Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America.

PURPOSE: To establish the incidence, etiology and risk factors for microbial keratitis (MK) in Hong Kong. METHODS: Two hundred and twenty-three new cases of presumed MK were recruited over a period of 17 months and comprehensive microbiologic studies performed. A nested case-control study was pursued for patients wearing contact lenses (CLW) to determine risk factors for MK with regards to types of CLW and hygiene practice. RESULTS: Of the 223 patients recruited, 59 (26%) wore contact lenses. Corneal scrapes yielded positive cultures from 77 patients (35% overall, 56 non-CLW, 21 CLW). Two hundred and six CLW volunteers were recruited to participate in the case-control study, of whom 135 were matched with 45 CLW patients. The annual incidence of MK was 0.63 per 10,000 population and 3.4 per 10,000 CLW with rates for daily, extended and rigid lens wear of 3.09, 9.30 and 0.44 per 10,000 CLW respectively. Pseudomonas aeruginosa was the dominant bacterial pathogen. Six cases of Acanthamoeba keratitis occurred, five in CLW (incidence 0.33 per 10,000 CLW) and one following corneal abrasion. Non-CLW developed MK at a peak age of 73, which is 10 years younger than expected for Scotland and USA. CONCLUSIONS: Previous ocular surface disease and trauma were the main risk factors for MK in Hong Kong. CLW appears at least as safe as that found in Scotland and the USA. Acanthamoeba keratitis was detected but with an incidence rate five times lower than Scotland. Factors predisposing hydrogel CLWs to MK, that were statistically significant, included overnight wear, poor hygiene and smoking.

18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

Microbial keratitis in Hong Kong: relationship to climate, environment and contact-lens disinfection.

Microbial keratitis has been studied in Hong Kong as a representative sub-tropical climate of south China. An 18-month investigation in 1997/98 of 223 cases of ulcerative keratitis (presumed microbial) was conducted in the 2 million population of Shatin and Kowloon at the Prince of Wales and Hong Kong Eye Hospitals respectively with comprehensive microbiology. A case-control study was pursued at the same time between 45 contact-lens wearers (CLW) developing microbial keratitis and 135 lens-wearing volunteers matched for age, sex, educational status and visual acuity. Home water supplies were sampled for Acanthamoeba. Previous ocular surface disease and trauma (preventable by wearing goggles for grinding) were common predisposing causes while cosmetic wear of contact lenses was responsible for 26% of cases overall. Pseudomonas aeruginosa was the commonest bacterium isolated, from both CLW and non-CLW, with infection being acquired within the community. These 28 pseudomonads remained fully sensitive to the third-generation cephalosporins, aminoglycosides and quinolone antibiotics, which is very encouraging. Fungi were isolated, predominantly Fusarium sp., but less commonly than expected. A fungal/bacterial ratio was obtained of 1/17, while in comparison, the expected ratio for a tropical climate ranges from 1/5 (Singapore) to 1/2 (South India). Acanthamoeba was the second commonest microbe isolated from keratitis of CLW. The domestic water environment of 8% of homes of both patients and controls wearing contact lenses was colonized with Acanthamoeba. Lack of hygiene, use of tap water for storing lenses, failure to air-dry lens-storage cases or use of one-step hydrogen peroxide disinfectant were identified as risk factors for keratitis in CLW. The study results commend use of multipurpose solutions by CLW in Hong Kong to achieve the lowest expected rates of infection.

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.

Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections.

A. baumannii is rarely recovered from the skin of patients or healthy European subjects as other genospecies predominate, but it isa significant nosocomial pathogen. The natural reservoir of this organism is therefore uncertain. We determined the isolation rates of Acinetobacter spp. from vegetables (as an indicator of the natural environment) using a selective technique and classified the genospecies by amplified ribosomal DNA restriction analysis (ARDRA). Of the 177 samples of vegetables examined, 30 yielded Acinetobacter, with genospecies 2 and 11 being the most common, each with a frequency of 27%. MIC assays showed that strains of genospecies 1, 2, 3, and 13TU (the A. calcoaceticus-A. baumannii complex) were significantly more resistant than other genospecies to ciprofloxacin and gentamicin. Vegetables may therefore be a natural habitat of A. baumannii and provide a route by which these bacteria are introduced into hospitals with obvious implications for infection control.

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.

Pyrolysis mass spectrometry of cephalosporin-resistant Enterobacter cloacae.

Thirteen clinical and four environmental isolates of third-generation cephalosporin-resistant Enterobacter cloacae (CREC) together with single isolates from the hands of a nurse and from a blood gas analyser were associated with two clusters of nosocomial infection. With an unrelated CREC isolate they had been typed by serotype, biotype, ribotype and phage-type and were examined by pyrolysis mass spectrometry (PYMS) as described here. PYMS data yielded two clusters, major and minor. All except one isolate in the major cluster corresponded to type group identity (serotype 07, biotype 62, ribotype D) which had caused neonatal sepsis and colonization. Multivariate analysis showed a homogeneous group consisting of this strain plus two outliers. The minor cluster included four different strains, one of which, serotype 03, biotype 62, ribotype C had caused excoriation of the buttocks and colonization.

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.

Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins.

After uneventful use of cefotaxime and ceftazidime as first line therapy for three years in our neonatal intensive care unit we isolated cephalosporin-resistant Enterobacter cloacae (CREC) strains which caused clusters of cases or colonization and/or serious neonatal infection. By using two or more typing methods, at least five different strains with similar patterns of antimicrobial sensitivities were identified. The results of a case-control study did not support the notion that the use of third generation cephalosporins was associated with colonization and infection by CREC. The outbreak was brought under control by interrupting the transmission of the epidemic strain D, by measures such as cohort nursing, diligent handwashing before and after procedures, and thorough environmental cleaning as well as by decontamination with glutaraldehyde after dismantling of the blood gas analyser believed to have acted as a persistent reservoir. Our experience highlights the danger of inadequate supervision and maintenance of equipment used for near-patient testing and the need to monitor such equipment not only in terms of its calibration and analytical performance but also microbiologically.