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.

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.

Multicenter evaluation of ethambutol susceptibility testing of mycobacterium tuberculosis by agar proportion and radiometric methods.

Reproducibility of ethambutol (EMB) susceptibility test results for Mycobacterium tuberculosis has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems, M. tuberculosis drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 micro g/ml in addition to the standard 2.5 micro g/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.

Multicenter evaluation of a nonweekend reading schedule for radiometric pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an integral component of the short-course chemotherapy regimen for tuberculosis. The BACTEC 460TB PZA susceptibility test for Mycobacterium tuberculosis with a daily (D) reading schedule has been available for more than 10 years, but weekend laboratory staffing is necessary. A nonweekend (NW) reading schedule has not been validated in a multicenter study. This prospective multicenter study compares the interlaboratory reproducibility of PZA susceptibility results by following both the D and NW schedules. A total of 181 cultures were shared among four laboratories. Isolates were selected based on resistance or borderline resistance to at least one streptomycin-isoniazid-rifampin-ethambutol drug or PZA. One laboratory used a D reading schedule, and three laboratories used a NW schedule. Both reading schedules are based on the standard BACTEC 460TB PZA protocol. With the NW schedule, the growth index (GI) is not available for test interpretation on Saturday, Sunday, and Monday. Of the 181 shared cultures, 154 were found to be susceptible by all laboratories, 19 were found to be resistant, and 8 had discordant results. The overall pairwise interlaboratory agreement was 97.7%. The discrepancies were not associated with the type of reading schedule used. However, the median control GI was significantly higher for the NW schedule (321) than for the D schedule (259) (P < 0.0001) although results were available on average in about 7 days from setup for both schedules. These results show that the NW schedule is a suitable alternative for laboratories that do not read and interpret PZA susceptibility tests on weekends.

Comparison of methods for Identification of Mycobacterium abscessus and M. chelonae isolates.

Mycobacterium abscessus and Mycobacterium chelonae are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either M. abscessus or M. chelonae that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be M. abscessus and 29 were identified as M. chelonae. Eight isolates originally submitted as M. chelonae were identified as M. abscessus, and one isolate submitted as M. abscessus was found to be M. chelonae. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of M. abscessus exhibited resistance to tobramycin (MIC of 8 to > or =16 microg/ml), while all isolates of M. chelonae were susceptible to this drug (MIC of < or = 4 microg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of M. abscessus and M. chelonae.

Mycobacterium kubicae sp. nov., a slowly growing, scotochromogenic Mycobacterium.

A previously uncharacterized, slowly growing, scotochromogenic Mycobacterium species was detected by HPLC analysis of the cell-wall-bound mycolic acids. The mycolic acid pattern standard was shown to be a late-eluting, contiguous peak cluster occurring at approximately 8-9 min. The mycolic acid pattern was noted to be most similar in number of peaks and range of elution to that reported previously for Mycobacterium asiaticum. However, the relative distribution of peaks within the elution range demonstrated a pattern with prominent peaks that started to emerge later than the characteristic M. asiaticum pattern. Standard biochemical identification test results were similar to those of the photochromogenic species M. asiaticum. Comparative 16S rRNA gene sequence analysis confirmed the genetic uniqueness of the strains and demonstrated the unclassified mycobacteria to be in a unique, intermediate position between slow and rapid growers in the phylogenetic tree of Mycobacterium. The name Mycobacterium kubicae sp. nov. is proposed for this taxon. The type strain is CDC 941078T (= ATCC 700732T = CIP 106428T).

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.

Tuberculosis in the inner city: impact of a continuing epidemic in the 1990s.

Tuberculosis cases have recently declined in the United States, renewing interest in disease elimination. We examined the epidemiology of tuberculosis from 1991 through 1997 at an inner-city public hospital and assessed population-based tuberculosis rates by ZIP code in the 8 metropolitan Atlanta counties. During the 7 years, 1378 new patients had tuberculosis diagnosed at our hospital (mean, 197 patients/year), accounting for 25% of tuberculosis cases in Georgia. Coinfection with human immunodeficiency virus (HIV) was common, but a significant decrease in the proportion of HIV-infected patients with tuberculosis was noted over time. Most patients were members of a minority group (93%) and were born in the United States (96%). Two inner-city ZIP code areas had annual tuberculosis rates >120 cases per 100,000 persons, and 8 ZIP code areas had annual rates of 47-88 cases per 100,000 persons between 1993 and 1997, compared with the annual national average of 8.7 cases per 100,000 persons. Our hospital continues to care for large numbers of tuberculosis patients, and rates of tuberculosis remain high in the inner city. These data mandate a concentration of efforts and resources in urban locations if tuberculosis control and elimination is to be achieved in the United States.

Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia.

Nine isolates of Escherichia coli were recovered from seven blood cultures over a period of 3 months from a 19-month-old female with aplastic anemia. Initial isolates were susceptible to extended-spectrum cephalosporins, including ceftazidime (MIC, < or = 0.25 microgram/ml), but gradually became resistant to this drug (MICs, > or = 128 micrograms/ml) and other cephalosporins and the monobactam aztreonam. Molecular typing methods, including plasmid profile analysis, pulsed-field gel electrophoresis, and arbitrarily primed PCR, indicated that the nine isolates were derived from a common ancestor. Dot blot hybridization and PCR analysis of total bacterial DNA using blaSHV- and blaTEM-specific DNA probes and primers identified the presence of a blaTEM beta-lactamase gene in all of the isolates and a blaSHV gene in the isolates with elevated ceftazidime MICs. Isoelectric focusing analysis of crude lysates showed that all nine isolates contained an enzyme with a pI of 5.4 corresponding to the TEM-1 beta-lactamase, and those isolates containing an SHV-type beta-lactamase demonstrated an additional band with a pI of 7.6. The first of the ceftazidime-resistant isolates appeared to hyperproduce the SHV enzyme compared to the other resistant isolates. DNA sequencing revealed a blaSHV-1 gene in the first ceftazidime-resistant isolate and a novel blaSHV gene, blaSHV-8, with an Asp-to-Asn substitution at amino acid position 179 in the remaining four isolates. Three of the ceftazidime-resistant isolates also showed a change in porin profile. The patient had received multiple courses of antimicrobial agents during her illness, including multiple courses of ceftazidime. This collection of blood isolates from the same patient appears to represent the in vivo evolution of resistance under selective pressure of treatment with various cephalosporins.

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.

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.

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.

Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels.

We compared penicillin MICs obtained with three different commercially available broth microdilution panels (MicroScan, Sensititre, and Pasco) with MICs obtained with reference microdilution panels for 20 well-characterized pneumococci with decreased susceptibilities to penicillin (7 resistant and 13 intermediate). All panels were supplemented with 2 to 5% lysed horse blood (LHB) prepared in-house. Additional supplements included fastidious inoculum broth (FIB) for MicroScan panels and commercially prepared LHB (Difco) for Pasco panels. The percentages of penicillin-resistant strains (MIC 2 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 0; MicroScan-LHB 0; Pasco in-house LHB, 71; and Sensititre-LHB, 100. The percentages of intermediate strains (MIC = 0.1 to 1.0 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 31; MicroScan-LHB 23; Pasco in-house LHB, 46; and Sensititre-LHB, 85. Difco LHB supplement failed to support the growth of 86% of the strains in the Pasco panels. Of the commercially available panels evaluated, only Sensititre, supplemented with LHB prepared in-house could reliably detect penicillin-resistant pneumococci.

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.

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.

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.

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.

Severe illness in African children with diarrhoea: implications for case management strategies.

To identify clinical disorders associated with severe illness in African children with diarrhoea, we studied a group of under-5-year-olds with diarrhoea who had been brought to a large public hospital in central Cote d'Ivoire. The general condition of children with diarrhoea was assessed and classified according to criteria recommended by WHO, and then used as a nonspecific indicator of severity. Of the 264 children with diarrhoea who were enrolled in the study, 196 had nonsevere illness and 68 severe illness. Children with severe illness were significantly more likely than those with nonsevere illness to be dehydrated (45% versus 11%), moderate-to-severely wasted (47% versus 29%), bacteraemic (26% versus 9%), severely anaemic (haemoglobin level <6 g/dl; 15% versus 6%), have Plasmodium falciparum parasitaemia (27% versus 14%), and have two or more of these five conditions (60% versus 14%). Nontyphoidal Salmonella spp. were present in 68% of the blood isolates but were not associated with seropositivity to human immunodeficiency virus (HIV). The study demonstrates the need for a more comprehensive approach to assessment and management of children with diarrhoea that ensures prompt recognition of bacteraemia, anaemia, wasting and malaria, as well as dehydration. Simple nonspecific observational criteria, such as those recommended by WHO for assessing and classifying general condition, are useful for identifying children with diarrhoea who are at high risk of having life-threatening clinical disorders, and can readily be used by health workers whose clinical training and access to diagnostic laboratory facilities are both limited.

PIP: Researchers prospectively studied 264 children aged less than 5 years with diarrhea who were admitted to the Bouake Regional Hospital Center in the Ivory Coast between June 10 and August 11, 1991, to identify clinical disorders associated with severe diarrhea. They compared data on the 196 children with non-severe diarrhea with data on 68 children with severe diarrhea. All but three of the children were breast fed. The severely ill children were more likely than the non-severely ill children to have dehydration (45% vs. 11%; p 0.01), severe wasting (22% vs. 7%; p 0.01), anemia (29% vs. 13%; p = 0.01), bacteremia (26% vs. 9%; p 0.01), and malarial parasitemia (27% vs. 14%; p = 0.02). 68% of the blood isolates had nontyphoidal Salmonella spp. 6% of children had HIV-1 or HIV-2 infection. The most common pathogens in the stool specimens were rotavirus (41 cases), Campylobacter jejuni (22), Shigella spp. (21), and Salmonella spp (10). These findings indicate a need for a more comprehensive approach to assessment and management of children with diarrhea that secures immediate recognition of bacteremia, anemia, wasting, malaria, and dehydration.