A screening algorithm for diagnosing bacterial gastroenteritis by real-time PCR in combination with guided culture.

We have introduced a real-time PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli and Yersinia enterocolitica in fecal samples in our routine laboratory. This new approach showed consistent results, with minimal inter-sample variation. When compared to conventional culture, the hands-on time decreased by 13 h/wk, and the median turnaround time drastically shortened from 73 to 29 h (P < .0001). Moreover, the detection rate of the targeted pathogens seemed to increase: the positivity rate registered over a twelve month period increased from 4.98% when using bacterial culture, compared to 8.56% when using real-time PCR (P < .0001). For antimicrobial susceptibility testing, samples that are found to be PCR positive are additionally cultured after the PCR result is known. Using this algorithm, we got a positive culture for 71.0% of the PCR positive samples. The samples missed by guided culture had significantly higher quantification cycle (Cq) values compared to the samples picked up by guided culture (P = .0003). Finally; we also tested the effect of extended sample storage on the performance of guided culture. Storage time prior to inoculation did have an effect on the positivity rate of culture; interestingly, these effects were clearly species-dependent.

In vitro activity of tigecycline against multidrug-resistant Enterobacteriaceae isolates from a Belgian hospital.

Bacterial resistance among Gram-negative pathogens is a challenging clinical problem. Tigecycline has been developed specifically to overcome resistance. The aim of this study was to assess the in vitro activity of tigecycline against ESBL-producing Escherichia coli, ESBL-producing Klebsiella spp., and multidrug-resistant Enterobacter spp. Between May 2007 and March 2008, 26 strains of ESBL-producing Escherichia coli, 10 strains of ESBL-producing Klebsiella spp., and 27 strains of multidrug-resistant Enterobacter spp. were isolated consecutively from inpatients with a documented infection in which the collected isolate was identified as the probable causative organism. The in vitro susceptibility against tigecycline was measured by the E-test method. MIC(50) values were 1 microg/ml, 2 microg/ml, and 3 microg/ml respectively. MIC(90) values were respectively 1.5 microg/ml, 4 microg/ml, and 12 microg/ml. Nonsusceptibility rates of 35%, 100%, and 96% respectively were found using EUCAST breakpoints. Despite the limited number of strains tested, our in vitro data suggest that tigecycline is unsuitable for the treatment of infections with multidrug-resistant Enterobacteriaceae in our setting. Therefore, we suggest that larger multicenter studies should be conducted to reconsider the value of tigecycline for the treatment of infections with multidrug-resistant, Gram-negative bacteria.

[Female patient with cutaneous anthrax in Belgium]. / Een patiënte met cutane anthrax in België.

A 23-year-old Turkish woman was admitted with an infection of the left thumb. The clinical picture was typical for cutaneous anthrax. Microbiological tests confirmed the diagnosis 'infection by Bacillus anthracis'. She recovered when treated with penicillin, although later tests revealed that the bacteria were resistant to this antibiotic. The patient became infected in Belgium as a result of wounding herself on the teeth of an illegally slaughtered sheep, which had possibly become infected in the pasture. Recognising the characteristic clinical picture of cutaneous anthrax is essential for prompt treatment and a favourable prognosis.

Burkholderia (Pseudomonas) cepacia and cystic fibrosis: the epidemiology in Belgium.

Burkholderia cepacia has become an increasingly recognized pathogen among cystic fibrosis (CF) patients and its potential role in declining pulmonary function or unexpected fatal outcome has caused widespread concern. Direct person-to-person transmission has been documented and a segregation policy of CF patients colonized with B.cepacia from non-colonized CF patients is widely adopted. Since this policy has a dramatic impact on social behaviour of CF patients it is imperative that clinical laboratories accurately isolate and identify B.cepacia in the respiratory secretions. In order to comprehend the epidemiology of B.cepacia in the Belgian CF population a multicentre study was conducted during a period of 1 year (March'93-February'94). B.cepacia was isolated in only 12 of 465 CF patients (2.6%). Routine biochemical tests identified these strains as authentic B.cepacia. However, the combined data from protein and DNA-DNA hybridization analyses revealed that the Belgian CF "B.cepacia" isolates showed patterns different from reference B.cepacia isolates and belong to 3 different, newly identified Burkholderia genomovars, but not to B.cepacia. Comparative analysis of the selective media used for recovery of these "B.cepacia" strains from respiratory secretions indicated that the commercial medium (Mast) containing polymyxin B and ticarcillin as the selective agents was the best and most user-friendly. Molecular typing of these Burkholderia isolates by arbitrarily-primed PCR (AP-PCR) and pulsed-field gel electrophoresis (PFGE) showed that spread of a single strain within a same centre occurred but the mode of transmission remains unknown; inter-centre spread of strains was not observed. Interestingly, neither colonization with a distinct or an epidemic strain (belonging to either of the three newly identified Burkholderia genomovars) nor colonization for a prolonged period of time, led to a rapid deterioration of lung function in these CF patients. It appears essential to determine the prevalence of these "new" Burkholderia genomovars in larger populations of CF patients and to evaluate their virulence and other features as this may have important clinical and practical implications.

Congenital pneumonia due to Mycoplasma pneumoniae.

A case of probable vertical transmission of Mycoplasma pneumoniae is presented. The presence of M pneumoniae was demonstrated by the polymerase chain reaction (PCR) in the nasopharyngeal aspirate of a newborn who developed pneumonia shortly after birth. This result was confirmed by performing a second PCR, amplifying another part of the genome of M pneumoniae. It is concluded that M pneumoniae can be added to the long list of pathogens known to cause congenital pneumonia.

Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplasma pneumoniae in clinical specimens.

The polymerase chain reaction (PCR) was used to amplify a 209 base-pair fragment of Mycoplasma pneumoniae DNA. The amplicon was transferred into a plasmid and a 680 base-pair piece of foreign DNA was inserted between the two amplimer sites. Plasmid DNA was added to the reaction mixture as an internal control for the polymerase chain reaction. Since the original hybridization target sites were included in this construction, one pair of amplimers could be used to amplify both the target DNA and the internal control DNA. Separation of internal control from target DNA after amplification was easily obtained on agarose gel electrophoresis. For the analysis of clinical samples with the polymerase chain reaction, the addition of internal control DNA allowed monitoring of the overall effectiveness of the amplification in each tube. With this technique approximately one-third of the tests were shown to be unsatisfactory due to technical errors or contaminating inhibitors. Adequate internal controls are necessary to avoid false-negative results with the polymerase chain reaction.

Prosthetic valve endocarditis due to methicillin-resistant Staphylococcus epidermidis and Micrococcus species successfully treated with rifampicin combined with other antibiotics.

Two patients with prosthetic valve endocarditis due to methicillin-resistant Gram-positive cocci (Staphylococcus epidermidis and Micrococcus spp.) are described. They were successfully treated with rifampicin combined first with an aminoglycoside and later with co-trimoxazole or co-trimoxazole plus vancomycin. The addition of rifampicin to these antibiotics resulted in enhanced serum bactericidal activity. High doses of rifampicin (1200-1800 mg) for 7-8 weeks did not cause any serious side-effect. Surgery was not required. During surveillance for more than 2 years endocarditis did not recur.

Isolation of Mycoplasma hominis from genital ulcerations of patients in Eastern and Southern Africa.

In three separate surveys involving 413 patients in Mbabane (Swaziland), Nairobi (Kenya), and Carletonville (South Africa), Mycoplasma hominis was isolated from the base of ulcers in 41 (16%) of 251 men with genital ulcer disease. Antibodies to M. hominis were detected by indirect hemagglutination in 89 (51%) of 176 such patients. Of these male patients, 15% and 6% had indirect hemagglutinating antibodies at titers of greater than or equal to 160 and greater than or equal to 640, respectively. The rate of isolation of M. hominis and the results of serologic tests for antibodies to this organism were the same whether or not a cause of genital ulcer disease was identified.

Etiology of genital ulcerations in Swaziland.

The etiology of genital ulcer disease was determined among 155 consecutive new cases in Mbabane, Swaziland. In contrast to genital ulcerations in industrialized countries, chancroid was the most common diagnosis (44% of cases), as established on clinical grounds and by exclusion of other etiologies. Primary syphilis and genital herpes accounted for only 17% and 12% of the cases, respectively. Lymphogranuloma venereum was found in 13% of the patients, and in 15% of cases no diagnosis was made.

Characterisation of an unusual bacterium isolated from genital ulcers.

The preliminary characterisation of an unusual gram-negative bacillus isolated from genital ulcers in Swaziland is reported. Like Haemophilus ducreyi, it is an oxidase positive, nitrate-reductase-positive gram-negative rod that forms streptobacillary chains in some circumstances; it was therefore called the "ducreyi-like bacterium" (DLB). Distinguishing features of DLB are production of alpha-haemolysis on horse-blood agar, stimulation of growth by a microaerophilic atmosphere and by a factor produced by Staphylococcus aureus, a strongly positive porphyrin test, and a remarkable ability to undergo autolysis. DLB had a guanine + cytosine value of c. 50 mole% but it cannot be classified, even at the genus level, until more taxonomic data are obtained.

Syphilis in Swaziland: a serological survey.

Sera from 536 adults and children in Swaziland were examined for their reactivity in the rapid plasma reagin (RPR) and Treponema pallidum haemagglutination (TPHA) tests. None of 130 sera from children was reactive in either test; 8.6% of sera from 185 healthy adults were reactive in the RPR test and 33% in the TPHA test; 24.5% of 220 sera from patients with genital ulcers were RPR-positive and 45.9% TPHA-positive. The RPR positivity rates were not related to age, but the percentage of RPR-negative, TPHA-positive sera increased with age in both the healthy adults and the patients with genital ulcers. Thus venereal syphilis appears to be responsible for these high positivity rates. Estimates of the yearly incidence of syphilis are identical for both groups--approximately 1.4%, an unusually high figure.

Epidemiology and aetiology of urethritis in Swaziland.

The annual incidence of urethritis can be estimated to be at lest 3750 per 100,000 population in Swaziland. In a study of 109 males with symptomatic urethritis 80% had gonorrhoea, 6% non-gonococcal urethritis (ngu) and 14% were classified as having no 'objective' urethritis (less than 5 polymorphonuclear leucocytes per highpower field in the urethral smear). The relative frequency of gonorrhoea was 80 to 95% and of non-gonococcal urethritis 5 to 20 according to which criteria are used for patient selection and/or diagnosis of ngu. Chlamydia trachomatis was cultured in 3.4% of the cases with urethritis, comprising one positive culture in 70 patients with gonorrhoea, one in 5 with ngu, and one in 12 with no 'objective' urethritis. Seventy-one percent of patients, with a comparable percentage in each diagnostic group, had chlamydial antibodies when tested by the micro immunofluorescence test to pooled chlamydial antigens. Interpretation of the chlamydial serologic results indicates that lymphogranuloma venereum is probably endemic in the country, and that oculogenital chlamydial infections are not a problem; this corresponds with the low isolation rate of Chlamydia trachomatis in the urethritis cases. The study shows that the epidemiology and causes of urethritis are clearly of a different pattern to that seen in industrialised countries. This type of study is a sound basis for a simplified but effective urethritis control programme which can be implemented in the para-urban and rural health centres in developing countries.

Antibiotic susceptibility of Neisseria gonorrhoeae strains from Europe and Africa.

The in vitro activities of 16 antimicrobial agents were tested by a plate dilution method against 268 unselected isolates of Neisseria gonorrhoeae from Belgium, Rwanda, Swaziland, and Zaire. Fifteen beta-lactamase-producing strains isolated in Europe from various origins were also tested. There were significant regional variations in antimicrobial agent susceptibility, even among the African isolates, with the Rwandan and Zairean strains being most resistant. Benzylpenicillin and ampicillin were equally active in all but the beta-lactamase-producing strains. Among the cephalosporins, cefotaxime was by far the most active, followed by cefuroxime, cefamandole, cefoxitin, and cefaclor, in that order. All strains were susceptible to spectinomycin, thiamphenicol, kanamycin, and rifampin, with the exception of one highly rifampin-resistant isolate and a moderately thiamphenicol-resistant strain. Twenty-six percent of the isolates were highly resistant to streptomycin. Six percent of the gonococci had a minimal inhibitory concentration for tetracycline greater than 2 mug/ml. Clavulanic acid inhibited the beta-lactamase activity of the gonococci tested and improved markedly the activities of ampicillin and amoxicillin against beta-lactamase-producing strains.