Rapid diagnosis of tuberculous meningitis by a simplified nested amplification protocol.

We have developed a simplified protocol based on nested polymerase chain reaction (PCR) for early diagnosis of tuberculous meningitis. Using this protocol, we detected the Mycobacterium tuberculosis genome within 24 hours in the CSF of 19 of 21 patients (90%) with clinically suspected tuberculous meningitis. The PCR results were negative in all 79 nontuberculous meningitis controls.

Comparison of polymerase chain reaction and pulsed-field gel electrophoresis for the epidemiological typing of Alcaligenes xylosoxidans subsp. xylosoxidans in a burn unit.

Eighteen isolates of Alcaligenes xylosoxidans subsp. xylosoxidans were collected from clinical specimens of 15 patients in a burn unit and a plastic surgery ward over a 16-month period. Pulsed-field gel electrophoresis and polymerase chain reaction (PCR) were compared for the epidemiologic typing of these 18 isolates and fifteen epidemiologically unrelated strains. These 18 isolates demonstrated an identical fingerprint pattern and were easily distinguished from the 15 epidemiologically unrelated strains by pulsed-field gel electrophoresis typing and both enterobacterial repetitive intergenic concensus and repetitive extragenic palindrome-primed PCR fingerprinting. We conclude that pulsed-field gel electrophoresis analysis of XbaI-digested genomic DNA is a highly discriminatory and reproducible method for epidemiological typing of A. xylosoxidans subsp. xylosoxidans isolates. However, poor resolution due to frequent cutting in the smaller fragments (< 145.5 Kb) may lead to difficulty in interpretation. PCR is a rapid and highly discriminatory, but less reproducible, technique with occasional loss of major bands. The fingerprints produced by repetitive extragenic palindrome primed PCR had more intense bands and were easier to read than those produced by enterobacterial repetitive intergenic concensus-primed PCR in this study.

Comparison of susceptibility to extended-spectrum beta-lactam antibiotics and ciprofloxacin among gram-negative bacilli isolated from intensive care units.

The in vitro activities of extended-spectrum beta-lactam antibiotics (including piperacillin, cefotaxime, ceftriaxone, ceftazidime, cefepime, imipenem, and meropenems) were assessed and compared with the activity of ciprofloxacin against 366 clinical Gram-negative bacilli isolates from the intensive care units of Taichung Veterans General Hospital. The most prevalent species isolated were Pseudomonas aeruginosa and Acinetobacter baumannii. The activities of ceftazidime, cefepime, imipenem, and meropenem against these isolates were comparable to that of ciprofloxacin. Meropenem was found to be the most potent extended-spectrum beta-lactam antibiotic tested and the MIC50s and MIC90s for most of these multiresistant strains were lower than those of imipenem, ceftazidime, and cefepime, except for Stenotrophomonas maltophilia. The extended-spectrum beta-lactam antibiotics that were still active against S. maltophilia were piperacillin and ceftazidime. More than 50% of Enterobacter spp. were resistant to third-generation cephalosporins and piperacillin, but they remained susceptible to carbapenems and cefepime.

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolated in central Taiwan.

Previous studies have suggested that penicillin-resistant pneumococcal isolates (especially those with MIC > 1 microgram/mL) usually are clonally related. To test this hypothesis, the molecular epidemiology of 29 clinical isolates of penicillin-resistant pneumococci (of which 83% were also resistant to either cefotaxime or ceftriaxone) collected in central Taiwan was investigated by pulsed field gel electrophoresis. Twenty-seven distinct patterns were identified. Our results indicate that an increase in penicillin-resistant S. pneumoniae between April 1993 and June 1994 in central Taiwan is not due to the clonal dissemination of a limited number of epidemic strains.

Comparison of polymerase chain reaction and pulsed-field gel electrophoresis for the epidemiological typing of Campylobacter jejuni.

Seventeen sporadic Campylobacter jejuni enteritis cases occurred in Taichung City, Taiwan between July 1995 and September 1995. Pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC-1) primed polymerase chain reaction (PCR) techniques were compared for the epidemiological typing of the 17 C. jejuni isolates. Fourteen distinct PFGE fingerprint patterns were observed. Fifteen distinct PCR fingerprint patterns were demonstrated. Two clusters of isolates (isolates 5 and 6; isolates 10, 11 respectively) were found to be genetically indistinguishable by both methods. In conclusion, we consider that PFGE is a highly reproducible method for determining the relatedness among the C. jejuni isolates in this study, although their limited numbers of restriction fragments may reduce the discriminatory power. Although less reproducible than PFGE typing, ERIC-1 primed PCR can be used as a simple and rapid tool to discriminate different strains of C. jejuni.

DNA fingerprinting of Pseudomonas aeruginosa serotype O11 by enterobacterial repetitive intergenic consensus-polymerase chain reaction and pulsed-field gel electrophoresis.

We report the use of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-based polymerase chain reaction (PCR) to characterize clinical isolates of Pseudomonas aeruginosa serotype O11 collected from an incident of hospital-acquired infection. Both typing techniques differentiated 20 different strain types among seven epidemiologically related isolates and 22 epidemiologically unrelated isolates. There was complete concordance between these two techniques. Our results indicate that the ERIC-based PCR technique represents a rapid and simple means for typing P. aeruginosa serotype O11 with a level of discrimination equivalent to that of PFGE.

Antimicrobial susceptibility of clinical isolates of enterococci.

To describe antimicrobial resistance patterns of Enterococcus spp., we measured minimal inhibitory concentrations (MICs) of five antimicrobial agents for 174 clinical isolates of enterococci collected in Taichung Veterans General Hospital from November, 1996 to January, 1998. Major sources included blood (48), the genitourinary tract (39), soft tissue and wounds (28), and the gynecological tract (16). The sensitivity test of vancomycin was performed using the broth microdilution method. The susceptibility of ampicillin, penicillin, gentamicin, and teicoplanin was tested by the agar dilution method. The MIC90 of penicillin, ampicillin, gentamicin, teicoplanin and vancomycin were 2, 1, > 2,048, 0.125 and 2 microg/mL, respectively. Ampicillin-resistant isolates represented only 4.6% of all enterococci tested. These results reveal that vancomycin-resistant enterococci were found in 1.7% of all enterococci isolates in this study. However, all isolates were sensitive to teicoplanin.

Serratia marcescens renal abscess with endophthalmitis: a case report.

A renal abscess, caused by Serratia marcescens with endophthalmitis in a 68-year-old diabetic female, is described. Endophthalmitis presented with visual loss, conjunctiva injection and lid edema with eye pain. Right costovertebral knocking pain was also noted. Sonography and computed tomography of abdomen showed a 4 cm hypoechoic lesion in the middle portion of the right kidney with marginal enhancement after contrast media injection. Percutaneous abscess drain was performed. Pus culture from the drain tube revealed S. marcescens, yet, vitreous cultures yielded no growth, which was ascribed to previous antibiotics use. Although vitrectomy, fortified eye drops, intravitreal and systemic intravenous antibiotics were administered, the visual function was still lost. To our knowledge, this is the first reported case of S. marcescens renal abscess complicated with endophthalmitis.

Pyogenic psoas abscess: analysis of 27 cases.

From 1993 to 1998, 29 pyogenic psoas abscesses occurring in 27 patients were seen in Taichung Veterans General Hospital. Their age range was 25 to 85 years. Diabetes mellitus was the leading underlying disease. Fever and pain in the flank area, back and hip were the usual manifestations. The duration of symptoms prior to the diagnosis ranged from 3 days to 6 months. Most abscesses were diagnosed by computed tomography (CT) images and proven by abscess cultures, which were divided into primary and secondary types. Eighteen of 29 abscesses were regarded as primary. Staphylococcus aureus was the most common pathogen in the primary abscesses, followed by Streptococcus agalactiae, Escherichia coli, viridans streptococci, S. epidermidis, and Salmonella spp.. In the secondary abscess category, E. coli was the leading organism in this series, followed by S. aureus, Klebsiella pneumoniae, viridans streptococci and Candida albicans. The associated conditions included epidural abscess, osteomyelitis, septic arthritis, perirenal abscess, pulmonary tuberculosis, empyema, hydronephrosis and trauma history. The initial empiric therapy comprised mostly of cefazolin or oxacillin with or without an aminoglycoside. Thirteen patients underwent percutaneous drainage, while six received surgical debridement, including two with a recurrent abscess. One patient had both drainage and debridement. Others received medical treatment only. Two of the patients with primary abscess died in spite of percutaneous drainage. Therefore, open drainage, besides appropriate antibiotic treatment, is still required to control complex abscesses with sepsis.

Invasive Streptococcus pneumoniae infections of children in central Taiwan.

We carried out a retrospective study on childhood invasive pneumococcal infections (IPI) diagnosed from the January 1990 through the April 2000 at a medical center in central Taiwan. Their clinical features, outcome of the patients and the resistance patterns of the isolates were analyzed. A total of 95 clinical isolates from 72 patients younger than 14 years of age were included in this study. Of these 72 patients, 51 had bacteremia, 28 meningitis, 14 bacteremic pneumonia, 12 pleural empyema, eight otitis media, four arthritis, three sinusitis, two periorbital abscesses, one deep neck infection, one psoas muscle abscess, one peritonitis, one urinary tract infection, and one cutaneous infection. Ancillary diagnostic tests, including Gram stain smears and latex agglutination tests, were applied and the sensitivities were 86.2% and 54.3%, respectively. The prevalence rate of penicillin nonsusceptible Streptococcus pneumoniae has increased dramatically since 1995 in central Taiwan, with rates of 5.6% and 74.1% before and after 1995, and the overall mortality rate was 20.8% and 53.3% respectively. Ten of 19 children (52.6%) with pneumococcal meningitis who survived had long-term sequelae.

Comparison of detection of extended-spectrum beta-lactamases by agar dilution method, E-test ESBL screen and double disk test.

The extended-spectrum beta-lactamases (ESBL) are derived from TEM-or SHV-enzymes. They mediate resistance to broad-spectrum beta-lactams and can cause infectious outbreaks in hospitals. Rapid recognition and diagnosis are important for the clinician to prescribe more effective treatment. In the present study, a group of 52 probable ESBL-producing Klebsiella pneumoniae and Escherichia coli having a suspected resistant antibiogram phenotype were included. The E-test ESBL screen and the double disk test were performed for these isolates for detection of ESBL-producing strains, as compared with the conventional agar dilution method. The agreement between the E-test ESBL screen or the double disk test and the conventional agar dilution method was good and the degree of agreement were 86.5% and 92.3% respectively. The results showed that both the E-test ESBL screen and the double disk test were useful and convenient for detection of ESBLs.

Antimicrobial susceptibility and beta-lactamase production of Moraxella catarrhalis isolates in Taiwan.

Between 1 August 1993 and 31 July 1994, 135 clinical isolates of Moraxella catarrhalis were collected from 12 large medical laboratories in Taiwan. The majority of specimens came from sputum (124 isolates). Other specimens included four isolates from throats, three isolates from wounds or pus, two isolates from eyes, one isolate from blood and one from cerebrospinal fluid. Epidemiologically, M. catarrhalis isolates were found frequently in winter and spring with a peak in February, and only sporadically from April to September. The overall rate of beta-lactamase producing isolates was 98.5% (132/135). All isolates were considered to be ampicillin-resistant, none were found to be resistant to other beta-lactam agents. Among other antimicrobial agents, all isolates were susceptible to chloramphenicol, erythromycin, roxithromycin, ofloxacin and ciprofloxacin, but uniformly resistant to trimethoprim (minimum inhibitory concentration (MIC) > or = 4 micrograms/mL, zone diameter < or = 19 mm). There were 12 isolates (8.8%) resistant to sulfamethoxazole (MIC > or = 32 micrograms/mL, zone diameter < or = 19 mm) and 19 isolates (14.4%) resistant to tetracycline (MIC > or = 16 micrograms/mL, zone diameter < or = 19 mm). The high level of resistance to ampicillin due to beta-lactamase production indicates that this is no longer a reliable agent for the treatment of M. catarrhalis infections. Among the beta-lactam agents tested, amoxicillin + clavulanate and the cephalosporins were active. These agents appear to be reliable first-line therapies when infection with M. catarrhalis is suspected. Misidentification of the species and difficulties in determining susceptibility to ampicillin are still widespread in Taiwanese laboratories. The application of the butyrate hydrolysis test and an appropriate test for beta-lactamase production is necessary for the resolution of these problems.

Screening for diseases in elderly persons: the correlation between physical checkup findings and chief complaints.

BACKGROUND: The effectiveness of a physical checkup program for screening for diseases in the elderly, in terms of correlation with their chief complaints has not been previously evaluated. OBJECTIVE AND METHODS: The study was to examine the correlation between physical checkup findings and chief complaints of elderly people. Study subjects were over 65 years of age. There were 792 males and 373 females. They all attended a 2-day physical checkup program at Taichung Veterans General Hospital from January 1, 1994, to December 31, 1995. RESULTS: The results showed a significant difference (p<0.05) between male (12.0%) and female (16.8%) subjects in the relationship of clinical findings to chief complaints. When the locations of chief complaints were compared, those concerning the neck and the limbs corresponded well to our clinical findings. However, there were differences between female and male subjects (42.4 and 24.4%, respectively; p<0.01) in this aspect. There was a good relationship (p<0.05) between physical checkup findings and complaints of lower back pain (75.2%), upper abdominal pain (46.2%) and knee joint pain (38.3%). However, we found that 43.7% of physical checkup findings were not linked to chief complaints. CONCLUSIONS: The results suggest that additional appropriate clinical tests may improve the effectiveness of physical checkups and thus result in health benefits for elderly persons.

Detection of mycobacterial antigens in sputum by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) was evaluated for its usefulness in detecting mycobacterial antigens in sputum. Using anti-mycobacterial saline extract (MSE) and commonly available anti-bacillus Calmette Guerin (BCG) antibodies, Mycobacterium tuberculosis antigens can be detected in sputum by the double-antibody sandwich ELISA. A total of 1904 sputum specimens were tested by acid-fast bacillus (AFB) smear, culture and ELISA. Of 249 pulmonary tuberculosis patients, 225 (90.4%), 167 (67.1%), 114 (45.8%), 103 (41.4%) and (36.9%) were positive by BACTEC 460, 7H11 culture, AFB smear, BCG-ELISA and MSE-ELISA, respectively. If BACTEC 460 positive culture only is considered as the standard, sensitivity of 43.6% and 36.9%, and specificity of 98.2% and 98.3% are achieved, with positive predictive value of 76.0% and 74.1%, and negative predictive value of 92.8% and 92.1% for BCG- and MSE-ELISA, respectively. In 7H11 culture positive cases: 46.7% (BCG) and 43.1% (MSE) for sensitivity, 97.1% (BCG) and 97.7% (MSE) for specificity, 60.5% (BCG) and 64.3% (MSE) positive, 95% (BCG) and 94.7% (MSE) negative predictive values were achieved. When there is an urgent need the ELISA can serve as an alternative aid for the presumptive differentiation of other mycobacteria from M. tuberculosis.

Detection of mycobacterial antigens in cerebrospinal fluid by enzyme-linked immunosorbent assay utilizing anti-mycobacterial saline extract antibodies.

Using anti-mycobacterial saline extract antibodies, Mycobacterium tuberculosis antigens can be detected in cerebrospinal fluid by a highly specific and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The ELISA was dose-dependent and capable of detecting as little as 6.0 ng of antigens. Specificity was demonstrated by negative results (0.063 +/- 0.008) with other bacterial and cryptococcal antigens (n = 9), and maximum cross-reactivity with other mycobacteria was less than 2.0%. Absorbance for six patients with confirmed tuberculous meningitis gave results ranging from 0.168 to 0.898 with a mean value of 0.393 +/- 0.248. For 172 nonmeningitis and nontuberculosis control patients and 6 treated tuberculous meningitis patients, optical densities were 0.064 +/- 0.001 and 0.057 +/- 0.007, respectively. Significant statistical difference (p less than 0.001) was found between the tuberculous meningitis and control groups.

Detection of mycobacterial antigens in cerebrospinal fluid by enzyme-linked immunosorbent assay.

By use of commonly available antibodies against Mycobacterium bovis BCG, Mycobacterium tuberculosis antigens can be detected by a rapid and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The ELISA was dose-dependent and capable of detecting as little as 4 ng of antigens. Absorbance for 5 patients with confirmed tuberculous meningitis ranged from 0.150 to 0.600 with a mean value of 0.271 +/- 0.190. For 134 non-meningitis control patients and 6 treated tuberculous meningitis patients, optical densities were 0.032 +/- 0.009 and 0.029 +/- 0.010, respectively. Specificity was demonstrated by the negative results (0.028 +/- 0.006) with bacterial and cryptococcal antigens. Maximum cross-reactivity with non-tuberculous mycobacterial antigens was less than 7%.

[Effect of implantation of an Ommaya reservoir on prognosis for cryptococcal meningitis].

Cryptococcal meningitis has a high mortality rate of central nervous infection. The patients usually die of the disease itself, or complications from increased intracranial pressure. Early diagnosis and treatment, including surgical drainage, will improve the results. In this series, twenty-one patients with high intracranial pressure (ICP > 300 mmH2 O) are presented. Fourteen received implantation of Ommaya reservoir to aspirate cerebrospinal fluid (CSF) for relief of symptoms of ICP. Meanwhile 4 of these 14 patients also received intraventricular injection of amphotericin B because of poor response to systemic drugs. Another seven patient received systemic drug therapy only. Survival during therapy occurred in 11 of 14 patients in the surgical group, compared with only 1 of 7 patients treated by drug therapy alone (P = 0.019). In the 14 patients who received implantation of an Ommaya reservoir, there was one complication of CSF leakage when the reservoir ruptured because of repeated aspiration. For patients with cryptococcal meningitis with high ICP, early implantation of an Ommaya reservoir will improve the survival rate.

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis.

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.

Bacteremia in renal transplant recipients: retrospective analysis of 60 episodes in a teaching hospital.

BACKGROUND: Bacteremia is a major cause of death in renal transplant recipients. However, an understanding of the clinical spectrum of bacteremia post kidney transplantation in Taiwan is lacking. This study was undertaken in an attempt to analyze the causative organisms, the sites of primary infection, and the possible significant determinants of mortality. METHODS: Eighty-six positive blood cultures from renal transplant recipients were seen at the Taichung Veterans General Hospital from August 1993 to April 1999. Sixty blood cultures obtained from episodes that occurred in 43 kidney recipients were regarded as significant and included. All the data were obtained by reviewing medical records. RESULTS: Forty-six episodes (76.7%) of bacteremia happened 6 months after transplantation. Gram-negative bacilli accounted for 73.3% (44/60) of the cases of bacteremia. Escherichia coli, constituting 26.7% (16/60) of the etiologic agents, was the most common pathogen. In 50.0% of all episodes, urinary tract infection was the main cause of bacteremia. The overall mortality rate in this series was 16.7%. Risk of death seemed higher if bacteremia occurred with a primary site of infection other than the urinary tract (26.7% vs 6.7%) and after methylprednisolone pulse therapy (33.3% vs 12.5%), though both correlations were not statistically significant. CONCLUSIONS: This study may shed light on the empirical therapy and better management needed for treating bacteremia in renal transplant recipients in Taiwan and provide a nidus for further prospective surveys.

In vitro antimicrobial activity of levofloxacin against Streptococcus pneumoniae.

BACKGROUND: Infections caused by Streptococcus pneumoniae continue to be a significant cause of mortality and morbidity in humans. Diseases caused by multi-resistant pneumococci are increasing rapidly worldwide. The fluoroquinolones have been widely used clinically to treat infectious diseases. The results of a study here on the five fluoroquinolones susceptibilities of S. pneumoniae are reported from the Taichung Veterans General Hospital. METHODS: Minimum inhibitory concentrations (MICs) of five quinolones (enoxacin, norfloxacin, ofloxacin, levofloxacin and ciprofloxacin) were determined for 106 strains of S. pneumoniae. All MICs were determined by the agar dilution method utilizing Mueller-Hinton agar supplemented with 5% sheep blood. RESULTS: MIC90 of levofloxacin was 1 microgram/ ml, and was unaffected by penicillin-susceptibility. MIC90 of ofloxacin and that of ciprofloxacin were 2 and 4 micrograms/ml, respectively, with 90.6% sensitive to ofloxacin. MIC90 of enoxacin and that of norfloxacin were higher than other compounds. CONCLUSIONS: The in vitro activity of levofloxacin is twice that of ofloxacin, 4-fold of ciprofloxacin, 16-fold of norfloxacin, and 64-fold of enoxacin. MICs of these five quinolones were unaffected by penicillin-susceptibility. The antibacterial activity of levofloxacin was better than that of ofloxacin and ciprofloxacin, norfloxacin, or enoxacin against S. pneumoniae.