Lung ultrasonography in pulmonary tuberculosis: A pilot study on diagnostic accuracy in a high-risk population.

OBJECTIVES: The validity of lung ultrasound (LUS) in the diagnosis of interstitial or focal lung pathologies is well documented, we assessed its accuracy in the diagnosis of pulmonary tuberculosis (PTB). METHODS: Sonographic signs suggestive of PTB and their diagnostic accuracy were evaluated in patients admitted with clinical suspicion of PTB. Consolidations, subpleural nodules, pleural thickenings or irregularities and pleural effusion were assessed. LUS signs significantly associated with PTB in the univariate analysis (p < .05) were entered in a multivariate logistic regression model. RESULTS: PTB was confirmed in 51 out of 102 patients. Multiple consolidations (OR 3.54, 95%CI 1.43-8.78), apical consolidations (OR 9.65, 95%CI 3.02-30.78), superior quadrant consolidations (OR 4.01, 95%CI 1.76-9.14), and subpleural nodules (OR 5.29, 95%CI 2.27-12.33) were significantly associated with PTB diagnosis. Apical consolidation (OR 9.67, 95%CI 2.81-33.25, p 0.003) and subpleural nodules (OR 5.30, 95%CI 2.08-13.52, p 0.005) retained a significant association in a multivariate model, with an overall accuracy of 0.799. CONCLUSIONS: Our data suggest a possible role of LUS in the diagnosis of PTB, a high burden pathological condition for which the delay in diagnosis still represents a critical point in the control of the disease.

De-escalation therapy among bacteraemic patients with community-acquired pneumonia.

There is no evidence supporting the use of de-escalation therapy (DET) among patients with community-acquired pneumonia (CAP). We assessed the outcomes associated with DET among bacteraemic CAP patients. We performed a secondary analysis of the Community-Acquired Pneumonia Organization database, which contains data on 660 bacteraemic patients hospitalized because of CAP in 35 countries (2001-2013). Exclusion criteria were death within 72 h from admission and an inappropriate empirical antibiotic regimen. DET was defined as changing an appropriate empirical broad-spectrum regimen to a narrower-spectrum regimen according to culture results within 7 days from hospital admission. Two study groups were identified: patients whose antibiotic therapy was de-escalated (the DET group), and patients whose antibiotic therapy was not de-escalated (the N-DET group). The primary study outcome was 30-day mortality. Two hundred and sixty-one bacteraemic CAP patients were included. Gram-positive bacteria were responsible for 88.1% of the cases (Streptococcus pneumoniae, 75.9%). Gram-negative bacteria were responsible for for 7.3% of the cases. DET was performed in 165 patients (63.2%). The N-DET group was characterized by a more severe presentation at admission. After adjustment for confounders, DET was not associated with an increased risk of 30-day mortality. DET seems to be safe among bacteraemic patients with CAP. Randomized clinical trials are warranted to further explore these findings.

A new predictive model for an improved respiratory isolation strategy in HIV-infected patients with PTB.

SETTING: Luigi Sacco Hospital, Milan, Italy, 1 January 2000-31 December 2010. OBJECTIVES: To develop a predictive score for identifying human immunodeficiency virus (HIV) infected patients with pulmonary tuberculosis (PTB). DESIGN: Retrospective study based on the medical charts of HIV-infected patients admitted consecutively on presumption of PTB. Patients with culture-positive TB were included in the TB group. Culture-negative subjects formed the non-TB group. Risk factors for PTB were identified and a predictive model was developed. The diagnostic test accuracy of the derived score and that of previously developed scores were analysed. RESULTS: A total of 65 patients were included in the TB group and 505 subjects in the non-TB group. An eight-variable model (age, origin, alcohol use, respiratory rate, weight loss, haemoglobin, white blood cell count, typical chest X-ray) was derived. When compared with the different scores, this model showed the greatest area under the receiver operating characteristic curve (0.880). This score was the only one to present a negative likelihood ratio of <0.2, which is the threshold for giving strong diagnostic evidence against TB. CONCLUSIONS: This model may be useful in predicting PTB in HIV patients in low-endemic countries. A validation study is necessary.

Genotyping analyses of tuberculosis transmission among immigrant residents in Italy.

We used DNA fingerprinting to analyse tuberculosis (TB) epidemiology in immigrant patients living in two major northern Italian urban areas. The study population included 1999 TB patients (1500 Italian-born and 499 immigrants). Univariate and multivariate logistic regression models were used to identify risk factors related to clustering similar proportions of immigrant and Italian-born patients (46%) had infection with TB strains that belonged to genetic clusters. This supports the hypothesis that the disease in foreign patients is more likely to have arisen from reactivation of latent infection acquired in the country of origin than from recent transmission. Gender, age, human immunodeficiency virus infection and drug resistance were not significantly linked to TB clustering. Risk factors associated with strain clustering were country of origin (Somalia, adjusted OR (AOR) 3.19, p 0.017; Peru, AOR 2.86, p 0.014; and Senegal, AOR 2.60, p 0.045) and city of residence. Immigrant status in the larger urban area was an independent risk factor for infection with clustered TB, as reinforced by a subanalysis of the Senegalese group. In conclusion, variations in TB transmission were observed among immigrants from different countries and even within national groups, where living conditions have been found to exert a profound impact. These results emphasize the importance of improving social integration of immigrant subjects in order to limit risks of TB transmission in developed countries.

Impact on rates and time to first central vascular-associated bloodstream infection when switching from open to closed intravenous infusion containers in a hospital setting.

An open-label, prospective cohort, active healthcare-associated infection surveillance sequential study was conducted in four Italian intensive-care units. The aim was to determine the effect of switching from open (glass) to closed fully collapsible plastic intravenous (i.v.) infusion containers (Viaflo) on rate and time to onset of central venous catheter-associated bloodstream infections (CVC-BSI). A total of 1173 adult patients were enrolled. The CVC-BSI rate during the open container period was significantly higher than during the closed container period (8.2 vs. 3.5 BSI/1000 CVC days, relative risk 0.43, 95% confidence interval 0.22-0.84, P=0.01). The probability of developing a CVC-BSI was assessed over time comparing open and closed i.v. infusion containers. In the closed container period, it remained fairly constant (0.8% at days 1-3 to 1.4% at days 7-9) whereas during the open container period it increased (2% at days 1-3 to 5.8% at days 7-9). Overall, the chance of acquiring a CVC-BSI significantly decreased by 61% in the closed container period (Cox proportional hazard ratio 0.39, P=0.004).

Interferon-gamma and granulocyte-macrophage colony stimulating factor therapy in three patients with pulmonary aspergillosis.

An immune response mediated by type 2 cytokines is thought to contribute to the development and unfavorable outcome of aspergillosis. Adjuvant therapy with interferon-gamma (IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) was added to antifungal treatment in three nonneutropenic patients (one HIV-positive and two HIV-negative patients) with culture proven aspergillosis refractory to classical antifungal therapy. Clinical improvement was observed concomitantly with an increase in peripheral blood leukocyte proliferation and type 1 cytokines production. Our findings suggest an association between the improvement in type 1 cytokine production observed during IFN-gamma and GM-CSF administration and a better control of Aspergillus infection in patients with progressive disease despite adequate antifungal therapy.

Nosocomial bacterial pneumonia in HIV-infected patients: risk factors for adverse outcome and implications for rational empiric antibiotic therapy.

BACKGROUND: Nosocomial bacterial pneumonia (NBP) was once considered a common cause of morbidity and mortality among advanced AIDS patients. However, clinical and microbiological characteristics and outcome-associated risk factors in this population are poorly defined. PATIENTS: We conducted a retrospective study of all HIV-infected patients admitted during the period 1988-2002 at the Infectious Diseases Clinic of Milan, Italy, to determine incidence rate and factors affecting mortality of NBP, and to gather clinical and microbiological findings about the condition. RESULTS: We identified 120 episodes of NBP among 4,967 admissions of HIV-infected individuals. A reduction of incidence became evident after the introduction of highly active antiretroviral therapy (HAART). The more common causative agents were Pseudomonas aeruginosa (33%) Staphylococcus aureus (25%) and Streptococcus pneumoniae (21%). Methicillin resistance was frequent among staphylococci (65%). The mortality rate of NBP was 25.8%. Non-statistically significant factors associated with shorter survival were: CD4(+) count < 10 cells/microl, concomitant lung neoplasm, and complicated roentgenographic picture. Only one factor was significantly associated with lower survival, both in univariate and multivariate analysis: a methicillin-resistant Staphylococcus serving as an etiologic agent of pneumonia (RR 4.05; 95% CI, 1.076-15.239; p = 0.039). CONCLUSION: A decline in incidence of NBP in HIV-infected individuals was observed after introduction of HAART. S. aureus and P. aeruginosa were the leading causes of NBP, but frequency of pneumococcal pneumonia was significant. The sole predictor for mortality was methicillin-resistant Staphylococcus as a pneumonia-causing agent.

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.

PCR-hybridization assay for Mycobacterium avium complex: optimization of detection in peripheral blood from humans.

We evaluated the sensitivity of a DNA amplification test for the detection of Mycobacterium avium in blood samples using different blood components and different DNA extraction methods. M. avium-inoculated blood samples were processed to obtain separate blood components: peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNCs), and whole-blood sodium dodecyl sulfate (SDS)-lysate pellets. The sensitivity for the detection of the lowest mycobacterial load (1 CFU/ml) was significantly greater (P < 0.01) with DNA extracted from SDS-lysate pellets than with DNA extracted from PBMCs or PMNCs. Subsequently, DNA extraction methods based on guanidine NaOH, and proteinase were compared. The sensitivity of the guanidine-based method was significantly greater (P < 0.01) than those of the others.

A case of costochondral abscess due to Corynebacterium minutissimum in an HIV-infected patient.

Corynebacterium minutissimum, known as the causative agent of erythrasma, has recently been reported as a clinically significant pathogen in the immunocompromised host. We report for the first time the possible involvement of a multidrug-resistant C. minutissimum strain in a costochondral abscess occurring in an HIV-infected patient.

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

Outcome of multidrug-resistant tuberculosis in human immunodeficiency virus-infected patients.

Among 324 cases of culture-proven tuberculosis from 1988 to 1996 in a hospital in Milan, Italy, 90 (27.8%) were due to Mycobacterium tuberculosis strains resistant to isoniazid and rifampin. Sixty-one of 69 isolates tested had identical restriction fragment length polymorphism patterns. The prevalent strain tested susceptible only to ethionamide and was also resistant to ethambutol, streptomycin, cycloserine, amikacin, kanamycin, terizodone, ofloxacin, rifabutin, rifapentin, and KRM 1648. The median survival time was 94 days. Multivariate analysis showed a trend toward better outcome in the period 1994-1996 (hazard ratio, 4.16; P<.001), and extrapulmonary localization of tuberculosis was the only other independent predictor of a negative outcome (hazard ratio, 2.1; P = .019). The delay from symptoms to beginning of therapy did not seem to be a determining factor in survival time. Standard antituberculosis therapy with four drugs (isoniazid, rifampin, ethambutol, and pyrazinamide) had a higher efficacy than did other regimens with fewer drugs but without a statistically significant difference.

Monitoring of transmission of tuberculosis between wild boars and cattle: genotypical analysis of strains by molecular epidemiology techniques.

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovis strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.

Tuberculosis among immigrants from developing countries in the province of Milan, 1993-1996.

SETTING: The Province of Milan, which has high rates of immigration from developing countries, and the Villa Marelli Institute (VMI), Reference Centre for Tuberculosis Control of Lombardy. OBJECTIVE: To describe epidemiology and clinical patterns of tuberculosis among immigrants from developing countries (IDCs) in the Province from 1993 to 1996. DESIGN: Retrospective analysis of the registries of the Regional Bureau for Public Health and of the VMI concerning immigrant patients with active TB living in the Province. Restriction fragment length polymorphism (RFLP) analysis of the available strains to detect recent transmission among immigrants. RESULTS: IDCs represented 22.8% of all TB cases. The standardised incidence rate was eight times higher in IDCs compared to Italians. Of 596 cases notified in IDCs, 524 (87.9%) had been referred at least once to the VMI. Of these, 77.2% were diagnosed within 5 years of arrival, and 86.6% were brought to medical attention because of symptoms. RFLP fingerprinting demonstrated that the mean period of stay in Italy was significantly higher in clustered than in non clustered patients (61.5 versus 37.3 months). Spread to the native population was episodic. CONCLUSIONS: The incidence of TB is higher among more recent immigrants (i.e., Peruvians). TB cases are largely due to reactivation of infection occurring in the country of origin. Preventive measures for early diagnosis of disease or chemoprophylaxis of dormant infection are not regularly performed, but should be implemented for those immigrants at high risk.

An outbreak of multidrug-resistant tuberculosis involving HIV-infected patients of two hospitals in Milan, Italy. Italian Multidrug-Resistant Tuberculosis Outbreak Study Group.

OBJECTIVE: To describe an outbreak of multidrug-resistant tuberculosis (MDR-TB), amongst HIV-infected patients, spread from one hospital in Milan to another. DESIGN: Descriptive epidemiological investigation and molecular typing. METHODS: All cases identified by intensive case-finding were described in terms of clinical characteristics, previous nosocomial exposure to an infectious MDR-TB patient, previous stays in other institutional settings where exposure to MDR-TB could have occurred, and restriction fragment length polymorphism (RFLP) pattern. RESULTS: Between October 1991 and July 1995, 116 cases of MDR-TB were identified (85 at hospital A and 31 at hospital B). A single case patient, infected at hospital A, introduced the strain into hospital B. Eighty-two of the 92 strains available for fingerprinting revealed an identical pattern; 10 strains had unique RFLP patterns. Nosocomial exposure to an infectious MDR-TB patient was ascertained for 39 of the 56 patients with the 'outbreak' RFLP strain at hospital A (69.6%) and for 24 of the 26 patients at hospital B (92.3%). The median duration of exposure was 32 days at hospital A and 40 days at hospital B. For eight patients with the outbreak strain, exposure was determined to have probably occurred in other hospitals, in the community or in prison. CONCLUSIONS: This is the largest nosocomial outbreak of MDR-TB reported in Europe. Exposure to MDR-TB cases in other institutions caring for HIV-infected patients probably contributed to the spread of this epidemic. Strict control measures should be immediately adopted in order to prevent the spread of TB amongst HIV-infected patients in institutional settings in Europe.

Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays.

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissues with granulomatous diseases for rapid diagnosis of tuberculosis.

We evaluated the sensitivity and specificity of a nested polymerase chain reaction (PCR) to the Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue samples from patients with tubercular and other granulomatous lesions. Five groups of patients and samples were studied: (1) 28 samples from HIV-positive patients with tuberculosis, (2) 8 samples from HIV-negative patients with histologically suspected tuberculosis (confirmed by culture in 5 cases), (3) lymph nodes from 5 HIV-positive patients with Mycobacterium avium-intracellulare infection, (4) lymph nodes from 30 patients with sarcoidosis, and (5) specimens from 17 patients with other granulomatous diseases. The DNA was extracted from sections with a total thickness of 60 microm, and PCR amplified an internal fragment of 123 base pairs. All of the cases with M. tuberculosis infection were PCR-positive, although this sensitivity was partially related to the initial concentration of the DNA used for amplification. Two of the group 4 samples also were repeatedly positive, thus reducing the specificity of the method. All of the cases with granulomatous diseases other than sarcoidosis were negative. We propose a simplified and highly sensitive nested PCR for the diagnosis of M. tuberculosis infection on archived material in HIV-positive and HIV-negative patients.