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Background: In the Netherlands, access to direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has been unrestricted for chronic infection since 2015. We evaluated whether the nationwide incidence of HCV infections in individuals with HIV has changed since 2015., Methods: In this retrospective cohort study, data from the ATHENA cohort of people with HIV aged 18 years or older attending any of the 24 HIV treatment centres in the Netherlands between 2000 and 2019 were assessed. We used parametric proportional hazards models with a piecewise exponential survival function to model HCV primary infection and reinfection incidence per 1000 person-years., Findings: Of the 23 590 individuals without previous HCV infection, 1269 cases of HCV primary infection were documented (incidence 5·2 per 1000 person-years [95% CI 5·0-5·5]). The highest incidence was observed in men who have sex with men (MSM; 7·7 per 1000 person-years [7·3-8·2]) and was lower in people who inject drugs (PWID; 1·7 per 1000 person-years [0·7-4·1]) and other key populations (1·0 per 1000 person-years [0·8-1·2]). In MSM, incidence increased in 2007 to 14·3 per 1000 person-years and fluctuated between 8·7 and 13·0 per 1000 person-years from 2008 to 2015. In 2016, incidence declined to 6·1 cases per 1000 person-years and remained steady between 4·1 and 4·9 per 1000 person-years from 2017 to 2019. Of the 1866 individuals with a previous HCV infection, 274 reinfections were documented (incidence 26·9 per 1000 person-years [95% CI 23·9-30·3]). The highest incidence rate was observed in MSM (38·5 per 1000 person-years [33·9-43·7]) and was lower in PWID (10·9 per 1000 person-years [3·5-33·8]) and other key populations (8·9 per 1000 person-years [6·3-12·5]). In MSM, reinfection incidence fluctuated between 38·0 and 88·9 per 1000 person-years from 2006 to 2015, reaching 55·6 per 1000 person-years in 2015. In 2016, reinfection incidence declined to 41·4 per 1000 person-years, followed by further decreases to 24·4 per 1000 person-years in 2017 and 11·4 per 1000 person-years in 2019., Interpretation: The sharp decline in HCV incidence in MSM with HIV shortly after restrictions on DAAs were lifted suggests a treatment-as-prevention effect. HCV incidence was already low in PWID and other groups before unrestricted access. Ongoing HCV transmission is occurring in MSM, as illustrated by a declining but high rate of reinfection, stressing the need for additional preventive measures., Funding: Dutch Ministry of Health, Welfare, and Sport., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Direct-acting antivirals effectively treat chronic hepatitis C virus (HCV) infection but there is a paucity of data on their efficacy for acute HCV, when immediate treatment could prevent onward transmission. We assessed the efficacy of grazoprevir plus elbasvir treatment in acute HCV infection and investigated whether treatment can be shortened during the acute phase of HCV infection., Methods: The Dutch Acute HCV in HIV study number 2 (DAHHS2) study was a single-arm, open-label, multicentre, phase 3b trial. Adult patients (≥18 years) with acute HCV genotype 1 or 4 infection (duration of infection 26 weeks or less, according to presumed day of infection) were recruited at 15 HIV outpatient clinics in the Netherlands and Belgium. All patients were treated with 8 weeks of grazoprevir 100 mg plus elbasvir 50 mg administered as one oral fixed drug combination tablet once daily. The primary efficacy endpoint was sustained virological response at 12 weeks after the end of treatment (SVR12; HCV RNA <15 IU/mL) in all patients who started treatment. Reinfection with a different HCV virus was not considered treatment failure in the primary analysis. This trial is registered with ClinicalTrials.gov, number NCT02600325., Findings: Between Feb 15, 2016, and March 2, 2018, we assessed 146 patients with a recently acquired HCV infection for eligibility, of whom 86 were enrolled and 80 initiated therapy, all within 6 months after infection. All patients who initiated treatment completed treatment and no patients were lost to follow-up. 79 (99%, 95% CI 93-100) of 80 patients achieved SVR12. All 14 patients who were infected with a virus carrying a clinically significant polymorphism in NS5A were cured. If reinfections were considered treatment failures, 75 (94%, 86-98) of 80 patients achieved SVR12. Two serious adverse events not considered related to the treatment were reported (traumatic rectal bleeding and low back surgery). The most common adverse event was a new sexually transmitted infection (19 [24%] of 80 patients). The most common reported possibly drug-related adverse events were fatigue (11 [14%] patients), headache (seven [9%] patients), insomnia (seven [9%] patients), mood changes (five [6%] patients), dyspepsia (five [6%] patients), concentration impairment (four [5%] patients), and dizziness (4 [5%] patients), all of which were regarded as mild by the treating physician. No adverse events led to study drug discontinuation., Interpretation: 8 weeks of grazoprevir plus elbasvir was highly effective for the treatment of acute HCV genotype 1 or 4 infection. The ability to treat acute HCV immediately after diagnosis might help physicians to reach the WHO goal of HCV elimination by 2030., Funding: Merck Sharp and Dohme and Health-Holland., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Direct-acting antivirals (DAAa) cure hepatitis C virus (HCV) infections in 95% of infected patients. Modeling studies predict that universal HCV treatment will lead to a decrease in the incidence of new infections but real-life data are lacking. The incidence of HCV among Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) has been high for >10 years. In 2015 DAAs became available to all Dutch HCV patients and resulted in a rapid treatment uptake in HIV-positive MSM. We assessed whether this uptake was followed by a decrease in the incidence of HCV infections., Methods: Two prospective studies of treatment for acute HCV infection enrolled patients in 17 Dutch HIV centers, having 76% of the total HIV-positive MSM population in care in the Netherlands. Patients were recruited in 2014 and 2016, the years before and after unrestricted DAA availability. We compared the HCV incidence in both years., Results: The incidence of acute HCV infection decreased from 93 infections during 8290 person-years of follow-up (PYFU) in 2014 (11.2/1000 PYFU; 95% confidence interval [CI], 9.1-13.7) to 49 during 8961 PYFU in 2016 (5.5/1000 PYFU; 4.1-7.2). The incidence rate ratio of 2016 compared with 2014 was 0.49 (95% CI, .35-.69). Simultaneously, a significant increase in the percentage positive syphilis (+2.2%) and gonorrhea (+2.8%) tests in HIV-positive MSM was observed at sexual health clinics across the Netherlands and contradicts a decrease in risk behavior as an alternative explanation., Conclusions: Unrestricted DAA availability in the Netherlands was followed by a 51% decrease in acute HCV infections among HIV-positive MSM.

Background: In adults with febrile urinary tract infection (fUTI), data on optimal treatment duration in patients other than non-pregnant women without comorbidities are lacking., Methods: A randomized placebo-controlled, double-blind, non-inferiority trial among 35 primary care centers and 7 emergency departments of regional hospitals in the Netherlands. Women and men aged ≥ 18 years with a diagnosis of fUTI were randomly assigned to receive antibiotic treatment for 7 or 14 days (the second week being ciprofloxacin 500 mg or placebo orally twice daily). Patients indicated to receive antimicrobial treatment for at least 14 days were excluded from randomization. The primary endpoint was the clinical cure rate through the 10- to 18-day post-treatment visit with preset subgroup analysis including sex. Secondary endpoints were bacteriologic cure rate at 10-18 days post-treatment and clinical cure at 70-84 days post-treatment., Results: Of 357 patients included, 200 were eligible for randomization; 97 patients were randomly assigned to 7 days and 103 patients to 14 days of treatment. Overall, short-term clinical cure occurred in 85 (90%) patients treated for 7 days and in 94 (95%) of those treated for 14 days (difference -4.5%; 90% CI, -10.7 to 1.7; P non-inferiority  = 0.072, non-inferiority not confirmed). In women, clinical cure was 94% and 93% in those treated for 7 and 14 days, respectively (difference 0.9; 90% CI, -6.9 to 8.7, P non-inferiority  = 0.011, non-inferiority confirmed) and, in men, this was 86% versus 98% (difference -11.2; 90% CI -20.6 to -1.8, P superiority  = 0.025, inferiority confirmed). The bacteriologic cure rate was 93% versus 97% (difference -4.3%; 90% CI, -9.7 to 1.2, P non-inferiority  = 0.041) and the long-term clinical cure rate was 92% versus 91% (difference 1.6%; 90% CI, -5.3 to 8.4; P non-inferiority  = 0.005) for 7 days versus 14 days of treatment, respectively. In the subgroups of men and women, long-term clinical cure rates met the criteria for non-inferiority, indicating there was no difference in the need for antibiotic retreatment for UTI during 70-84 days follow-up post-treatment., Conclusions: Women with fUTI can be treated successfully with antibiotics for 7 days. In men, 7 days of antibiotic treatment for fUTI is inferior to 14 days during short-term follow-up but it is non-inferior when looking at longer follow-up., Trial Registration: The study was registered at ClinicalTrials.gov [ NCT00809913 ; December 16, 2008] and trialregister.nl [ NTR1583 ; December 19, 2008].

Background & Aims: Acute hepatitis C virus infections (AHCV) are prevalent among HIV positive men having sex with men and generally treated with pegylated interferon-alpha (PegIFN) and ribavirin (RBV) during 24weeks. The addition of a protease inhibitor could shorten therapy without loss of efficacy., Methods: We performed an open-label, single arm study to investigate the efficacy and safety of a 12-week course of boceprevir, PegIFN and RBV for AHCV genotype 1 infections in 10 Dutch HIV treatment centers. The primary endpoint of the study was achievement of sustained virological response rate at week 12 (SVR12) in patients reaching a rapid viral response at week 4 (RVR4) and SVR12 in the intent to treat (ITT) entire study population was the most relevant secondary endpoint., Results: One hundred twenty-seven AHCV patients were screened in 16 months, of which 65 AHCV genotype 1 patients were included. After spontaneous clearance in six patients and withdrawal before treatment initiation in two, 57 started therapy within 26 weeks after infection. RVR4 rate was 72%. SVR12 rate was 100% in the RVR4 group. SVR12 rate in the ITT group was 86% and comparable to the SVR12 rate of 84% in 73 historical controls treated for 24 weeks with PegIFN and RBV in the same study centers., Conclusion: With the addition of boceprevir to PegIFN and RBV, treatment duration of AHCV genotype 1 can be reduced to 12 weeks without loss of efficacy. Given the high drug costs and limited availability of interferon-free regimens, boceprevir PegIFN and RBV can be a considered a valid treatment option for AHCV. ClinicalTrials.gov, number NCT01912495., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Notification of 2 imported cases of infection with Middle East respiratory syndrome coronavirus in the Netherlands triggered comprehensive monitoring of contacts. Observed low rates of virus transmission and the psychological effect of contact monitoring indicate that thoughtful assessment of close contacts is prudent and must be guided by clinical and epidemiologic risk factors.

Bacterial infections such as febrile urinary tract infection (fUTI) may run a complicated course that is difficult to foretell on clinical evaluation only. Because the conventional biomarkers erythrocyte sedimentation rate (ESR), leucocyte count, C-reactive protein (CRP) and procalcitonin (PCT) have a limited role in the prediction of a complicated course of disease, a new biomarker-plasma midregional pro-adrenomedullin (MR-proADM)-was evaluated in patients with f UTI. We conducted a prospective multicentre cohort study including consecutive patients with f UTI at 35 primary-care centres and eight emergency departments. Clinical and microbiological data were collected and plasma biomarker levels were measured at presentation to the physician. Survival was assessed after 30 days. Of 494 fUTI patients, median age was 67 (interquartile range 49-78) years, 40% were male; two-thirds of them had significant co-existing medical conditions. Median MR-proADM level was 1.42 (interquartile range 0.67-1.57) nM; significantly elevated MR-proADM levels were measured in patients with bacteraemia, those admitted to the intensive care unit, and in 30-day and 90-day non-survivors, compared with patients without these characteristics. The diagnostic accuracy for predicting 30-day mortality in fUTI, reflected by the area-under-the-curve of receiver operating characteristics were: MR-proADM 0.83 (95% CI 0.71-0.94), PCT 0.71 (95% CI 0.56-0.85); whereas CRP, ESR and leucocyte count lacked diagnostic value in this respect. This study shows that MR-proADM assessed on first contact predicts a complicated course of disease and 30-day mortality in patients with fUTI and in this respect has a higher discriminating accuracy than the currently available biomarkers ESR, CRP, PCT and leucocyte count., (© 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.)

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.

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.

Background: Radiologic evaluation of adults with febrile urinary tract infection (UTI) is frequently performed to exclude urological disorders. This study aims to develop a clinical rule predicting need for radiologic imaging., Methods: We conducted a prospective, observational study including consecutive adults with febrile UTI at 8 emergency departments (EDs) in the Netherlands. Outcomes of ultrasounds and computed tomographs of the urinary tract were classified as "urgent urological disorder" (pyonephrosis or abscess), "nonurgent urologic disorder," "normal," and "incidental nonurological findings." Urgent and nonurgent urologic disorders were classified as "clinically relevant radiologic findings." The data of 5 EDs were used as the derivation cohort, and 3 EDs served as the validation cohort., Results: Three hundred forty-six patients were included in the derivation cohort. Radiologic imaging was performed for 245 patients (71%). A prediction rule was derived, being the presence of a history of urolithiasis, a urine pH ≥7.0, and/or renal insufficiency (estimated glomerular filtration rate, ≤40 mL/min/1.73 m(3)). This rule predicts clinically relevant radiologic findings with a negative predictive value (NPV) of 93% and positive predictive value (PPV) of 24% and urgent urological disorders with an NPV of 99% and a PPV of 10%. In the validation cohort (n = 131), the NPV and PPV for clinically relevant radiologic findings were 89% and 20%, respectively; for urgent urological disorders, the values were 100% and 11%, respectively. Potential reduction of radiologic imaging by implementing the prediction rule was 40%., Conclusions: Radiologic imaging can selectively be applied in adults with febrile UTI without loss of clinically relevant information by using a simple clinical prediction rule.

Introduction: Guidelines recommend that two blood cultures be performed in patients with febrile urinary tract infection (UTI), to detect bacteremia and help diagnose urosepsis. The usefulness and cost-effectiveness of this practice have been criticized. This study aimed to evaluate clinical characteristics and the biomarker procalcitonin (PCT) as an aid in predicting bacteremia., Methods: A prospective observational multicenter cohort study included consecutive adults with febrile UTI in 35 primary care units and 8 emergency departments of 7 regional hospitals. Clinical and microbiological data were collected and PCT and time to positivity (TTP) of blood culture were measured., Results: Of 581 evaluable patients, 136 (23%) had bacteremia. The median age was 66 years (interquartile range 46 to 78 years) and 219 (38%) were male. We evaluated three different models: a clinical model including seven bed-side characteristics, the clinical model plus PCT, and a PCT only model. The diagnostic abilities of these models as reflected by area under the curve of the receiver operating characteristic were 0.71 (95% confidence interval (CI): 0.66 to 0.76), 0.79 (95% CI: 0.75 to 0.83) and 0.73 (95% CI: 0.68 to 0.77) respectively. Calculating corresponding sensitivity and specificity for the presence of bacteremia after each step of adding a significant predictor in the model yielded that the PCT > 0.25 μg/l only model had the best diagnostic performance (sensitivity 0.95; 95% CI: 0.89 to 0.98, specificity 0.50; 95% CI: 0.46 to 0.55). Using PCT as a single decision tool, this would result in 40% fewer blood cultures being taken, while still identifying 94 to 99% of patients with bacteremia.The TTP of E. coli positive blood cultures was linearly correlated with the PCT log value; the higher the PCT the shorter the TTP (R(2) = 0.278, P = 0.007)., Conclusions: PCT accurately predicts the presence of bacteremia and bacterial load in patients with febrile UTI. This may be a helpful biomarker to limit use of blood culture resources.

Background: Current guidelines on the management of urinary tract infection recommend treating febrile urinary tract infection or acute pyelonephritis with antimicrobials for at least 14 days. Few randomized trials showed the effectiveness of treatment durations of 5 to 7 days but this has only been studied in young previously healthy women., Methods/design: A randomized placebo-controlled double-blind multicenter non-inferiority trial in which 400 patients with community acquired febrile urinary tract infection will be randomly allocated to a short treatment arm (7 days of ciprofloxacin or 7 days of empirical beta-lactams +/- gentamicin intravenously with early switch to oral ciprofloxacin followed by 7 days of blinded placebo) or standard treatment arm (7 days of ciprofloxacin or 7 days of empirical beta-lactams +/- gentamicin intravenously with early switch to oral ciprofloxacin followed by 7 days of blinded ciprofloxacin). The study is performed in the Leiden region in which one university hospital, 6 general hospitals and 32 primary health care centers are clustered. Patients eligible for randomization are competent patients aged 18 years or above with a presumptive diagnosis of acute pyelonephritis as defined by the combination of fever, one or more symptoms of urinary tract infection and a positive urine nitrate test and/or the presence of leucocyturia. Exclusion criteria are known allergy to fluoroquinolones, female patients who are pregnant or lactating, polycystic kidney disease, permanent renal replacement therapy, kidney transplantation, isolation of ciprofloxacin-resistant causal uropathogen, renal abscess, underlying chronic bacterial prostatitis, metastatic infectious foci and inability to obtain follow-up. The primary endpoint is the clinical cure rate through the 10- to 18-day post-treatment visit. Secondary endpoints are the microbiological cure rate 10- to 18-day post-treatment, the 30- and 90-day overall mortality rate, the clinical cure rate 70- to 84-day post-treatment, relapse rate of UTI and adverse events or complications during 90 days of follow-up., Discussion: This study aims to demonstrate that 7 days of antimicrobial treatment is non-inferior as compared with 14 days of treatment in patients with febrile urinary tract infection. In addition, it will generate insights into the side-effects of antimicrobial treatment in relation to its duration. The study population will also include men, the elderly and patients with significant co-morbidity. Reflecting daily practice of primary health care and emergency departments, the results of this study can be generalized to other locations.

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.

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 (kappa = 0.71) and between TST and LST (kappa = 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.

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/K(b) and HLA-DR3.Ab(0) 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.

Recently, interferon-gamma 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-gamma 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.

Background: The tuberculin skin test (TST) has low specificity. QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB are based on interferon (IFN)-gamma responses to Mycobacterium tuberculosis-specific antigens. A novel in-tube format of QFT-G (QFT-GIT) offers logistical advantages., Objective: To compare TST, QFT-GIT, and T-SPOT.TB in bacillus Calmette-Guérin unvaccinated contacts and correlate results with measures of recent exposure., Methods: When a supermarket employee with smear-positive tuberculosis had infected most close contacts, a contact investigation among more than 20,000 customers was performed. We recruited subjects randomly on the day of TST administration (n = 469) and subjects with TST of more than 0 mm on the day of TST reading (n = 316). QFT-GIT and T-SPOT.TB were performed. Demographic data and measures of exposure were collected. TST results were analyzed at a cutoff of 10 or 15 mm. Blood tests were interpreted following the manufacturers' criteria and by varying cutoff levels., Results: Among 785 study participants, TST results were associated with age, whereas positive IFN-gamma responses were significantly associated with cumulative shopping time, most markedly for QFT-GIT. Among participants with a TST of 15 mm or greater, sensitivity of QFT-GIT and T-SPOT.TB was 42.2 and 51.3%, respectively. Interassay agreement was 89.6% (kappa = 0.59). By varying cutoff values, agreement between the IFN-gamma assays was optimal at 93.6% (kappa = 0.71) using a cutoff of 0.20 IU/ml for QFT-GIT and 13 spots for T-SPOT.TB., Conclusions: Blood test results were associated with exposure, whereas the TST was not. A possible lack of sensitivity of IFN-gamma assays in detecting individuals with TST of 15 mm or greater, despite negative bacillus Calmette-Guérin vaccination status, warrants further investigation into alternative cutoff values.

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.

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.

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 alpha-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Enterotoxigenic Escherichia coli (ETEC), which produces heat labile toxin (LT) and/or heat stable toxin (ST), is considered to be the most common known cause of travellers' diarrhoea (TD). Owing to the antigenic similarity between cholera toxin and LT, immunization with inactivated oral B-subunit/whole-cell cholera vaccine (BS-WC) offers short term (3 months) but significant (>67%) protection against TD caused by LT-related ETEC. Since it expresses the cholera toxin B (CTB) subunit, the live attenuated oral cholera vaccine strain CVD 103-HgR, may induce similar protection. A trial was performed to determine if CVD 103-HgR live oral cholera vaccine would provide a protective efficacy of at least 50% against TD. In addition, the protective efficacy of the vaccine against TD specifically due to LT-ETEC and LT/ST-ETEC was determined. Volunteers (n=134) travelling to Indonesia, India, Thailand or West-Africa were randomised to receive either a placebo (n=65) or the vaccine (n=69). In the placebo group, 46% reported an episode of diarrhoea, compared to 52% in the vaccine group. No significant group differences were found with regard to incidence, duration or severity of all caused TD or ETEC-associated TD. However, ETEC-associated TD occurred earlier in the placebo group (median 5 days), compared to the vaccine group (median 15 days). In conclusion, CVD 103-HgR live oral cholera vaccine failed to provide a 50% protection against TD. This study does not exclude that the vaccine may offer a short-lived protection against ETEC-associated TD. However, the power of the study was limited by the unexpected low incidence of LT-ETEC-associated diarrhoea (9% of all TD) compared to ST-associated TD (24% of all TD).

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%.

A cross-sectional study of reverse smoking and its association with pre-malignant and malignant lesions of the palate was conducted in the north coastal areas of Andhra Pradesh, India. A total of 480 randomly selected persons were interviewed. Information about smoking status, diet and access to mass media was obtained in each case and an examination of the oral cavity was performed. Reverse smoking of chutta was practised by 33% of the total rural population. The prevalence rate of all palatal lesions was 55%. The prevalence rates of the separate lesions: leukoplakia palatii, palatal keratosis and palatal cancer, were 9.8%, 18.1% and 1.9%, respectively. The presence of these (pre-)malignant lesions was strongly associated with reverse smoking and also associated with conventional chutta smoking. Reverse smoking induced significantly more lesions than conventional chutta smoking, and was a major determinant of subsequent palatal cancer: all 9 newly diagnosed palatal cancers were observed within the group of reverse smokers. There was an inverse relationship between the incidence of palatal lesions and vitamin A intake. The study of access to mass media indicated that the most favourable medium for promoting a prevention campaign would be the cinema.