Use of DNA fingerprinting for primary surveillance of nosocomial tuberculosis in a large urban hospital: detection of outbreaks in homeless people and migrant workers

SETTING: A large urban teaching hospital in the southeast of Paris. OBJECTIVE: Primary surveillance of nosocomial transmission of tuberculosis (TB) by systematic restriction fragment length polymorphism analysis (RFLP) of isolates (n = 161) recovered from smear-positive pulmonary TB patients identified from 1 March 1993 to 28 February 1994, and from all TB patients (with any form of tuberculous infection) identified from 1 March 1994 to 30 April 1995. RESULTS: Systematic RFLP analysis revealed 12 clusters of patients (n = 40) infected by strains of Mycobacterium tuberculosis showing matching RFLP patterns. None of the isolates were multidrug-resistant. Compared with non-clustered patients, clustered patients were more likely to be homeless (55% vs 19%, P < or = 0.001), or Africans living in hostels for migrant workers (20% vs 6%, P = 0.01), and had fewer previous admissions to hospital (12% vs 28%, P = 0.05). Further epidemiological investigations showed that the clustered TB cases actually resulted not from nosocomial transmission, but from transmission in the community, very likely in homeless shelters and hostels for migrant workers. CONCLUSION: No nosocomial transmission of TB was identified among the patients included during the study period. Systematic RFLP analysis using hospital-based sampling can detect the spread of TB in specific environments in the community where transmission is occurring.     

Tuberculosis among urban health care workers: a study using restriction fragment length polymorphism typing

Cases of tuberculosis identified during 1992-1994 through an active tuberculosis surveillance network among six hospitals that serve New York City (the TBNetwork) were analyzed according to the occupational status of the patients. Clinical data were obtained by review of medical records, and restriction fragment length polymorphism (RFLP) typing of Mycobacterium tuberculosis isolates was performed. No known nosocomial outbreaks of tuberculosis occurred at these hospitals in the study period. Occupational status was known for 142 of 201 patients whose isolates were available for strain typing. Patients infected by organisms with a clustered strain typing pattern, as determined by RFLP analysis, were presumed to have recently acquired disease. RFLP typing revealed that isolates from 13 (65%) of 20 health care workers and 50 (41%) of 122 non-health care workers had a clustered RFLP pattern. The strains infecting eight (89%) of nine health care workers seropositive for human immunodeficiency virus (HIV) had a clustered RFLP pattern. Multivariate analysis of 75 patients with known HIV and occupational status revealed that HIV status (P = .03) and health care worker status (P = .02; RR = 2.77) were independent risk factors for a clustered RFLP strain. These findings suggest that many of the apparently sporadic cases of tuberculosis among health care workers may be due to unrecognized occupational transmission.     

M. tuberculosis molecular variation in CNS infection: evidence for strain-dependent neurovirulence

OBJECTIVE: To determine the molecular diversity among Mycobacterium tuberculosis isolates associated with central nervous system tuberculosis (CNS TB) in a defined cohort of HIV uninfected patients. DESIGN/METHODS: A retrospective analysis was performed of clinical and laboratory data for all patients with CNS TB diagnosed in Manitoba, Canada, between 1979 and 1996. Restriction fragment-length polymorphisms (RFLP) of archival isolates of M. tuberculosis from CNS TB patients were determined and interpreted against the frequency of different isolates from all TB patients in the years 1992 to 1996. RESULTS: Among 2,334 patients with active TB, CNS TB was diagnosed in 42 (1.8%); meningitis with or without tuberculoma in 76%; and tuberculoma alone in 24%. CNS TB patients were significantly more likely to be young (<40 years old), female, and of Aboriginal origin. Morbidity (fixed/recurrent CNS deficit) rate was 29% and mortality rate was 26%. An adverse outcome, either morbidity or mortality, was significantly more common in those with meningitis. RFLP analysis of isolates (n=19) from CNS TB patients revealed 13 distinct restriction patterns with a predominance of the type 1 pattern (n=6). The frequency of type 1 restriction pattern was significantly greater in patients with CNS TB compared to all TB patients in Manitoba. CONCLUSIONS: CNS TB continues to have a high morbidity and mortality despite modern methods of detection and treatment. Although several strains of M. tuberculosis cause CNS TB, the current study suggests that the occurrence of CNS TB may be strain-dependent.     

DNA restriction fragments length polymorphism of Mycobacterium tuberculosis isolates in Pisa, Italy

A total of 60 Mycobacterium tuberculosis strains isolated in the area of Pisa, Italy, over a period from April 1993 to December 1995, were analyzed for the IS6110-based restriction fragments length polymorphism (RFLP). Isolates were found to show a great heterogeneity and only few isolates shared identical DNA banding patterns. In particular, 55 distinct IS6110 patterns were found (average number of isolates per pattern: 1.09) and only 9 strains (15%) occurred in 4 clusters of 2-3 identical clones. Computer analysis of genetic similarities among the strains revealed a family of 17 isolates including the clustered clones implicated in recently acquired infections. No correlation was found between the RFLP DNA patterns of the isolates and drug susceptibility. Of the 5 isolates from immigrants only one showed abnormal DNA fingerprinting. Our data indicate that the patterns of M. tuberculosis isolates in Pisa area are comparable to those of countries with low-prevalence TB and that a low level of TB transmission occurs in this area.     

Thyroid hormone receptor is a negative regulator in p53-mediated signaling pathways

SETTING: Molecular typing has become an important tool for examining the extent of active transmission of tuberculosis. OBJECTIVES: To examine transmission of tuberculosis in Cuba using IS6110 restriction fragment length polymorphism (RFLP) typing and to evaluate the utility of spoligotyping. DESIGN: One hundred and sixty Mycobacterium tuberculosis strains isolated over a one year period in Cuba were subjected to RFLP and spoligotyping. RESULTS: Forty-eight percent of the isolates were found in 19 clusters of strains with identical RFLP patterns. In general, cluster sizes were limited, except for two large institutional outbreaks. Age was strongly inversely correlated to clustering. Most streptomycin-resistant isolates were found in clusters. Fifteen spoligotype clusters comprised 78% of the isolates. Significantly different IS6110 RFLP types subdivided 11 spoligotype clusters, whereas none of the IS6110 clusters were subdivided by spoligotyping. CONCLUSIONS: Considering the short study period, 48% clustering is high, indicating that recent transmission plays an important role in Cuba. Although resistance is still a minor problem, transmission of streptomycin-resistant strains occurs. The high polymorphism observed with IS6110 RFLP indicates that this marker is useful for future molecular epidemiological studies in Cuba. Spoligotyping appeared less suitable for population-based studies.     

Histamine phase shifts the hamster circadian pacemaker via an NMDA dependent mechanism

SETTING: The Netherlands, 1993 and 1994. OBJECTIVE: To determine 1) rates of drug resistance in relation to nationality and country of birth, 2) risk factors for drug resistance, 3) treatment outcome of drug-resistant cases, and 4) rates of primary and acquired drug resistance. DESIGN: Retrospective study of all cases notified with bacillary tuberculosis in The Netherlands in 1993 and 1994. RESULTS: Drug resistance to one or more drugs was reported in 268 (14.6%) of all 1836 cases, of whom 203 (76%) were foreign born. In Dutch patients rates of isoniazid (H) (2.9%) and streptomycin resistance (3.6%) were lower than in foreign patients (8.6% and 10.6% respectively, P < 0.001). Multidrug (H and rifampicin [R]) resistance was reported in 0.5% of Dutch-born and 1.4% of foreign cases (P = 0.055). Rates of acquired resistance to H (11.4%) and HR (5.7%) were higher than rates of primary resistance to these drugs (5.2% and 0.7% respectively, P < 0.05), but the number of retreatment cases was low (6.8% of all cases). Drug resistance was associated with immigration but not with drug use, homelessness or human immunodeficiency virus (HIV) co-infection. One fifth (20%) of drug-resistant cases was diagnosed by active case finding. Treatment outcome in sensitive and resistant cases was compared. CONCLUSION: These findings suggest that drug resistance is imported, but it is unclear to what extent drug resistance among foreigners has been transmitted or created in The Netherlands. Drug resistance data should be monitored in Dutch and foreign patients separately.     

Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.     

Characterization of the rpoB gene mutations in clinical isolates of rifampicin-resistant Mycobacterium tuberculosis

OBJECTIVES: Characterization and frequency of the rpoB gene mutations associated with rifampin resistance in Mycobacterium tuberculosis clinical isolates in Sevilla. METHODS: Characterization of rpoB mutations in 21 rifampicin-resistant strains of M. tuberculosis isolated during a three-year period (1994-1996) by three different molecular methods: a nonradioactive Single-strand conformation polymorphism (SSCP) analysis, DNA sequence analysis and a commercial method the line probe assay InnoLiPA. RESULTS: Five distinct rpoB mutations were identified. Ser531-->Leu mutation was detected in 14 strains (66.7%), H526-->Asp in 3 strains (14.3%), Ans512-->Ser in 1 strain (4.8%), Glu513-->Leu in 1 strain (4.8%). A nine nucleotide deletion (codon 510-513) was found in one strain (4.8%) while in the remaining resistant strain (4.8%) no mutation was detected. CONCLUSIONS: The frequency of the different mutations found in the rpoB gene, associated with rifampicin resistance in Mycobacterium tuberculosis clinical isolates in Seville, are similar to those previously reported. However, two new mutations has been detected: a nine nucleotide deletion (codon 510-513), and the Asn512-->Ser point mutation. The characterization of the mutations in the rpoB gene could serve as epidemiological marker for the rifampicin resistant clinical isolates of M. tuberculosis.     

Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 1994 and 1995. Report of the Australian Mycobacterium Reference Laboratory Network

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on isolates of Mycobacterium tuberculosis reported during 1994 and 1995. The total number of confirmed isolates was 708 in 1994 and 705 in 1995. This represents an annual incidence of approximately 4 cases of laboratory confirmed tuberculosis per 100,000 population. These figures are similar to those reported in previous years and confirms that the incidence of tuberculosis in Australia remains stable. The incidence rate varied between States. Overall the male:female ratio fell, and there were signs of a downward shift in the median age. We were unable to assess the impact of HIV infection on the number of isolates reported. Positive microscopy was obtained in 55-60% of patients with pulmonary disease. Approximately 8% of isolates had in vitro resistance to at least one of the four standard anti-tuberculosis drugs. Over the two year period seven strains were found to be multi-drug resistant. Overall, the data from 1994-1995 gives no indication of a significant change in the drug susceptibility profiles of isolates from Australian patients with tuberculosis.     

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.     

Modification of natural immunity in mice by imipenem/cilastatin

SETTING: A tuberculosis programme in hill and mountain districts of Nepal supported by an international non-governmental organisation (NGO). OBJECTIVE: To evaluate under programme conditions the effectiveness of unsupervised monthly-monitored treatment using an oral short-course regimen. DESIGN: In this prospective cohort study, outcomes for new cases of smear-positive tuberculosis starting treatment over a two-year period in four districts in which a 6-month rifampicin-containing regimen was introduced as first-line treatment (subjects) were compared to outcomes for similarly defined cases in four districts where a 12-month regimen with daily streptomycin injections in the intensive phase continued to be used (controls). RESULTS: Of 359 subjects started on the 6-month regimen, 85.2% completed an initial course of treatment compared to 62.8% of 304 controls started on the 12-month regimen (P < 0.001); 78.8% of subjects and 51.0% of controls were confirmed smear-negative at the end of treatment (P < 0.001). The case-fatality rate during treatment was 5.0% among subjects and 11.2% among controls (P = 0.003). Among those whose status was known at two years, 76.9% of subjects were smear-negative without retreatment, compared to 60.9% of controls (P < 0.001). CONCLUSION: In an NGO-supported tuberculosis control programme in remote districts of Nepal, patient-responsible short-course therapy supported by rapid tracing of defaulters achieved acceptable outcomes. Where access and health care infrastructure are poor, district-level tuberculosis teams responsible for treatment planning, drug delivery and programme monitoring can be an appropriate service model.     

Microcolony observation for rapid detection and identification of cultures of Mycobacterium tuberculosis

219 sputa were seeded on Kuang's agar plates. A total of 112 isolates of Mycobacterium tuberculosis were detected in 219 specimens. Of these 112 isolates, 104 (92.8%) were detected in Kuang's agar media and 108 (96.4%) were detected by microcolony observation. The detection time of microcolony observation and culture method needed 11 and 18.6 days respectively. The detection time of microcolony method is much shorter (P < 0.001). The results of conventional tests of different species of Mycobacterium and microcolony differentiation were identical in 99% of isolates of Mycobacterium tuberculosis.     

Glucose intolerance in chronic respiratory failure

A 75-g oral glucose tolerance test (OGTT) was performed on 18 patients with chronic respiratory failure and without fasting hyperglycemia, positive urine glucose, or hepatic/pancreatic disorders. Underlying diseases in these patients were pulmonary emphysema (11 cases, 61%), pulmonary tuberculosis (5 cases, 28%), and chronic bronchial asthma (2 cases, 11%). The body mass index (mean +/- SD, 17.6 +/- 2.2 kg/m2, P < 0.001) in these patients was significantly lower than that (23.8 +/- 3.1 kg/m2) in normal subjects. The OGTT results showed an impaired glucose tolerance pattern in 9 cases (50%) and a diabetes mellitus pattern in 6 cases (34%). The mean two-hour plasma glucose value in the patients was 9.8 mmol/L. However, insulin secretion responded well to glucose loading. These results suggest that a high proportion of chronic respiratory failure patients may have an intolerance for glucose loading but a normal insulin secretion pattern.     

Use of the BacT/Alert blood culture system for culture of sterile body fluids other than blood

Studies have demonstrated that large-volume culture methods for sterile body fluids other than blood increase recovery compared to traditional plated-medium methods. BacT/Alert is a fully automated blood culture system for detecting bacteremia and fungemia. In this study, we compared culture in BacT/Alert standard aerobic and anaerobic bottles, BacT/Alert FAN aerobic and FAN anaerobic bottles, and culture on routine media for six specimen types, i.e., continuous ambulatory peritoneal dialysate (CAPD), peritoneal, amniotic, pericardial, synovial, and pleural fluids. Specimen volumes were divided equally among the three arms of the study. A total of 1,157 specimens were tested, with 227 significant isolates recovered from 193 specimens. Recovery by method was as follows: standard bottles, 186 of 227 (82%); FAN bottles, 217 of 227 (96%); and routine culture, 184 of 227 (81%). The FAN bottles recovered significantly more gram-positive cocci (P < 0.001), Staphylococcus aureus (P = 0.003), coagulase-negative staphylococci (P = 0.008), gram-negative bacilli (P < 0.001), Enterobacteriaceae (P = 0.005), and total organisms (P < 0.001) than the routine culture. There were no significant differences in recovery between the standard bottles and the routine culture. The FAN aerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), coagulase-negative staphyococci (P = 0.003), and total organisms (P < 0.001) than the standard aerobic bottle, while the FAN anaerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), Enterobacteriaceae (P = 0.03), and total organisms (P < 0.001) than the standard anaerobic bottle. For specific specimen types, significantly more isolates were recovered from the FAN bottles compared to the routine culture for synovial (P < 0.001) and CAPD (P = 0.004) fluids. Overall, the FAN bottles were superior in performance to both the standard bottles and the routine culture for detection of microorganisms from the types of sterile body fluids included in this study.     

Chondrodysplasia in Australian Dexter cattle

To evaluate the current prevalence of initial and acquired resistance to the main antituberculosis drugs in Yaounde, isolates of M. tuberculosis complex obtained from sputum cultures of 602 adult patients with pulmonary tuberculosis (516 new cases and 86 old cases) consecutively admitted into the tuberculosis centre of H?pital JAMOT from July 1994 to December 1995 were studied. The susceptibility of isolates to the major antituberculosis drugs was tested by the indirect proportion method. The overall resistance rate (1 or more drugs) was 35.2%, with initial resistance 31.8% (164 of 516) and acquired resistance 55.8% (48 of 86). Initial resistance to streptomycin was the most frequent (20.5%), followed by isoniazid 12.4%), thiacetazone (5.6%), rifampicine (0.8%) and ethambutol (0.4%). Initial resistance was noted as 25% to 1 drug, 5.8% to 2 drugs, 0.8% to 3 drugs and 0.2% to 4 drugs. Acquired resistance to isoniazid was the most frequent (45.3%), followed by streptomycin (40.7%), rifampicine (30.2%), thiacetazone (10.5%) and ethambutol (9.3%). Acquired resistance was found as 13.9% to one drug, 19.8% to 2 drugs, 12.8% to 3 drugs and 9.3% to 4 drugs. A combined resistance to rifampicine and isoniazid in the same patient was noted in 0.8% of the new cases and in 26.7% of the old cases. These high rates af antituberculosis drug resistance in Yaounde underline the urgent need to reestablish a tuberculosis control programme in Cameroon.     

Typing of human Mycobacterium avium isolates in Italy by IS1245-based restriction fragment length polymorphism analysis

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.     

Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 1996. Report of the Australian Mycobacterium Reference Laboratory Network

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new diagnoses of infection with Mycobacterium tuberculosis complex during 1996. A total of 750 cases were identified, representing an annual incidence of 4.1 cases of laboratory confirmed tuberculosis per 100,000 population. The incidence rate varied between States, reflecting differences in the distribution of persons belonging to 'high-risk' categories for tuberculosis. Incidence statistics were almost identical to those recorded by the Network in 1994 and 1995. The male:female ratio remained at around 1.2:1. As was the case in 1995, the median age group for males was 45-49 years and for females 35-39 years. The frequency of positive microscopy in pulmonary samples was stable at around 55%. Lymphatic disease accounted for 19% of the total cases in 1996 compared with 15% in the previous year, confirming that lymphadenitis is becoming more common in females with tuberculosis in Australia. Approximately 11% of isolates had in vitro resistance to at least one of the four standard anti-tuberculosis drugs, an increase from 8% in 1994-95. Fifteen isolates were multi-drug resistant, compared with a total of only 38 during the previous seven years. Thus, the 1996 data points to an increasing frequency of multi-drug resistant strains among isolates from Australian patients with tuberculosis.     

Transmission of tuberculosis in the metropolitan area of Zurich: a 3 year survey based on DNA fingerprinting

Between 1991 and 1993, 444 inhabitants of the metropolitan area of Zurich were reported as confirmed or suspected cases of tuberculosis (TB). Overall, isolates of Mycobacterium tuberculosis of 361 patients (90% of the bacteriologically confirmed cases) were available to study the frequency of transmission of the strains on a molecular level. Restriction fragment length polymorphism (RFLP) analysis was performed by using IS6110 and the polymorphic GC-rich sequence (PGRS) as genetic markers. Ninety nine isolates shared by 77 patients (21.3%) were associated with 28 IS6110-defined clusters. However, secondary typing of low copy number isolates decreased the number of clusters to 25, encompassing 81 isolates from 63 (17.5%) patients. By deoxyribonucleic acid (DNA) fingerprinting plus conventional contact tracing, definite transmission of TB was proven in only five patients (1.4%) and assumed in 20 patients (5.6%). In all other cluster-associated isolates, no epidemiological connections between the patients could be found using the clinical and sociodemographic data available. The present study demonstrates that in the time period studied only minor transmission occurred.