Genomic and epidemiological report of the recombinant XJ lineage SARS-CoV-2 variant, detected in northern Finland, January 2022.

Recombinant sequences of the SARS-CoV-2 Omicron variant were detected in surveillance samples collected in north-western Finland in January 2022. We detected 191 samples with an identical genome arrangement in weeks 3 to 11, indicating sustained community transmission. The recombinant lineage has a 5'-end of BA.1, a recombination breakpoint between orf1a and orf1b (nucleotide position 13,296-15,240) and a 3'-end of BA.2 including the S gene. We describe the available genomic and epidemiological data about this currently circulating recombinant XJ lineage.

Identification of urinary tract pathogens after 3-hours urine culture by MALDI-TOF mass spectrometry.

Complicated urinary tract infections, such as pyelonephritis, may lead to sepsis. Rapid diagnosis is needed to identify the causative urinary pathogen and to verify the appropriate empirical antimicrobial therapy. We describe here a rapid identification method for urinary pathogens: urine is incubated on chocolate agar for 3h at 35°C with 5% CO2 and subjected to MALDI-TOF MS analysis by VITEK MS. Overall 207 screened clinical urine samples were tested in parallel with conventional urine culture. The method, called U-si-MALDI-TOF (urine short incubation MALDI-TOF), showed correct identification for 86% of Gram-negative urinary tract pathogens (Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae), when present at >10(5)cfu/ml in culture (n=107), compared with conventional culture method. However, Gram-positive bacteria (n=28) were not successfully identified by U-si-MALDI-TOF. This method is especially suitable for rapid identification of E. coli, the most common cause of urinary tract infections and urosepsis. Turnaround time for identification using U-si-MALDI-TOF compared with conventional urine culture was improved from 24h to 4-6h.

Combined Expression of IFN-γ, IL-17, and IL-4 mRNA by Recall PBMCs Moderately Discriminates Active Tuberculosis from Latent Mycobacterium tuberculosis Infection in Patients with Miscellaneous Inflammatory Underlying Conditions.

New biomarkers are needed for discriminating active tuberculosis (TB) from latent TB infection (LTBI), especially in vulnerable groups representing the major diagnostic challenge. This pilot study was carried out to explore the diagnostic potential of selected genes, IFN-γ, IL-17, IL-4, and FoxP3, associated with TB immunity and immunopathology. IFN-γ, IL-17, IL-4, and FoxP3 mRNA expression levels were measured by quantitative reverse transcription PCR (RT-qPCR) from antigen-stimulated peripheral blood mononuclear cells of patients with active TB (n = 25); patients with miscellaneous inflammatory disorders and concomitant LTBI (n = 20), rheumatoid arthritis (RA) being the most predominant in the group (n = 11); and in healthy Bacillus Calmette-Guérin (BCG) vaccinees (n = 8). While the levels of FoxP3 mRNA did not differ between the tested groups, the cumulative expression levels of purified protein derivative-stimulated IFN-γ, IL-17, and IL-4 mRNAs were found to distinguish active TB from the whole group of LTBI with 48% sensitivity and 85% specificity. When restricting the LTBI group to RA cases only, the sensitivity was 56% and specificity 100%. When interpreting the result as positive in at least one of the mRNAs IFN-γ, IL-17, or IL-4, sensitivity of 64% and specificities of 75% (heterogeneous group of LTBI) or 100% (LTBI with RA) were achieved. Moderate discrimination of active TB from LTBI with miscellaneous inflammatory underlying conditions by using combined quantitative expression of IFN-γ, IL-17, and IL-4 mRNA seems not to be of high diagnostic potential.

Capillary electrophoresis as a method to determine underivatized urinary lipoarabinomannans, a biomarker of active tuberculosis caused by Mycobacterium tuberculosis.

Tuberculosis is a devastating contagious disease caused by Mycobacterium tuberculosis. This is the first report describing the development of novel capillary electrophoresis methods to detect lipoarabinomannans shed into the blood circulation by replicating bacteria. The novelty of the methods is the detection without derivatization. The lipoarabinomannan is detected owing to the ionization of the diverse functional groups of the structure, such as the multibranched mannan domain or the phosphatidyl group. Four alkaline solutions were used; normal polarity in three of them and reversed polarity in one. Urinary lipoarabinomannans by saccharide domains were identified with direct absorbance detection. The accuracy and the analytical sensitivity were then validated with cello-, manno- and xylooligosaccharides. Lipoarabinomannan detection was feasible within 20 min (RSD 2.1%). This method worked at the dynamic range of 0.1-10 µg/mL. With reversed polarity, indirect absorbance detection, and pH 9.0 electrolyte were used, the analytes migrated already within 5 min (RSD 0.01%). Inorganic nonabsorbing ions were used for this method optimization. This improvement resulted in the detection limit of 1 pg/mL in water and in the linear dynamic range of 1 pg/mL to 10 ng/mL. In conclusion, the described method has great potential as a point-of-care assay for clinical use.

Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly.

BACKGROUND: Since protective efficacy of the current typhoid vaccines-oral whole-cell Salmonella Typhi Ty21a and parenteral Vi-capsular polysaccharide preparation-is not optimal, and no vaccines are available against paratyphoid or non-typhoidal Salmonella (NTS) serotypes, new approaches deserve to be explored. The immunological mechanisms elicited by the two typhoid vaccines are mainly targeted against different structures. We studied whether these vaccines would enhance S. Typhi-specific immune response and cross-reactivity against other Salmonellae, if administered concomitantly. MATERIALS AND METHODS: Volunteers were immunized simultaneously with Ty21a and Vi vaccines (Ty21a+Vi group) or with either of the two singly (Ty21a and Vi groups). All volunteers were investigated for circulating specific and cross-reactive plasmablasts, identified by ELISPOT as IgA, IgG or IgM antibody-secreting cells (ASC) reactive with S. Typhi, S. Paratyphi A/B/C, or selected NTS serotypes (S. Enteritidis, S. Typhimurium). RESULTS: In the Ty21a+Vi group, no specific or cross-reactive plasmablasts were detected before vaccination. After vaccination, the number of S. Typhi-specific plasmablasts (878 ASC/10(6) PBMC, 95%CI 554-1201) proved higher than in the Ty21a (339 ASC/10(6) PBMC; p<0.001) and Vi (149 ASC/10(6) PBMC; p<0.001) groups. Likewise, cross-reactive responses in the Ty21a+Vi group were higher than in the Ty21a and Vi groups (Ty21a+Vi vs Ty21a: ASC against S. Paratyphi A/B, S. Enteritidis and S. Typhimurium p<0.05, against S. Paratyphi C p<0.01; Ty21a+Vi vs Vi: against S. Paratyphi C not significant, others p<0.0001). A gut-directed homing profile was seen among O antigen-specific and a systemic one among Vi antigen-specific plasmablasts. CONCLUSIONS: Concomitant administration of Ty21a and Vi vaccines is well tolerated and induces an additive immune response to the two vaccines. Thus it enhances the magnitude of both typhoid-specific plasmablast responses and those cross-reacting with paratyphoid and most important NTS serotypes. The data encourage concomitant use of Ty21 and Vi vaccines for those at risk.

Cytotoxic response persists in subjects treated for tuberculosis decades ago.

BACKGROUND: Data exploring the potential use of effector molecules produced by cytotoxic T lymphocytes (CTLs) in the immunodiagnostics of tuberculosis (TB) are scarce. The present study focused a) to gain an insight into the discriminatory power of CTLs in patients with acute pulmonary or extra-pulmonary TB, or latent tuberculosis infection (LTBI); and b) to evaluate the influence of various anti-TB therapeutic schemes on the immunological profiles of residual CTLs. METHODS: Immunological signatures of antigen-specific CTLs were explored in patients with active pulmonary and extra-pulmonary TB, LTBI and in those treated for TB decades ago by using ELISPOT, intracellular flow cytometry and extracellular CD107a detection. RESULTS: No difference was seen between active TB, LTBI or any of those treated for TB in the ELISPOT analysis of antigen-specific Granzyme B (GrB), Perforin (Prf) and interferon-gamma (IFN-γ) producing lymphocytes, the FACS analysis of the intracellular expression of IFN-γ, or the surface expression of CD107a degranulation factor of both CD8+ and CD4+ antigen-specific T cell subsets. The effector memory (TEM) phenotype proved predominant in the surface marker profiling both in active TB and LTBI. The proportion of the CD107a degranulation factor proved higher in the central memory (TCM) than in the other cell subsets in all the study groups. Interestingly, functionally and phenotypically similar CTLs profiles were observed in active TB, LTBI and in all the three groups treated for TB. CONCLUSION: The phenotypic and functional profiling of CTLs has a limited potential in the immunodiagnostics of active TB. Antigen-specific CTLs persist in patients treated for TB decades ago regardless of the efficacy of implemented and completed anti-TB therapy.

Modification of clearview tuberculosis (TB) enzyme-linked immunosorbent assay for TB patients not infected with HIV.

Diagnosis of active tuberculosis by detection of urinary lipoarabinomannan (uLAM) from Mycobacterium tuberculosis is an attractive approach. Concentrating urine 100-fold allowed quantitation of uLAM at levels equal to picograms/ml of nonconcentrated urine. The approach of concentrating urine 100-fold improved the clinical sensitivity of the Clearview TB enzyme-linked immunosorbent assay (ELISA) from 7% to 57% yet impaired its specificity from 97% to 89%. (This study has been registered at University Hospital of Turku under registration no. 47/180/2009, Helsinki University Central Hospital under 149/2010, University Hospital of Kuopio under 105/2010, and China Medical University Hospital, Taichung, under DMR-99-IRB-075-2.).

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.

Pilot study of diagnostic potential of the Mycobacterium tuberculosis recombinant HBHA protein in a vaccinated population in Finland.

BACKGROUND: In recent years T cell based interferon gamma release assays (IGRA) have been developed for immunodiagnosis of M. tuberculosis infection. At present these assays do not discriminate between disease and latency. Therefore, more promising antigens and diagnostic tools are continuously being searched for tuberculosis immunodiagnostics. The heparin binding hemagglutinin (HBHA) is a surface protein of M. tuberculosis which promotes bacterial aggregation and adhesion to non-phagocytic cells. It has been previously assumed that native, methylated form of this protein would be a promising antigen to discriminate latent from active infection. METHODOLOGY AND PRINCIPAL FINDINGS: We performed a pilot investigation to study humoral and T-cell mediated immunological responses to recombinant HBHA produced in M. smegmatis or to synthetic peptides in patients with recent or past tuberculosis, with atypical mycobacteriosis, or in healthy vaccinated individuals. The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie. Early Secretory Antigen Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well. We observed a positive correlation between the reactivities to HBHA and non-specific PPD among all studied subjects. As expected, ESAT-6 and CFP-10 were the most powerful antigens to discriminate disease from immunity caused by vaccination. CONCLUSIONS: On the basis of results of this exploratory investigation we raise concerns that in countries like Finland, where BCG vaccination was routinely used, HBHA utility might not be sufficient for diagnostics because of inability to explicitly discriminate tuberculosis infection from immunoreactivity caused by previous BCG vaccination.