood culture bottles, than the traditional method. Thus, new national recommendations recommend collection of four blood culture bottles from the same venipuncture as the routine blood culture method in Sweden.

Evaluation of within-host evolution of methicillin-resistant Staphylococcus aureus (MRSA) by comparing cgMLST and SNP analysis approaches.

Whole genome sequencing (WGS) of methicillin-resistant Staphylococcus aureus (MRSA) provides high-resolution typing, facilitating surveillance and outbreak investigations. The aim of this study was to evaluate the genomic variation rate in MRSA, by comparing commonly used core genome multilocus sequencing (cgMLST) against single nucleotide polymorphism (SNP) analyses. WGS was performed on 95 MRSA isolates, collected from 20 carriers during years 2003-2019. To assess variation and methodological-related differences, two different cgMLST schemes were obtained using Ridom SeqSphere+ and the cloud-based 1928 platform. In addition, two SNP methods, 1928 platform and Northern Arizona SNP Pipeline (NASP) were used. The cgMLST using Ridom SeqSphere+ and 1928 showed a median of 5.0 and 2.0 allele variants/year, respectively. In the SNP analysis, performed with two reference genomes COL and Newman, 1928 showed a median of 13 and 24 SNPs (including presumed recombination) and 3.8 respectively 4.0 SNPs (without recombination) per individual/year. Accordantly, NASP showed a median of 5.5 and 5.8 SNPs per individual/year. In conclusion, an estimated genomic variation rate of 2.0-5.8 genetic events per year (without recombination), is suggested as a general guideline to be used at clinical laboratories for surveillance and outbreak investigations independently of analysis approach used.

RT-PCR cycle threshold value in combination with visual scoring of chest computed tomography at hospital admission predicts outcome in COVID-19.

BACKGROUND: COVID-19 has a most variable prognosis. Several risk factors for an unfavourable outcome have been identified including extensive lung involvement on chest CT and high viral load estimated by RT-PCR cycle threshold (Ct) values. We investigated Ct value for outcome prediction, relation between Ct value and extent of lung involvement on chest CT and the combination of Ct value and chest CT lung involvement to predict outcome in COVID-19. METHODS: Population-based retrospective study on all patients (n = 286) hospitalised for COVID-19 in Örebro Region, Sweden, between 1 March and 31 August 2020. Nasopharyngeal samples and chest CT at hospital admission were evaluated in relation to outcome of COVID-19. RESULTS: Both Ct value and chest CT lung involvement were independently associated with risk for ICU admission or death. Lung involvement was superior as a single parameter, but addition of Ct value increased the prediction performance. Ct value was especially useful to identify patients with high risk for severe disease despite limited lung involvement. CONCLUSIONS: The addition of RT-PCR Ct value to the assessment of lung involvement on chest CT adds valuable prognostic information in COVID-19. We believe that this information can be used to support clinical decision-making when managing COVID-19 patients.

Potential impact of the COVID-19 pandemic on the national and regional incidence, epidemiology and diagnostic testing of chlamydia and gonorrhoea in Sweden, 2020.

The COVID-19 pandemic has challenged the societies and health care systems globally, and resulted in many social and physical distancing restrictions to limit the spread of SARS-CoV-2. These restrictions have also likely affected the frequency of intimate contacts and the spread of sexually transmitted infections (STIs). Compared to most other countries, Sweden especially in Spring-Autumn 2020 pursued mainly milder voluntary, that is, not mandatory enforced by laws, recommended restrictions and the impacts of these on society and spread of STIs remain largely unknown. We describe the potential impact of the COVID-19 pandemic on the national and regional incidence, epidemiology and diagnostic testing of chlamydia and gonorrhoea in Sweden in 2020. Compared to 2019, we found a significant decrease in incidence of chlamydia (-4.5%) and gonorrhoea (-17.5%), and in diagnostic testing (-10.5% for chlamydia, -9.4% for gonorrhoea) in 2020. However, the decrease in chlamydia incidence, which has mainly been decreasing in the last 10 years, was not significant when compared with the average incidence in 2017-2019. The largest decrease in national incidence of both infections was observed among young and heterosexual patients, however, some Swedish regions showed an increased incidence, particularly of chlamydia. Increased "internet-based self-sampling" testing approach partly compensated for a decreased attendance at STI clinics. Studies, including sexual behaviour, prevention, reasons for attending STI health care, STIs in different anatomical sites and management of STIs, are required to elucidate the impact of COVID-19-associated social and physical distancing restrictions on sexual activity and the incidence and epidemiology of chlamydia and gonorrhoea in Sweden.

CXCL13 in laboratory diagnosis of Lyme neuroborreliosis-the performance of the recomBead and ReaScan CXCL13 assays in human cerebrospinal fluid samples.

The chemokine CXCL13 is used as complement to serology in the diagnostics of Lyme neuroborreliosis (LNB). We evaluated and compared the semi-quantitative, cassette-based ReaScan CXCL13 assay with the quantitative recomBead CXCL13 assay using a collection of 209 cerebrospinal fluid samples. The categorical agreement between results interpreted as negative, grey zone, and positive by the two methods was 87%. The diagnostic sensitivity was higher using the recomBead assay, whereas specificity was higher using ReaScan. Few manual steps, and a short turn-around time with no batching of samples makes the ReaScan CXCL13 assay an attractive complement to serology in the diagnostics of LNB.

Seasonal variations in use and outcome of rapid antigen detection tests and cultures in pharyngotonsillitis: a register study in primary care.

BACKGROUND: Diagnosis and treatment of pharyngotonsillitis are commonly focused on group A streptococci (GAS), although the disease is often associated with other pathogens. While the incidence of pharyngotonsillitis is known to vary with season, seasonal variations in the prevalence of potential pathogens are sparsely explored. The aim of this study was to explore any seasonal variations in the use and outcome of rapid antigen detection tests (RADTs) for GAS and throat cultures among patients diagnosed with pharyngotonsillitis in primary care. METHODS: We retrieved and combined retrospective data from the electronic medical record system and the laboratory information system in Kronoberg County, Sweden. Primary care visits resulting in a diagnosis of tonsillitis or pharyngitis were included, covering the period 2013-2016. The monthly rate of visits was measured, along with the use and outcome of RADTs for GAS and throat cultures obtained on the date of diagnosis. The variations between calendar months were then analysed. RESULTS: We found variations between calendar months, not only in the mean rate of visits resulting in a diagnosis of pharyngotonsillitis (p < 0.001), but in the mean proportion of RADTs being positive for GAS among the diagnosed (p < 0.001), and in the mean proportion of visits associated with a throat culture (p < 0.001). A lower mean rate of visits in August and September coincided with a lower proportion of RADTs being positive for GAS among them, which correlated with a higher proportion of visits associated with a throat culture. CONCLUSIONS: This study suggests that the role of GAS in pharyngotonsillitis in Sweden is less prominent in August and September than during the rest of the year.

The aetiology of pharyngotonsillitis in primary health care: a prospective observational study.

BACKGROUND: Few studies on pharyngotonsillitis have examined the clinical presentation of different aetiologies where pathogens have been detected using molecular methods. We aimed to assess how well clinical signs and symptoms can predict (1) the presence or absence of a broad range of viruses and bacteria, and (2) reconsultations for a sore throat or a complication. METHODS: In this descriptive observational prospective study in primary health care 220 patients aged 15-45 with suspected pharyngotonsillitis were sampled from nose, throat and blood and screened for 20 bacteria and viruses using polymerase chain reaction (PCR), culture and serology. Odds ratios (OR) and predictive values with 95% confidence intervals (CI) were used to show association between microbiological findings and clinical signs and symptoms. Patients were followed up after 3 months by reviewing electronic medical records. RESULTS: Both cough and coryza were more common in patients with only viruses (67%) than in patients with only bacteria (21%) (p < 0.001), whereas tonsillar coating was more common in patients with only bacteria (53%) than in patients with only viruses (29%) (p = 0.006). Tonsillar coating (adjusted OR 6.0; 95% CI 2.5-14) and a lack of cough (adjusted OR 3.5; 95% CI 1.5-8.0) were significantly associated with Streptococcus pyogenes (group A streptococci; GAS) and with any bacterial finding. A Centor score of 3-4 had a positive predictive value of 49% (95% CI 42-57) for GAS and 66% (95% CI 57-74) for any bacterial findings. The use of rapid antigen detection test for GAS increased the positive predictive value for this group to 93%. CONCLUSIONS: Signs and symptoms, both single and combined, were insufficient to rule in GAS or other pathogens. However, both cough and coryza were useful to rule out GAS. The results support the clinical approach of restricting rapid antigen detection testing to patients with 3-4 Centor criteria. The low carriage rate of bacteria among asymptomatic controls implied that most detections in patients represented a true infection.

Association between bacterial finding, antibiotic treatment and clinical course in patients with pharyngotonsillitis: a registry-based study in primary healthcare in Sweden.

BACKGROUND: The role of non-group A streptococci and Fusobacterium necrophorum in pharyngotonsillitis has been disputed and few prospective studies have evaluated the effect of antibiotic treatment. This study uses registry data to investigate the relation between antibiotic prescription for pharyngotonsillitis in primary healthcare and return visits for pharyngotonsillitis, complications, and tonsillectomy. METHODS: Retrospective data were extracted from the regional electronic medical record system in Kronoberg County, Sweden, for all patients diagnosed with pharyngotonsillitis between 2012 and 2016. From these data, two cohorts were formed: one based on rapid antigen detection tests (RADT) for group A streptococci (GAS) and one based on routine throat cultures for ß-haemolytic streptococci and F. necrophorum. The 90 days following the inclusion visit were assessed for new visits for pharyngotonsillitis, complications, and tonsillectomy, and related to bacterial aetiology and antibiotic prescriptions given at inclusion. RESULTS: In the RADT cohort (n = 13,781), antibiotic prescription for patients with a positive RADT for GAS was associated with fewer return visits for pharyngotonsillitis within 30 days compared with no prescription (8.7% vs. 12%; p = 0.02), but not with the complication rate within 30 days (1.5% vs. 1.8%; p = 0.7) or with the tonsillectomy rate within 90 days (0.27% vs. 0.26%; p = 1). In contrast, antibiotic prescription for patients with a negative RADT was associated with more return visits for pharyngotonsillitis within 30 days (9.7% vs. 7.0%; p = 0.01). In the culture cohort (n = 1 370), antibiotic prescription for patients with Streptococcus dysgalactiae ssp. equisimilis was associated with fewer return visits for pharyngotonsillitis within 30 days compared with no prescription (15% vs. 29%; p = 0.03). CONCLUSIONS: Antibiotic prescription was associated with fewer return visits for pharyngotonsillitis in patients with a positive RADT for GAS but with more return visits in patients with a negative RADT for GAS. There were no differences in purulent complications related to antibiotic prescription.

Evaluation of the eazyplex® EHEC complete (Amplex) assay for the differentiation of stx1 and stx2 positivity in stool samples.

The performance of the eazyplex® EHEC complete (Amplex) for the detection of Shiga toxin genes in stool samples was evaluated. The assay performed well in distinguishing between stx1 and stx2 but suboptimal sensitivity may limit its use to complementary testing rather than primary diagnosis of Shiga toxin-producing Escherichia coli infections.

Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome.

BACKGROUND: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method). METHODS: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order. RESULTS: The standard method resulted in a significantly higher proportion of positive culture outcomes among the 94 included episodes of infectious keratitis (61%; 57/94) than the indirect inoculation method (44%; 41/94) (p = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen's kappa of 0.38 (95% CI: 0.28-0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method. CONCLUSION: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

Atypical presentation of Neisseria meningitidis serogroup W disease is associated with the introduction of the 2013 strain.

Since 2015, the incidence of invasive meningococcal disease (IMD) caused by serogroup W (MenW) has increased in Sweden, due to the introduction of the 2013 strain belonging to clonal complex 11. The aim of this study was to describe the clinical presentation of MenW infections, in particular the 2013 strain, including genetic associations. Medical records of confirmed MenW IMD cases in Sweden during the years 1995-2019 (n = 113) were retrospectively reviewed and the clinical data analysed according to strain. Of all MenW patients, bacteraemia without the focus of infection was seen in 44%, bacteraemic pneumonia in 26%, meningitis in 13% and epiglottitis in 8%, gastrointestinal symptoms in 48% and 4% presented with petechiae. Phylogenetic analysis was used for possible links between genetic relationship and clinical picture. The 2013 strain infections, particularly in one cluster, were associated with more severe disease compared with other MenW infections. The patients with 2013 strain infections (n = 68) were older (52 years vs. 25 years for other strains), presented more often with diarrhoea as an atypical presentation (P = 0.045) and were more frequently admitted for intensive care (P = 0.032). There is a risk that the atypical clinical presentation of MenW infections, with predominantly gastrointestinal or respiratory symptoms rather than neck stiffness or petechiae, may lead to delay in life-saving treatment.