Jaap T. van Dissel

Clostridium difficile infection in Europe: a hospital-based survey

BACKGROUND Little is known about the extent of Clostridium difficile infection in Europe. Our aim was to obtain a more complete overview of C difficile infection in Europe and build capacity for diagnosis and surveillance. METHODS We set up a network of 106 laboratories in 34 European countries. In November, 2008, one to six hospitals per country, relative to population size, tested stool samples of patients with suspected C difficile infection or diarrhoea that developed 3 or more days after hospital admission. A case was defined when, subsequently, toxins were identified in stool samples. Detailed clinical data and stool isolates were collected for the first ten cases per hospital. After 3 months, clinical data were followed up. FINDINGS The incidence of C difficile infection varied across hospitals (weighted mean 4·1 per 10,000 patient-days per hospital, range 0·0-36·3). Detailed information was obtained for 509 patients. For 389 of these patients, isolates were available for characterisation. 65 different PCR ribotypes were identified, of which 014/020 (61 patients [16%]), 001 (37 [9%]), and 078 (31 [8%]) were the most prevalent. The prevalence of PCR-ribotype 027 was 5%. Most patients had a previously identified risk profile of old age, comorbidity, and recent antibiotic use. At follow up, 101 (22%) of 455 patients had died, and C difficile infection played a part in 40 (40%) of deaths. After adjustment for potential confounders, an age of 65 years or older (adjusted odds ratio 3·26, 95% CI 1·08-9·78; p=0·026), and infection by PCR-ribotypes 018 (6·19, 1·28-29·81; p=0·023) and 056 (13·01; 1·14-148·26; p=0·039) were significantly associated with complicated disease outcome. INTERPRETATION PCR ribotypes other than 027 are prevalent in European hospitals. The data emphasise the importance of multicountry surveillance to detect and control C difficile infection in Europe. FUNDING European Centre for Disease Prevention and Control.

Anti-inflammatory cytokine profile and mortality in febrile patients

BACKGROUND An anti-inflammatory cytokine profile on whole-blood stimulation in vitro is associated with fatal outcome of meningococcal disease. We investigated whether an anti-inflammatory cytokine profile in the circulation is associated with adverse outcome in other infectious diseases. METHODS We enrolled 464 consecutive patients (272 men, 192 women) who presented to hospital with fever (> or = 38.2 degrees C). On admission we measured plasma interleukin 10 (IL-10) and tumour necrosis factor alpha (TNF alpha), and collected clinical and microbiological data on the febrile illness, then followed up all patients for clinical outcome. FINDINGS In at least 399 of the 464 patients fever was caused by infection. 33 patients died after a median hospital stay of 11 days (interquartile range 3-20). Concentrations of IL-10 were significantly higher in non-survivors (median 169 pg/mL [IQR 83-530]) than in survivors (median 88 pg/mL [42-235], p=0.042). When dichotomised around the median, the mortality risk was two times higher in patients who had high concentrations of IL-10 than in those with low concentrations (relative risk 2.39 [95% CI 1.07-5.33]), in patients with low and high concentrations of TNF alpha. In the 406 patients without haemodynamic deterioration in the first 24 h, IL-10 was higher and TNF alpha lower in patients who died than in those who survived. The ratio of IL-10 to TNF alpha was higher in non-survivors (median 6.9 [3.0-21.0]) than in survivors (median 3.9 [2.0-7.0], p=0.040). This ratio was highest in patients who died without underlying disease (median 21.5 [5.0-25.0]). Age, sex, and duration of fever before admission did not explain the differences in IL-10 and TNF alpha. INTERPRETATION An anti-inflammatory cytokine profile of a high ratio of IL-10 to TNF alpha is associated with fatal outcome in febrile patients with community-acquired infection. Our findings caution against a widespread use of proinflammatory cytokine inhibition in patients with sepsis.

Clinical features of dominant and recessive interferon γ receptor 1 deficiencies

BACKGROUND Interferon gamma receptor 1 (IFNgammaR1) deficiency is a primary immunodeficiency with allelic dominant and recessive mutations characterised clinically by severe infections with mycobacteria. We aimed to compare the clinical features of recessive and dominant IFNgammaR1 deficiencies. METHODS We obtained data from a large cohort of patients worldwide. We assessed these people by medical histories, records, and genetic and immunological studies. Data were abstracted onto a standard form. FINDINGS We identified 22 patients with recessive complete IFNgammaR1 deficiency and 38 with dominant partial deficiency. BCG and environmental mycobacteria were the most frequent pathogens. In recessive patients, 17 (77%) had environmental mycobacterial disease and all nine BCG-vaccinated patients had BCG disease. In dominant patients, 30 (79%) had environmental mycobacterial disease and 11 (73%) of 15 BCG-vaccinated patients had BCG disease. Compared with dominant patients, those with recessive deficiency were younger at onset of first environmental mycobacterial disease (mean 3.1 years [SD 2.5] vs 13.4 years [14.3], p=0.001), had more mycobacterial disease episodes (19 vs 8 per 100 person-years of observation, p=0.0001), had more severe mycobacterial disease (mean number of organs infected by Mycobacterium avium complex 4.1 [SD 0.8] vs 2.0 [1.1], p=0.004), had shorter mean disease-free intervals (1.6 years [SD 1.4] vs 7.2 years [7.6], p<0.0001), and lower Kaplan-Meier survival probability (p<0.0001). M avium complex osteomyelitis was more frequent in dominant than in recessive patients (22/28 [79%] vs 1/8 [13%], p=0.002), and this disorder without other organ involvement arose only in dominant patients (9/28 [32%]). Disease caused by rapidly growing mycobacteria was present in more recessive than dominant patients (7/22 [32%] vs 1/38 [3%], p=0.002). INTERPRETATION Recessive complete and dominant partial IFNgammaR1 deficiencies have related clinical phenotypes, but are distinguishable by age at onset, dissemination, and clinical course of mycobacterial diseases. A strong correlation exists between IFNGR1 genotype, cellular responsiveness to interferon gamma, and clinical disease features.

Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae

Host genetic factors are important in determining the outcome of infections caused by intracellular pathogens, including mycobacteria and salmonellae, but until now have been poorly characterized. Recently, some individuals with severe infections due to otherwise weakly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis bacille Calmette-Guérin) or Salmonella species have been shown to be unable to produce or respond to interferon-γ. This inability results from mutations in any of five genes encoding essential proteins of the type 1 cytokine cascade: interleukin-12p40, interleukin-12Rβ1, interferon-γR1, interferon-γR2 or STAT1. Ten syndromes have thus far been identified. Recent insights in genetically controlled host defense and susceptibility to mycobacterial disease are discussed.

Antibody response to influenza, tetanus and pneumococcal vaccines in HIV-seropositive individuals in relation to the number of CD4+ lymphocytes.

Objective:To establish when the formation of antibodies against T-lymphocyte-dependent and -independent antigens is impaired during HIV infection. Design:Prospective study on antibody formation before and 30 days and 60 days after vaccination with tetravalent influenza vaccine, tetanus toxoid and pneumococcal vaccine; booster with influenza vaccine was administered 30 days after initial vaccination. Setting:Outpatient clinic of University Hospital Leiden. Participants:Fifty-one HIV-infected individuals and 10 healthy controls. Results:In HIV-infected individuals with <100×106/l CD4+ lymphocytes almost no influenza antibodies were formed; CD4+ counts between 100 and 300 x 106/l correlated with suboptimal antibody formation; CD4+ counts ≤300x106/l yielded more individuals with protective antibody titres. Thirty days after vaccination, protective antibody titres against the four influenza strains had been achieved in 24% of all HIV-infected individuals for A/Beijing (H3N2) (controls, 90%), 59% for A/Taiwan (H1N1) (controls, 80%), 18% for B/Beijing (controls, 30%) and 37% for B/Panama (controls 90%). Booster vaccination after 1 month did not increase antibody levels. Anti-tetanus toxin antibody formation, which is also T-lymphocyte-dependent, was correlated with the number of CD4+ lymphocytes. After pneumococcal vaccination (T-lymphocyte-independent), normal antibody formation was observed in HIV-infected individuals, including those with low CD4+ counts. Conclusions:Influenza vaccination should not be administered to HIV-infected individuals with CD4+ counts <100×106/l; pneumococcal vaccination can be offered to all HIV-infected individuals and a tetanus toxoid booster should be administered when indicated.

The effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia. A randomized, double-blind, multicenter crossover trial.

Patients with primary hypogammaglobulinemia, such as X-linked agammaglobulinemia and common variable immunodeficiency, have recurrent infections, predominantly of the respiratory and intestinal tract (1, 2). Most respiratory infections in these patients are caused by Haemophilus influenzae and Streptococcus pneumoniae and, without proper treatment, may lead to severe pneumonia, bronchiectasis, decreased pulmonary function, and death (1, 3-6). Recurrent Giardia lamblia infections may result in chronic diarrhea, whereas chronic Campylobacter jejuni infections may cause recurrent bacteremia and cellulitis (1, 3, 4, 7, 8). In patients with X-linked agammaglobulinemia, persistent enterovirus infections, notably those caused by echovirus, are associated with chronic meningoencephalitis (3, 6). Since immunoglobulin therapy was introduced for the treatment of immunodeficiency diseases, the frequency and severity of infections have decreased (1, 9, 10). However, repeated long-term use of intramuscular immunoglobulin has serious limitations. Injections cause pain at the injection site, and their volume is limited, in turn limiting the prospective increase in IgG level (11). With subcutaneous infusion of immunoglobulin, normal IgG concentrations can be achieved. In most patients, however, local tissue reactions, such as swelling, induration, and soreness, are observed; long-term subcutaneous infusion can result in fibrosis at the injection site. These disadvantages were overcome when intravenous immunoglobulin preparations became available in the 1980s (12). Several studies have compared the clinical efficacy of various immunoglobulin products and dosage regimens in patients with primary hypogammaglobulinemia. Results have shown that intravenous immunoglobulin, when compared with subcutaneous or intramuscular administration, reduces the incidence of infections because it offers increased bioavailability and allows administration of higher doses (5, 12, 13). Furthermore, it has been suggested that when the serum IgG trough level exceeds 5 g/L, protection against bacterial infections is improved (12, 14). However, even in patients who have trough IgG levels greater than 5 g/L during replacement therapy, infections continue to be present. We performed this crossover study to determine whether doubling the commonly advised (standard) dose of intravenous immunoglobulin (300 mg/kg of body weight every 4 weeks in adults, 400 mg/kg every 4 weeks in children) decreases the incidence and duration of infections. Methods Patients Between September 1995 and February 1998, 46 patients with established humoral primary immunodeficiency who had X-linked agammaglobulinemia and common variable immunodeficiency (as defined by the World Health Organization) and an IgG trough level of 4 g/L or less at the time of diagnosis were studied in 15 hospitals in the Netherlands (15). They represented 40% to 50% of the total number of patients with X-linked agammaglobulinemia and common variable immunodeficiency in the country. Exclusion criteria were age younger than 1 year; anti-IgA antibodies; chronic active diseases, such as hepatitis, AIDS, and malignant conditions; history of anaphylactic reactions to intravenous immunoglobulin; and participation in a clinical trial 3 months before the start of the study. Thirteen of the patients in the study group (28%) had clinical and radiographic evidence of preexisting chronic bronchopulmonary disease. Before the start of the study, all patients received regular replacement therapy with intravenous immunoglobulin, with the exception of four patients who were treated with subcutaneously administered 16% immunoglobulin. The ethics committee of each participating hospital approved the study protocol. Before enrollment in the study, all patients or their legal representatives provided written informed consent. Study Design and Treatment Protocol The study was a multicenter, double-blind, randomized, crossover trial. After providing written informed consent, patients were randomly divided into two groups according to a computer-generated randomization list. During the first 9 months of the study, one group was treated with standard-dose intravenous immunoglobulin followed by a 3-month washout period, during which patients received the dose of intravenous immunoglobulin used before the study began. Patients were then treated with high-dose immunoglobulin for 9 months. In the second group, the treatment sequence was reversed (Figure 1). The time schedule was chosen to prevent seasonal variations from influencing the infection rate. Figure 1. Flow of patients through the study. To ensure that patients, nurses, and physicians remained unaware of the dosages of intravenous immunoglobulin, each hospital pharmacy provided the study product after dissolving the freeze-dried immunoglobulin in the required volume of sterile water. An equal volume of sodium chloride (NaCl 0.9%) was added to the solution of the standard dose immunoglobulin to mimic the high-dose volume. The two preparations did not differ in appearance. Intravenous immunoglobulin (Immunoglobuline I.V., Sanquin Blood Supply Foundation, Amsterdam, the Netherlands) was manufactured from the pooled plasma of at least 1000 voluntary, nonremunerated donors by using cold ethanol fractionation according to the Cohn method. At the end of the purification process, immunoglobulin was treated by using mild proteolysis at a pH of 4. After the freeze-dried product is reconstituted with water, Immunoglobuline I.V. is composed of at least 95% IgG and 0.24 mol of glucose per L as stabilizer. The distribution of IgG subclasses in the product (by mean percentage [SD]) is as follows: IgG1, 61.9% 4.8%; IgG2, 32.6% 4.8%; IgG3, 2.1% 0.4%; and IgG4, 3.3% 0.4%. The product contains only small amounts of IgA and IgM. The standard dose of intravenous immunoglobulin was 300 mg/kg every 4 weeks for adults and 400 mg/kg every 4 weeks for children (those 20 years of age). High-dose therapy was 600 mg/kg every 4 weeks for adults and 800 mg/kg every 4 weeks for children. For patients who were receiving regular treatment (one infusion every 2 to 3 weeks) before the start of the study, the dose and frequency were adjusted to ensure that a similar total quantity of intravenous immunoglobulin was received. Follow-up and Outcome Measures Before each infusion of intravenous immunoglobulin, we reviewed each patients health status using his or her diary, medical record, and an interview. We recorded previous hospital admissions (diagnosis, frequency, and duration); number, type, and duration of infections (as defined by the Infectious Diseases Society of America [16]); prophylactic and therapeutic use of antibiotics; febrile periods (temperature 38.5 C); and absence from school or work. Duration of infection was determined from the number of days with symptoms. The attending physician recorded whether infections were considered to be associated with immunodeficiency. We measured the peak expiratory flow rate at the onset of each study period, as well as 6 and 9 months thereafter, because previous studies have shown that lung function can improve in patients with hypogammaglobulinemia who receive higher doses of intravenous immunoglobulin (14, 17). During and after each infusion of intravenous immunoglobulin, vital signs (blood pressure, temperature, pulse rate) were measured and side effects were monitored. Laboratory Analysis Trough levels of IgG; levels of subclasses IgG, IgA, and IgM; levels of antibodies against a variety of microorganisms; and safety measures such as blood cell count and chemistry values (for example, kidney and liver function) were determined immediately before the intravenous immunoglobulin infusion at the onset of each study period and at 6 and 9 months. We quantified levels of immunoglobulins and IgG subclasses by nephelometry and levels of antibodies to microorganisms by specific enzyme-linked immunosorbent assays. If possible, sputum and stools for culture were obtained at the same time periods. The trough levels of both immunoglobulins and levels of antibodies to microorganisms were withheld from each patients physician during the study period. Statistical Analysis The primary end point, occurrence of infection, was used to calculate the required sample size. From the results of the study by Bernatowska and colleagues (17), we assumed that infections would develop at a rate of approximately 200 days per 1000 observation-days during standard-dose treatment, compared with 180 days per 1000 observation-days during high-dose treatment. This implied that a minimum of 30 patients was required to allow us to detect a 10% difference between the two groups (17). The final analysis was conducted on an intention-to-treat basis. All patients were included in the study, and those who had at least one efficacy result while receiving high-dose therapy as well as standard-dose therapy were analyzed. Before the analysis of efficacy, the data were investigated for a carryover effect by using the sequence of treatment (high-dose vs. standard-dose and standard-dose vs. high-dose) as one of the factors and the primary variable of efficacy (number of infections related to immunodeficiency) as the dependent variable. Thus, we created a multivariate model in which the number of infections was the dependent variable and dosage, sequence of treatment, and patient were the independent variables. We then determined whether the sequence of treatment was statistically significant. The total number of acute infections was compared by using the paired t-test. In addition, the total duration of all infections per patient and per dosing for all patients was computed. These results were compared by using the paired t-test, as were differences in time period from the start of treatment until occurrence of first infection. For the primary efficacy variables, we conducted subanalyses for both diagnoses

Detection of Active Tuberculosis Infection by T Cell Responses to Early-Secreted Antigenic Target 6-kDa Protein and Culture Filtrate Protein 10

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.

Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides.

ABSTRACT The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96,P < 0.0001 for ESAT-6; r = 0.98,P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6;r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.

Ag85B-ESAT-6 adjuvanted with IC31® promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naïve human volunteers.

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients.

Tuberculin Skin Testing and In Vitro T Cell Responses to ESAT-6 and Culture Filtrate Protein 10 after Infection with Mycobacterium marinum or M. kansasii

T cell responses to ESAT-6 and culture filtrate protein 10 (CFP-10), antigens expressed by Mycobacterium tuberculosis but not by M. bovis bacille Calmette-Guérin (BCG), were found to discriminate reliably between infection with M. tuberculosis and BCG vaccination. Because the esat-6 and cfp-10 genes occur in M. kansasii and M. marinum, T cell responses to ESAT-6 and CFP-10 were investigated in patients infected with M. kansasii or M. marinum, persons intensively exposed to environmental mycobacteria, and unexposed control subjects. Tuberculin skin tests were performed, and peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, peptide mixtures of ESAT-6 and CFP-10, and control antigens. When enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) were used to measure interferon-gamma production, most M. kansasii- or M. marinum-infected patients and several persons exposed to environmental mycobacteria were found to respond to ESAT-6 and/or CFP-10. ELISA and ELISPOT yielded comparable results, as did whole antigen and peptides (P<.0001). These results may be relevant for the development of novel assays for diagnosis of tuberculosis.