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Successful treatment of Helicobacter pylori infection is becoming compromised by emerging resistance. We report the prevalence rates of H. pylori resistance to metronidazole, clarithromycin, amoxycillin, tetracycline and trovafloxacin in The Netherlands. A total of 231 H. pylori clinical isolates were collected throughout the country over a period of 6 months during 1997-1998. The MICs of the above-mentioned antibiotics were determined in a single laboratory. The overall percentage of resistance for clarithromycin and metronidazole was 1.7% and 21.2%, respectively. None of the strains was resistant to amoxycillin or tetracycline. The primary resistance rate of trovafloxacin was as high as 4.7%. Since trovafloxacin has not yet been introduced on to the Dutch market, the resistance is probably induced by the use of other quinolones. Our data indicate that treatment outcome would benefit from susceptibility testing before starting therapy, especially when prescribing metronidazole. [ABSTRACT FROM PUBLISHER]

Purpose: Infectious complications occur following pulmonary resections preceded or not by induction chemoradiotherapy. We aimed to investigate whether bacterial colonization of the bronchial tree at the time of surgery was associated with postoperative complications., Patients and Methods: A retrospective analysis of all patients who underwent open anatomical pulmonary resections for malignancies at a single center was performed. Demographical data of the included patients, intraoperative data, and data on the postoperative course of patients were collected. Outcome of patients with a positive intraoperative bronchial culture was compared to patients with a negative bronchial culture. Relations between the presence of potential bacterial pathogens in the bronchial tree and other possible risk factors for the development of postoperative infectious and non-infectious complications, were analyzed using uni- and multivariate analysis., Results: Between January 2010 and January 2012, a total of 121 consecutive patients underwent open anatomical pulmonary resections for malignancy, of whom 45 were preceded by induction chemoradiotherapy and 5 by induction chemotherapy. Intraoperative bronchial cultures were taken from 58 patients (48 %). Patients with a positive bronchial culture developed significantly more infectious (88 % vs. 20 %, p < 0.001) and non-infectious complications (63 % vs. 12 %, p = 0.001). Positive intraoperative bronchial cultures showed the strongest association with the development of infectious and non-infectious postoperative complications (OR 24.8 and 12.2, respectively). After multivariate analysis, only BMI less than 20 kg/m(2) and the presence of a positive intraoperative bronchial culture were found to be independent risk factors for the development of infectious complications. Chemoradiotherapy was not associated with postoperative complications in the present study., Conclusions: Bacterial colonization of the bronchial tree assessed intraoperatively, appears to be associated with higher rates of infectious and non-infectious complications after pulmonary resection. Whether early starting of appropriate antibiotics based on intraoperative-taken culture findings will reduce the infectious complication rate in a subcategory of patients needs to be investigated.

Background: Invasive aspergillosis is the most common life-threatening opportunistic invasive mycosis in immunocompromised patients. A test for invasive aspergillosis should neither be too invasive nor too great a burden for the already weakened patient. The serum galactomannan enzyme-linked immunosorbent assay (ELISA) seems to have the potential to meet both requirements., Objectives: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of invasive aspergillosis., Search Methods: We searched MEDLINE, EMBASE and Web of Science with both MeSH terms and text words for both aspergillosis and the sandwich ELISA. We checked the reference lists of included studies and review articles for additional studies. We conducted the searches in February 2014., Selection Criteria: We included cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of invasive aspergillosis in patients with neutropenia or patients whose neutrophils are functionally compromised. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG)., Data Collection and Analysis: Two review authors independently assessed quality and extracted data. We carried out meta-analysis using the bivariate method. We investigated sources of heterogeneity by adding potential sources of heterogeneity to the model as covariates., Main Results: We included 54 studies in the review (50 in the meta-analyses), containing 5660 patients, of whom 586 had proven or probable invasive aspergillosis. When using an optical density index (ODI) of 0.5 as a cut-off value, the sensitivity of the test was 82% (73% to 90%) and the specificity was 81% (72% to 90%). At a cut-off value of 1.0 ODI, the sensitivity was 72% (65% to 80%) and the specificity was 88% (84% to 92%). At a cut-off value of 1.5 ODI, the sensitivity was 61% (47% to 75%) and the specificity was 93% (89% to 97%). None of the potential sources of heterogeneity had a statistically significant effect on either sensitivity or specificity., Authors' Conclusions: If we used the test at a cut-off value of 0.5 ODI in a population of 100 patients with a disease prevalence of 9% (overall median prevalence), two patients who have invasive aspergillosis would be missed (sensitivity 82%, 18% false negatives), and 17 patients would be treated unnecessarily or referred unnecessarily for further testing (specificity 81%, 19% false negatives). If we used the test at a cut-off value of 1.5 in the same population, that would mean that four invasive aspergillosis patients would be missed (sensitivity 61%, 39% false negatives), and six patients would be treated or referred for further testing unnecessarily (specificity 93%, 7% false negatives). These numbers should, however, be interpreted with caution because the results were very heterogeneous.

Background and Purpose: For prosthetic joint-associated infection (PJI), a regimen of debridement, antibiotics, irrigation, and retention of the prosthesis (DAIR) is generally accepted for acute infections. Various risk factors associated with treatment success have been described. The use of local antibiotic carriers (beads and sponges) is relatively unknown. We retrospectively analyzed risk factors in a cohort of patients from 3 hospitals, treated with DAIR for PJI., Patients and Methods: 91 patients treated with DAIR for hip or knee PJI in 3 Dutch centers between 2004 and 2009 were retrospectively evaluated. The mean follow-up was 3 years. Treatment success was defined as absence of infection after 2 years, with retention of the prosthesis and without the use of suppressive antibiotics., Results: 60 patients (66%) were free of infection at follow-up. Factors associated with treatment failure were: a history of rheumatoid arthritis, late infection (> 2 years after arthroplasty), ESR at presentation above 60 mm/h, and infection caused by coagulase-negative Staphylococcus. Symptom duration of less than 1 week was associated with treatment success. The use of gentamicin sponges was statistically significantly higher in the success group, and the use of beads was higher in the failure group in the univariate analysis, but these differences did not reach significance in the logistic regression analysis. Less surgical procedures were performed in the group treated with sponges than in the group treated with beads., Interpretation: In the presence of rheumatoid arthritis, duration of symptoms of more than 1 week, ESR above 60 mm/h, late infection (> 2 years after arthroplasty), and coagulase-negative Staphylococcus PJI, the chances of successful DAIR treatment decrease, and other treatment methods should be considered.

Background: Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus., Methods: Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles., Results: Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR46/Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR46/Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR46/Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR46/Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites., Conclusions: We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasive aspergillosis. Recovery of TR46/Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.

Objectives: Fever suggests the presence of microbial infection in critically ill patients. The aim was to compare the role of old and new biomarkers in predicting absence or presence of microbial infection, its invasiveness and severity in critically ill patients with new onset fever., Methods: We prospectively studied 101 patients in the intensive care unit with new onset fever (>38.3 °C). Routine infection parameters, lactate, procalcitonin (PCT), midregional pro-adrenomedullin (MR proADM), midregional pro-atrial natriuretic peptide (MR proANP) and copeptin (COP) were measured daily for three days after inclusion. Likelihood, invasiveness (by bloodstream infection, BSI) and severity of microbial infection were assessed by cultures, imaging techniques and clinical courses., Results: All patients had systemic inflammatory response syndrome; 45% had a probable or proven local infection and 12% a BSI, with 20 and 33% mortality in the ICU, respectively. Only peak PCT (cutoff 0.65 ng/mL at minimum) was of predictive value for all endpoints studied, i.e. BSI, septic shock and mortality (high risk infection) and infection without BSI, shock and mortality (low risk infection), at areas under the receiver operating characteristic curves varying between 0.67 (P = 0.003) and 0.72 (P < 0.001). In multivariable analysis, the combination of C-reactive protein and lactate best predicted high risk infection, followed by PCT. For low risk infection, PCT was the single best predictor., Conclusions: In critically ill patients with new onset fever, plasma PCT as a single variable, among old and new biomarkers, best helps, to some extent, to predict ICU-acquired, high risk microbial infection when peaking above 0.65 ng/mL and low risk infection when peaking below 0.65 ng/mL., (Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Arthrographis kalrae is a rare isolate in clinical specimens. Only ten cases of infection with this species have been described so far. To our knowledge, we report the first case of a pulmonary infection caused by A. kalrae in a patient with a past history of stage IIA Hodgkin's lymphoma and demonstrate that this organism can act as an opportunistic human pathogen.

The prevalence and spread of azole resistance in clinical Aspergillus fumigatus isolates in the Netherlands are currently unknown. Therefore, we performed a prospective nationwide multicenter surveillance study to determine the effects of resistance on patient management strategies and public health. From June 2007 through January 2009, all clinical Aspergillus spp. isolates were screened for itraconazole resistance. In total, 2,062 isolates from 1,385 patients were screened; the prevalence of itraconazole resistance in A. fumigatus in our patient cohort was 5.3% (range 0.8%-9.5%). Patients with a hematologic or oncologic disease were more likely to harbor an azole-resistant isolate than were other patient groups (p<0.05). Most patients (64.0%) from whom a resistant isolate was identified were azole naive, and the case-fatality rate of patients with azole-resistant invasive aspergillosis was 88.0%. Our study found that multiazole resistance in A. fumigatus is widespread in the Netherlands and is associated with a high death rate for patients with invasive aspergillosis.

The treatment of choice of H. pylori infections is a 7-day triple-therapy with a proton pump inhibitor (PPI) plus amoxicillin and either clarithromycin or metronidazole, depending on local antibiotic resistance rates. The data on efficacy of eradication therapy in a group of rheumatology patients on long-term NSAID therapy are reported here. This study was part of a nationwide, multicenter RCT that took place in 2000-2002 in the Netherlands. Patients who tested positive for H. pylori IgG antibodies were included and randomly assigned to either eradication PPI-triple therapy or placebo. After completion, follow-up at 3 months was done by endoscopy and biopsies were sent for culture and histology. In the eradication group 13% (20/152, 95% CI 9-20%) and in the placebo group 79% (123/155, 95% CI 72-85%) of the patients were H. pylori positive by histology or culture. H. pylori was successfully eradicated in 91% of the patients who were fully compliant to therapy, compared to 50% of those who were not (difference of 41%; 95% CI 18-63%). Resistance percentages found in isolates of the placebo group were: 4% to clarithromycin, 19% to metronidazole, 1% to amoxicillin and 2% to tetracycline.

A carbapenem-resistant Pseudomonas stutzeri strain isolated from a Dutch patient was analyzed in detail. This isolate produced a metallo-beta-lactamase (MBL) whose gene, with 43.5% GC content, was cloned and expressed in Escherichia coli. beta-Lactamase DIM-1 (for Dutch imipenemase) was weakly related to other Ambler class B beta-lactamases, sharing <52% amino acid identity with the most closely related MBL, GIM-1, and 45% identity with IMP-type MBLs. The beta-Lactamase DIM-1 significantly hydrolyzed broad-spectrum cephalosporins and carbapenems and spared aztreonam. This MBL gene was embedded in a class 1 integron containing two other gene cassettes, encoding resistance to aminoglycosides and disinfectants, that was located on a 70-kb plasmid.

We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL(+)) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL(+) bacteria detected in samples obtained from patients in our intensive care unit.

Background: Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mycosis in immunocompromized patients. A test for IA needs to be not too invasive and not too big a burden for the already weakened patient. The serum galactomannan ELISA seems to have potential for both requirements., Objectives: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of IA., Search Strategy: We searched MEDLINE, EMBASE and Web of Science with both Medical Headings and text words for both aspergillosis and the sandwich ELISA. We checked reference lists of included studies and review articles for additional studies., Selection Criteria: Cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of IA in patients with neutropenia or patients whose neutrophils are functionally compromised were included. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG)., Data Collection and Analysis: Two review authors independently assessed quality and extracted data, Main Results: Thirty studies were included in the meta-analyses, with a median prevalence of IA (proven or probable) of 7.7%. Seven of these (901 patients) reported results for an Optical Density Index (ODI) of 0.5 as cut-off value. The overall sensitivity in these studies was 78% (61% to 89%) and overall specificity was 81% (72% to 88%). Twelve studies (1744 patients) reported the results for cut-off value of 1.0 ODI, overall sensitivity was 75% (59% to 86%) and mean specificity 91% (84% to 95%). Seventeen studies (2600 patients) reported the results for cut-off value 1.5 ODI, sensitivity was 64% (50% to 77%) and mean specificity 95% (91% to 97%)., Authors' Conclusions: At a cut-off value 0.5 ODI in a population of 100 patients with a disease prevalence of 8% (overall median prevalence), 2 patients who have IA, will be missed (sensitivity 78%, 22% false negatives), and 17 patients will be treated or further referred unnecessarily (specificity of 81%, 19% false negatives). If we use the test at cut-off value 1.5 in the same population, that will mean that 3 IA patients will be missed (sensitivity 64%, 36% false negatives) and 5 patients will be treated or referred unnecessarily (specificity of 95%, 5% false negatives). These numbers should however be interpreted with caution, because the results were very heterogeneous.

A Salmonella enterica serotype Typhi strain was cultured from blood and fecal samples from a 54-year-old man with fever and diarrhea. He had returned from travel to the Philippines a few days earlier. Phenotypic and genotypic analysis confirmed the production of the SHV-12 extended-spectrum beta-lactamase.

During recent years, a rising incidence of invasive pulmonary aspergillosis (IPA) in non-neutropenic critically ill patients has been reported. Critically ill patients are prone to develop disturbances in immunoregulation during their stay in the ICU, which render them more vulnerable for fungal infections. Risk factors such as chronic obstructive pulmonary disease (COPD), prolonged use of steroids, advanced liver disease, chronic renal replacement therapy, near-drowning and diabetes mellitus have been described. Diagnosis of IPA may be difficult and obtaining histo- or cytopathological demonstration of the fungus in order to meet the gold standard for IPA is not always feasible in these patients. Laboratory markers used as a non-invasive diagnostic tool, such as the galactomannan antigen test (GM), 1,3-beta-glucan, and Aspergillus PCR, show varying results. Antifungal therapy might be considered in patients with persistent pulmonary infection who exhibit risk factors together with positive cultures or sequentially positive GM and Aspergillus PCR in serum, in whom voriconazole is the drug of choice. The benefit of combination antifungal therapy lacks sufficient evidence so far, but this treatment might be considered in patients with breakthrough infections or refractory disease.

Three hundred sixty Enterobacteriaceae and nonfermenting gram-negative bacilli, isolated during one week in May 2004 at five hospitals in Amsterdam, The Netherlands, were evaluated for the presence of extended-spectrum beta-lactamases (ESBLs). A prevalence of 7.8% was found, in contrast to the 1% observed in 1997. CTX-M ESBLs dominated, and four types were identified in 18 isolates.

Objective: To determine the prevalence of carriers of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and gentamicin-resistant Gram-negative bacilli (GGNB) in patients repatriated from foreign hospitals to The Netherlands., Design: Determination of prevalence., Method: In the period May 1998-August 2001, 1167 patients were repatriated. Swab specimens, demographic data and clinical data were obtained during the transfer., Results: The prevalence of carriers of resistant microorganisms was 18.2%. MRSA was carried by 2.7% of the total repatriated group and by 4.7% of patients transferred to a Dutch hospital. Risk factors were antimicrobial treatment (odds ratio (OR): 3.4; 95% CI: 1.2-9.7), length of stay in a foreign hospital > or = 14 days (OR: 5.4; 95% CI: 2.3-12) and artificial ventilation (OR: 8.5; 95% CI: 1.8-41). VRE and GGNB were isolated from 2.7% and 14.1% of patients, respectively. Transfer from Asia or southern, south-eastern and eastern Europe were risk factors for carrying GGNB., Conclusion: Carriership of resistant microorganisms was high among repatriated patients. The highest risk of GGNB was more closely associated with the country from which the patient was transferred than the antimicrobial treatment received in the foreign hospital.

In Ecuador, Helicobacter pylori infections are highly prevalent. A total of 42 H. pylori clinical isolates from 86 patients attending the outpatient clinic of the gastroenterology department of the university hospital of Guayaquil in Ecuador were characterized. Their susceptibility, and cagA and vacA status were determined. Resistance to metronidazole and clarithromycin was found in 80.9% and 9.5% of strains, respectively. Neither amoxicillin- nor tetracycline-resistant strains were found. The most prevalent genotype was the cagA(+), vacA s1b,m1 type. This genotype was associated with gastric cancer and peptic ulcer. Typing by random amplified polymorphic DNA showed no genetic relationship among the strains.

Background: Haemodialysis catheters used for vascular access are frequently complicated by infection and catheter-related thrombosis. Improvement of interdialytic locking solutions could reduce these problems. Trisodium citrate (TSC) has been advocated in recent years because it might have antimicrobial qualities., Methods: Antimicrobial efficacy of four concentrations of TSC (2.2, 7.5, 15 and 30%) was compared with three equi-osmolal sodium chloride (NaCl) concentrations, unfractionated heparin 5000 IU/ml and a solution of gentamicin 1 mg/ml in TSC 7.5%. We analysed antimicrobial properties by two classical in vitro susceptibility tests. All tests were performed in triplicate by incubation of test fluids with Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans., Results: Increasing TSC concentrations effectively killed the staphylococcal strains in both assays. For E.coli and P.aeruginosa complete killing was achieved only with TSC 30%. TSC 30% was also the only solution that significantly inhibited growth of C.albicans. Heparin manifested no antimicrobial effect of any significance. Adding gentamicin to TSC provided superior bacterial growth inhibition but had no effect on yeast growth. TSC solutions manifested superior antimicrobial activity compared with iso-osmolal NaCl solutions in both assays., Conclusion: This in vitro study demonstrates superior antimicrobial activity of TSC, especially in higher concentrations, in contrast to heparin. The mechanism seems to differ from hyperosmolality. Ca(2+) and Mg(2+) chelating effects are probably more important. Adding gentamicin provided the most potent antimicrobial solution. However, for reasons concerning development of bacterial resistance and sensitization of the patient, continuous exposition to aminoglycosides seems not advisable.

A premature infant in a neonatal intensive care unit (NICU) developed a bloodstream infection caused by coagulase-negative staphylococci (CoNS) sensitive to vancomycin. The infection persisted for 3 weeks, despite therapy with vancomycin and replacement of all intravenous catheters. The neonate died due to necrotizing enterocolitis which developed during the ongoing sepsis. We screened this strain and 216 other strains of CoNS from cultures of blood obtained from neonates between 1997 and 2000 for heteroresistance to vancomycin. Forty-eight isolates, including the strain that caused ongoing sepsis, proved heteroresistant. All isolates were identified as Staphylococcus capitis and were identical, just as their resistant stable subcolonies were, when they were genetically fingerprinted by amplified-fragment length polymorphism analysis. The heteroresistant phenotype of this endemic strain was confirmed by population analysis. We conclude that heteroresistance to vancomycin occurs in S. capitis and might be the cause of therapeutic failures in NICUs. Moreover, heteroresistant strains can become endemic in such units.

Two patients, a 34-year old man-to-woman transsexual and a 32-year-old man, with aids presented with pulmonary symptoms, fever, serious weight loss and an oral ulcer. A third patient, a 16-year-old boy, had signs of transverse myelitis and meningitis without immunodeficiency. All were South American citizens and had disseminated histoplasmosis. After antifungal treatment they recovered, although the third patient remained a wheelchair user. If pulmonary or miliary tuberculosis is suspected in a patient originating from South America, histoplasmosis should be considered. Oral ulcers and skin lesions can be diagnostic clues. Specific stainings of direct preparations and longer-lasting cultures of various materials, especially of biopsy samples, then provide the diagnosis.

We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porgammadeltaalphabeta), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations in rdxA. However, the mechanism of rdxA inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.

The prevalence of ESBL was determined among isolates of Escherichia coli (n = 571) and Klebsiella spp. (n = 196) collected during a 1-week study period in 8 university and 3 large regional laboratories all over the Netherlands. 18 isolates were positive for at least one of the screening tests used, i.e., VITEK-ESBL, E-test ESBL and MIC ratio of ceftazidime/ceftazidime-clavulanic acid, cefotaxime/cefotaxime-clavulanic acid. In 5 of these 18 putative ESBLs no betalactamase production was detectable. A TEM type was found in three E. coli and two Klebsiella spp. An SHV type was present in five Klebsiella spp. In one E. coli and one Klebsiella pneumoniae both enzymes were present. In one Klebsiella oxytoca neither of the two enzymes was present. Using PCR for both ESBL TEM and ESBL SHV, an SHV ESBL was found in one E. coli and four Klebsiella isolates. The mutations at position 238 and 240 were already described. In one E. coli isolate a TEM ESBL was found with three mutations, at position 21, 164 and 265. These mutations were already described in other ESBLs but not in this combination suggesting a new TEM ESBL. The overall prevalence of ESBL producing E. coli and Klebsiella spp. was less than 1% (6 out of 767).

A PCR-based reverse hybridization system (research prototype kit INNO-LiPA for H. pylori resistance) was developed and evaluated for simultaneous detection of 23S ribosomal DNA point mutations, associated with macrolide resistance in Helicobacter pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains) were tested by PCR-LiPA (detecting mutations A2115-->G, G2141-->A, A2142-->G, A2142-->C, A2143-->G, A2143-->C, and A2143-->T), DNA sequencing, restriction fragment length polymorphism, and/or hybridization to oligonucleotide probes. Results were highly concordant, but PCR-LiPA appears to be more sensitive for the simultaneous detection of multiple mutants.

A single point mutation in the 23S rRNA gene of Helicobacter pylori is known to confer resistance to clarithromycin. Most prevalent among clarithromycin-resistant clinical H. pylori isolates are the mutations from A-2142 to G and A-2143 to G in the 23S rRNA gene. The bias in the 23S rRNA gene mutations conferring clarithromycin resistance may result from the higher MIC, stability of resistance, and growth rate found for the strains with the above-mentioned mutations.

The intra- and interlaboratory reproducibilities of a commercial sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Aspergillus galactomannan in serum (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) were evaluated in six laboratories of university hospitals. Twenty serum samples were obtained from 12 neutropenic patients including 6 with invasive aspergillosis. These samples were blinded and sent to each center together with eight blinded ELISA-negative serum samples spiked with known concentrations of galactomannan. The centers were provided with ELISA microtiter plates from a single batch and a detailed protocol. Ten clinical samples showed ELISA reactivity, while 10 samples were ELISA negative. The mean coefficient of variation (CV) of the optical density values was 4.24% within a single assay and 25.6% between runs. The interassay CV of the ratios for the serum samples tested was 18.6%. Analysis of ordinal interpretation of the ELISA result (i.e., negative, gray zone, or positive) showed excellent reproducibility. Recalculation of the cutoff values for positive and negative samples suggested that the cutoff level recommended by the manufacturer could be lowered from 1.0 to 0.8 for negative samples and from 1.5 to 1.0 for positive samples. The intra- and interlaboratory reproducibilities were excellent when the ELISA results were interpreted as ordinal data, but considerable variation in optical density values and, to a lesser extent, in the ratios for the serum samples tested, was observed between runs. High assay variability was also found for serum samples spiked with known concentrations of galactomannan. Therefore, antigen titers in serum samples from a single patient, measured in different runs, should be compared with caution.

Seventy-three Helicobacter pylori-positive patients were treated with a combination of clarithromycin and ranitidine in order to eradicate the bacterium. Eradication was successful in 79.5%. In 15 patients eradication failed, and in 11 cases this was due to clarithromycin resistance. In one patient the infecting strain was resistant at the onset of treatment, while in the remaining 10 patients resistance developed during therapy. These isolates had also become resistant to various other antibiotics. Random amplified polymorphic DNA and restriction fragment end-labeling analysis of the isolates showed close genetic relatedness between pre- and post-treatment isolates, indicating that resistance was the result of selection of variants of the infecting strain rather then infection with an exogenous resistant strain. Nucleotide sequence comparisons revealed that all resistant isolates had a single base pair mutation in the 23S rRNA. Since this single point mutation results in co-resistance to various antibiotics at high frequencies, caution should be taken when using clarithromycin as a single antibiotic.

The susceptibility of 30 clinical isolates of Helicobacter pylori to trospectomycin, ampicillin, metronidazole, clarithromycin, azithromycin and clindamycin under varying pH conditions was evaluated. An acidic environment was shown to affect unfavourably the activity of all the antimicrobial agents tested. This pH effect was most marked for the two macrolides and for clindamycin.