Comparison of tuberculin skin test and Quantiferon immunological assay for latent tuberculosis infection.

BACKGROUND: Correct identification of individuals with latent tuberculosis infection (LTBI) is a crucial element of the elimination strategy, allowing their adequate treatment. In addition to tuberculin skin test (TST), the Quantiferon test (QFT, based on whole blood gamma-interferon release) had been recently proposed. Aim of the study is to compare this test to TST for identification of LTBI in a non-selected population, in order to verify their value in identifying truly infected individuals (entitled to receive preventive chemotherapy), and to exclude from treatment those having a positive TST for other reasons (e.g. after BCG vaccination). METHODS: 136 consecutive persons (78 males, mean age 34 +/- 9 years) referred to the clinic for TST were recruited (78 born in low--or middle--income countries). Based on their history, the cases were divided into 4 groups: 1) recently traced contacts of whom 18 TST negative and 28 TST positive; 2) 22 screening subjects, all TST negative; 3) BCG vaccinated subjects (14); and 4) 54 subjects already undergoing treatment of LTBI for exposure to TB. RESULTS: The overall agreement between TST and QFT was 72% (64% in TST positive and 88.4% in TST negative subjects). The proportion of TST positive/QFT negative BCG vaccinated individuals was 23.1%. The K coefficient was 0.474 in recently traced contacts, 0.366 in BCG vaccinated individuals and 0.451 overall. CONCLUSIONS: The study results suggest that agreement between TST and QFT is lower in TST positive than in negative subjects, being lower in individuals treated for LTBI. Quantiferon does not seem to have brought significant improvement in the diagnosis of LTBI.

Direct rapid diagnosis of rifampicin-resistant M. tuberculosis infection in clinical samples by line probe assay (INNO LiPA Rif-TB).

M. tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant Tuberculosis (MDR-TB) is associated with a high case-fatality rate. Rapid identification of resistant strains is crucial to institute prompt appropriate therapy, and prevent the development of further resistance and spreading of MDR strains. The INNO-LiPA Rif. TB is a commercial reverse hybridisation line probe assay designed for rapid detection of rpoB gene mutations in clinical isolates. We applied this test directly to 44 smear-positive and 45 smear-negative clinical specimens collected from patients suspected of active TB. The capability of this technique to correctly identify local MDR-TB strains was tested on 50 MDR strains isolated in Italy. Results of the test were compared to conventional antibiogram performed on isolated strains. The concordance rate of the LiPA test results on clinical specimens with those obtained with "in vitro" sensitivity was 100%. These results show that the LiPA test can be useful in rapid detection and prompt management of tuberculosis when MDR disease is suspected.

Two-year population-based molecular epidemiological study of tuberculosis transmission in the metropolitan area of Milan, Italy.

A 2-year, population-based, molecular epidemiological study was conducted in Milan, Italy, to determine the proportion of tuberculosis (TB) cases attributable to recent transmission. All strains were typed by restriction fragment length polymorphism (RFLP) analysis; clustering was considered indicative of recent transmission. Of the 581 cases, 239 (41.1%) belonged to clusters that consisted of 2 to 11 patients; 28.1% were attributable to recent transmission (number of clustered patients minus 1). Clustering was associated with multidrug-resistant Mycobacterium tuberculosis strains (74.2% of cases), AIDS (60.2%), and a history of incarceration (67.4%). The frequency of multidrug-resistant Mycobacterium tuberculosis was 5.3% overall (15.4% among AIDS patients). Among AIDS patients, infection with a resistant strain was independently associated with clustering (odds ratio, 1.32; 95% confidence interval, 1.07-1.163), while among non-AIDS patients, three factors were associated with clustering: history of incarceration (odds ratio, 2.03; 95% confidence interval, 1.41-2.92), age <30 years (odds ratio, 1.43; 95% confidence interval, 1.05-1.94), and native-born Italian nationality (odds ratio, 1.44; 95% confidence interval, 1.08-1.92). Of the 118 patients who belonged to either the smallest or the largest cluster, 19 (16.1%) reported an epidemiological link with another study patient. The results of this study highlight the need for control programs that focus on selected high-risk groups consisting primarily of HIV-infected individuals and persons with social and lifestyle risks for TB. These programs should be aimed at reducing the probability of transmission of drug-resistant TB through early identification of cases and provision of effective treatment until the individual is cured.

Molecular epidemiology study of exogenous reinfection in an area with a low incidence of tuberculosis.

In geographical areas with a low incidence of tuberculosis, recurrent tuberculosis is generally due to reactivation of the disease. However, the relative contribution of tuberculosis reinfection increases in parallel with the incidence of disease and is likely to depend on the epidemiological context: factors such as the spread of multidrug resistance, human immunodeficiency virus (HIV) infection, and immigration from developing countries could modify disease transmission in areas at low risk for tuberculosis. A molecular epidemiology study was performed in Lombardy, Northern Italy, where the incidence of tuberculosis is 17.5 cases per 100,000 persons. A total of 2,452 cases of culture-confirmed tuberculosis in 2,127 patients were studied. A group of 32 patients (1.5%), each of whom had two episodes of tuberculosis with cure as the outcome of the first episode and with more than 6 months between the two episodes, were studied by means of restriction fragment length polymorphism DNA fingerprinting analysis. For 5 of the 32 patients (16%), the DNA fingerprinting patterns of Mycobacterium tuberculosis strains responsible for the second episode did not match those of the corresponding isolates of the first episode, indicating exogenous reinfection. Two of these patients developed multidrug-resistant tuberculosis during the second episode, and in three cases the isolates belonged to clusters of M. tuberculosis strains spreading in the community. A fourfold-increased risk for reinfection was observed in immigrant patients compared to Italian subjects. In contrast, a higher risk of relapse rather than reinfection was evidenced in HIV-positive subjects and in patients infected with multidrug-resistant tuberculosis. Episodes of tuberculosis reinfection in areas with a low incidence of tuberculosis are rare compared to those in high-incidence geographical regions. In populations that have immigrated from high-risk areas, reinfection may represent a considerable contributor to the rate of recurrent tuberculosis. This finding emphasizes the importance of containing the spread of epidemic strains in close communities, in order to prevent changes in global tuberculosis trends for developed countries.

Surveillance of anti-tuberculosis drug resistance: results of the 1998/1999 proficiency testing in Italy. SMIRA (Italian Multicentre Study on Antituberculosis Drug Resistance) Study Group.

OBJECTIVE: To determine the accuracy of drug-susceptibility testing (DST) for isoniazid, rifampicin, ethambutol and streptomycin in a provisional network of 22 regional laboratories in Italy. METHODS: Methods, definitions and reference Mycobacterium tuberculosis strains were derived from the WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance. The laboratories were selected based on technical skills required by the project, the number of DST performed annually and geographic localisation. The results (sensitive/resistant strain) were compared with the gold standard (global project results). Sensitivity (ability to detect true resistance), specificity (ability to detect true susceptibility), positive predictive values for resistance and susceptibility, efficiency and reproducibility were calculated in two rounds. RESULTS: Eighteen of 22 laboratories completed the first round of proficiency testing for the four drugs. Sensitivity was 76.6%, specificity 97.2%, predictive value of a resistant test 89.8% and of a susceptible test 86.8%, efficiency 87.8% and reproducibility 92.8%. A second round was performed by all those laboratories that did not achieve > or = 90% agreement with the results of the Global Project. Overall, after the second round, all the parameters except specificity improved, exceeding 90%. CONCLUSIONS: A network of 15 regional laboratories that fulfil the quality criteria for determining the susceptibility of M. tuberculosis to the four primary antituberculosis drugs was established in Italy.

Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the "gold standard." Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.

Pulmonary infection due to Mycobacterium szulgai, case report and review of the literature.

We describe the case of a patient with a chronic pulmonary infection due to a mycobacterium tentatively identified as Mycobacterium flavescens, but finally shown to be Mycobacterium szulgai; this is the first M. szulgai infection reported in Italy. The patient responded to treatment with multiple antituberculosis drugs only after two cycles of 10 and 6 months, respectively. The literature concerning previous case reports in which M. szulgai is involved is revised and the difficulty concerning the identification of this rare mycobacterium, along with its in vitro and in vivo susceptibility, are discussed.

Characterization of mycobacterial isolates phylogenetically related to, but different from Mycobacterium simiae.

The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade resistance to the majority of antimycobacterial drugs. Three of the isolates were clinically significant according to stringent criteria. Sophisticated techniques, like genetic sequencing or HPLC, by now seem indispensable for differentiating unusual and new mycobacteria from well-established ones.

Feline leprosy: spontaneous remission in a cat.

A two-year-old, male domestic shorthair with a solitary lesion of the right forelimb was presented for fine-needle aspiration biopsy of a suspected cutaneous, neoplastic process. Neutrophils, lymphocytes, and acid-fast bacilli packed in the cytoplasm of foamy macrophages and giant cells were seen on cytological examination. Bacteriological culture of the material from the skin lesion was negative for Mycobacterium spp. Intraperitoneal injection of homogenized material from the lesion resulted in generalized mycobacteriosis in one mouse after 10 months. Based on these results, a diagnosis of feline leprosy was made. No medical or surgical therapy was performed. Nonetheless, the lesion showed progressive and complete spontaneous remission within 3.5 months from the time of diagnosis; after 14 months, the cat still is free of disease.

Identification of the newly described Mycobacterium poriferae from tuberculous lesions of snakehead fish (Channa striatus).

A mycobacterium isolated form a cultured snakehead with nodular lesions was identified on the basis of high performance liquid chromatography (HPLC) profile of cell wall mycolic acids, and confirmed by conventional tests, as Mycobacterium poriferae, a species previously isolated only from a marine sponge. The profiles of M. poriferae, Mycobacterium aurum and Mycobacterium parafortuitum are here reported for the first time.

Mycobacterium malmoense in Italy.

A case of mycobacterial lymphadenitis due to Mycobacterium malmoense was recently diagnosed in a 5-year-old girl. The organism was isolated from pus and tissue fragments obtained by surgical excision of the affected nodes. This is the first documented case of human infection due to this organism in Italy.

Tentative evidence of AIDS-associated biotype of Mycobacterium kansasii.

Previous studies revealed heterogeneous behavior within the species Mycobacterium kansasii against commercially available DNA probes (Accuprobe M. kansasii culture identification test; Gen-Probe); several isolates, conventionally identified as M. kansasii, failed in fact to hybridize. Looking for a possible association with phenotypic features, we tested a fully characterized panel of 69 clinical isolates of M. kansasii (19 of which were Accuprobe negative) with a semiquantitative micromethod which tests for 19 enzymatic activities (Api Zym; BioMérieux). The strains were from 25 hospitals in 18 Italian towns; 20 isolates came from human immunodeficiency virus type 1-positive patients who fulfilled the Centers for Disease Control criteria for AIDS diagnosis. On the basis of the whole set of phenotypic traits, our strains clustered in two groups, allowing the differentiation of biotypes within the species. There was a perfect association between biotype 2 and hybridization failures with Accuprobe and a very significant association between this novel biotype 2 and AIDS status, which suggests that it differs in virulence.

Evaluation of a commercial DNA probe assay for the identification of Mycobacterium kansasii.

A commercially available DNA probe (the AccuProbe Mycobacterium kansasii culture identification test, Gen-Probe, USA) for the identification of Mycobacterium kansasii was tested on a panel of 143 fully characterized mycobacterial strains. The isolates included 70 Mycobacterium kansasii and 73 mycobacteria other than kansasii. The specificity was 100% while the sensitivity was 72.8%. This sensitivity is unusually low in comparison with that of commercial DNA probes for other mycobacteria and confirms a previous study that found genetic heterogeneity within the species Mycobacterium kansasii. Strains that do not hybridize with the AccuProbe are particularly prevalent in Italy and perhaps elsewhere in Europe.

Anti-mycobacterial activity of amikacin.

We have tested in the IUTM egg medium the susceptibility against amikacin (AKC) of 147 strains of Mycobacterium tuberculosis (M.t.) isolated from the sputum of patients affected with pulmonary Tb. Only five strains showed resistance at 5 and 10 mcg/ml: they were also resistant to other main anti-tubercular drugs (MATD). The other 59 strains resistant to MATD and the 82 ones without any resistance to MATD were found normally susceptible to AKC at 5 mcg/ml in IUTM m. The parallel test performed on a synthetic medium (7H10) with 10 firstly-observed strains, allowed us to define that AKC undergoes a 60% inactivation in the egg medium. Two patients suffering from chronic pulmonary Tb, with M.t. constantly isolable in their sputum, assumed a twice-weekly treatment (TWT) with AKC alone (total 26 gr). This management induced in them improvement of the radiological and bacteriological findings, without alteration of the checking parameters.