- Volume 53, Issue 7, 2004

Between January 2002 and July 2003, 173 bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87.0 % for conventional PCR and 100 and 84.9 % for real-time PCR, respectively. The use of a concentration of 103 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84.9 to 98.6 % without reducing the sensitivity of the technique. This technique is rapid (<3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with <103 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.

Most past studies of cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients in the UK suggest that it is a rare occurrence. However, two recent reports of highly transmissible strains in patients in regional centres in England (Liverpool and Manchester) have raised questions as to the extent of the problem and prompted a nationwide survey to establish the distribution of P. aeruginosa strain genotypes among these patients. Isolates of P. aeruginosa were requested from over 120 hospitals in England and Wales and a sample size of approximately 20 % of the CF patient population in each centre was recommended. In total, 1225 isolates were received from 31 centres (range 1 to 330). Single patient isolates were typed by SpeI macrorestriction and PFGE. A panel of strains of the common genotypes including representatives of reported transmissible strains was assembled and further characterized by fluorescent amplified fragment length polymorphism (FAFLP) genotyping. At least 72 % of all patients harboured strains with unique genotypes. Small clusters of related strains were evident in some centres, presumably indicating limited transmission of local strains. The most prevalent strain was indistinguishable from that previously described as the ‘Liverpool’ genotype, and accounted for approximately 11 % of patient isolates from 15 centres in England and Wales. The second most common genotype (termed Midlands 1) was recovered from 86 patients in nine centres and the third genotype, which matched closely the PFGE profile of Clone C, a genotype originally described in Germany, was found in eight centres and was isolated from 15 patients. A fourth genotype, identical to the published Manchester strain, was found in three centres. FAFLP analysis revealed some microheterogeneity among strains of the Liverpool genotype but all isolates of this genotype were positive by PCR for a strain-specific marker. These data suggest that cross-infection with P. aeruginosa has occurred both within and widely between CF centres in England and Wales. The two most common genotypes accounted for more than one-fifth of patients’ isolates examined and transmissible genotypes were found in all but three centres studied. These results emphasize the need for continued surveillance of P. aeruginosa genotypes in CF patients to provide informed infection control policy in treatment centres.

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40 %) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70 %) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18 %) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6 %) patients were found to be positive by PCR analysis.

Stool antigen-testing allows non-invasive detection of Helicobacter pylori that is indicative of active infection. Three commercial kits are currently marketed in the UK for stool antigen-testing. The aim of this study was to conduct a comparative evaluation of the performances of each of these tests, compared with culture and histological examination of gastric biopsies, for pre-treatment diagnosis of infection in an adult dyspeptic population in south-east England. Examination of 112 stool samples by the Premier Platinum HpSA ELISA (Meridian Diagnostics) and by the Amplified IDEIA HpStAR ELISA (DakoCytomation) kits demonstrated that the latter was more sensitive (81.3 versus 93.8 %, respectively) and specific (91.7 versus 100.0 %, respectively). Additionally, the IDEIA HpStAR was easier to interpret, with OD readings of positive and negative results being far from the recommended cut-off, whereas equivocal results that were generated by the HpSA kit were difficult to interpret. Additional testing of 87 of the 112 stools by the ImmunoCard STAT! HpSA kit (Meridian Diagnostics) demonstrated that this test was easier to perform than ELISA and was more sensitive than the HpSA kit but, compared with the IDEIA HpStAR kit, the ImmunoCard test was less sensitive (87.8 versus 95.9 %, respectively) and specific (89.4 versus 100.0 %, respectively). Furthermore, the ImmunoCard test generated weakly positive results, correlating with lower OD readings for both ELISA kits, that were difficult to interpret. The Amplified IDEIA HpStAR kit is therefore the most sensitive and specific of the three tests that are available for pre-treatment, non-invasive detection of H. pylori in stool samples in an English adult dyspeptic population.

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify Toxoplasma gondii DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed T. gondii DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of T. gondii DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of T. gondii DNA in some samples.

Candida dubliniensis is an emerging pathogen capable of causing oropharyngeal, vaginal and bloodstream infections. Although C. dubliniensis is similar to Candida albicans in several phenotypic characteristics, it differs from it with respect to epidemiology, certain virulence factors and the ability to develop resistance to fluconazole rapidly. In this study, the first seven isolations of C. dubliniensis from Kuwait are described, all originating from non-human immunodeficiency virus (HIV)-infected patients. The isolates were initially identified by the Vitek 2 yeast identification system, positive germ tube test, production of rough colonies and chlamydospores on Staib agar and by their inability to assimilate xylose, trehalose or methyl α-d-glucoside. The species identity of the isolates was subsequently confirmed by specific amplification of rDNA targeting the internally transcribed spacer 2 (ITS2), restriction endonuclease digestion of the amplified DNA and direct DNA sequencing of the ITS2. Using the E-test method, the MICs of C. dubliniensis test isolates were in the range 0.125–0.75 μg ml−1 for fluconazole, 0.002–0.75 μg ml−1 for itraconazole, 0.006–0.125 μg ml−1 for ketoconazole, 0.002–0.5 μg ml−1 for amphotericin B and 0.002–0.016 μg ml−1 for voriconazole. Two of the isolates were resistant to 5-flucytosine (>32 μg ml−1), but none against fluconazole. The study reinforces the current view that C. dubliniensis has a much wider geographical and epidemiological distribution.

The aims of this study were to determine the levels of procalcitonin (PCT) and C-reactive protein (CRP) in Helicobacter pylori-positive (HP+) patients diagnosed with duodenal and gastric ulcer and to evaluate the correlation of PCT and CRP levels with other invasive and non-invasive diagnostic methods for determination of H. pylori eradication in post-treatment follow-up. Thirty-five HP+ patients with dyspepsia were included in this study. Serum samples (5 ml) were collected at admission and after 24 h. Antimicrobial therapy (omeprazole, amoxycillin and clarithromycin) was given for 1 week to HP+ patients who were positive only by culture or by urease test plus pathology. After 1 month, serum samples (5 ml) were collected again and culture, urease and pathology investigations were performed on endoscopic samples. PCT and CRP levels were measured in the collected blood samples. Thirty-five H. pylori-negative (HP−) cases with dyspepsia, 38 cases with bacteraemia and 35 healthy blood donors were included in this study as control groups. The mean and minimum–maximum levels of PCT were 1.39 (0.25–6.75), 0.35 (0.12–0.71), 7.45 (0.68–51.5) and 0.40 (0.12–0.71) ng ml−1 for the groups of HP+, HP− and bacteraemia patients and healthy donors, respectively. Mean CRP levels were 1.00 (<0.5–8.11), 0.62 (<0.5–3.2), 11.5 (3.2–43.5) and 0.63 (<0.5–5.46) mg dl−1 for the same groups. A statistically significant difference was found between HP+ patients and both HP− cases and healthy blood donors for PCT levels, and higher PCT levels were found on admission in cases of bacteraemia than in the other groups (P < 0.05). PCT levels of HP+ cases decreased significantly (from 1.39 to 0.86) between admission and the post-treatment period (30 days); however, PCT levels remained higher than the cut-off value (0.5 ng ml−1). Similar ranges of CRP levels were found over the same time-period. The sensitivity of PCT was found to be higher than that of CRP on admission, but the specificity of PCT was found to be lower than that of CRP on the day of admission (65 and 74 %, respectively). The sensitivity of PCT was the same as that of CRP for the post-treatment period, but specificity of PCT was higher than that of CRP for the post-treatment period (83 and 76 %, respectively). It was concluded that PCT and CRP are not very effective markers for H. pylori infection in primary diagnosis or in eradication follow-up after therapy when used in parallel with conventional diagnostic methods, even if there is a difference in PCT and CRP levels between HP+ and HP− cases on admission.

This study was designed to investigate the prevalence of carbapenem-resistant Acinetobacter calcoaceticus–baumannii complex (Acb complex) and to type carbapenemases. The relatedness of 45 isolates of carbapenem-resistant Acb complex collected from a clinical setting was analysed by PFGE. The carbapenemases produced by these isolates were typed by IEF, a three-dimensional test, 2-mercaptopropanoic acid inhibition assay, PCR and DNA cloning and sequencing. Results showed that all 45 isolates were resistant to multiple antibiotics including meropenem. The resistance rates to cefoperazone/sulbactam and ampicillin/sulbactam were 2.2 and 6.5 %, respectively. About 71.7–78.3 % of these isolates were intermediately resistant to cefepime, ceftazidime and cefotaxime. Forty-five isolates were classified into type A (98 %) and B (2 %) based on their PFGE patterns. Most of type A isolates were from the ICU. Type A was the dominant isolate, including subtypes A1 (22 %), A2 (71 %), A3 (2 %) and A4 (2 %). Only one isolate, from the haematology department, belonged to type B. Forty-three isolates (96 %) were positive for carbapenemase. One isolate had two bands by IEF, the pIs of which were 6.64 and 7.17. The band with the pI of 6.64 was OXA-23. The other 42 isolates produced two bands with pIs of 6.40 and 7.01 which could not be inhibited by clavulanic acid, cloxacillin or 2-mercaptopropanoic acid. It can be concluded that the prevalent carbapenem-resistant Acb complex isolates from this hospital all had similar β-lactamase patterns.

Analysis of 182 Neisseria meningitidis strains isolated over the past 30 years in Japan by serogroup typing and multilocus sequence typing (MLST) was performed. The serogroups of the 182 Japanese isolates were B (103 isolates), Y (39), W135 (1) and non-groupable (39). By MLST analysis, 65 different sequence types (ST) were identified, 42 of which were not found in the MLST database as of January 2004 and seemed to be unique to Japan. Statistical analysis of the MLST results revealed that, although the Japanese isolates seemed to be genetically divergent, they were classified into six major clonal complexes and other minor complexes. Among these isolates, well-documented ST complexes found worldwide were present, such as ST-23 complex (49 isolates), ST-44 complex (41 isolates) and ST-32 complex (8 isolates). On the other hand, a new clonal complex designated ST-2046 complex (28 isolates), which has not been identified in other countries, was also found, suggesting that this clone was indigenous to Japan. Taken together, it was speculated that meningococcal isolates in Japan comprised heterogeneous clones, which were derived both from clones identified in other countries and clones unique to Japan.

Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79 % of patients were infected with Burkholderia cenocepacia (genomovar III), 18 % with Burkholderia multivorans (genomovar II) and less than 5 % of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested.

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.