IMP-4, a novel metallo-beta-lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998.

Between 1994 and 1998, 97 imipenem-resistant Acinetobacter isolates were identified at the Prince of Wales Hospital, Hong Kong, China. A bla(IMP) PCR product was obtained from 23 of 35 viable cultures; 12 isolates belonged to genomic DNA group 3, 8 belonged to group 2 (Acinetobacter baumannii), 2 belonged to group 13TU, and 1 belonged to group 1. The bla(IMP) homologues were sequenced from two isolates from genomic DNA group 2 and one isolate each from groups 3 and 13TU. The four sequences included an identical 738-bp open reading frame, predicted to encode a polypeptide of 246 amino acids, with 95.6% homology to IMP-1 and 89.3% homology to IMP-2. The new enzyme, designated IMP-4, was partially purified. It had a pI of 8.0 and was strongly active against imipenem and meropenem, with V(max) values 53 and 8% of that for penicillin G, respectively. Strong activity was also seen against oxyimino-aminothiazolyl cephalosporins but not against aztreonam. Hydrolytic activity was inhibited by EDTA but not by clavulanate or tazobactam. Carbapenem MICs for most bla(IMP)-positive isolates were 4 to 32 microg/ml, but one isolate with the intact gene was susceptible, with imipenem and meropenem MICs of 0.25 and 0.5 microg/ml, respectively. The latter isolate did not produce the band with a pI of 8.0, and gene expression was inferred to have been lost. None of the isolates studied in detail contained extrachromosomal DNA, and carbapenem resistance was not transmissible to Escherichia coli. Nevertheless, the presence of bla(IMP-4) in different genomic DNA groups implies horizontal transfer, and sequences resembling a GTTRRRY integrase-dependent recombination motif were identified in the flanking regions of bla(IMP-4).

Epidemiology and infection control implications of Acinetobacter spp. in Hong Kong.

In a previous study, we showed that Acinetobacter genomic DNA group 3 was the most common species among blood culture isolates and was commonly found on superficial carriage sites of the healthy and the sick, which are different findings from those reported in Europe and North America. We used amplified ribosomal DNA restriction analysis and pulsed-field gel electrophoresis to study further the molecular epidemiology of acinetobacters in our region. Over a study period of 6 weeks with 136 consecutive routine clinical isolates (1.33% of all specimens), genomic DNA groups 2 (Acinetobacter baumannii), 3, and 13TU were obtained from 59 of 69 positive patients. There is a significant difference in the specimen sources of the three genomic DNA groups, with group 13TU being significantly associated with the respiratory tract (chi-square exact test, P = 0.0064). Settle plates showed a significantly heavier environmental load from the intensive care unit (ICU) than from the four surgical wards examined (22 of 70 versus 76 of 120 plates with <5 colonies; chi-square test, P < 0. 0001). Genomic group 3 accounted for 6 of 12 clusters of possibly related strains among patients, between patients and the ICU environment, and in the ICU environment. Genomic groups 2 and 3 accounted for 21% of the 132 genomically identified isolates recovered from 21 of 41 local vegetables, 53 of 74 fish and meat samples, and 22 of 60 soil samples. Group 13TU was present only in patients' immediate surroundings. The role played by the environment and by human carriage should be evaluated in order to devise a cost-effective infection control program pertinent to our situation of acinetobacter endemicity.

The use of CHROMagar Orientation as a primary isolation medium with presumptive identification for the routine screening of urine specimens.

The aim of the study was to compare the use of a novel differential culture medium CHROMagar, for both primary isolation and presumptive identificaton, with the method currently used in our laboratory for screening mid-stream-urine samples (MSU). Routine methods (RM) included blotting paper imprinting of all specimens and additional quantitative culture on cysteine lactose electrolyte-deficient agar (CLED) for selected samples together with Microbact 12E for further identification. The CHROMagar method (CH) relied on the use of blotting paper imprints, colonial colour and morphology on CHROMagar only. With respect to the 3390 MSU specimens examined, both methods yielded similar results in 3240, including > or = 87% of Escherichia coli, Pseudomonas spp., Staphylococcus spp., Proteus mirabilis/Morganella morganii and Enterobacter/Serratia/Klebsiella/Citrobacter spp. Of the 52 discordant identifications, yeasts were reported as staphylococci on CHROMagar in 10. The overall cost of materials per specimen was US$ 0.30 by RM and $ 0.24 by CH. It took about 3 min to perform each Microbact test. Thus, CHROMagar plus Gram stain and other simple bench tests gave results similar to those using our current method, but had the advantage of saving time and materials.

Skin carriage of acinetobacters in Hong Kong.

We studied the carriage of Acinetobacter spp. at five superficial sites in 79 patients from two hospitals, in 133 healthy controls from the community (medical students and new nurses), and in 198 student nurses in different classes. A total of 431 isolates from 364 positive sites of 201 subjects and 124 blood culture isolates (1997 to 1998) were genospeciated by amplified ribosomal DNA restriction analysis. Genospecies 3 was the most common species. The carriage rate of student nurses (42 of 131) was significantly lower than that of new nurses from the community (25 of 38) (chi-square test, P = 0.0004; odds ratio [OR], 4.08; 95% confidence limits, 1.78 to 9.41) but not significantly different (P = 0.1) from that of patients in the same hospital (20 of 42). Genospecies from blood cultures and subjects (acute patients and student nurses) from Prince of Wales Hospital were similar to one another but different from subjects from the community or from another hospital (chi-square test, P < 0.0001). Half of the subjects who were positive at at least two sites had different genospecies. Of the 28 sites examined, 68% showed strain variation among isolates of the same genospecies by random amplified polymorphic DNA analysis. Half of the 106 subjects who had samples taken again within 6 weeks or 6 months later were positive only once. In the 17 subjects who were positive on at least two occasions, each occasion yielded different genospecies in 13 subjects. Our results indicate that skin carriage in the majority of healthy subjects is characterized by low density, variation in genospecies and strains, short-term duration, and the typicality of a given locality.

Simple, reliable, and cost-effective yeast identification scheme for the clinical laboratory.

The appearance of colonies on the chromogenic medium CHROMagar Candida combined with observation of morphology on corn meal-Tween 80 agar was used for the identification of 353 clinical yeast isolates. The results were compared with those obtained with API yeast identification kits. The accuracy of identification and the turnaround time were equivalent for each method, and our cultural method was less expensive.

Study of the relatedness of isolates of Shigella flexneri and Shigella sonnei obtained in 1986 and 1987 and in 1994 and 1995 from Hong Kong.

We used pulsed-field gel electrophoresis (PFGE) to study the genetic relatedness of 235 isolates of Shigella flexneri and Shigella sonnei collected in Hong Kong (97 isolates from 1986 and 1987 and 138 isolates from 1994 and 1995). Altogether, 13 gels were run with bacteriophage lambda ladder DNA (Pharmacia) as an external reference in every sixth lane, standardized reagents and methods, and isolates randomized for species and years. For quantitative illustration of the relationships within a large body of isolates, computer-generated dendrograms were used to determine the number of isolates in pulsotypes at Dice coefficients of similarity of 75% (PT75) and 50% (PT50). For S. flexneri, there was a significant difference in the distribution of isolates collected during the two periods in both PT75 and PT50, with 68% of isolates collected in 1994 and 1995 sharing a coefficient of similarity of >/=68%. For S. sonnei, a significant difference was observed in PT50 only. We also used Upholt's formula for an approximation of the fraction of nucleotide difference between isolates and Molecular Evolutionary Genetics Analysis to determine relative genetic distances. For both species, the relative genetic distances between isolates of the earlier collection period were significantly greater (P < 0.0001), i. e., they were further apart and therefore more diverse than those of the later period. We conclude that it is possible for a typical clinical laboratory to analyze a large amount of PFGE information on Shigella isolates obtained under controlled conditions. Such data analysis should enhance surveillance capabilities and give indications of further work to be done on various aspects of bacterial pathogenicity of the species.

Antimicrobial resistance in Shigella flexneri and Shigella sonnei in Hong Kong, 1986 to 1995.

Three hundred and thirty-three Shigella isolates obtained in 1986 to 1995 were tested for their susceptibilities to 19 antimicrobial agents. Nalidixic acid resistance had emerged in 59.6% of Shigella flexneri isolates during 1994 to 1995, with all tested resistant isolates having the mutation in gyrA encoding the Ser-83 alteration. Multiresistance (resistance to four or more agents) was more common in S. flexneri than in Shigella sonnei.

Effect of desiccation on the ultrastructural appearances of Acinetobacter baumannii and Acinetobacter lwoffii.

An Acinetobacter baumannii isolate survived desiccation beyond 30 days and an Acinetobacter lwoffii isolate up to 21 days. For both species, desiccation resulted in a significant increase in the proportion of round cells (A baumannii, 40% to 80%; A lwoffii, 51% to 63%) and a significant decrease in rod shaped cells (A baumannii, 58% to 13%; A lwoffii, 46% to 34%). Electronmicroscopic examination showed that there was also a corresponding significant increase in the cell wall thickness (A baumannii, up to 53%; A lwoffii, up to 26%). Desiccated A baumannii cells became more electron-dense and had significantly thicker cell walls (x1.3) than those of A lwoffii. Cell wall structures of A baumannii strains with different abilities to resist desiccation deserve further study.

Use of CHROMagar Candida for genital specimens in the diagnostic laboratory.

OBJECTIVE: To evaluate CHROMagar Candida (CA), a new yeast differential medium, for yeast isolation in a clinical laboratory for the routine examination of high vaginal swabs. METHODS: Results of high vaginal swab cultures processed in a standard manner on plates containing equal halves of Sabouraud dextrose agar (SDA) and CA were compared. Non-Candida albicans yeast isolates were further speciated with API 20C AUX or API 32C. To assess the ease of use of CA, laboratory staff lacking in experience of the medium were asked to identify 23 unlabelled yeast cultures on CA by referring to six labelled reference plates. RESULTS: Of the 1784 swab cultures processed, yeasts were isolated from 373 SDA and 368 CA. Of the 78 non-albicans isolates further speciated, CA identified correctly all cultures of C krusei and C tropicalis, and 82% of C glabrata. All the 38 inexperienced laboratory staff achieved 100% accuracy for C albicans and over 90% for C krusei and C tropicalis. CONCLUSIONS: CA is a satisfactory isolation medium for genital specimens, allowing immediate and correct identification of the commonly encountered yeasts and easy recognition of mixed cultures.

Anonymous questionnaire survey on the knowledge and practices of hospital staff in infection control.

We carried out an anonymous questionnaire survey to assess the extent to which hospital medical and nursing staff were familiar with the written policies and procedures of infection control and their intended course of action in situations where no formal policies were available. All 25 full-time medical staff below the grade of consultant and 70 of 163 permanent nursing staff caring for inpatients at one day shift were sampled. Nineteen (76%) medical and 56 (80%) nursing staff returned their copies. Nurses were significantly more familiar with all written policies and procedures than medical staff. They were also more likely to seek advice in situations where there were no written guidelines. Many hospital staff were uncertain about the practical details of policies and procedures for infection control. Ways to educate and motivate staff to comply with infection control measures are urgently required; some degree of national standardization of policies and procedures in infection control is desirable.

Successful treatment of four patients with recalcitrant vaginal trichomoniasis with a combination of zinc sulfate douche and metronidazole therapy.

BACKGROUND AND OBJECTIVES: There is no certain cure available for patients suffering from recalcitrant trichomoniasis. Zinc sulfate is reported to have antitrichomonal properties. We report our experience in treating four patients empirically with a combination of zinc sulfate douche and metronidazole. STUDY DESIGN: A retrospective case analysis. Patients who presented to the tertiary referral clinic with chronic recurrent trichomoniasis without evidence of reinfection were treated empirically with a combination of zinc sulfate douching (1%) followed by a metronidazole 500 mg suppository per vaginale twice daily and 200 to 400 mg three times a day orally. The douching and suppository were used prophylactically for 3 nights after menstruation for some months. RESULTS: Four patients who had a history of 4 months to 4 years of culture-positive symptomatic trichomoniasis and received a variety of therapies before referral were treated successfully. At the review of 2 to 5 months after therapy, all had remained asymptomatic and the results of clinical and laboratory examinations were normal. CONCLUSIONS: The exact role of zinc sulfate douching in the successful outcome is not certain, but the combination therapy requires only a moderate dose of metronidazole (1.6-2.2 g/day), avoiding the side effects of larger doses. The combination therapy therefore merits further evaluation.

Species identification of members of the Streptococcus milleri group isolated from the vagina by ID 32 Strep system and differential phenotypic characteristics.

The importance of bacterial vaginosis as a risk factor in obstetric and gynecological infections has recently been recognized. The bacterial vaginosis group of organisms includes members of the Streptococcus milleri group, the identification of which has caused much confusion. We prospectively surveyed the rates of carriage of S. milleri group organisms in 397 high vaginal swabs received in our laboratory. For the identification of 99 clinical isolates and 23 control strains, we compared the results obtained by the rapid ID 32 Strep system (Analytab Products) and by a scheme utilizing six differential phenotypic characteristics (presence of beta-N-acetylglucosaminidase, alpha-glucosidase, beta-D-fucosidase, beta-galactosidase, beta-N-acetylgalactosaminidase, and beta-glucosidase) as described by Whiley et al. (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). We identified Streptococcus anginosus in 18% and Streptococcus constellatus in 0.05% of the specimens examined. Of the isolates of S. anginosus that reacted with grouping antisera, 20 of 25 belonged to Lancefield group F. The incubation conditions for bacterial cultures and for reaction mixtures affected the results of phenotypic characterization in the production of alpha-glucosidase, beta-galactosidase, and beta-glucosidase. However, by using bacterial cultures grown under hypercapnic conditions and incubating the reaction mixtures aerobically, consistent phenotypic characteristics were obtained, allowing identification similar to that obtained by the ID 32 Strep system. We therefore recommend the phenotypic scheme as an inexpensive, reliable, and convenient method for the initial identification of species of the S. milleri group.

Intraoperative wound contamination during abdominal hysterectomy.

Qualitative bacterial culture of specimens taken from several operative sites during abdominal hysterectomy has not been of value in predicting postoperative infection. We have therefore studied the relationship between the magnitude of contamination and the likelihood of the development of postoperative infection, in the course of a trial comparing the efficacy of cefotetan with ampicillin-plus-metronidazole for chemoprophylaxis in 163 women undergoing abdominal hysterectomy. Forty women who did not receive chemoprophylaxis were also studied. From each patient, an intraoperative sample of pelvic fluid was obtained after closure of the vaginal vault and examined quantitatively for anaerobic, aerobic and microaerophilic bacteria. The incidence of postoperative infectious morbidity (wound infection and febrile morbidity) was correlated with bacterial counts. Analyses by step-up multiple logistic regressions were performed on all the variables and only the total and microaerophilic bacterial counts were significant. Of the 40 patients with total bacterial counts greater than or equal to 10(4) cfu ml-1, 42.5% developed postoperative infectious morbidity, compared with 12.6% of 135 of patients with counts of less than or equal to 10(3) cfu ml-1. The contaminating bacteria were similar to those of the vaginal and skin flora, with anaerobes occurring in 52.9% and microaerophiles in 63.9% of positive fluid samples. Thus, we have concluded that the magnitude of contamination during abdominal hysterectomy is an important determinant in the development of postoperative infectious morbidity.

Comparison of vaginal flora after treatment with a clotrimazole 500 mg vaginal pessary or a fluconazole 150 mg capsule for vaginal candidosis.

The effect of antifungal therapy on the vaginal microbial flora was studied in 23 patients suffering from culture-positive, symptomatic vaginal candidosis. They were randomly allocated to receive either a 500 mg clotrimazole vaginal pessary or a 150 mg fluconazole capsule. Quantitative microbiological examination was carried out on samples of vaginal secretions obtained prior, and at intervals up to 10 days after, treatment. No significant difference was found in the vaginal flora before or after therapy in individual patients or between the treatment groups. In patients with C glabrata or C krusei, the yeasts persisted longer in the vagina with poorer response to either of the medications.

Antibiotic prophylaxis in hysterectomy and induced abortion. A review of the evidence.

By the early 1980s, perioperative prophylaxis in vaginal hysterectomy had been shown consistently to be of such value in reducing postoperative infection that some authors maintained that further placebo-controlled studies were no longer ethical. The benefit of prophylaxis in abdominal hysterectomy was less uniformly demonstrated, in studies which were prospective, placebo-controlled, double-blind and randomised. Prophylaxis may significantly reduce the incidence of febrile morbidity and/or wound/pelvic infection, the duration of hospital stay, or the total usage of antibiotics. It is therefore generally agreed that each centre should itself scientifically evaluate the efficacy of prophylaxis before a decision on its routine use in abdominal hysterectomy is made. In comparative studies, agents which were active against both anaerobic and aerobic organisms were more efficacious than those active against anaerobes only. Antibiotics with similar spectra of activity showed similar efficacy in both types of hysterectomy. Multiple- and single-dose regimens of the same antibiotics also showed equal efficacy. The new cephalosporins with a longer half-life were attractive theoretically as agents in single-dose regimens; ceftriaxone, however, has been shown to have an adverse effect on the normal gut flora. With the increased numbers of induced abortions carried out in the UK and other parts of the world in recent years, the need to reduce postabortal infection is generally appreciated. The results of early studies using tetracyclines as the prophylactic agents were difficult to evaluate because of the incomplete follow-up and different definitions of pelvic infections. No benefit was demonstrated in 2 studies using a single preoperative dose of tinidazole, whereas oral metronidazole in 3 doses and penicillin/pivampicillin for 4 days were shown to be efficacious in reducing postabortal infection. In a recent study with doxycycline, significant benefit was shown in patients with negative preoperative screening for gonococcal and chlamydial infection. These genital infections, together with a history of previous pelvic inflammatory disease (PID)/gonorrhoea, nulliparity with multiple partners, young age of the patient and gestational age have been described as significant risk factors. Some researchers hold the view that selective prophylaxis based on these risk factors should be practised instead of mass prophylaxis. All agree that an antibiotic regimen that is both efficacious and well tolerated has yet to be found.

Fluconazole levels in plasma and vaginal secretions of patients after a 150-milligram single oral dose and rate of eradication of infection in vaginal candidiasis.

Mean peak concentrations of fluconazole in plasma and vaginal secretions of females after a 150-mg single oral dose were shown to be 2.82 micrograms/ml and 2.43 micrograms/g, respectively. Our results indicate that clinically efficacious concentrations of fluconazole in vaginal secretions are easily achieved after this single oral dose.

Single dose therapy of vaginal candidiasis: a comparative trial of fenticonazole vaginal ovules versus clotrimazole vaginal tablets.

An open, randomized comparative clinical trial was performed in 153 patients suffering from symptomatic vaginal candidiasis confirmed by mycological tests. Patients were allocated at random into two groups: the first group (consisting of 75 subjects) was treated with a single vaginal ovule of fenticonazole (600 mg) and the second group (consisting of 78 subjects) was treated with a single vaginal tablet of clotrimazole (500 mg). Therapeutic efficacy was assessed by microbiological and clinical criteria 7 days and 1 month (when possible) after the single dose treatment. At the first follow-up visit, complete disappearance of the signs and symptoms or a highly significant reduction of their intensity was observed in both treatment groups. No significant difference was evident between the two drugs. At 7 days, the mycological tests gave negative results in 92% of the patients in the fenticonazole group and in 88.5% of the patients in the clotrimazole group. The difference between the two treatment groups was again not statistically significant. The second follow-up visit was performed in 55 (73.3%) patients of the fenticonazole group and in 52 (66.7%) patients of the clotrimazole group. The results indicate that 83.6% of patients in the fenticonazole group and 69.2% of patients in the clotrimazole group were still disease free at the time of this visit. Both drugs were well tolerated. Mild, local and short lasting side-effects were reported in only 5 cases of the group treated with fenticonazole.(ABSTRACT TRUNCATED AT 250 WORDS)