Monitoring the performance of mycobacteriology laboratories: a proposal for standardized indicators.

SETTING: Thailand Tuberculosis (TB) Active Surveillance Network: Bangkok, Chiang Rai, Phuket, Tak and Ubon-Ratchathani, Thailand. BACKGROUND: Mycobacteriology laboratories in resource-limited, high TB burden settings are expanding to perform conventional solid media culture and broth-based mycobacteriology culture. Indicators that measure how well a laboratory performs sputum microscopy have been developed and broadly implemented. Routine monitoring of sputum culture performance, however, is not as common. DESIGN: We implemented indicators for monitoring the quality of laboratory services in five province-level mycobacteriology culture facilities in Thailand. These indicators were derived from literature review, consultation with subject matter experts and our program experience. CONCLUSIONS: We believe that an international consensus document providing monitoring guidelines for mycobacteriology laboratories is urgently needed.

Third generation cephalosporin-resistant gram-negative bacilli in the feces of hospitalized children.

In view of the widespread use of third generation cephalosporins in hospitalized infants, we attempted to determine whether their use was associated with the emergence of resistance in fecal Gram-negative bacilli. Stools from infants hospitalized for varying durations were cultured on MacConkey agar containing 4 micrograms/ml of cefotaxime. All isolates growing on this medium were identified and their susceptibilities to 29 antimicrobial agents were determined. Sixty-five infants were studied of whom 44 were receiving a third generation cephalosporin, 7 another antibiotic and 14 no antibiotic. Thirty-one strains resistant to third generation cephalosporins (minimal inhibitory concentrations > or = 16 micrograms/ml) to cefotaxime, ceftriaxone or ceftazidine) were isolated from 26 infants. The proportions of infants with resistant strains were not significantly different whether they were: (1) receiving a third generation cephalosporin or not; (2) hospitalized for longer or shorter than 2 days or not; (3) older or younger than 3 months or not. Notably 8 infants harbored resistant strains within 24 hours of admission. The commonest resistant strains isolated belonged to the genera Enterobacter (10), Citrobacter (6), Serratia (3), Cedecea (3) and Chromobacterium (3). In conclusion hospitalized infants had a high incidence of fecal colonization with Gram-negative bacilli resistant to third generation cephalosporins. These bacteria were predominantly those known to produce broad spectrum beta-lactamases. This colonization was not necessarily associated with the infant receiving such antibiotics or with prolonged hospitalization.

Basic microbiologic support for hospital epidemiology.

The laboratory plays a major role in the epidemiology program's efforts to minimize nosocomial infections in healthcare institutions. This article will describe some of the interactions between the laboratory and the epidemiology program, and will identify resources and procedures that the laboratory needs to achieve epidemiologic goals.

Evaluation of modified MicroScan screening tests for high-level aminoglycoside resistance in Enterococcus faecalis.

Enterococcus faecalis strains that are refractory to aminoglycoside-penicillin synergistic killing can be predicted by their ability to grow in high concentrations of aminoglycoside (2,000 mg/L) or by disk diffusion tests using high content aminoglycoside disks. Forty-eight well-characterized clinical isolates of E. faecalis were used to evaluate recently reformulated gentamicin and streptomycin synergy screening tests on both overnight and rapid MicroScan (MS) Pos MIC panels (Baxter, West Sacramento, CA). The results obtained with the MS screening tests were compared with those from modified agar disk diffusion results. Discrepancies were resolved by an in-house broth microdilution method. The MS overnight tests detected 25 of 25 (100%) gentamicin high-level resistant strains and 23 of 24 (96%) streptomycin high-level resistant strains. No false high level resistance was found with the MS overnight tests. The MS rapid tests detected 100% of the gentamicin and 100% of the streptomycin high-level resistant strains, but one isolate was falsely high-level resistant to gentamicin (96% specific). The reformulated MS synergy screening tests were acceptable alternatives to disk diffusion for detection of high level aminoglycoside resistance in E. faecalis.

The impact of a clinic for adults with HIV infection on the microbiology laboratory.

The Infectious Diseases Clinic (IDC) discussed serves adults who are seropositive for human immunodeficiency virus. The authors reviewed the outpatient and inpatient microbiology tests of a three-month period during 1989 for a systematic sample of IDC patients. The 249 patients in the sample had 682 microbiology tests performed during the period (mean 2.7 tests per patient). Tests most frequently requested were mycobacterial culture, routine blood culture, and cryptococcal antigen determination. Patients with acquired immunodeficiency syndrome (43% of IDC patients) accounted for 63% of the requested IDC tests. IDC patients comprised about 2.4% of patients served but accounted for 3.9% of the requested microbiology tests and 6.6% of the microbiology work load for reported tests. Using Centers for Disease Control case projections, the authors estimated that services to IDC patients in 1993 would comprise 6.6% of all microbiology tests and 10.6% of the microbiology work load. The implications of these data for microbiology probably also apply to other laboratory testing and emphasize the need for more efficient ways to use and perform diagnostic studies required by patients with HIV infection.

Rapid detection of mycobacteria in inflammatory necrotizing granulomas from formalin-fixed, paraffin-embedded tissue by PCR in clinically high-risk patients with acid-fast stain and culture-negative tissue biopsies.

A collection of inflammatory necrotizing granulomas (INGs) negative by acid-fast stain and culture (AFSC) were analyzed by polymerase chain reaction (PCR) for the presence of mycobacteria. Forty-two paraffin-embedded specimens with INGs were collected from patients at high risk for contracting tuberculosis. Twenty biopsies were positive and 22 were negative for mycobacteria by AFSC. Two universal primers specific for all mycobacteria were used to detected a 414 base pair (bp) fragment of 16S rRNA gene. Twenty of 20 biopsies were positive for mycobacteria by both AFSC and PCR (100%), whereas 19 of 22 biopsies negative by AFSC were positive by PCR (86%). Follow-up of patients who were PCR positive but AFSC negative identified nine patients who had subsequent biopsies. Specimens from eight of these nine patients eventually grew Mycobacterium tuberculosis. Our results demonstrate that the detection of mycobacterial DNA by this method should be used in conjunction with AFSC for the initial diagnosis of mycobacterial infection.

Childhood mortality during and after hospitalization in western Kenya: effect of malaria treatment regimens.

Plasmodium falciparum infection is an important cause of the high childhood mortality rates in sub-Saharan Africa. Increasingly, the contribution of P. falciparum-associated severe anemia to pediatric mortality is being recognized while the impact of chloroquine resistance on mortality has not been evaluated. To address the issues of pediatric mortality, causes of death among hospitalized children less than five years of age in western Kenya were identified using standardized clinical examinations and laboratory evaluations. Follow-up examinations were conducted to determine the child's clinical status posthospitalization. Of the 1,223 children admitted to Siaya District Hospital from March to September 1991, 293 (24%) were severely anemic (hemoglobin level < 5.0 g/dL). There were 265 (22%) deaths; 121 (10%) occurred in-hospital and 144 (13%) occurred out-of-hospital within eight weeks after admission; 32% of all deaths were associated with malaria. Treatment for malaria with chloroquine was associated with a 33% case fatality rate compared with 11% for children treated with more effective regimens (pyrimethamine/sulfa, quinine, or trimethoprim/sulfamethoxazole for five days). The risk of dying was associated with younger age (P < 0.0001) and severe anemia (relative risk [RR] = 1.52, 95% confidence interval [CI] = 1.22, 1.90), and was decreased by treatment with an effective antimalarial drug (RR = 0.33, 95% CI = 0.19, 0.65). Effective drug therapy for P. falciparum with regimens that are parasitocidal in areas with a high prevalence of severe anemia and chloroquine resistance can significantly improve the survival of children in Africa.

PIP: Plasmodium falciparum infection is an important cause of the high childhood mortality rates in sub-Saharan Africa. Causes of death among hospitalized children less than age 5 years in western Kenya were identified using standardized clinical examinations and laboratory evaluations. Follow-up examinations were then conducted to determine the child's clinical status posthospitalization. 293 of the 1223 children admitted to Siaya District Hospital during March-September 1991 were severely anemic. 265 children died; 32% of the deaths were associated with malaria. 121 of the deaths occurred in-hospital and 144 out-of-hospital within 8 weeks after admission. The treatment of malaria with chloroquine was associated with a 33% case fatality rate compared with 11% for children treated with more effective regimens of pyrimethamine/sulfa, quinine, or trimethoprim/sulfamethoxazole for 5 days. The risk of dying was associated with younger age and severe anemia, and was decreased by treatment with an effective antimalarial drug.

Clinical and programmatic mismanagement rather than community outbreak as the cause of chronic, drug-resistant tuberculosis in Buenaventura, Colombia, 1998.

SETTING: Buenaventura, Colombia. OBJECTIVE: To assess whether antituberculosis drug resistance was generated by poor management or community transmission. DESIGN: Treatment-failure and new tuberculosis (TB) patients identified between May 1997 and June 1998 were interviewed and their treatment histories reviewed. Bacteriologic testing, including drug susceptibility profiles (DSP) and DNA fingerprinting by restriction fragment length polymorphism (RFLP), was performed and human immunodeficiency virus (HIV) testing was offered. RESULTS: DSP and RFLP fingerprints were obtained for isolates from 34 of 64 treatment-failure patients; 25 (74%) were resistant to > or = one drug. Fifteen of the 25 patients consented to HIV testing; none were positive. An average of 2.8 major treatment errors per patient was identified. RFLP from the treatment-failure patients revealed 20 unique isolates and six clusters (isolates with identical RFLP); 4/6 clusters contained isolates with different DSP. Analysis of the RFLP from both treatment-failure and new patients revealed that 44/111 (40%) isolates formed 18 clusters. Four of 47 (9%) new patients had multidrug-resistant TB (MDR-TB). Eleven isolates belonged to the Beijing family, related to the MDR strain W. CONCLUSION: Drug resistance in Buenaventura results from both poor management and community transmission. Dependence on DSP to identify TB transmission is inadequate when programmatic mismanagement is common.

Penicillin-resistant pneumococci--an emerging threat to successful therapy.

Pneumococci highly resistant to penicillin G [minimum inhibitory concentration (MIC) > or = 2 mg L-1] have become prevalent in many parts of the world since their emergence and spread in the late 1970s. In the USA, such organisms are seen primarily in two populations: infants and children, and adults with AIDS. Surveys in both rural and urban areas have revealed presence of these organisms, as well as an increasing frequency of Streptococcus pneumoniae strains relatively resistant to penicillin (MIC 0.1-1.0 mg L-1--now defined by some as 'intermediate' resistance). Predisposing factors are not yet clear. Prior antimicrobial therapy was given to some of the children and most of the adults who are colonized or infected with resistant strains. Prior or concurrent use of cotrimoxazole prophylaxis for Pneumocystis carinii pneumonia has been frequent in our cases in adults, most of whom had a concurrent diagnosis of AIDS. Children with disease often have a history of long-term prophylaxis with a beta-lactam drug (for sickle cell disease, etc). Many strains are also resistant to newer cephalosporins like cefotaxime and ceftriaxone (MIC > or = 2 mg L-1). The organisms are frequently multi-resistant, with high MIC values common as well for chloramphenicol and variable for tetracycline, macrolides, cotrimoxazole, and fluoroquinolones. Only to vancomycin are the organisms consistently susceptible. These findings raise alarms about the future of pneumococcal disease in both community and nosocomial disease. Increasing prevalence in otitis and pneumonia in children and in community-acquired pneumonia in adults may lead to use of vancomycin as empirical therapy for these clinical situations. This would increase the selective pressure for emergence of vancomycin-resistant organisms, whether S. pneumoniae or others. Moreover, the pneumococcus was a common cause of hospital infection prior to the introduction of penicillin. The potential now exists for nosocomial pneumococcal infection again to become a feared and ominous occurrence.

Short-course treatment of typhoid fever with ciprofloxacin in south India.

This study assessed the performance of short-course ciprofloxacin for the treatment of 34 adult patients with culture-positive typhoid fever. Patients received ciprofloxacin, 750 mg orally twice daily for 7 d. Measurement of response was based upon time from initial treatment to fever lysis, to afebrile state, and to symptom resolution. Ciprofloxacin-treated patients defervesced in a mean of 3.21 d (+/- 0.56), with stabilization of temperature in 4.0 +/- 0.73 d. After 90 d follow-up, no relapse or carrier was identified. Side effects during therapy were minimal.

Laboratory diagnosis of tuberculosis: past, present, and future.

Resurgence of tuberculosis justifies extraordinary efforts to expedite TB diagnosis and susceptibility testing. This demands that laboratory support expand to a "second generation" of methods and procedures, including rapid availability of fluorochrome smears of concentrated specimens, faster techniques for detection (e.g., the BACTEC radiometric broth system and microcolony detection), quicker identification (e.g., high-pressure liquid chromatography, nonisotopic genetic probes), more rapid susceptibility testing methods (e.g., BACTEC), and reporting of these results as critical values. Guidelines have been established for turnaround time for results of smears, TB organism identification, and susceptibility testing to usual first-line drugs. A "third generation" of laboratory techniques soon will make testing not only more effective but also more efficient. These methods include direct testing of respiratory specimens through nonisotopic genetic probes as well as nucleic acid amplification techniques utilizing polymerase chain reaction (PCR) and other molecular procedures. These new procedures and protocols place heavy demands on laboratory test volume, technologist time and costs. For the healthcare system or clinical laboratory without the resources to deal with these new demands, referral of TB specimens represents a reasonable alternative, as long as transport is adequate to meet current CDC and other guidelines for turnaround time.

Rifampicin-resistant Mycobacterium tuberculosis: susceptibility to isoniazid and other anti-tuberculosis drugs.

Based on data from 14 Supranational Tuberculosis (TB) Reference Laboratories worldwide, the proportion of rifampicin (RMP) resistant isolates that were isoniazid (INH) susceptible by phenotypic drug susceptibility testing varied widely (0.5-11.6%). RMP-resistant isolates that were INH-susceptible had significantly lower rates of resistance to other first- and second-line anti-tuberculosis drugs (except rifabutin) compared to multidrug-resistant isolates. RMP resistance is not always a good proxy for a presumptive diagnosis of multidrug-resistant TB, which has implications for use of molecular assays that identify only RMP resistance-associated DNA mutations.

In-vitro activity of azithromycin compared with other macrolides and oral antibiotics against Salmonella typhi.

The in-vitro activity of azithromycin against 60 clinical isolates of Salmonella typhi was determined by broth microdilution and compared with eight macrolides, including erythromycin, and with other orally administered antimicrobial agents (ampicillin, amoxycillin, cefaclor, trimethoprim/sulphamethoxazole, chloramphenicol, tetracycline, and ciprofloxacin). Azithromycin was more potent (MIC range 4-16 mg/l; MIC90 8 mg/l) than erythromycin (MIC range 32- greater than 128 mg/l; MIC90 greater than 128 mg/l). Of the other macrolides, only rosaramicin showed increased activity against Salm. typhi (MIC range 16-32 mg/l; MIC90 32mg/l) when compared with erythromycin. All 60 Salm. typhi were susceptible to ciprofloxacin (MIC greater than 0.5 mg/l). In 22 isolates, resistance to one or more of the following compounds occurred: ampicillin, amoxycillin, cefaclor, tetracycline, chloramphenicol, trimethoprim/sulphamethoxazole.

Algorithm for use of nucleic acid probes for identifying Mycobacterium tuberculosis from BACTEC 12B bottles.

Nucleic acid probes (Gen-Probe, San Diego, Calif.) can be used to identify mycobacteria in BACTEC 12B broth cultures prior to detection of growth on solid media. We developed an algorithm that can be used to make an initial choice of a probe (either Mycobacterium tuberculosis complex [MTB] or M. avium complex [MAC]) for use in testing respiratory specimens. The algorithm was based on both the fluorochrome smear result of the concentrated specimen and the time from inoculation until the BACTEC 12B broth culture is flagged (growth index 10) as presumptively positive. The MTB probe is used first for all 4+ smear specimens, 3+ smear specimens positive in 5 days, 2+ and 1+ smear specimens positive in 7 days, and smear-negative specimens positive in 11 days. The MAC probe is used for all other specimens. The algorithm is used when other information about the culture (e.g., previous positive cultures and colonial morphology of growth on solid media) is unknown. Use of the algorithm to probe 102 respiratory BACTEC 12B broth cultures (35 with MTB; 1 with MTB, MAC, and M. gordonae; 47 with MAC; and 19 with other mycobacterial species) from 1 September through 30 November 1992 resulted in the initial use of the MTB probe for 35 (97%) of the cultures positive for MTB and the use of the MAC probe for 35 (73%) of the cultures positive for MAC. Use of the algorithm aided in the efficient use of laboratory resources without delaying the time to identification of MTB isolates.

Evaluation of the Vitek GPS-TA card for laboratory detection of high-level gentamicin and streptomycin resistance in enterococci.

The Vitek GPS-TA card (Vitek Systems, Hazelwood, Mo.) was compared with single-concentration broth microdilution and disk diffusion methods using high-content disks for the detection of high-level resistance to gentamicin and streptomycin in 99 isolates of enterococci (81 Enterococcus faecalis isolates and 18 Enterococcus faecium isolates). The GPS-TA card accurately detected high-level resistance to gentamicin, but not streptomycin, in E. faecalis. When streptomycin is being considered for therapy, either disk diffusion or time-kill studies should be used to confirm susceptible results obtained by Vitek testing. Additional studies are needed to determine the best method for testing E. faecium isolates.

Determination of growth value thresholds for BACTEC PLUS aerobic blood culture vials.

Growth value thresholds used to identify positive blood culture vials can be defined by users for each BACTEC NR-660 bacteremia detection instrument. Growth values were compared with the recovery of organisms from vials flagged as positive during the testing of 3.056 high-volume vials containing aerobic (BACTEC PLUS 26) medium over a 2-month period. Results showed that optimal threshold values for our use of these vials varied from those recommended by the manufacturer; if the thresholds defined from these data had been used during the study period, total vials flagged as positive from which no organisms were recovered (false alarms) would have been reduced from 181 (5.9/100 vials tested) to 71 (2.3/100 vials tested), with a minimal decrease in the identification of vials containing usual or occasional pathogens (hits). Adjustments of growth value thresholds by the individual user can make the use of BACTEC instruments more efficient by decreasing further processing of vials from which no organisms are recovered.

Microplate phosphocellulose binding assay for aminoglycoside-modifying enzymes.

We modified the phosphocellulose binding assay for aminoglycoside-modifying enzymes (AMEs) by use of microdilution plates and a multichannel micropipette. Batteries of aminoglycoside substrates for screening organisms for the presence of AMEs as well as for subclassifying enzymes were prepared and stored in microdilution plates. When tested in parallel with the conventional tube reaction assay, the microplate assay yielded comparable radioactive counts and therefore equally correct identifications of AMEs in 32 isolates representing nine bacterial species. Other modifications, such as multichannel dispensing of crude enzyme preparations and radioisotopic precursors, provided a more rapid, convenient, and less expensive means of examining large collections of organisms for AMEs.

Yersinia enterocolitica: a frequent seasonal stool isolate from children at an urban hospital in the southeast United States.

From 1 December 1988 through 28 February 1991, 7,290 rectal swab specimens received in our laboratory were screened for Yersinia enterocolitica. A total of 76 patients had Y. enterocolitica isolated from their stool samples. Of these patients, 59 (77.6%) were 12 months old or younger. Y. enterocolitica was second only to Salmonella spp. in this age group. Routine screening for Y. enterocolitica may be warranted in hospitals serving large pediatric populations.