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Background: There has not been a comprehensive multi-center study investigating the microbial profile of community acquired pneumonia (CAP) in Taiwan., Methods: A prospective study of adult CAP patients requiring hospitalization between December 2001 and April 2002 was carried out in 13 hospitals in Taiwan. Etiology was determined based on laboratory data from blood and sputum cultures plus serology from paired serum and urine antigen detection tests., Results: Etiology was assigned to 99 (58.9%) of the 168 patients having the most complete data for etiology determination, with mixed infection in 21 (12.5%) patients. More than half (51.8%) of the patients were>60 years and 63.7% of the patients were males. The most common etiologic agent was Streptococcus pneumoniae (40, 23.8%), the majority (60%, 24 cases) of which was detected by positive urine antigen test. Other common agents included Mycoplasma pneumoniae (24, 14.3%), Chlamydia pneumoniae (12, 7.1%), Influenza A virus (11, 6.5%), Klebsiella pneumoniae (8, 4.8%) and Haemophilus influenzae (8, 4.8%). The prevalence of S. pneumoniae and M. pneumoniae was highest in patients>60 years (25/87, 28.7%), and<44 years (12/59, 19%), respectively; while K. pneumoniae comprised a larger proportion (4/22, 18%) in the 45-59 years group., Conclusions: S. pneumoniae was the most common etiology agent in adult patients hospitalized due to CAP in Taiwan and the spectrum of other major pathogens was similar to studies conducted elsewhere in the world. Empiric treatment recommendations developed in other parts of the world may be appropriately adapted for local use after taking into account local resistance profiles. Our data also support the recommendation that urine antigen test be added as an adjunct to adult CAP etiology diagnosis protocol.

We report the prevalence and characteristics of Salmonella strains resistant to ciprofloxacin and extended-spectrum cephalosporins in Taiwan from January to May 2004. All isolates resistant to extended-spectrum cephalosporins carried blaCMY-2, and all ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates were genetically related.

To understand the Candida colonization of human immunodeficiency virus (HIV)-infected outpatients in Taiwan, we have conducted a prospective cohort study of Candida colonization and its risk factors at the National Taiwan University Hospital from 1999 to 2002. More than 50% of the patients were colonized with Candida species, and 12% developed symptomatic candidiasis. Patients colonized with fluconazole-resistant strains of Candida species had a higher prevalence of candidiasis than those colonized with susceptible strains. Our analysis found that antibiotic treatment and lower CD4(+) counts (<200 cells/mm(3)) increased the rate of oropharyngeal candidiasis in HIV-infected patients, while antiretroviral therapy protected patients from the development of candidiasis.

Klebsiella are well-recognized community and nosocomial pathogens responsible for septicemias, urinary tract infections, pneumonia, and soft tissue infections. The emergence of multi-drug resistant Klebsiella is becoming a global concern. Since fluoroquinolones are excellent antibiotics for clinical therapy of complicated infections, their consumptions have increased rapidly. Coincidental with the increased usage of fluoroquinolones, the incidences of drug resistance have increased, which highlights the need for understanding the molecular mechanisms of fluoroquinolone resistance. Alterations in targets and reduction of intracellular drug accumulation are two major mechanisms involved in fluoroquinolone resistance. This review focuses on the mechanisms of action of fluoroquinolones and molecular mechanisms of drug resistance, including known and proposed ones, and the discussion on clinical impact of multi-drug resistance in Klebsiella.

In the Taiwan Surveillance of Antimicrobial Resistance programme, isolates were collected from 21 hospitals over a 3-month period in 2000 (TSAR II) and rates of resistance in Gram-positive pathogens were determined. Resistance rates were high including oxacillin resistance in Staphylococcus aureus (60%) and coagulase-negative staphylococci (80%), high-level gentamicin resistance (HLGR) in Enterococcus faecalis (60%) and penicillin non-susceptibility in Streptococcus pneumoniae (69%). Oxacillin resistance had increased from 1998 (TSAR I) and may be spreading into outpatient settings. In contrast, less than 2% enterococci were vancomycin-resistant. No linezolid resistance was found in either staphylococci or enterococci.

In a nationwide surveillance of antimicrobial resistance (Taiwan Surveillance of Antimicrobial Resistance, TSAR), isolates were collected from 21 medical centers and regional hospitals throughout Taiwan over a three-month period in 2000 (TSAR II). This report summarizes susceptibility data of 7 common Gram-negative bacilli (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii) in the TSAR II collection and compared selected key forms of resistance by epidemiologic factors and with isolates collected in 1998 (TSAR I) as well as with data from international surveillance studies. Resistance of the 5 Enterobacteriaceae species to most of the commonly prescribed "first-line" antimicrobials in Taiwan, such as ampicillin (78% in E. coli, 68% in P. mirabilis), gentamicin (19% in K. pneumonia to 66% in S. marcescens), and trimethoprim/sulfamethoxazole (29% in K. pneumoniae to 70% in P. mirabilis), was high, several of which are higher than other countries. Resistance to certain broad-spectrum antimicrobials is also more acute in Taiwan than most Western countries, such as ceftazidime resistant A. baumannii (73%) and ciprofloxacin resistant E. coli (12%). Differences in geographic regions and specimen types were associated with certain forms of resistance in TSAR II; however, the resistance problem is prevalent among both inpatients and outpatients of not only medical centers but also regional hospitals throughout Taiwan.

In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters of < or = 15 mm, 16-26 mm, and > or = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.

To better understand the epidemiology of bacterial pathogens with particular public health importance in Taiwan, we determined the prevalence of nasal colonization with methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in a cohort of HIV-infected patients attending two hospital outpatient departments. All HIV-infected patients followed regularly between May and September 1999 were enrolled and cultures of the anterior nares were performed using a dry sponge swab. All confirmed S. aureus isolates underwent antimicrobial susceptibility testing using disk diffusion according to recommendations of the National Committee for Clinical Laboratory Standards. Of a total of 162 outpatients studied, 48 (30%) were found colonized with S. aureus including 39 (24%) colonized with MSSA and 9 (6%) colonized with MRSA. The only factor associated with MSSA colonization was receipt of trimethoprim-sulphamethoxazole which appeared protective (relative risk 0.4, 95% confidence interval [CI(95)] 0.2-0.78, P = 0.006). In contrast, ciprofloxacin use was an independent risk factor for MRSA colonization (conditional odds ratio [OR] 11.9, CI(95) 1.8-77.8, P = 0.010) along with a one-quartile reduction in CD(4) count (OR 3.9, CI(95) 1.1-14.3, P = 0.04). Although MRSA colonization was not associated with hospitalization within the previous three months, the multi-drug resistance pattern of MRSA isolates suggests strains were at some point acquired in the healthcare setting. Our study shows that the rate of S. aureus colonization in Taiwanese HIV-infected outpatients is 30%. Low CD4+ counts are most likely associated with other unmeasured risk factors for MRSA. Antimicrobial use may function alternatively as a protective or risk factor for colonization with S. aureus, depending upon the drugs involved and resistance encountered. Fluoroquinolone use may have an important role in the spread of MRSA from inpatient to outpatient settings.

Vancomycin resistant enterococci (VRE) with VanB phenotype-vanA genotype incongruence were found in all 39 VRE isolated from chicken carcasses and four human VRE isolates in Taiwan. Three identical mutations in the vanS gene were found in the VanB phenotype-vanA genotype VRE sequenced. This finding indicates possible transmission of glycopeptide resistance among different hosts.

A survey of 1,203 Escherichia coli isolates from 44 hospitals in Taiwan revealed that 136 (11.3%) isolates were resistant to fluoroquinolones and that another 261 (21.7%) isolates had reduced susceptibility. Resistance was more common in isolates responsible for hospital-acquired (mostly in intensive care units) infections (17.5%) than in other adult inpatient (11.4%; P = 0.08) and outpatient isolates (11.9%; P > 0.1). Similarly, reduced susceptibility was more common in isolates responsible for hospital-acquired infections (30.9%) than in other adult inpatient (21.0%; P = 0.04) and outpatient (21.4%; P = 0.06) isolates. Isolates from pediatric patients were less likely to be resistant (1.3 versus 12.0%; P < 0.01) but were nearly as likely to have reduced susceptibility (17.7 versus 21.9%; P > 0.1) as nonpediatric isolates. There was an inverse relationship in the proportion of isolates that were resistant versus the proportion that had reduced susceptibility among isolates from individual hospitals (R = 0.031; P < 0.05). In an analysis of isolates from two hospitals, all 9 resistant strains possessed double point mutations in gyrA and all 19 strains with reduced susceptibility strains had single point mutations; no mutations were found among fully susceptible strains. Risk factors for resistance included underlying cancer (odds ratio [OR], 83; 95% confidence interval [CI(95)], 7.3 to 2,241; P < 0.001), exposure to a quinolone (OR, undefined; P = 0.02), and exposure to a nonquinolone antibiotic (OR, 20; CI(95), 2.2 to 482; P < 0.001); underlying cancer was the only independent risk factor (OR, 83; CI(95), 8.6 to 807; P < 0.001). There were no significant associations between any of these factors and reduced susceptibility. Whereas acute and chronic quinolone use in cancer patients is a major selective pressure for resistance, other undetermined but distinct selective pressures appear to be more responsible for reduced susceptibility to fluoroquinolones in E. coli.

There are increasing public health concerns about antibiotics used in food-producing animals that may contribute to the development of resistance in human pathogens. Such resistance may be critical to human medicine when resistance develops to drugs that treat certain pathogens of which there is no good alternative therapy. We surveyed 10 farms, eight feed mills, and one animal drug distributor in Taiwan to determine the major antibiotics used in food-producing animals, and the extent of use of five drugs that may select for resistance to antibiotics that are critical for human medicine. The five animal drugs, and the resistance of human drug/class they may select for, included avoparcin (vancomycin/glycopeptides), avilomycin (ziracin/envirninomycins), enrofloxacin (ciprofloxacin/fluoroquinolones), virginiamycin (quinupristin and dalfopristin combination/streptogramins), and kanamycin (gentamicin/aminoglycosides). Tetracyclines were the class of antibiotic that was most widely used in the greatest amounts. Over the past 12 months, the number of farms, chicken feed mills, and pig feed mills, that have respectively reported the use of avoparcin was 1 (10%), 5 (63%), 0; avilomycin 0, 0, 3 (50%); enrofloxacin 4 (40%), 1 (13%), 3 (50%); virginiamycin 2 (20%), 5 (63%), 0; and kanamycin 3 (30%), 1 (13%), 1 (17%). We conclude that although the most commonly used antibiotics (ie tetracyclines) have little effect on human medicine, there is a widespread use of antibiotics that may select for critical forms of resistance in human pathogens in food-producing animals.

Nine isolates of Klebsiella pneumoniae, obtained from one colonized and eight bacteraemic patients on a paediatric ward, were shown to be identical by PFGE, indicating an outbreak. Screening for extended-spectrum beta-lactamase (ESBL) production using the double-disc synergy test, Etest for ESBLs and agar diffusion tests indicated ESBL production. The isolates showed reduced susceptibility to cefotaxime but not to other third-generation cephalosporins. Molecular studies revealed production of TEM-1 and SHV-1 but no ESBLs were identified. Deficiency in expression of an outer membrane protein (OmpK35) was also observed. These observations led us to postulate that the extremely low level of OmpK35 expression and the co-existence of TEM-1 and SHV-1 resulted in an increased MIC of cefotaxime and the false designation of the isolates as ESBL producers. All the infected infants were treated with either third-generation cephalosporins alone or multiple antibiotics including a third-generation cephalosporin, and recovered and were discharged without sequelae.

For the first national surveillance of antibiotic resistance in Taiwan, we collected in 1998 from 22 hospitals (6 medical centers, 15 regional hospitals, and 1 local hospital) 3,211 isolates in all parts of the country. Besides 50 random successive isolates from inpatients, each hospital was requested to collect 25 isolates each from positive blood cultures, hospital-acquired infections, outpatients and the pediatric department. We re-speciated all the submitted specimens and determined their antibiotic susceptibility patterns. The most common isolates were Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae), Staphylococcus aureus, and Pseudomonas aeruginosa. Among hospital-acquired infections, Acinetobacter spp. were among those which accounted for over 10% of the isolates. The oxacillin resistance of S. aureus was 82% in isolates from hospital-acquired infections, and 40% from outpatients. Among Enterococcus spp., 85% were either E. faecalis or E. faecium. They were 14% resistant to vancomycin. Among gram-negative bacteria, K. pneumoniae and Acinetobacter baumanii were hospital-acquired isolates that were most clearly more resistant than community acquired isolates. This difference was less apparent in the case of Enterobacter cloacae, Serratia marcescens, and P. aeruginosa. These bacteria were generally more resistant from all sources. Fifty-one percent of Salmonella were resistant to ampicillin; however, these were all sensitive to ciprofloxacin. Isolates from the East were least resistant. Plotting the disc zone diameters of antibiotics within the susceptible range, we identified subpopulations with smaller diameters in the case of vancomycin against S. aureus, ciprofloxacin against E. coli, and ciprofloxacin against Salmonella spp. These findings represent one of the purposes of this surveillance as they may portend developing resistances which bear careful watching in the future.

Screening for sexually transmitted diseases (STDs) in a greater proportion of sexually active patients has become an accepted protocol by most health care providers. The purpose of this study was to compare the current test methods for detection of Chlamydia trachomatis used at the University of South Alabama, the PACE 2 assay (Gen-Probe) and the Clearview EIA (Wampole Laboratories), with two amplification technologies, the AMP CT (Gen-Probe) and LCx (Abbott) assays. In addition, a number of demographic parameters were ascertained by asking questions at the time of examination as well as for health care provider concerns and preferences. One urine and four endocervical swab specimens were collected in random order from 787 female patients attending one of four obstetrics-gynecology clinics. Eighty-seven percent of patients had no STD-related symptoms. Patients were considered positive for C. trachomatis if three or more assays (swab and/or urine) were positive. Abbott and Gen-Probe confirmed discrepant results by alternate amplified assays. A total of 66 true-positive specimens were detected by use of the combination of endocervical swabs and urine specimens. After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Sensitivities for the LCx and AMP CT assays with urine specimens were 98 and 81%, respectively. The prevalence of C. trachomatis was 8.4%, as determined by amplification technology. Overall, the amplification technologies were the most sensitive methods with either swab (AMP CT assay) or urine (LCx assay) specimens. The PACE 2 assay offered the advantage of a simpler and less expensive assay with acceptable sensitivity. The clearview CT EIA, while yielding a rapid in-office result, had unacceptably low sensitivity. The wide variation in performance with amplification assays with urine specimens as reported in both this study and the literature obviates the need to clarify optimal parameters for this specimen type.

The Air Thermal Cycler (ATC) (Idaho Technology, Idaho Falls, Idaho) utilizes the unique technology of small-volume glass capillary tubes and high-velocity air for the heating and cooling medium for the PCR. Standard heat block thermal cycler (HBTC) and ATC performance characteristics were compared for the detection of Mycobacterium tuberculosis. Sensitivity was 100% for all smear-positive, M. tuberculosis culture-positive specimens for both the HBTC and the ATC. Of smear-negative, M. tuberculosis culture-positive specimens, sensitivity was 42.9% with the HBTC and 22.0% with the ATC. Specificity was 100% for both assay systems. Total assay time was 6.5 and 4 h and the reagent cost was 84 and 32 cents for the HBTC and ATC, respectively. The ATC offered an excellent alternative to the traditional HBTC for diagnosis of M. tuberculosis in smear-positive specimens by PCR.

A comparison of the Bactec 9240 (Becton-Dickinson, Sparks, Md.) and Difco ESP (Difco Laboratories, Detroit, Mich.) instruments for the detection of organism growth from vials whose entry was delayed was evaluated. The instruments' capabilities for organism recovery, time to detection, rates of false-positive results, and numbers of vials in which growth was not detected were made by using seeded blood culture vial pairs and controls with and without delayed entry. Bactec 9240 and Difco ESP aerobic and anaerobic vials were inoculated with human blood and were seeded with organism growth from 18 species, including obligate aerobic, anaerobic, and facultative anaerobic organisms. Each organism was tested in triplicate at 0, 8, 24, 36, and 48 h and was incubated at both room temperature (RT) and 35 degrees C. Two separate phases of the study were performed, each with a different version of Bactec 9240 software. Overall, detection of growth in vials with delayed entry into either the Bactec 9240 or the Difco ESP instrument resulted in an increased total time to detection with incubation at both RT and 35 degrees C compared with the total time to detection for nondelayed vials. However, false-positive results and vials in which growth was not detected were minimal, and delayed entry did not require routine entry or exit subcultures for either system. Analysis of individual time points and incubation temperatures for the detection of all organisms suggested that Difco ESP vials delayed by up to 8 h may be incubated at 35 degrees C (100% detection) and vials delayed for longer than 8 h may remain at RT. Bactec 9240 vials may be incubated at 35 degrees C for up to 24 h with a minimal loss of detection (97.9% detection), and vials delayed for more than 24 h should remain at RT for optimal recovery of organism growth.

Six hundred and sixty-six bovine and fifty-seven swine clinical isolates of E. coli from New York state were examined for the presence of enterotoxins (STaP, STb, LT, SLT-I, and SLT-II) and adhesins (K88, K99, F41, and 987P) using colony hybridization techniques. Three hundred and sixty-seven of the bovine isolates (45.2%) hybridized with at least one gene probe. Of these, two hundred and twenty-three (33.2%) hybridized with F41, one hundred twelve (16.7%) with K99, eighty-two (12.2%) with 987P, ninety-six (14.3%) with STaP, seven (1.1%) with STb, and none (0.0%) with LT and K88. A total of thirty-three (4.7%) of the isolates hybridized with SLT-I, and one (0.1%) with SLT-II. The major pathotypes among the 666 isolates from bovine were K99/F41/StaP (9.8%), K99/F41 (2.5%), p87P/F41 (2.1%) and 987P/K99/F41/StaP (1.4%). Of the swine clinical isolates, twenty-two hybridized with at least one gene probe. The major pathotypes among the isolates from piglets were K88/K99/F41/StaP (5.3%) and K88/F41 (5.3%).

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi. This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD. The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.