Nontuberculous mycobacteria infection in solid organ transplant recipients.

Although advances in surgical technique, drug-induced immunosuppression, and supportive medical therapy have led to improved survival and quality of live after solid organ transplantations, infections still represent a major threat for transplant recipients.Infections with non tuberculous mycobacteria (NTM) are infrequent, but can be associated with relevant morbidity and mortality. With the exception of few comprehensive studies, the available literature consists predominantly of case reports and institutional experiences. This article reviews NTM infection in the setting of solid organ transplant (SOT) recipients dealing with clinical features, diagnosis, treatment and outcome. Published studies have documented a protean presentation of NTM infection in SOT recipients which may include skin and soft tissue lesions, osteoarticular infections and pleuropulmonary disease. An aggressive approach including histopathologic examination and acid-fast bacilli culture of aspirates or biopsy specimens from involved sites are essential for diagnosis. A combined treatment of debridement surgery, reduction of immunosuppressive medications and/or antimycobacterial drugs is frequently associated with a favorable outcome. It is strongly emphasized that a high index of suspicion for NTM infection should be maintained when managing SOT recipients with unusual clinical manifestations. Early diagnosis and initiation of therapy are essential to prevent morbidity and mortality in this vulnerable population.

Performance of a commercial nucleic acid amplification test with extrapulmonary specimens for the diagnosis of tuberculosis.

The laboratory diagnosis of tuberculosis (TB) on extrapulmonary specimens is particularly challenging. A number of commercial nucleic acid amplification tests able to detect and identify Mycobacterium tuberculosis (MTB) complex directly from respiratory secretions have been developed, but their use on extrapulmonary samples still calls for validation. The BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was applied to 918 consecutive extrapulmonary specimens (collected from 863 patients), including 84 gastric aspirates, 145 urine, 136 sterile body fluids, 83 cerebrospinal (CSF) fluids, 237 fine-needle aspirates, 175 pus, 56 biopsies, and two stool specimens. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the gold standard. Ninety-two specimens yielded culture positive for MTB and 24 (smear- and culture-negative) were from patients with TB clinical diagnosis. Of these, 96 were DTB-positive, including all of those from culture-negative TB cases. From 26 specimens, nontuberculous mycobacteria were grown. Two of these specimens were positive by the DTB assay. Finally, of the 776 samples that were smear- and culture-negative for acid-fast bacilli (AFB), collected from patients for whom the diagnosis of TB was excluded, six were DTB-positive. The overall sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of extrapulmonary samples were 82.7, 99.0, 92.3, and 97.8%, respectively. Although, at present, amplification assays cannot replace culture techniques, DTB proved to be rapid and specific for the detection of MTB in extrapulmonary samples.

Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and metaregression.

In-house PCR (hPCR) could speed differential diagnosis between tuberculosis (TB) and nontuberculous mycobacterial disease in patients with positive smears and pulmonary infiltrates, but its reported accuracy fluctuates across studies. We conducted a systematic review and meta-analysis of hPCR sensitivity and specificity for smear-positive TB diagnosis, using culture as the reference standard. After searching English language studies in MEDLINE and EMBASE, we estimated cumulative accuracy by means of summary receiver operating characteristic analysis. The possible influence of hPCR procedures and study methodological features on accuracy was explored by univariate metaregression, followed by multivariate adjustment of items selected as significant. Thirty-five articles (1991 to 2006) met the inclusion criteria. The pooled estimates of the diagnostic odds ratio, sensitivity, and specificity (random-effect model) were, respectively, 60 (confidence interval [CI], 29 to 123), 0.96 (CI, 0.95 to 0.97), and 0.81 (CI, 0.78 to 0.84), but significant variations (mainly in specificity) limit their clinical applicability. The quality of the reference test, the detection method, and real-time PCR use explained some of the observed heterogeneity. Probably due to the limited study power of our meta-analysis and to the wide differences in both laboratory techniques and methodological quality, only real-time PCR also displayed a positive impact on accuracy in the multivariate model. Currently, hPCR can be confidently used to exclude TB in smear-positive patients, but its low specificity could lead to erroneous initiation of therapy, isolation, and contact investigation. As the inclusion of samples from treated patients could have artificially reduced specificity, future studies should report mycobacterial-culture results for each TB and non-TB sample analyzed.

Pyrazinamide susceptibility testing: proposed new standard with the BACTECTM MGITTM 960 system.

The susceptibility of 253 Mycobacterium tuberculosis complex isolates to pyrazinamide (PZA) was assessed using the BACTECTM MGITTM 960 (M960) system. Resistant strains underwent paired repeat testing using 1) a critical concentration of 200 g/ml (PZA-200), and 2) a reduced inoculum of 0.25 ml. They were also examined using the BACTEC 460 (B460) reference method and investigated for pncA mutations. On M960, 37 isolates were resistant. In the PZA-200 assay, 20 of these were resistant and 17 susceptible, while 18 were resistant and 19 susceptible with reduced inoculum. The B460 assay and pncA sequencing confirmed results with reduced inoculum.

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.

Synthesis and in vitro anti-mycobacterium activity of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides. Preliminary toxicity and pharmacokinetic evaluation.

Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired human immunodeficiency syndrome (AIDS). A series of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard or isolated from infected patients, have been determined. Preliminary tests show a good activity and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very rapid elimination from the body after intravenous administration and a poor absorption after oral administration.

Enteropathogens associated with childhood diarrhea in Italy. The Italian Study Group on Gastrointestinal Infections.

BACKGROUND: Infectious diarrheal diseases remain an important cause of childhood morbidity in industrialized countries. The knowledge of the etiology and epidemiology of childhood diarrhea in a given area is needed to plan any measure designed to prevent or ameliorate diarrheal illness and to develop practice guidelines for the most appropriate stool examination procedures. METHODS: We evaluated 618 children with diarrhea and 135 controls prospectively for viral, bacterial and parasitic enteric pathogens. Diarrheagenic Escherichia coli was identified by gene probes specific to different virulence factors. Stool filtrates were examined for the presence of free bacterial toxins by a cell culture cytotoxicity assay. Clinical and epidemiologic data were recorded and analyzed in relation to microbiologic findings. RESULTS: Enteropathogens were identified in 59% of children with diarrhea and in 10.4% of asymptomatic controls. The agents mainly associated with disease were rotavirus (23.6%), Salmonella (19.2%) and Campylobacter (7.9%). Rotavirus was significantly more frequent among children observed as inpatients whereas Campylobacter was significantly more common in outpatients. Infections with diarrheagenic E. coli, Shigella flexneri, yersinia enterocolitica, Cryptosporidium and Giardia were observed in a limited number of patients. The clinical presentation of children was not sufficiently characteristic to permit presumptive diagnosis of a specific pathogen. conversely the presence of blood and/or leukocytes in stools had a high positive predictive value for Salmonella or Campylobacter infection. CONCLUSION: The results of this study will be useful for planning strategies to prevent and control diarrheal diseases in our country.

Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis.

The in vitro activity of 16 antimicrobial agents against 46 drug-resistant strains of Mycobacterium tuberculosis recently isolated from Italian patients was determined. As for first-line antituberculosis drugs, while isoniazid was ineffective against all the strains tested, resistance to streptomycin, rifampicin, pyrazinamide, and ethambutol was 80.4%, 71.7%, 39.1%, and 8.7%, respectively. Among second-line antituberculous drugs, resistance to ciprofloxacin, ofloxacin, and sparfloxacin and to amikacin and kanamycin was around 20%. About 10% of the strains were resistant to capreomycin and cycloserine and 4.3% were resistant to ethionamide; no strain was found to be resistant to thiacetazone, para-aminosalicylic acid, and viomycin. Although all strains displayed a rather continuous distribution of minimal inhibitory concentrations (MICs), a bimodal distribution was observed for rifampicin, amikacin, and kanamicin, with very high MIC values for resistant strains; relatively low MICs were found for fluoroquinolone-resistant strains. Among the small number of strains resistant to second-line agents, low resistant levels were observed. Restriction fragment length polymorphism analysis showed few strain clusters with resistance to first-line antituberculous drugs and aminoglycosides, fluoroquinolones, or both. Altogether, these results showed that second-line agents were still active against the isoniazid-resistant and multiply first-line resistant strains tested, with none or low resistance levels; these observations can be of importance for the treatment of multidrug-resistant tuberculosis in Italy.

Multicenter evaluation of the MB-Redox medium compared with radiometric BACTEC system, mycobacteria growth indicator tube (MGIT), and Löwenstein-Jensen medium for detection and recovery of acid-fast bacilli.

MB-Redox is a new manual culture system designed for the recovery of mycobacteria from clinical specimens. It consists of a liquid medium (modified Kirchner medium) containing a redox indicator, a colorless tetrazolium salt, which is reduced to colored formazan by actively growing mycobacteria. Acid fast bacilli (AFB) are easily detected in the medium as pink to purple pinhead-sized particles. We report the results of a multicenter study (involving four Italian microbiology laboratories processing 2370 clinical specimens) aiming to evaluate the recovery rates of AFB and time required for their detection by using the MB-Redox medium. Two different protocols were set up: in Protocol A (1580 specimens) the performance of MB-Redox was compared with those of the radiometric BACTEC 460 TB system (B460) and Löwenstein-Jensen medium (L-J), whereas in Protocol B (790 specimens) it was compared with those of the Mycobacteria Growth Indicator Tube (MGIT) and L-J. A total of 213 mycobacteria were recovered, including 172 Mycobacterium tuberculosis complex (MTB) isolates and 41 nontuberculous mycobacteria (NTM) isolates. In Protocol A, recovery rates were 81% for MB-Redox system, 84% for B460 system, and 77% for L-J. In Protocol B the recovery rates by individual system were 87, 83, and 76% for MB-Redox, MGIT, and L-J, respectively. Differences in both the protocols were not statistically significant. The MB-Redox system plus L-J (Combination 1) recovered 94% of the isolates in Protocol A and 93% in Protocol B, while B460 plus L-J (Combination 2) and MGIT plus L-J (Combination 3) detected 91 and 89% of all mycobacteria isolates respectively. No statistically significant differences were found among the combinations. The mean time to detection of mycobacteria was 16.3 days in Protocol A and 19.1 days in Protocol B with the MB-Redox system, 22.4 and 25.9 days with L-J, 13.2 days with B460, and 18.2 days with MGIT. The contamination rates were 2.1, 2.0, 1.9, and 3.6 for MB-Redox, B460, MGIT, and L-J respectively. The MB-Redox is a reliable, nonradiometric system for growth and detection of mycobacteria. When used in combination with a solid medium it proved to be an effective replacement for B460. The MB Redox system is a labor-intensive method requiring much handling during the visual reading procedures.

Mycobacterial testing in hospital laboratories: results from a questionnaire survey in Italy.

Between 1999 and 2001, 355 hospital laboratories in Italy were asked to complete a questionnaire addressing mycobacterial test methods, 1-year workloads and laboratory safety features. Analysis of the data showed that rapid methods for mycobacterial testing were being used by most larger laboratories; however, sub-optimal methods were still in use in small and medium-size laboratories. In a country such as Italy, which has a low prevalence of tuberculosis cases, implementation of rapid technologies, combined with regionalisation of mycobacterial diagnostic services, seems to be the most reasonable and cost-effective strategy.

Type frequency and antimicrobial susceptibility of Mycobacterium avium-intracellulare complex strains isolated in Italy from AIDS and non-AIDS patients.

Typing of the glycopeptidolipid antigens performed by thin layer chromatography on 59 Mycobacterium avium-intracellulare (MAC) strains isolated in Italy from AIDS patients showed that the most frequent types were 1, 4, 3, 8, and 21 (24, 19, 14, 14 and 8% of the strains, respectively). Among non-AIDS patients, types 1, 4 and 8 were also frequently found. The antimicrobial susceptibility tested in agar and/or liquid media to a panel of drugs indicated in clofazimine and rifabutin effective agents against both AIDS and non-AIDS strains. The data obtained show that MAC type distribution in Italy appears to be different from that reported for other countries. No major differences in drug susceptibility between AIDS and non-AIDS related strains were found.

Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity.

Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections. Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level. SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A. hydrophila, 7 A. sobria and 18 A. caviae) from patients with gastroenteritis and from healthy controls. Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A. caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests. Data obtained in analyzing A. sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species. Strains biochemically identified as A. hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment. Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A. sobria and A. hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium bovis in bovine tissue samples.

The commercial LCx amplification assay, usually employed to detect the Mycobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Lowenstein-Jensen solid medium and pathological findings in 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.

Mycobacterial infections in solid organ transplant recipients.

Mycobacterial infections represent a growing challenge for solid organ transplant recipients (SOT). The adverse effects of tuberculosis (TB) therapy present a major difficulty, due to the interactions with immunosuppressive drugs and direct drug toxicity. While TB may be donor-transmitted or community-acquired, it usually develops at a latent infection site in the recipient. Pre-transplant prevention efforts will improve transplant outcomes and avoid the complications associated with post-transplant diagnosis and treatment. The present review and consensus manuscript is based on the updated published information and expert recommendations. The current data about epidemiology, diagnosis, new regimens for the treatment of latent TB infection (LTBI), the experience with rifamycins for the treatment of active TB in the post-transplant period and the experience with isoniazid for LTBI in the liver transplant population, are also reviewed. We attempt to provide useful recommendations for each transplant period and problem concerning mycobacterial infections in SOT recipients.

Bacteriostatic and bactericidal activities of paromomycin against Mycobacterium avium complex isolates.

The MICs and MBCs of paromomycin for 32 Mycobacterium avium complex (MAC) strains isolated from patients with the acquired immunodeficiency syndrome were determined by a radiometric broth dilution method. The MICs for the majority of strains were either 8 or 16 mg/L and the MBCs were four- to eight-fold higher. Paromomycin merits further evaluation as oral prophylaxis against disseminated MAC infection.