PCR-RFLP typing of ureC from Helicobacter pylori isolated from gastric biopsies during a European multi-country clinical trial.

A multi-country clinical trial was conducted in ten European countries to determine the efficacy of clarithromycin-omeprazole dual therapy for treating Helicobacter pylori infection in peptic ulcers. Gastric biopsies were cultured for H. pylori before and after treatment. PCR-RFLP was used to determine the genetic heterogeneity of 100 H. pylori isolates from pretreatment and posttreatment biopsies. An 820 bp amplified fragment of the ureC gene was digested with the restriction enzymes Sau3A and Hhal. Fourteen different Sau3A patterns and 15 different Hhal patterns were identified among the pretreatment isolates. In combination, 42 different RFLP types were identified. Comparison of isolates before treatment with those after treatment showed that five of ten patients on clarithromycin-omeprazole dual therapy had the same RFLP type and that all 12 patients on omeprazole therapy alone had the same RFLP type. All isolates were susceptible to clarithromycin prior to treatment, while seven of ten patients on clarithromycin-omeprazole therapy had H. pylori that was resistant to clarithromycin after therapy and 11 of 12 patients on omeprazole therapy had isolates susceptible to clarithromycin after treatment. In addition to PCR-RFLP typing, the presence of the cytotoxin-associated gene (cagA) and the vacuolating gene (vacA) was determined; 79% of the isolates were cagA-positive and all were vacA-positive. The results of this study indicate that infection of H. pylori in Europe is not restricted to a few RFLP types.

A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori.

We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was > or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was < or = 32 micrograms per ml.

The value of serum IgG titres against Pseudomonas aeruginosa in the management of early pseudomonal infection in cystic fibrosis.

We report the results of a clinical trial. Patients enrolled had serum IgG titres against Pseudomonas aeruginosa above the control range. Assignment to the observation or treatment group was by minimisation. Significant signs or symptoms in any patient prompted antipseudomonal treatment. In addition, the treatment group received antipseudomonal treatment at intervals of four months until the serum IgG titre returned to the control range. P aeruginosa was isolated intermittently from patients in the main trial. Nineteen patients were enrolled (12 observation, seven treatment). After one year in the trial changes in parameters studied, including forced expiratory volume in one second, IgG titre, serum IgG concentrations, and frequency of P aeruginosa isolation had improved in the treated group and worsened in the observation group.

Aztreonam therapy in children with febrile neutropenia: a randomized trial of aztreonam plus flucloxacillin versus piperacillin plus gentamicin.

In a prospective, randomized trial in 100 febrile neutropenic children, aztreonam plus flucloxacillin was compared with piperacillin plus gentamicin. At the 72 h clinical assessment there was no statistically significant difference between the two groups. However, in microbiologically documented infections there was a higher response rate in the piperacillin/gentamicin group (57%) than in the aztreonam/flucoxacillin group (41%). This was contributed to by the poorer Gram-positive cover of the aztreonam/flucloxacillin combination. In clinically documented infections and unexplained fevers the response rate of the two antibiotic regimens was identical. There were two deaths; one early death (in the piperacillin/gentamicin arm) and one late death. At the final assessment a successful outcome was obtained in the remaining patients. In the aztreonam/flucloxacillin group 75% of the episodes required modification compared with 59% in the piperacillin/gentamicin group.

Pseudomonas cepacia: a new pathogen in patients with cystic fibrosis referred to a large centre in the United Kingdom.

Pseudomonas cepacia infection has become increasingly common among patients with cystic fibrosis in North America. In a large cystic fibrosis centre in the United Kingdom 11 cases have been identified during the last six years, with a maximum prevalence of 7% in 1988. Three patients have died, two of whom deteriorated rapidly shortly after acquisition of the organism despite intensive treatment with appropriate antibiotics. Analysis of possible causes of the increase in P cepacia infection suggested that neither patient to patient transmission nor the use of nebulised antibiotics was associated with an increased risk of infection.

Serum IgA antibodies against Pseudomonas aeruginosa in cystic fibrosis.

Serum IgA antibodies to Pseudomonas aeruginosa cell surface antigens were estimated by ELISA. Titres in patients with and without cystic fibrosis and with no pseudomonal infection were low (less than 105 to less than 261). Titres in patients with cystic fibrosis who were chronically infected with P aeruginosa were very high (1200-163,000), and patients who grew the organism intermittently had intermediate titres. Longitudinal studies suggested increasing tissue invasion or involvement of the lower respiratory tract, or both, with increasing time of infection and identified patients with a good prognosis after the onset of pseudomonal infection. Detection of an increased serum IgA titre can give an earlier indication than measurement of the serum IgG titre of the presence of P aeruginosa in the respiratory tract in a proportion of patients. IgA measurement seems to be better than IgG measurement at predicting the reappearance of P aeruginosa after apparent eradication of early infection. These results suggest that this assay may be a valuable additional indicator of the presence of P aeruginosa at the beginning of infection, and of the reappearance of the organism after treatment in the early stages of infection.

An outbreak of acinetobacter septicaemia in a neonatal intensive care unit.

We describe an outbreak of Gram-negative septicaemia due to a rare, non-fermenting, aerobic organism, Acinetobacter calcoaceticus var. lwoffi. The outbreak occurred on a neonatal unit and was confined to babies who were receiving parenteral nutrition. Seven babies developed septicaemia within 24 hours. The source of the outbreak was never firmly established, but contamination of the parenteral nutrition fluid was considered most likely. All 7 babies recovered uneventfully after a week's course of intravenous ceftazidime. Thrombocytopenia was an unexpected feature of this infection.

An ELISA to detect antipseudomonal IgA antibodies in sera of patients with cystic fibrosis.

An enzyme linked immunosorbent assay (ELISA) to measure free serum IgA antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis is described. Results were reproducible and there was no interference from crossreacting antibodies directed against other Gram negative bacteria. Titres were high in patients with Pseudomonas aeruginosa infection and differed according to the stage of the infection. These preliminary results suggest that this assay may be of value in assessing the state of Pseudomonas aeruginosa infection in patients with cystic fibrosis.

Prediction and diagnosis of early Pseudomonas aeruginosa infection in cystic fibrosis: a follow-up study.

Immunoglobulin G (IgG) antibodies to Pseudomonas aeruginosa surface antigens in serum were estimated by enzyme-linked immunosorbent assay for all patients from whom P. aeruginosa was isolated for the first time during a study period of 3 years (33 patients). The titer of IgG antibodies was greater than control values at or up to 24 months before the first isolation of P. aeruginosa in 24 patients. Another five patients had titers that were within the control range before isolation of P. aeruginosa but increased to above the control range within the following 2 months. In these 29 patients, continuing intermittent isolations of P. aeruginosa were accompanied by further increases in titer. The presence of a systemic immune response above the control range indicates tissue invasion and hence infection. Four patients were deemed to have no infection: one or two isolations of P. aeruginosa were accompanied by no increase in specific antibodies to above the control range throughout the entire study period. Fifteen patients received intravenous antipseudomonal chemotherapy. Eradication of the organism and a return of titer to control values, suggesting complete removal of the organisms, occurred in 5 patients, while continued isolations and only a partial decrease in titer occurred in 10 patients. The 15 patients who received treatment improved clinically, in contrast to untreated patients, whose clinical state worsened during the study period. Continuous steroid treatment, given to two patients, was accompanied by a dramatic decrease in both serum IgG concentration and titer, despite continuing intermittent isolations of P. aeruginosa. These results confirm and extend our earlier finding that this assay appears to detect P. aeruginosa infection at a very early stage and helps in differentiating between early infection and harmless colonization. It also appears to be a useful monitor of the progress of infection and the response to intravenous antibiotic treatment in these early stages of infection, before any clinical changes are sufficiently large to be detected, in patients who were not on continuous steroid therapy. The effect of steroid treatment on the immunological response and clinical outcome of patients with early P. aeruginosa infection requires further investigation.

Serum IgG antibodies in patients with cystic fibrosis with early Pseudomonas aeruginosa infection.

Serum IgG antibodies to Pseudomonas aeruginosa surface antigens were measured by enzyme linked immunosorbent assay in all patients with cystic fibrosis from whom P. aeruginosa was isolated for the first time during a study period of 18 months. In 15 patients the titre of serum IgG antibodies was greater than control values before or at the time of the first bacteriological isolation of P. aeruginosa. The presence of serum antibodies specific to P. aeruginosa suggests exposure to infection by that organism for some months before its isolation in significant numbers from the respiratory tract. In the other two patients serum titres were within the control range before isolation of P. aeruginosa but had increased to above the control range within the next month. Longitudinal studies on the entire group of patients showed further increases in titre concurrently with further isolations of P. aeruginosa. These results suggest that this assay may be an indicator of the beginning of pulmonary infection by P. aeruginosa and may prove to be a sensitive monitor of the progress of infection, and response to treatment, during the first months of infection by that organism.

Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis.

Serum IgG antibodies to Pseudomonas aeruginosa cell surface antigens were determined by enzyme linked immunosorbent assay. Titres in patients without cystic fibrosis were low (140-235). Those in patients with cystic fibrosis who were chronically infected by P. aeruginosa were very high (1100-20,500), while patients who grew the organism intermittently had lower titres (160-4400). Longitudinal studies showed that raised titres were observed at a very early stage of infection. High titres were associated with a poor clinical state, while low titres were associated with a better clinical state in both chronic and intermittently infected patients with cystic fibrosis. These results suggest that this test is a specific and sensitive measure of the severity and progress of the different stages of pulmonary infection by P. aeruginosa in patients with cystic fibrosis.

Development of enzyme linked immunosorbent assay (ELISA) to detect antibodies to Pseudomonas aeruginosa cell surface antigens in sera of patients with cystic fibrosis.

An enzyme linked immunosorbent assay (ELISA) to measure free serum IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis was developed. Seven strains of P aeruginosa cells, treated with glutaraldehyde and representing the most commonly isolated serotypes in our cystic fibrosis unit, were used. The specificity of the test was confirmed by the absence of cross reacting antibodies to other Gram negative bacteria. The results showed differences in the titres of antibodies at different stages of P aeruginosa infection. Because of its reproducibility, specificity, and sensitivity these preliminary results suggest that this test may be of value in monitoring the progress of P aeruginosa infection in patients with cystic fibrosis.

Defective neutrophil function and microbicidal mechanisms in the myelodysplastic disorders.

Neutrophil function studies have been carried out in a series of 44 patients with primary myelodysplastic syndromes (MDS). In vitro tests of phagocytosis and killing of Candida guilliermondii and Staphylococcus aureus identified 13 patients with abnormal neutrophil function at presentation and a further 10 who developed abnormalities during the course of their disease. The incidence of defective function in the five disease categories in this series was: refractory cytopenia (RC) 8/17; refractory cytopenia with sideroblastic change (RC + SC) 5/8; acquired idiopathic sideroblastic anaemia (AISA) 2/4; refractory anaemia with excess blasts (RAEB) 7/11; chronic myelomonocytic leukaemia (CMML) 1/4. Eleven of 23 patients with defective neutrophil function experienced severe infective complications; in only three of these patients were neutrophil counts less than 1 X 10(9)/l and susceptibility to infection was considered to reflect, at least partially, qualitative neutrophil abnormalities. There was no correlation between absolute neutrophil count and defective function. Abnormal overall neutrophil microbicidal activity was equally associated with impaired and normal phagocytosis. Some patients with intracellular killing defects had reduced myeloperoxidase (MPO) activities and one had reduced hexose monophosphate shunt (HMPS) activity. In two patients, whose neutrophils showed markedly impaired candidacidal activity, levamisole corrected function when added in vitro at 10(-7) M and also when administered in therapeutic dosage. It is suggested that deranged function, probably reflecting abnormalities in maturation of the granulocyte series, occurs across the myelodysplastic spectrum and that several microbicidal mechanisms may be defective.

Comparison of the effects of filtration leucapheresis and discontinuous flow centrifugation leucapheresis on granulocyte microbicidal function.

In an investigation of the in vitro phagocytic and microbicidal function of granulocytes collected by filtration leucapheresis (FL) from 18 donors and by discontinuous flow centrifugation leucapheresis (DFC) from six donors, comparison was made with the function of granulocytes obtained from the same donors by venepuncture and density gradient centrifugation over Ficoll-Isopaque (FI). No significant impairment of the phagocytosis or killing of Candida guilliermondii by either FL- or DFC-granulocytes was observed. Although the ability of FL-granulocytes to phagocytose and kill Staphylococcus aureus did not differ significantly from the function of control FI-granulocytes, DFC-granulocytes were significantly less active.

Establishment of minimum inhibitory concentrations of cefoperazone for control and reference anaerobic organisms.

The minimum inhibitory concentrations of cefoperazone were determined in a collaborative study for eight National Committee for Clinical Laboratory Standards control and reference strains of anaerobic bacteria by agar and microdilution techniques with several types and sources of media. Recommended minimum inhibitory concentrations for the control strains, Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741, are 32 to 64 micrograms/ml and 64 micrograms/ml, respectively. Clostridium perfringens ATCC 13124 gave inconsistent results, and no value is recommended. Recommended values for reference strains are presented. Modification of media did not significantly change the minimum inhibitory concentrations.

Antimicrobial susceptibilities of bacteria associated with periodontal disease.

A total of 193 bacterial strains were tested for their susceptibilities to 14 antimicrobial agents. Penicillin G was active at 2 U/ml against 98% of the oral isolates. Other antibiotics with good activity were cefoperazone, moxalactam, Sch 29,482, and clindamycin. Metronidazole was active against more than 90% of the anaerobic bacteria and Capnocytophaga but was inactive against most other microaerophilic and facultative strains.

Serial C-reactive protein measurements in infective complications following cardiac operation: evaluation and use in monitoring response to therapy.

Serum C-reactive protein (CRP) was studied serially in 100 patients who underwent cardiac operation and in another 17 patients in whom serious infections including prosthetic valve endocarditis developed in the early postoperative period. Eleven patients with late onset of prosthetic valve endocarditis and infective endocarditis were also investigated. The assay method used was radial immunodiffusion. Patients without postoperative infective complications showed a rapid increase in CRP levels, which reached a peak within 72 hours after operation followed by a progressive decline. The differences between the CRP levels in infected and uninfected patients were significant (p less than 0.01). Serial measurements were of prognostic value in evaluating the response to chemotherapy and in predicting the outcome of the disease.

Assessment of chemoprophylaxis in open-heart surgery patients.

Regular assay of antibiotics in the sera of patients may be helpful in open-heart surgery patients, neurosurgery patients, immunosuppressed individuals, patients on antineoplastic therapy, or other high-risk patient groups. It may be especially helpful in regulating their antibiotic dosages and in giving forewarning about impending bacterial infections. A method for simultaneous serum assay of two antibiotics belonging to the same penicillin group of drugs is described here. Such combined assay procedures are usually not undertaken, largely due to the technical difficulties involved. The procedures described here should prove helpful in monitoring antibiotic therapy in patients on combined Ampi-Clox regimen, whenever necessary.