Eliane M. S. Leyten

Recognition of Stage-Specific Mycobacterial Antigens Differentiates between Acute and Latent Infections with Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or α-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Discrepancy between Mycobacterium tuberculosis-Specific Gamma Interferon Release Assays Using Short and Prolonged In Vitro Incubation

ABSTRACT The sensitivities of various gamma interferon release assays (IGRAs) for the detection of past latent Mycobacterium tuberculosis infection are not known. In this study, we aimed to assess the effects of various IGRA formats and in vitro incubation periods on test outcome. The results of the tuberculin skin test (TST) were compared with those of the QuantiFERON-TB Gold in-tube (QFT-GIT) test, an overnight enzyme-linked immunospot assay (ELISPOT), and a 6-day lymphocyte stimulation test (LST) by using the same M. tuberculosis-specific peptides and samples from 27 TST-positive persons with a history of exposure to M. tuberculosis, 4 patients cured of tuberculosis (TB), and 9 TST-negative controls. Among the TST-positive persons, the LST was more frequently positive (92%; P < 0.01) than either the QFT-GIT test (33%) or ELISPOT (46%). While good agreement was observed between the QFT-GIT test and ELISPOT (κ = 0.71) and between TST and LST (κ = 0.78), the agreement between TST or LST, on the one hand, and the QFT-GIT test or ELISPOT, on the other, was poor. These data indicate that the QFT-GIT test and overnight ELISPOT are less sensitive for the detection of past latent TB than the 6-day LST. The observed discrepancies between these IGRAs are most likely related to differences in incubation periods. Whether TST-positive persons with positive LST results but negative QFT-GIT and ELISPOT results are at risk for the development of TB needs to be elucidated before short-incubation IGRAs can be used for the screening of individuals for latent TB before immunosuppressive treatment.

T-Cell Recognition of the HspX Protein of Mycobacterium tuberculosis Correlates with Latent M. tuberculosis Infection but Not with M. bovis BCG Vaccination

ABSTRACT During stationary growth or in vitro conditions mimicking relevant aspects of latency, the HspX protein (Rv2031c) is specifically upregulated by Mycobacterium tuberculosis. In this study we compared T-cell responses against HspX and the secreted M. tuberculosis protein Ag85B (Rv1886c) in tuberculosis (TB) patients, tuberculin skin test-positive individuals, M. bovis BCG-vaccinated individuals, and healthy negative controls. Gamma interferon responses to HspX were significantly higher in M. tuberculosis-exposed individuals than in M. tuberculosis-unexposed BCG vaccinees. In contrast, no such differences were found with respect to T-cell responses against Ag85B. Therefore, BCG-based vaccines containing relevant fragments of HspX may induce improved responses against this TB latency antigen. To identify relevant major histocompatibility complex class I- and class II-restricted HspX-specific T-cell epitopes, we immunized HLA-A2/Kb and HLA-DR3.Ab0 transgenic (tg) mice with HspX. Two new T-cell epitopes were identified, p91-105 and p31-50, restricted via HLA-A*0201 and HLA-DRB1*0301, respectively. These epitopes were recognized by human T cells as well, underlining the relevance of HspX T-cell recognition both in vivo and in vitro. In line with the data in humans, BCG immunization of both tg strains did not lead to T-cell responses against HspX-derived epitopes, whereas nonlatency antigens were efficiently recognized. These data support the notion that BCG vaccination per se does not induce T-cell responses against the latency antigen, HspX. Thus, we suggest that subunit vaccines incorporating HspX and/or other latency antigens, as well as recombinant BCG strains expressing latency antigens need to be considered as new vaccines against TB.

Risk factors for fluoroquinolone-resistant Escherichia coli in adults with community-onset febrile urinary tract infection

OBJECTIVES To assess risk factors for fluoroquinolone resistance in community-onset febrile Escherichia coli urinary tract infection (UTI). METHODS A nested case-control study within a cohort of consecutive adults with febrile UTI presenting at primary healthcare centres or emergency departments during January 2004 through December 2009. Resistance was defined using EUCAST criteria (ciprofloxacin MIC >1.0 mg/L). Cases were subjects with fluoroquinolone-resistant E. coli, and controls those with fluoroquinolone-susceptible isolates. Multivariable logistic regression analysis was used to identify potential risk factors for fluoroquinolone resistance. RESULTS Of 787 consecutive patients, 420 had E. coli-positive urine cultures. Of these, 51 (12%) were fluoroquinolone resistant. Independent risk factors for fluoroquinolone resistance were urinary catheter [odds ratio (OR) 3.1; 95% confidence interval (CI) 0.9-11.6], recent hospitalization (OR 2.0; 95% CI 1.0-4.3) and fluoroquinolone use in the past 6 months (OR 17.5; 95% CI 6.0-50.7). Environmental factors (e.g. contact with animals or hospitalized household members) were not associated with fluoroquinolone resistance. Of fluoroquinolone-resistant strains, 33% were resistant to amoxicillin/clavulanate and 65% to trimethoprim/sulfamethoxazole; 14% were extended-spectrum β-lactamase (ESBL) positive compared with <1% of fluoroquinolone-susceptible isolates. CONCLUSIONS Recent hospitalization, urinary catheter and fluoroquinolone use in the past 6 months were independent risk factors for fluoroquinolone resistance in community-onset febrile E. coli UTI. Contact with animals or hospitalized household members was not associated with fluoroquinolone resistance. Fluoroquinolone resistance may be a marker of broader resistance, including ESBL positivity.

Effect of tuberculin skin testing on a Mycobacterium tuberculosis-specific interferon-γ assay

Recently, interferon-γ release assays (IGRA) for specific diagnosis of Mycobacterium tuberculosis infection have become available. In recent UK tuberculosis (TB) guidelines, it has been advised to screen for latent M. tuberculosis infection using the tuberculin skin test (TST), followed by IGRA if the TST is positive. Since TST can boost immune responses to tuberculin, the present authors evaluated whether TST administration affects the result of QuantiFERON®-TB Gold in-tube (QFT-GIT), a whole blood-based IGRA. QFT-GIT was performed on the day of TST administration and the day of reading in 15 TST-negative subjects, 46 TST-positive subjects with recent or remote exposure to M. tuberculosis and five cured TB patients. No systematic boosting of QFT-GIT responses from negative to positive was observed. Only in a few TST-positive persons did TST enhance pre-existing QFT-GIT responses. Screening for latent Mycobacterium tuberculosis infection using tuberculin skin testing followed by interferon-γ release assays on the day of reading is a reliable approach, as the specificity of QuantiFERON®-TB Gold in-tube is not affected by prior tuberculin skin test administration.

A Patient with De Novo Tuberculosis during Anti-Tumor Necrosis Factor—α Therapy Illustrating Diagnostic Pitfalls and Paradoxical Response to Treatment

In 2005, a 24-year-old man with Crohn disease who had been treated with infliximab for several months was exposed to an individual with smear-positive tuberculosis. Soon after exposure, he complained of malaise, dry cough, and weight loss. Despite normal chest radiograph findings and negative tuberculin skin test results, tuberculosis was considered to be the most likely diagnosis. The results of a whole-blood assay for detection of interferon- gamma production in response to Mycobacterium tuberculosis-specific antigen were positive. Acid-fast staining and polymerase chain reaction of bronchoalveolar lavage fluid samples had negative results, but M. tuberculosis was cultured. After the initiation of 4 antitubercular drugs and the discontinuation of infliximab therapy, the patient developed an immune reconstitution syndrome accompanied by enlarged mediastinal lymph nodes and multiple intrapulmonary miliary lesions. This case of de novo tuberculosis during anti-tumor necrosis factor alpha treatment illustrates the uncharacteristic presentation of the disease and the elusiveness of the diagnosis, as well as the fact that discontinuation of anti-tumor necrosis factor alpha treatment can be accompanied by an immune reconstitution syndrome similar to that observed in human immunodeficiency virus-infected individuals with tuberculosis.

Middle east respiratory syndrome coronavirus (MERS-CoV) infections in two returning travellers in the Netherlands, May 2014

Two patients, returning to the Netherlands from pilgrimage in Medina and Mecca, Kingdom of Saudi Arabia, were diagnosed with Middle East respiratory syndrome coronavirus (MERS-CoV) infection in May 2014. The source and mode of transmission have not yet been determined. Hospital-acquired infection and community-acquired infection are both possible.

ESAT-6 and CFP-10 in Clinical versus Environmental Isolates of Mycobacterium kansasii

Mycobacterium kansasii consists of 5 genetically distinct groups, of which 2 are associated with human disease. Determinants of the differences in virulence are unknown. Potential genes of interest are esat-6 and cfp-10, which are associated with virulence of Mycobacterium tuberculosis and Mycobacterium bovis but are lacking in bacille Calmette-Guérin and in most environmental mycobacteria (M. kansasii is an exception). We investigated esat-6 and cfp-10 genes in 22 clinical and 14 environmental isolates of M. kansasii. Both were present in all isolates; each genetic group had its own characteristic Southern-blot pattern corresponding to a highly conserved fingerprint pattern. Nucleotide sequences of the genes differed 12.6% and 10.1%, respectively, from the M. tuberculosis homologues, but the deduced amino acid sequences were <5% different. In vitro, clinical and environmental genotypes of M. kansasii expressed CFP-10 and ESAT-6. Thus, virulence of M. kansasii is not directly related to esat-6 and cfp-10 genes or gene expression.

A Randomized Controlled Study of Accelerated Versus Standard Hepatitis B Vaccination in HIV-Positive Patients

BACKGROUND In human immunodeficiency virus (HIV)-infected patients, the immunogenicity of hepatitis B vaccines is impaired. The primary and secondary aims of our study were to investigate the effectiveness and compliance of 2 different vaccination regimen in an HIV-infected population. METHODS A noninferiority trial with a 10% response margin was designed. Included were patients ≥ 18 years old, with negative HBsAg/anti-HBc serology, and not previously vaccinated against hepatitis B. Patients were stratified according to CD4(+) cell count: <200, 200-500, >500. Participants received 10 μg HBvaxPRO intramuscularly according to a 0-1-3 week schedule or the standard 0-4-24 week schedule. Anti-HBs levels were measured at week 28, considered protective ≥ 10 IU/L. RESULTS Modified intention to treat analysis in 761 patients was performed. Overall response difference was 50%(standard arm) versus 38.7% (accelerated arm) =11.3% (95% confidence interval [CI], [4.3, 18.3]), close to the 10% response margin. In CD4(+) cell count group 200-500 cells/mm(3,) the response difference was 20.8% (95% CI [10.9, 30.7]). However, the response difference in CD4(+)cell count group >500 cells/mm(3) was -1.8% (95% CI [-13.4,+9.7]). Compliance was significantly superior with the accelerated schedule, 91.8% versus 82.7% (P ≤ .001). CONCLUSION In HIV-infected patients, compliance with an accelerated hepatitis B vaccination schedule is significantly better. The efficacy of an accelerated schedule proved to be non-inferior in CD4(+) cell count group >500 cells/mm(3). CLINICAL TRIALS REGISTRATION CT00230061.

Use of Enzyme-Linked Immunospot Assay with Mycobacterium tuberculosis- Specific Peptides for Diagnosis of Recent Infection with M. tuberculosis after Accidental Laboratory Exposure

ABSTRACT This report of an accidental exposure to Mycobacterium tuberculosis in a microbiological laboratory illustrates the value of gamma interferon enzyme-linked immunospot assay using peptides of ESAT-6, CFP-10, TB37.6, and TB7.7 for the diagnosis of latent infection. In particular, positive responses to peptides 2 to 6 of TB37.6 were observed exclusively in recently infected persons.