Gonorrhoea on the rise in Denmark since 2022: distinct clones drive increase in heterosexual individuals.

A surge in gonorrhoea in Denmark has occurred since 2022, a 46% increase from 2021. National surveillance, leveraging mandatory reporting and epidemiological data, highlights three distinct clades linked to heterosexual transmission. Despite the rise, these exhibit high susceptibility, contrasting MSM-associated strains. Geographical hotspots and age-specific patterns further illuminate transmission dynamics. The combination of genomic and epidemiological data provides novel insights into the evolving landscape of gonorrhoea, indicating potential shifts in infection dynamics and transmissibility.

Identification of Rv0222 from RD4 as a novel serodiagnostic target for tuberculosis.

Forty-seven Mycobacterium tuberculosis genes from the 'regions of difference' RD2-7, RD9-13 and RD15 were cloned and expressed, and the purified recombinant proteins were screened for their serodiagnostic potential. Evaluation of six selected proteins in serum samples from Danish resident tuberculosis patients and healthy controls led to identification of Rv0222 as the most promising serodiagnostic antigen. Recognition of the Rv0222 was compared with the 38 kDa protein and a fusion protein of the RD1 proteins ESAT-6 and CFP10 in a serum panel from pulmonary tuberculosis (TB) patients from Uganda. The highest overall sensitivity was observed for Rv0222 compared to BCG-vaccinated non-endemic healthy controls as well as symptomatic endemic controls. Importantly, Rv0222 identified human immuno deficiency (HIV) virus-positive patients and HIV-negative patients with the same overall sensitivity. The results emphasize the importance of cut-off values in TB endemic regions based on endemic control individuals to diagnose active TB, and identify Rv0222 as a promising new antigen for serodiagnosis of TB in both HIV-negative and HIV-positive patients.

Latent tuberculosis in HIV positive, diagnosed by the M. tuberculosis specific interferon-gamma test.

BACKGROUND: Although tuberculosis (TB) is a minor problem in Denmark, severe and complicated cases occur in HIV positive. Since the new M. tuberculosis specific test for latent TB, the QuantiFERON-TB In-Tube test (QFT-IT) became available the patients in our clinic have been screened for the presence of latent TB using the QFT-IT test. We here report the results from the first patients screened. METHODS: On a routine basis the QFT-IT test was performed and the results from 590 HIV positive individuals consecutively tested are presented here. CD4 cell count and TB risk-factors were recorded from patient files. MAIN FINDINGS: 27/590(4.6%) of the individuals were QFT-IT test positive, indicating the presence of latent TB infection. Among QFT-IT positive patients, 78% had risk factors such as long-term residency in a TB high endemic area (OR:5.7), known TB exposure (OR:4.9) or previous TB disease (OR:4.9). The prevalence of latent TB in these groups were 13%, 16% and 19% respectively. There was a strong correlation between low CD4 T-cell count and a low mitogen response (P < 0.001;Spearman) and more patients with low CD4 cell count had indeterminate results. CONCLUSION: We found an overall prevalence of latent TB infection of 4.6% among the HIV positive individuals and a much higher prevalence of latent infection among those with a history of exposure (16%) and long term residency in a high endemic country (13%). The QFT-IT test may indeed be a useful test for HIV positive individuals, but in severely immunocompromised, the test may be impaired by T-cell anergy.

Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells.

BACKGROUND: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment. A candidate for such regulation could be the chemokine CXCL10. CXCL10 is mainly produced by human monocytes, but a few reports have also found CXCL10 production by human B cells. The objective of this study was to investigate CXCL10 production by human B cells in response to in vitro stimulation with Mtb antigens. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed human blood samples from 30 volunteer donors using multiparameter flow cytometry, and identified a subgroup of B cells producing CXCL10 in response to in vitro stimulation with antigens. T cells did not produce CXCL10, but CXCL10 production by B cells appeared to be mediated via IFN-γ and dependent on contact with antigen-specific T cells recognizing the antigen. CONCLUSION: Human B cells are able to produce CXCL10 in an IFN-γ and T cell contact-dependent manner. The present findings suggest a possible mechanism through which B cells in part may influence granuloma formation in human tuberculosis (TB) and participate in infection control.

[First Danish case of Clostridium difficile ribotype 027]. / Første påvisning af Clostridium difficile type 027 i Danmark.

Increasing rates of Clostridium difficile-associated diarrhoea (CDAD) with an unusual, severe course have been reported in Canada, USA and several European countries since 2003. A new virulent strain, PCR ribotype 027 (CD027), is associated with this increase. We report the first Danish case of CDAD caused by CD027. A 85-year-old woman was admitted to hospital with pneumonia. Following treatment initially with penicillin, secondly with moxifloxacin she developed bloody diarrhoea. A stool specimen showed CD027. She was treated with metronidazol for ten days and recovered completely.

[Comparison of QuantiFERON and tuberculin skin test in possible suspected tuberculosis infection]. / Sammenligning af QuantiFERON og hudtest ved mulig tuberkuloseinfektion.

INTRODUCTION: Two cases of tuberculosis were diagnosed at a high school. A contact investigation using Mantoux test (TST) and QuantiFERON TB in Tube test (QFT) was conducted in order to compare QFT with TST. METHODS: A total of 787 individuals were invited to participate, of whom 734 had a TST applied and 490 a QFT test done. RESULTS: Of the skin test applied 689 (94%) were read, and 41 (5.9%) were positive. TSTs were positive in 17/603 (2.8%) students and 24/87 (28%) staff members. A total of 490 (62%) had a QFT performed; 11 (2.2%) were positive and four (0,8%) inconclusive. QFT positive results were found in 8/419 (1.9%) students and 3/71 (4.2%) staff members. Agreement between the TST and the QFT was moderate 97% (Kappa 0.407; confidence interval (CI) 0.124-0.689) among students and poor among staff 68% (Kappa 0.036; CI -0.102-0.175). The majority of the TST-positive and all the QFT positive students were found in relation to the index case. Positive TST results and BCG vaccination (p < 0,05) were associated. Only 5/10 (50%) of the QFT-positive individuals were TST positive, which indicates a suboptimal TST sensitivity. CONCLUSION: The two tests identified an equal amount of Mycobacterium tuberculosis infected students, whereas a high proportion of the staff were TST positive. The discordant results underline that the two tests do not measure the same and that their sensitivity is not identical. Choice of test should rest on a case-by-case analysis and should be tailored to the desired level of safety and the cost for society and for the patients.

Risk for tuberculosis among children.

Contacts of adults with tuberculosis (TB) are at risk for infection. Tests based on interferon-gamma (IFN-gamma) expression in response to Mycobacterium tuberculosis antigens may be more sensitive than the tuberculin skin test (TST). Risk for infection was assessed by using TST and an IFN-y-based assay (QuantiFERON Gold in Tube [QFT-IT] test) for 207 children in Nigeria in 1 of 3 groups: contact with adults with smear-positive TB, contact with adults with smear-negative TB, and controls. For these 3 groups, respectively, TST results were >10 mm for 38 (49%) of 78, 13 (16%) of 83, and 6 (13%) of 46 and QFT-IT positive for 53 (74%) of 72, 8 (10%) of 81, and 4 (10.3%) of 39 (p < 0.01). Most test discrepancies were TST negative; QFT-IT positive if in contact with TB-positive persons; and TST positive, QFT-IT negative if in contact with TB-negative persons or controls. TST may underestimate risk for infection with TB in children.

Comparison of tuberculin skin test and new specific blood test in tuberculosis contacts.

The tuberculin skin test used to detect latent Mycobacterium tuberculosis infection has many drawbacks, and a new diagnostic test for latent tuberculosis (QuantiFERON-TB [QTF-TB]) has recently been introduced. This test measures the production of IFN-gamma in whole blood upon stimulation with purified protein derivative (PPD). The QTF-TB test addresses the operational problems with the tuberculin skin test, but, as the test is based on PPD, it still has a low specificity in populations vaccinated with the Bacille Calmette-Guérin (BCG) vaccine. We have modified the test to include the antigens ESAT-6 and CFP-10, which are not present in BCG vaccine strains or the vast majority of nontuberculous mycobacteria. This test was used to detect infection in contacts in a tuberculosis outbreak at a Danish high school. The majority of the contacts were BCG-unvaccinated, which allowed a direct comparison of the skin test and the novel blood test in individuals whose skin test was not confounded by vaccination. An excellent agreement between the two tests was found (94%, kappa value 0.866), and in contrast to the blood test based on PPD, the novel blood test was not influenced by the vaccination status of the subjects tested.

Mapping immune reactivity toward Rv2653 and Rv2654: two novel low-molecular-mass antigens found specifically in the Mycobacterium tuberculosis complex.

New tools are urgently needed for the detection of latent tuberculosis (TB). We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs. Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors. However, peptides from Rv2653 were recognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule. Rv2654 also induced M. tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs. Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.

The currently used method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules was evaluated by sensitive testing of the T-cell responses of peripheral blood mononuclear cells derived from M. bovis BCG-vaccinated healthy individuals to synthesized overlapping peptides. Three of the four molecules contained regions with significant specificity problems (Rv2653, Rv3873, and Rv3878). We selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis BCG-vaccinated controls. The combination of the most promising stretches from this analysis showed a sensitivity level (57%) comparable to the level found with the two well-known M. tuberculosis-specific proteins ESAT-6 and CFP-10 (75 and 66%, respectively). The combination of ESAT-6, CFP-10, and the novel specific peptide stretches gave an overall sensitivity of 84% at a specificity of 97%. In a validation experiment with new experimental groups, the sensitivities obtained were 57% for the combination of peptides and 90% for the combination of the peptides, ESAT-6, and CFP-10. This combination gave a specificity of 95%.

Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2.

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes "protective immunity" is one of the holy grails of M. tuberculosis immunology. It is known that IFN-gamma is essential for protection, but it is also apparent that IFN-gamma levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4delta2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.

PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family.

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family.

The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the esat-6 family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the Mycobacterium tuberculosis complex, including BCG, and M. kansasii, whereas other atypical mycobacteria had either one (M. avium, M. intracellulare, and M. marinum) or none (M. scrofulaceum, M. fortuitum, and M. szulgai) of the genes. The fine specificity of the T-cell response to the three closely related esat-6 family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.

Human T-cell responses to the RD1-encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis.

In recent years, there has been considerable focus on the discovery and characterization of proteins derived from Mycobacterium tuberculosis leading to the identification of a number of candidate antigens for use in vaccine development or for diagnostic purposes. Previous experiments have demonstrated an important immunological role for proteins encoded by the RD1 region, which is absent from all strains of bacillus Calmette-Guérin (BCG) but present in the genomes of virulent M. bovis and M. tuberculosis. Herein, we have studied human T-cell responses to the antigen encoded by the putative open reading frame (rv3878) of the RD1 region. Immunoblot analysis revealed that rv3878 was expressed and the native protein was designated TB27.4. Immunological evaluations demonstrate that TB27.4 elicits a prominent immune response in human tuberculosis patients with a dominant region in the C-terminal part of the molecule. In contrast, very limited responses were seen in M. bovis BCG-vaccinated donors. This study therefore emphasizes the diagnostic potential of proteins encoded by the RD1 region.