Diagnostic benefits of adding EspC, EspF and Rv2348-B to the QuantiFERON Gold In-tube antigen combination.

Interferon (IFN)-γ release assays (IGRAs) are used to diagnose latent tuberculosis (TB) infection (LTBI). To improve the accuracy of these tests, different approaches, such as alternative cytokine detection and using different antigens, are considered. Following this purpose, this study aims to evaluate the addition of EspC, EspF and Rv2348-B to those present in the QuantiFERON-TB Gold In-Tube (QFN-G-IT). We included 115 subjects: 74 active TB patients, 17 LTBI individuals and 24 healthy controls. Whole blood samples were collected in QFN-G-IT and in-house tubes containing different combinations of EspC, EspF and Rv2348-B, together with ESAT-6, CFP-10, and TB7.7. After overnight incubation at 37 ºC, plasma was harvested and IFN-γ quantified. IFN-γ levels in the QFN-G-IT and in-house tubes correlated very good (Spearman Rho(r) > 0.86). In-house antigen combinations distinguished healthy individuals from those with active TB and LTBI (specificities and sensitivities higher than 87.5% and 96.3%, respectively [AUC > 0.938]). Adding EspC, EspF and Rv2348-B, increased the sensitivity of the test, being the addition of EspC and Rv2348-B the combination that yielded a higher sensitivity with no specificity loss. Addition of these antigens could improve diagnosis in patients with impaired or immature immune response who are at high risk of developing TB.

Use of IP-10 detection in dried plasma spots for latent tuberculosis infection diagnosis in contacts via mail.

The aim of this study was to test the use of IP-10 detection in dried plasma from contact studies individuals (contacts of smear positive patients), by comparing it with IP-10 and IFN-γ detection in direct plasma, to establish IP-10 detection in DPS as a useful assay for LTBI diagnosis. Whole blood samples were collected from 80 subjects: 12 with active tuberculosis (TB), and 68 from contact studies. The amount of IFN-γ produced by sensitized T cells was determined in direct plasma by QuantiFERON Gold In-Tube test. IP-10 levels were determined in direct and dried plasma by an in-house ELISA. For dried plasma IP-10 determination, two 25 µl plasma drops were dried in Whatman903 filter paper and sent by mail to the laboratory. Regarding TB patients, 100.0%, 91.7% and 75.0% were positive for IFN-γ detection and IP-10 detection in direct and dried plasma, respectively. In contacts, 69.1%, 60.3% and 48.5% had positive results after IFN-γ and IP-10 in direct and dried plasma, respectively. The agreement among in vitro tests was substantial and IP-10 levels in direct and dried plasma were strongly correlated (r = 0.897). In conclusion, IP-10 detection in dried plasma is a simple and safe method that would help improve LTBI management.

Identification of oestrogen, progesterone receptor and human epidermal growth factor receptor 2 expression in mediastinal metastases of breast cancer obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

BACKGROUND: In breast cancer patients, the expression statuses of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are crucial in the choice of treatment. Receptor expression in metastatic lesions can differ from the primary tumour. The aim of our study was to analyse the utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain samples allowing the identification of ER, PR and HER2 expression in patients with mediastinal metastases of breast cancer. PATIENTS AND METHODS: The clinical files of all patients with a final diagnosis of breast cancer mediastinal metastases diagnosed by EBUS-TBNA in our institution were retrospectively analysed. The ability of EBUS-TBNA to obtain samples that allowed hormone receptor and HER2 expression analysis was calculated. RESULTS: Twenty-four patients were included. ER, PR and HER2 assessments could be performed in 22, 20 and 22 patients, respectively. In 20 of the 24 patients it was possible to investigate all three types of receptor expression. In the remaining four cases, where ER, PR or HER2 expression tests could not be performed, it was due to a lack of tissue. In cases with adequate results for EBUS-TBNA and the primary tumour agreement was greater for ER (16/19) and HER2 (12/14) than PR (8/17). Based on receptor status, there was a change in the choice of treatment for five patients. CONCLUSION: In patients with breast cancer mediastinal metastases, ER, PR and HER2 expression can be assessed in samples obtained by EBUS-TBNA whenever a sufficient tissue sample is collected.

PyroTyping, a novel pyrosequencing-based assay for Mycobacterium tuberculosis genotyping.

We developed a novel method, PyroTyping, for discrimination of Mycobacterium tuberculosis isolates combining pyrosequencing and IS6110 polymorphism. A total of 100 isolates were analysed with IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, mycobacterial interspersed repetitive units - variable number tandem repeats (MIRU-VNTR), and PyroTyping. PyroTyping results regarding clustering or discrimination of the isolates were highly concordant with the other typing methods performed. PyroTyping is more rapid than RFLP and presents the same discriminatory power, thus, it may be useful for taking timely decisions for tuberculosis control.

Factors Associated with Fatality during the Intensive Phase of Anti-Tuberculosis Treatment.

OBJECTIVE: To determine the case-fatality rate (CFR) at the end of the intensive phase of tuberculosis (TB) treatment, and factors associated with fatality. METHODS: TB patients diagnosed between 2006 and 2013 were followed-up during treatment. We computed the CFR at the end of the intensive phase of TB treatment, and the incidence of death per 100 person-days (pd) of follow-up. We performed survival analysis using the Kaplan-Meier method and Cox regression, and calculate hazard ratios (HR) and 95% confidence intervals (CI). RESULTS: A total of 5,182 patients were included, of whom 180 (3.5%) died; 87 of these deaths (48.3%) occurred during the intensive phase of treatment, with a CFR of 1.7%. The incidence of death was 0.028/100 pd. The following factors were associated with death during the intensive phase: being >50 years (HR = 36.9;CI:4.8-283.4); being retired (HR = 2.4;CI:1.1-5.1); having visited the emergency department (HR = 3.1;CI:1.2-7.7); HIV infection (HR = 3.4;CI:1.6-7.2); initial standard treatment with 3 drugs (HR = 2.0;CI:1.2-3.3) or non-standard treatments (HR = 2.68;CI:1.36-5.25); comprehension difficulties (HR = 2.8;CI:1.3-6.1); and smear-positive sputum (HR = 2.3-CI:1.0-4.8). CONCLUSION: There is a non-negligible CFR during the intensive phase of TB, whose reduction should be prioritised. The CFR could be a useful indicator for evaluating TB programs.

Evaluation of GenoFlow DR-MTB Array Test for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis.

The aim of this study was to evaluate the GenoFlow DR-MTB array test (DiagCor Bioscience, Hong Kong) on 70 cultured isolates and 50 sputum specimens. The GenoFlow array test showed good sensitivity and specificity compared to the phenotypic Bactec 460TB. This array accurately detected mutations inrpoB,katG, andinhAassociated with resistance to rifampin and isoniazid.

Diagnostic accuracy study of multiplex PCR for detecting tuberculosis drug resistance.

OBJECTIVE: To study the diagnostic accuracy of a multiplex real-time PCR (Anyplex II MTB/MDR/XDR, Seegene, Corea) that detects Mycobacterium tuberculosis resistant to isoniazid (INH), rifampicin (RIF), fluoroquinolones (FLQ) and injectable drugs (kanamycin [KAN], amikacin [AMK] and capreomycin [CAP]) in isolates and specimens. METHODS: One hundred fourteen cultured isolates and 73 sputum specimens were retrospectively selected. Results obtained with multiplex PCR were compared with those obtained with BACTEC. Discordant results between multiplex PCR and BACTEC were tested by alternative molecular methods. RESULTS: Sensitivity and specificity of multiplex PCR for detecting drug resistance in isolates were 76.5% and 100%, respectively, for INH; 97.2% and 96.0%, respectively, for RIF; 70.4% and 87.9%, respectively, for FLQ; 81.5% and 84.8%, respectively, for KAN; 100% and 60%, respectively, for AMK, and 100% and 72.3%, respectively, for CAP. Sensitivity and specificity of Anyplex for detecting drug resistance in specimens were 93.3% and 100%, respectively, for INH; 100% and 100%, respectively, for RIF; 50.0% and 100%, respectively, for FLQ; and 100% and 94.4%, respectively, for both KAN and CAP. Among the discordant results, 87.7% (71/81) of results obtained with the multiplex PCR were concordant with at least one of the alternative molecular methods. CONCLUSIONS: This multiplex PCR may be a useful tool for the rapid identification of drug resistant tuberculosis in isolates and specimens, thus allowing an initial therapeutic approach. Nevertheless, for a correct management of patients, results should be confirmed by a phenotypic method.

AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples.

OBJECTIVES: To determine the sensitivity and specificity of AID TB Resistance line probe assay (AID Diagnostika, Germany) to detect Mycobacterium tuberculosis and its resistance to first- and second-line drugs in clinical samples using BACTEC 460TB as the reference standard. METHODS: The test consists on three strips to detect resistance to isoniazid/rifampicin, fluoroquinolones/ethambutol, and kanamycin/amikacin/capreomycin/streptomycin, respectively. This test was performed on 65 retrospectively selected clinical samples corresponding to 32 patients. RESULTS: A valid result was obtained for 92.3% (60/65), 90.8% (59/65) and 78.5% (51/65) of the samples tested, considering the three strips, respectively. Global concordance rates between AID and BACTEC for detecting resistance to isoniazid, rifampicin, fluoroquinolones, ethambutol, kanamycin/capreomycin and streptomycin were 98.3% (59/60), 100% (60/60), 91.5% (54/59), 72.9% (43/59), 100% (51/51) and 98.0% (50/51), respectively. Regarding the discordant results obtained between AID and BACTEC, the alternative molecular methods performed (GenoType MTBDRplus, GenoType MTBDRsl [Hain Lifescience, Germany] and/or pyrosequencing) confirmed the genotypic result in 90.9% (20/22) of the cases. CONCLUSIONS: AID line probe assay is a useful tool for the rapid detection of drug resistance in clinical samples enabling an initial therapeutic approach. Nevertheless, for a correct management of drug resistant tuberculosis patients, molecular results should be confirmed by a phenotypic method.

Monitoring changes in anti-tuberculosis treatment: associated factors determined at the time of diagnosis.

OBJECTIVES: To determine predictive factors for changes in standard anti-tuberculosis chemotherapy at the time of diagnosis. METHODS: A prospective study was performed among tuberculosis (TB) patients treated at specialised centres during 2008-2009. Treatment outcome was monitored per standard guidelines. Treatment was considered successful if the patient was cured or completed treatment. Factors associated with treatment modification were analysed at the bivariate and multivariate levels using logistic regression. RESULTS: A total of 427 patients were included in the study. The initial standard treatment regimen was retained for 249 patients (58.3%), extended to 9 months for 36 (8.4%) and changed for 142 (33.3%). Factors associated with a change of regimen at the multivariate level were female sex, age ≥ 50 years, human immunodeficiency virus infection, comorbidities, alcoholism, hospitalisation and culture-positive sputum. Drug resistance and toxicity were analysed independently. Treatment outcome was successful in 97.2% of cases without a regimen change and in 87.3% of those with a changed regimen (P < 0.001). CONCLUSION: Factors associated with changes in the initial anti-tuberculosis regimen should be considered for rigorous follow-up. Results obtained through individualised treatment provided by specialists were good despite the complexity of the cases treated.

Anaemia in chronic obstructive pulmonary disease. Does it really matter?

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent chronic diseases, with an increasing rate in morbidity and mortality. In recent years, there has been a greater awareness about the clinical importance of systemic effects and other chronic conditions associated with COPD, as these significantly impact on the course of disease. The most studied extrapulmonary manifestations in COPD include the presence of concomitant cardiovascular disease, skeletal muscle wasting, osteoporosis and lung cancer. Anaemia is a recognised independent marker of mortality in several chronic diseases. Recent studies have shown that anaemia in patients with COPD may be more frequent than expected, with a prevalence ranging from 5% to 33%. Some evidence suggests that systemic inflammation may play an important pathogenic role, but anaemia in COPD is probably multifactorial and may be caused by others factors, such as concealed chronic renal failure, decreased androgenic levels, iron depletion, angiotensin-converting enzyme inhibitor treatment and exacerbations. Low levels of haemoglobin and haematocrit in COPD patients have been associated with poor clinical and functional outcomes as well as with mortality and increased healthcare costs. Despite the potential clinical benefit of successfully treating anaemia in these patients, evidence supporting the importance of its correction on the prognosis of COPD is uncertain.

Initial management of pneumonia and sepsis: factors associated with improved outcome.

Processes of care and adherence to guidelines have been associated with improved survival in community-acquired pneumonia (CAP). In sepsis, bundles of processes of care have also increased survival. We aimed to audit compliance with guideline-recommended processes of care and its impact on outcome in hospitalised CAP patients with sepsis. We prospectively studied 4,137 patients hospitalised with CAP in 13 hospitals. The processes of care evaluated were adherence to antibiotic prescription guidelines, first dose within 6 h and oxygen assessment. Outcome measures were mortality and length of stay (LOS). Oxygen assessment was measured in 3,745 (90.5%) patients; 3,024 (73.1%) patients received antibiotics according to guidelines and 3,053 (73.8%) received antibiotics within 6 h. In CAP patients with sepsis, the strongest independent factor for survival was antibiotic adherence (OR 0.4). In severe sepsis, only compliance to antibiotic adherence plus first dose within 6 h was associated with lower mortality (OR 0.60), adjusted for fine prognostic scale and hospital. Antibiotic adherence was related to shorter hospital stay. In sepsis, antibiotic adherence is the strongest protective factor of care associated with survival and LOS. In severe sepsis, combined antibiotic adherence and first dose within 6 h may reduce mortality.

Usefulness of consecutive biomarkers measurement in the management of community-acquired pneumonia.

The aim of this study was to investigate whether procalcitonin (PCT), neopterin, C-reactive protein (CRP), and mid regional pro-atrial natriuretic peptide (MR-proANP) levels at admission and during the clinical course can be useful for the management of patients with pneumonia. The study population consisted of 75 patients with clinical and radiological diagnosis of pneumonia. Serum samples were collected at admission and during hospitalization. Complications were defined as intensive care unit (ICU) admission or death. The levels of PCT were significantly higher in pneumonia of definite bacterial origin in comparison to probable bacterial or unknown origin. The PCT levels were higher in pneumococcal pneumonia. The PCT and MR-proANP levels increased significantly according to the Pneumonia Severity Index (PSI). All biomarkers levels are higher in patients developing complications and who were dying. The serial levels of MR-proANP remain significantly elevated in patients developing complications and in patients classified in PSI and CURB-65 risk groups. In patients not developing complications, there is a significant decrease in the PCT levels. PCT can be useful for identifying pneumonia etiology. PCT and MR-proANP levels correlate with pneumonia severity rules. PCT and MR-proANP serial measurements can be useful for predicting short-term prognosis. Systemic biomarkers can provide additional information regarding clinical evolution, because these are dynamic and can be measured daily.

GenoType MTBDRsl for molecular detection of second-line-drug and ethambutol resistance in Mycobacterium tuberculosis strains and clinical samples.

The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

Pyrosequencing for rapid molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical specimens.

The aim of this study was to evaluate a pyrosequencing method for the detection of Mycobacterium tuberculosis isolates resistant to rifampin and isoniazid using both clinical strains and clinical samples, comparing the results with those of the Bactec 460TB and GenoType MTBDRplus assays. In comparison to Bactec 460TB as the gold standard, the sensitivity of pyrosequencing for detecting isoniazid and rifampin resistance was 76.9% and 97.2%, respectively, for clinical strains, and the specificity was 97.2 and 97.9%, respectively. For clinical specimens, the sensitivity and specificity for both drugs were 85.7% and 100%, respectively. The overall concordance between pyrosequencing and the GenoType MTBDRplus assay for clinical strains was 99.1%, and for clinical samples, it was 98.2%. Pyrosequencing is a valuable tool for rifampin and isoniazid resistance detection.

Diagnosing TB infection in children: analysis of discordances using in vitro tests and the tuberculin skin test.

The aim of the present study was to compare the performance of the interferon (IFN)-γ tests (QuantiFERON®-TB Gold In-Tube (QFT-G-IT) and T-SPOT®.TB) with the tuberculin skin test (TST) in diagnosing tuberculosis (TB) infection in children, and to analyse discordant results. This was a prospective study including 98 children from contact-tracing studies and 68 children with TST indurations ≥ 5 mm recruited during public health screenings. Positive IFN-γ tests results were associated with risk of exposure (p<0.0001). T-SPOT.TB was positive in 11 (78.6%) out of 14 cases with active TB and QFT-G-IT in nine (64.3%) out of 14 cases. Sensitised T-cells against Mycobacterium avium were detected in six out of 12 children not vaccinated with bacille Calmette-Guérin (BCG), a TST induration 5-9 mm in diameter and both IFN-γ tests negative. In concordant IFN-γ tests results, a positive correlation was found (p = 0.0001) between the number of responding cells and the amount of IFN-γ released. However, in discordant IFN-γ tests results this correlation was negative (p = 0.371): an increase in the number of spot-forming cells correlated with a decrease in the amount of IFN-γ released. The use of IFN-γ tests is helpful for the diagnosis of TB infection, avoiding cross-reactions with BCG immunisation and nontuberculous mycobacterial infections. The analysis of highly discordant results requires further investigation to elucidate possible clinical implications.

Evaluating the non-tuberculous mycobacteria effect in the tuberculosis infection diagnosis.

The aim of the present study was to determine the role of previous non-tuberculous mycobacteria sensitisation in children as a factor of discordant results between tuberculin skin test (TST) and an in vitro T-cell based assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille Calmette-Guérin-vaccinated paediatric patients for suspicious of latent tuberculosis infection (LTBI). These patients yielded a positive TST and a negative T-SPOT.TB. Cells were stimulated with Mycobacterium avium sensitin (having cross-reaction with Mycobacterium intracellulare and Mycobacterium scrofulaceum) and the presence of reactive T-cells was determined by an ex vivo ELISPOT. From the 21 patients, in 10 cases (47.6%), we obtained a positive ELISPOT result after stimulation with M. avium sensitin, in six (28.6%) cases, the result was negative and in the remaining five (23.8%) cases, the result was indeterminate. In conclusion, previous non-tuberculous mycobacteria sensitisation induces false-positive results in the TST for diagnosing LTBI and the use of gamma-interferon tests could avoid unnecessary chemoprophylaxis treatment among a child population.

Persistence of Streptococcus pneumoniae urinary antigen excretion after pneumococcal pneumonia.

The aim of this study was to determine the duration of Streptococcus pneumoniae antigen excretion in urine after pneumococcal pneumonia. Urinary antigen detection remained positive in nonconcentrated urine in 18 (52.9%) of the 34 patients in the first month after pneumonia diagnosis. In 12 of these positive cases, the test was still positive in the second month, in six patients after 4 months, and in two cases 6 months after the diagnosis of pneumonia. Using concentrated urine, antigenuria remained positive in all patients for at least 3 months, with antigen detected in three cases more than one year later. We did not observe a relation between age, gender, immunosuppression, underlying diseases, pneumonia severity, positive blood culture, or X-ray presentation and longer-term antigenuria excretion. However, the small number of patients evaluated is a limitation for statistical analysis. In order to correctly analyse a positive urinary antigen test result in patients with pneumonia, it is necessary to know which patients have recently had a previous episode of pneumonia.

GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples.

The purpose of this study was to evaluate the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 62 clinical strains characterized with the Bactec 460TB system were included. For the INH-resistant strains, the MIC was measured and sequencing was performed. Sixty-five clinical samples from 28 patients (39 smear-positive samples and 26 smear-negative samples) were also tested directly. The corresponding isolates of the clinical specimens were studied with the Bactec 460TB system. The overall rates of concordance of the MTBDRplus assay and the Bactec 460TB system for the detection of RIF and INH susceptibility in clinical strains were 98.3% (61/62) and 79% (49/62), respectively. The rate of concordance between the Bactec 460TB system and the MTBDRplus test for the detection of INH resistance in the group of 27 strains with low-level resistance was 62.9% (17/27), and that for the detection of INH resistance in the group of 21 strains with high-level resistance was 85.71% (18/21). Valid test results were obtained for 78.45% (51/65) of the clinical samples tested. The rates of concordance between both assays for the detection of drug resistance in these samples were 98% (50/51) for RIF and 96.2% (49/51) for INH. Taking into account only one sample per patient, the overall rate of concordance between both tests was 92.85% (26/28). The GenoType MTBDRplus assay is easy to perform and is a useful tool for the management of tuberculosis, as it allows the detection of resistance to RIF and INH in M. tuberculosis strains and also in clinical samples.

The tuberculin skin test increases the responses measured by T cell interferon-gamma release assays.

RUTI is a vaccine consisting of Mycobacterium tuberculosis bacilli grown in stress conditions that is fragmented, detoxified and liposomed. RUTI was designed to shorten the treatment of latent tuberculosis infection (LTBI) with isoniazid from 9 months to just 1 month, by additional treatment with two inoculations of RUTI 4 weeks apart. During the validation process for monitoring the immunogenicity of administration of RUTI in a Phase I clinical trial, the question arose whether to introduce the tuberculin skin test (TST) in the screening of non-LTBI volunteers. This study was designed to evaluate the effect of TST on subsequent different T-cell interferon-gamma release assay (TIGRA) responses, using a spectrum of M. tuberculosis-related antigens (ESAT-6, CFP-10, 16 kDa, 19 kDa, MPT64, Ag 85B, 38 kDa, hsp65, PPD and BCG). The results showed an increase in post-TST response even in non-LTBI subjects for most antigens tested, as measured both by whole blood assay (WBA) and ELISPOT. Increased ELISPOT response decreased toward pre-TST levels within 1 month whereas the WBA response did not. Taking into account that there is no definitive correlation between TST and TIGRA tests to diagnose LTBI and the feasibility that TST might alter the immune monitoring included in clinical trials, these data suggest that TST determination should be carefully planned to avoid any interference with TIGRA.