- Volume 55, Issue 9, 2006

A total of 71 enteropathogenic Escherichia coli (EPEC) strains isolated from children with diarrhoea in Montevideo, Uruguay, were characterized in this study. PCR showed that 57 isolates carried eae and bfp genes (typical EPEC strains), and 14 possessed only the eae gene (atypical EPEC strains). These EPEC strains belonged to 21 O : H serotypes, including eight novel serotypes not previously reported among human EPEC in other studies. However, 72 % belonged to only four serotypes: O55 : H− (six strains), O111 : H2 (13 strains), O111 : H− (14 strains) and O119 : H6 (18 strains). Nine intimin types, namely, α1 (two O142 strains), β1 (29 strains, including 13 O111 : H2 and 14 O111 : H−), γ1 (three O55 : H− strains), θ (five strains, including three strains with H40 antigen), κ (two strains), ε1 (one strain), λ (one strain), μB (six strains of serotypes O55 : H51 and O55 : H−) and ξR/β2B (22 strains, including 18 O119 : H6) were detected among the 71 EPEC strains. The authors have identified two novel intimin genes (μB and ξR/β2B) in typical EPEC strains of serotypes O55 : H51/H− and O119 : H6/H−. The complete nucleotide sequences of the novel μB and ξR/β2 variant genes were determined. PFGE typing after XbaI DNA digestion was performed on 44 representative EPEC strains. Genomic DNA fingerprinting revealed 44 distinct restriction patterns and the strains were clustered in 12 groups. Only 15 strains clustered in six groups of closely related (similarity >85 %) PFGE patterns, suggesting the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.

Following antigen preparation procedures similar to those of the direct agglutination test (DAT), an IgG ELISA employing intact β-mercaptoethanol (β-ME)-treated Leishmania donovani promastigotes was developed. The performance of the β-ME ELISA thus developed was assessed in patients with confirmed visceral leishmaniasis (VL), revealing slightly lower sensitivity (39/40=97.5 %) than that of the DAT (40/40=100 %). When challenged with sera of individuals with non-VL conditions, including leukaemia and African trypanosomiasis, the specificity of the β-ME ELISA was 100 % (158/158), compared to 98.8 % (156/158) for DAT. In an endemic population (n=145) manifesting a clinical suspicion of VL, results obtained with the β-ME ELISA were highly concordant with those of DAT, both in the seropositive (65/68=95.6 %) and seronegative (77/80=96.3 %) groups. Furthermore, the incorporated intact antigen demonstrated higher sensitivity in ELISA (16/18=88.9 %) than the water-soluble equivalent (13/18=72.2 %). The stability of the formaldehyde-fixed antigen (2 months at 4 °C) in β-ME ELISA, as well as the option for direct testing of whole-blood samples and visual reading of results (within 2 h, compared to 18 h for DAT), advocate the simultaneous application of the technique with DAT for confirmation of VL in laboratories with limited facilities.

Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as ‘microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10 % of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1–3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 103 oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91 % versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100 % accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

Bordetella pertussis infection is being increasingly recognized as a cause of prolonged, distressing cough (without whooping symptoms) in children and young adults. Diagnosis of infection in this population is important for treatment and surveillance purposes, and may also prove useful in reducing transmission to unvaccinated babies, for whom disease can be fatal. Serum IgG titres against pertussis toxin (PT) are routinely used as a marker of recent or persisting B. pertussis infection. However, collection of serum from young children is difficult, and compliance amongst these subjects to give samples is low. To circumvent these problems, an IgG-capture ELISA capable of detecting anti-PT IgG in oral fluid was devised. The assay was evaluated by comparison to a serum ELISA, using 187 matched serum and oral fluid samples from children (aged 5–16 years) with a history of prolonged coughing, whose serum anti-PT titre had already been determined (69 seropositive, 118 seronegative). The results showed that, using a cutoff of 70 arbitrary units (AU), the oral fluid assay detected seropositive subjects with a sensitivity of 79.7 % [95 % confidence interval (CI) 68.3–88.4] and a specificity of 96.6 % (95 % CI 91.5–99.1). Thus, oral fluid titres of ⩾70 AU would possess a positive predictive value of 76.2–93.2 % for pertussis amongst children with chronic coughs when used as a surrogate for the serum ELISA (assuming disease prevalence of 12–37 %). This oral fluid ELISA will greatly assist in the convenience of B. pertussis disease diagnosis and surveillance.

Pneumocystis jiroveci pneumonia (PCP) is an opportunistic infection affecting immunocompromised patients. While conventional diagnosis of PCP by microscopy is cumbersome, the use of PCR to diagnose PCP has great potential. Nevertheless, inter-laboratory validation and standardization of PCR assays is lacking. The aim of this study was to evaluate the inter-laboratory agreement of three independently developed real-time PCR assays for the detection of P. jiroveci in bronchoalveolar lavage fluid samples. Therefore, 124 samples were collected in three tertiary care laboratories (Leiden University Medical Center, Maastricht Infection Center and Radboud University Nijmegen Medical Centre) and were tested by both microscopy and real-time PCR. Of 41 samples positive for P. jiroveci by microscopy, 40 were positive in all three PCR assays. The remaining sample was positive in a single assay only. Out of 83 microscopy-negative samples, 69 were negative in all three PCR assays. The other 14 samples were found positive, either in all three assays (n=5), in two (n=2) or in one of the assays (n=7). The data demonstrate high inter-laboratory agreement among real-time PCR assays for the detection of P. jiroveci.

Two Salmonella enterica serovar Typhimurium isolates and one S. enterica serovar Enteritidis isolate that were resistant to 4 μg cefotaxime ml−1 and produced CTX-M-type extended-spectrum β-lactamases (ESBLs) were characterized from patients in Hong Kong during 2003–2004. The S. Typhimurium strain isolated in 2003 produced a CTX-M-9 ESBL and harboured a bla CTX-M-9 gene that was associated with a class I integron-containing gene cassette and orf513 similar to those of In60. The second S. Typhimurium strain and the S. Enteritidis strain, both isolated in 2004, produced CTX-M-14; the former also produced TEM-1. The bla CTX-M-14 gene in these two isolates was associated with the insertion sequence ISEcp1. The CTX-M genes were present on a transferable plasmid of 62, 70 or 92 kb. PFGE of XbaI-restricted total DNA from the two S. Typhimurium isolates indicated that they were not clonally related. These three isolates were also resistant to one of the other non-β-lactam antimicrobial agents tested. This is the first report of a CTX-M-9 ESBL in Salmonella in Hong Kong and the presence of bla CTX-M-9 and bla CTX-M-14 in S. Typhimurium.

In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100 % resistance to ciprofloxacin and 98.9 % non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1 % of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95→Ala and the parC mutations Ser-87→Arg and Ser-87→Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D 10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D 10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D 10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of DNA in viable bacterial cells has little effect on amplifiable DNA, was not able to eliminate amplifiable 16S rRNA genes at a dose of up to 12 kGy and cannot therefore be used for elimination of DNA contamination of PCR reaction components or laboratory equipment when this DNA is present in microbial cells. This finding has practical implications for those using molecular diagnostic techniques in microbiology.

The aim of this study was to assess the incidence and susceptibility to antibacterial agents of anaerobic strains in 118 patients with head and neck abscesses (31) and cellulitis (87). Odontogenic infection was the most common identified source, occurring in 73 (77.7 %) of 94 patients. The incidence of anaerobes in abscesses and cellulitis was 71 and 75.9 %, respectively, and that in patients before (31 patients) and after (87) the start of empirical treatment was 80.6 and 72.4 %, respectively. The detection rates of anaerobes in patients with odontogenic and other sources of infection were 82.2 and 71.4 %, respectively. In total, 174 anaerobic strains were found. The predominant bacteria were Prevotella (49 strains), Fusobacterium species (22), Actinomyces spp. (21), anaerobic cocci (20) and Eubacterium spp. (18). Bacteroides fragilis strains were isolated from 7 (5.9 %) specimens. The detection rate of Fusobacterium strains from non-treated patients (32.2 %) was higher than that from treated patients (13.8 %). Resistance rates to clindamycin and metronidazole of Gram-negative anaerobes were 5.4 and 2.5 %, respectively, and those of Gram-positive species were 4.5 and 58.3 %, respectively. One Prevotella strain was intermediately susceptible to ampicillin/sulbactam. In conclusion, the start of empirical treatment could influence the frequency or rate of isolation of Fusobacterium species. The involvement of the Bacteroides fragilis group in some head and neck infections should be considered.