Assessment of Interferon-Gamma Release Assay in Patients with Non-Tuberculous Mycobacteria Pulmonary Disease.

BACKGROUND: Interferon-gamma release assay (T-SPOT.TB) has the theoretical possibility of discriminating TB from most non-tuberculous mycobacteria (NTM) infections, but there are limited reports on the use of T-SPOT.TB for diseases due to NTM in high TB burden country. The aim of the present study was to assess the utility of T-SPOT.TB in patients with NTM pulmonary disease. METHODS: Clinical parameters and laboratory characteristics of patients with NTM pulmonary disease between July 2011 and Jan 2017 were investigated retrospectively and comprehensively reviewed. RESULTS: A total of 127 patients with NTM pulmonary disease were retrospectively reviewed. Seven NTM species were isolated from 115 patients, and the most common species were M. intracellulare (48.7%, 56/115) and M. abscessus (34.8%, 40/115). NTM isolates were mainly prevalent in people aged 50 years or older (73.0%). The overall positive rate of T-SPOT.TB test was 29.6% (24/81). In patients infected with NTM sharing the RD1 region of Mycobacterium tuberculosis (M. TB), 50% (3/6) were positive in the T-SPOT.TB test, whereas 28.0% (21/75) was positive in the group with NTM not sharing the RD1 region of M. TB. No significant difference was detected in the positive rate of T-SPOT.TB between definite (28.3%, 15/53) and probable disease (32.1%, 9/28). CONCLUSIONS: Our data indicated a relatively high positive rate of T-SPOT.TB test in patients infected with NTM not sharing the RD1 region of M. TB. Thus, T-SPOT.TB test displays a limited ability in differentiating TB infection from NTM disease in a high TB burden country.

Serum Zinc Concentrations in Patients with Pulmonary Tuberculosis.

Bangladesh is a tuberculosis (TB) burden country. It is one of the most important causes of mortality and morbidity and a major barrier of social and economic development. Zinc is a major trace element and an essential component of the body immune system. It's an important determinant of resistance to infection by maintaining cell mediated immunity. This analytical case control study was conducted in the Department of Internal Medicine, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh to see the association of serum zinc concentrations with pulmonary tuberculosis (PTB) in adult population (18-60 years) from January 2015 to January 2016. Freshly diagnosed PTB patients before initiating anti-TB chemotherapy as cases (N=43) and TB negative subjects as controls (N=48) were included conveniently in this study with a rigid selection criteria. Serum zinc concentrations were estimated by using atomic absorption spectrophotometer. The mean±SD age and BMI of the case group and control group were 33.30±14.71 and 32.69±11.60 years, 19.88±2.31 and 22.08±2.80 kg/m2 respectively. The concentrations of serum zinc were significantly lower (P=0.01) in PTB group (840.9±230.0 µgm/l) compared with the control group (965.6±219.9 µgm/l). There was marked variation of mean±SD serum zinc concentrations between male (1008.95±246.16 µgm/l) and female (937.24±200.35 µgm/l) in control group (P=0.182) though the variation is minimal in PTB group (P=0.724). The serum zinc concentrations showed positive correlation with BMI (P=0.642) but negative correlation with age (P=0.023) in both case and control. The lower serum zinc concentrations (12.06%) in PTB patients indicate relative immune deficiency. Routine assessment of serum zinc concentration of PTB patients should be considered and further outcome should be assessed with zinc supplementation.

Effect of SLCO1B1 Polymorphisms on Rifabutin Pharmacokinetics in African HIV-Infected Patients with Tuberculosis.

Rifabutin, used to treat HIV-infected tuberculosis, shows highly variable drug exposure, complicating dosing. Effects of SLCO1B1 polymorphisms on rifabutin pharmacokinetics were investigated in 35 African HIV-infected tuberculosis patients after multiple doses. Nonlinear mixed-effects modeling found that influential covariates for the pharmacokinetics were weight, sex, and a 30% increased bioavailability among heterozygous carriers of SLCO1B1 rs1104581 (previously associated with low rifampin concentrations). Larger studies are needed to understand the complex interactions of host genetics in HIV-infected tuberculosis patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT00640887.).

Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa.

BACKGROUND: Autopsy studies of HIV/AIDS-related hospital deaths in sub-Saharan Africa reveal frequent failure of pre-mortem diagnosis of tuberculosis (TB), which is found in 34-64 % of adult cadavers. We determined the overall prevalence and predictors of TB among consecutive unselected HIV-positive adults requiring acute hospital admission and the comparative diagnostic yield obtained by screening urine and sputum samples obtained on day 1 of admission with Xpert MTB/RIF (Xpert). METHODS: To determine overall TB prevalence accurately, comprehensive clinical sampling (sputum, urine, blood plus other relevant samples) was done and TB was defined by detection of Mycobacterium tuberculosis in any sample using Xpert and/or mycobacterial liquid culture. To evaluate a rapid screening strategy, we compared the diagnostic yield of Xpert testing sputum samples and urine samples obtained with assistance from a respiratory study nurse in the first 24 h of admission. RESULTS: Unselected HIV-positive acute adult new medical admissions (n = 427) who were not receiving TB treatment were enrolled irrespective of clinical presentation or symptom profile. From 2,391 cultures and Xpert tests done (mean, 5.6 tests/patient) on 1,745 samples (mean, 4.1 samples/patient), TB was diagnosed in 139 patients (median CD4 cell count, 80 cells/µL). TB prevalence was very high (32.6 %; 95 % CI, 28.1-37.2 %; 139/427). However, patient symptoms and risk factors were poorly predictive for TB. Overall, ≥1 non-respiratory sample(s) tested positive in 115/139 (83 %) of all TB cases, including positive blood cultures in 41/139 (29.5 %) of TB cases. In the first 24 h of admission, sputum (spot and/or induced samples) and urine were obtainable from 37.0 % and 99.5 % of patients, respectively (P <0.001). From these, the proportions of total TB cases (n = 139) that were diagnosed by Xpert testing sputum, urine or both sputum and urine combined within the first 24 h were 39/139 (28.1 %), 89/139 (64.0 %) and 108/139 (77.7 %) cases, respectively (P <0.001). CONCLUSIONS: The very high prevalence of active TB and its non-specific presentation strongly suggest the need for routine microbiological screening for TB in all HIV-positive medical admissions in high-burden settings. The incremental diagnostic yield from Xpert testing urine was very high and this strategy might be used to rapidly screen new admissions, especially if sputum is difficult to obtain.

[ASSESSMENT OF INDICATORS FOR INTERFERON SYSTEM IN PATIENTS WITH COMMON FORMS OF PULMONARY TUBERCULOSIS RECEIVING COMPLEX THERAPY WITH CYCLOFERON].

We have evaluated the efficacy of cycloferon inclusion in the complex therapy of newly diagnosed patients with common forms of pulmonary tuberculosis, based on monitoring of the number of monocytes with receptors to interferon-gamma (flow cytometry) and the concentration of interferon-gamma in the serum (ELISA). For this purpose, a group of 36 patients (18 patients received standard chemotherapy, and 18 additionally received 600 mg cycloferon tablets 3 times per week) was examined for 3 months. Control group consisted of 18 apparently healthy patients. The study did not include patients with multiple or extensively drug-resistant M. tuberculosis strains. The analysis of results showed a statistically significant positive dynamics of the level of monocyte receptors to interferon-gamma in patients receiving cycloferon as manifested by an increase in their number in the first 2 months of therapy (period of clinical manifestations of the disease), followed by a decrease in the 3rd months of treatment, which corresponds to clinical improvement, in contrast to patients treated with standard chemotherapy alone.

Development of a POC test for TB based on multiple immunodominant epitopes of M. tuberculosis specific cell-wall proteins.

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.

A whole blood assay as a simple, broad assessment of cytokines and chemokines to evaluate human immune responses to Mycobacterium tuberculosis antigens.

In vitro stimulation of whole blood or isolated peripheral blood cells with specific antigens is used for several purposes. We sought to identify a reliable, reproducible, fast and feasible in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis. In contrast to peripheral blood mononuclear cell (PBMC) culture, a whole blood assay (WBA) provides a more physiological environment, which may provide a broader assessment of serum biomarker, biosignature profiles. Twenty-three asymptomatic individuals with M. tuberculosis infection were recruited. Total cells from the WBA (diluted 1:3 in completed RPMI) and PBMC (2×10(5)cells/ml) plus M. tuberculosis Ag85A, Ag85B, ESAT-6 and Mycobacterium bovis 65kDa were characterized by flow cytometry, then added in 96-well plates and on day 5 plasma and supernatants were harvested for detection of 17 cytokines by a Luminex array system. There was agreement between PBMC and WBA for IL-2, IL-5, IL-6, IL-7, IL-13, IFN-γ, TNF-α, MCP-1 and MIP-1ß. There was evidence toward higher IL-10 (p≤0.049) and G-CSF (p≤0.012) plasma production, and higher IL-1ß (p≤0.048), IL-4 (p≤0.044), IL-12p70 (p≤0.006), IL-17 (p≤0.002) and GM-CSF (p≤0.049) production for PBMC vs. WBA. Both methods provided virtually no reaction to the internal, negative control. Due to technical issues linked to data out of range, IL-8 data were not considered. These results suggest that, depending on the method employed, PBMC and/or WBA techniques provide fine conditions for the model proposed and thus whole blood cultures are well-suited low-cost proxy-measures during search for serum biomarkers.

Assessment of serum KL-6 as a prognostic marker in pulmonary tuberculosis patients.

Although serum Krebs von den Lungen-6 (KL-6) levels are reported to increase in pulmonary tuberculosis (PTB) patients according to disease activity, the relationship between serum KL-6 levels and prognosis remains unclear. In this study, we prospectively examined serum KL-6 levels in 188 PTB patients and assessed 60-day mortality. Univariate and multivariate logistic regression analysis demonstrated that serum KL-6 levels were not significantly associated with prognosis. For receiver operating characteristic analysis, the area under the curve had low accuracy for predicting mortality. These findings indicate that serum KL-6 levels do not perform adequately for use as a prognostic marker in patients with PTB.

[The clinical laboratory comparison in the assessment of treatment prognosis in patients with infiltrative tuberculosis of lungs].

The comprehensive prospective examination of 66 patients with first established non-treated infiltrative tuberculosis of lungs was used to analyze the possibility of optimization of assessment of course of specific inflammatory process on the basis of levels of proteins-reactants of acute phase. The characteristics of dynamics of clinical roentgenologic data and terms of coming of abacillarization as a result of three months anti-tuberculosis therapy has been used as grouping factors. It is established that the constellation of 3 out of 12 basic (before treatment) analyzed level indicators are the most prognostic informative--haptoglobin, ceruloplasmin and blood albumin. Their combined application according the proposed decisive rule provides 90.6% of effectiveness of prognosis of the results of treatment in the discussed category of patients.

Serological tests for the diagnosis of active tuberculosis: relevance for India.

Diagnostic tests for active tuberculosis (TB) based on the detection of antibodies (serological tests) have been commercially available for decades, although no international guidelines have recommended their use. An estimated 1.5 million serological TB tests, mainly enzyme-linked immunosorbent assays, are performed in India alone every year, mostly in the private sector. The cost of serological tests in India is conservatively estimated at US $15 million (`825 million) per year. Findings from systematic reviews on the diagnostic accuracy of serological tests for both pulmonary and extra-pulmonary TB suggest that these tests are inaccurate and imprecise. A cost-effectiveness modelling study suggests that, if used as a replacement test for sputum microscopy, serology would increase costs to the Indian TB control sector approximately 4-fold and result in fewer disability-adjusted life years averted and more false-positive diagnoses. After considering all available evidence, the World Health Organization issued a strong recommendation against the use of currently available commercial serological tests for the diagnosis of TB disease. The expanding evidence base continues to demonstrate that the harms/risks of serological tests far outweigh the benefits. Greater engagement of the private sector is needed to discontinue the use of serological tests and to replace these tests with WHO-endorsed new diagnostics in India. The recent ban on import or sale of TB serological tests by the Indian health ministry is a welcome step in the right direction.

Assessment of serum IL-1, IL-2 and IFN-γ levels in untreated pulmonary tuberculosis patients: role in pathogenesis.

BACKGROUND AND AIMS: Tuberculosis (Tb) infection is controlled by cell-mediated immunity through mediation of IL-1, IL-2 and IFN-γ. In this study IL-1, IL-2 and IFN-γ were determined in serum samples of untreated pulmonary Tb and control group including apparently healthy individuals or contacts and normal healthy blood donors with an objective of understanding defect(s), if any, in synthesis of any of these cytokines that may lead to a diseased state of Tb. METHODS: IL-1, IL-2 and IFN-γ were measured in serum samples of untreated Tb patients (n=33), contacts (n=19) and healthy individuals (n=20) by commercially available monoclonal antibody-based ELISA. RESULTS: Statistically significant differences in IL-1 and IFN-γ concentrations between groups of pulmonary Tb and controls were observed, whereas no significant difference in IL-2 was seen. CONCLUSIONS: In the present study, increased levels of cytokines in patients with pulmonary Tb are indicative of Th1 response. An increased level of cytokine (IFN-γ) in patients with untreated pulmonary Tb appears to be functionally defective.

[Is the tuberculin skin test still suitable to diagnose tuberculosis infection ?]. / ¿Es suficiente la prueba tuberculínica para el diagnóstico de la infección tuberculosa ?

Tuberculosis (TB) infection is currently being diagnosed by the tuberculin skin test (TST) with PPD. Some Mycobacterium tuberculosis PPD components are present in BCG, which can be the cause of false positive TST results in BCG vaccinated persons. New IFN-g release assays (IGRAs) are based on the ex vivo release of IFN-g by peripheral blood cells in presence of M. tuberculosis antigens ESAT-6 and CFP-10, which should be absent in BCG. These assays consist in either quantifying released IFN-g or enumerating IFN-g producing cells. In principle, IGRAs should differentiate true TB infection from vaccination and results of several studies suggest that these assays display lower positivity than TST. Whether the lower positivity could be attributed to higher specificity or to lower sensitivity as compared with PPD is still unclear. BCG vaccination, if not recently applied, cannot be blamed for false positive TST reactions. Strong TST reactions (> or = 10 mm or > or =15 mm) are highly correlated with TB infection. In settings where TB continues being a serious health problem, cost-effectiveness evaluations would privilege TST under certain conditions: supply of quality-assured PPD reagent, standardized criteria for TST application, reading and interpretation, and regular availability in health centers countrywide. In view of current limitations in the supply of imported PPD in Argentina, its production/quality assurance should be considered a public health priority. Still, key questions remain to be addressed concerning the role of IGRAs and TST in predicting risk of TB disease, in other words, in identifying persons who will benefit most from chemoprophylaxis.

[Serum nitric oxide level in the assessment of systemic inflammation in patients with drug-resistant pulmonary tuberculosis].

Two hundred and forty-three patients with active pulmonary tuberculosis were examined. Their sera were tested for the level of stable nitric oxide (NO) metabolites, by using the Griess reagent after previous reduction of nitrates to nitrites by a copper-impregnated cadmium reducer. The frank active pulmonary tuberculosis was ascertained to follow the lower serum levels of NO metabolites. The serum NO level did not correlate with inflammatory markers, but reduced when the process was recurrent or chronic. By taking into account the role of NO in the performance of the body's different systems, its serum reduction in patients with pulmonary tuberculosis should be probably referred to as the manifestations of metabolic decompensation as the suppressed endothelial NOS activity that determines the level of NO in circulation.

¿Es suficiente la prueba tuberculínica para el diagnóstico de la infección tuberculosa? / Is the tuberculin skin test still suitable to diagnose tuberculosis infection?

La infección tuberculosa (TB) se determina por la prueba tuberculínica (PTC) con PPD, un extracto de proteínas/péptidos de Mycobacterium tuberculosis, algunos compartidos con otras micobacterias como BCG, lo cual origina falsos resultados positivos en vacunados/no infectados. La nuevas pruebas ex vivo miden el interferón ? (IFN- ?) liberado en sangre, o la cantidad de células que lo producen, en presencia de los péptidos ESAT-6 y CFP-10 de M. tuberculosis. Como estos antígenos no existirían en BCG, las pruebas IFN-? diferenciarían infección TB de vacunación. Numerosos estudios han comparado estas pruebas con la PTC con resultados aún no concluyentes. Las pruebas IFN-? tendrían menor sensibilidad que la PTC, aunque su menor positividad en poblaciones vacunadas podría interpretarse como mayor especificidad. Por otra parte, la vacunación BCG, si no es reciente, no es causa de falsos positivos a la PTC: reacciones =10 mm o =15 mm indican infección TB con altísima probabilidad. Donde la incidencia de TB es mediana o alta, la PTC aventaja en costo-eficiencia a las pruebas IFN-?, siempre que se emplee PPD de calidad garantizada, disponible en todos los centros de salud del país, con aplicación, lectura e interpretación estandarizadas. Como existen en la Argentina problemas de abastecimiento de PPD importado, es preciso producirlo localmente y asegurar su control de calidad. También es necesaria la investigación aplicada al desarrollo de nuevos métodos y la evaluación de su capacidad de predecir la evolución de infección a TB activa, es decir, de identificar las personas que más se beneficiarían con quimioprofilaxis.

Tuberculosis (TB) infection is currently being diagnosed by the tuberculin skin test (TST) with PPD. Some Mycobacterium tuberculosis PPD components are present in BCG, which can be the cause of false positive TST results in BCG vaccinated persons. New IFN-? release assays (IGRAs) are based on the ex vivo release of IFN-? by peripheral blood cells in presence of M. tuberculosis antigens ESAT-6 and CFP-10, which should be absent in BCG. These assays consist in either quantifying released IFN-? or enumerating IFN-? producing cells. In principle, IGRAs should differentiate true TB infection from vaccination and results of several studies suggest that these assays display lower positivity than TST. Whether the lower positivity could be attributed to higher specificity or to lower sensitivity as compared with PPD is still unclear. BCG vaccination, if not recently applied, cannot be blamed for false positive TST reactions. Strong TST reactions (=10 mm or =15 mm) are highly correlated with TB infection. In settings where TB continues being a serious health problem, cost-effectiveness evaluations would privilege TST under certain conditions: supply of quality-assured PPD reagent, standardized criteria for TST application, reading and interpretation, and regular availability in health centers countrywide. In view of current limitations in the supply of imported PPD in Argentina, its production/quality assurance should be considered a public health priority. Still, key questions remain to be addressed concerning the role of IGRAs and TST in predicting risk of TB disease, in other words, in identifying persons who will benefit most from chemoprophylaxis.

The assessment of IFN-gamma and its regulatory cytokines in the plasma and bronchoalveolar lavage fluid of patients with active pulmonary tuberculosis.

OBJECTIVES: To assess the role of IFN-gamma and its regulatory cytokines in active pulmonary tuberculosis (TB). DESIGN: Cytokines were measured in the plasma of TB patients and healthy subjects with different risk for TB exposure. In addition, cytokine profile was assessed in the bronchoalveolar lavage fluid (BALf) of six TB patients and nine normal controls. RESULTS: Circulating IFN-gamma, IL-10 and IL-18 were higher in TB patients than in control groups. Plasma IL-12 levels were extremely variable, and no difference was observed among study groups. An inverse correlation between plasma IFN-gamma and IL-10 levels was found in TB patients. Furthermore, circulating IL-18 correlated with IL-10 but not with IFN-gamma levels. Finally, IFN-gamma, IL-18 and IL-12 were increased in the BALf of TB patients, whereas no difference was observed in IL-10 levels. CONCLUSIONS: In human TB, at least at certain disease stages, there is a differential compartmentalization of the IFN-gamma-regulatory factors IL-12 and IL-10, the former being concentrated in the lungs and the latter being present in peripheral circulation. In addition, our findings address more critically the role of IL-18 in the host response to tuberculosis infection in humans.

Is there a value of mantoux test and erythrocyte sedimentation rate in pre-employment screening of health care workers for tuberculosis in a high prevalence country?

SETTING: Pre-employment screening of health care workers (HCWs) is practiced widely. Research needs to be carried out to evaluate the screening procedure in developing countries. OBJECTIVE: To evaluate the efficacy of Mantoux test and erythrocyte sedimentation rate (ESR) for the diagnosis of active tuberculosis (TB), in pre-employment screening of HCWs, in a high prevalence country. DESIGN: Pre-employment screening of all new employees was reviewed from June to September 2000. The screening consisted of history, physical examination, blood and urine tests, Mantoux test and a chest radiograph. Patients with clinical, laboratory or radiological features suggestive of active TB were referred to a specialist. RESULTS: Out of 207 employees, a Mantoux reaction of > or = 10 mm and ESR of > or = 25 mm/first hour was noted in 90 (43.5%) and 21 (10.1%), respectively. One person had symptoms suggestive of TB and was already on anti-tuberculosis therapy at the time of screening. All other employees were asymptomatic. Based on radiographic findings, four (2%) cases were referred and one was given anti-tuberculosis therapy. An additional 48 (23.1%) employees were referred on the basis of positive Mantoux or elevated ESR; none were found to have active TB. CONCLUSION: In high prevalence countries use of Mantoux test and ESR in pre-employment screening of HCWs is not recommended for detection of active TB.

[Scanning electron microscopy in the assessment of erythrocyte shape in peripheral blood of patients with anthracotic tuberculosis and pulmonary tuberculosis]. / Rastvovaia élektronnaia mikroskopiia v otsenke konfiguratsii éritrotsitov perifericheskoi krovi bol'nykh koniotuberkulezom i tuberkulezom legkikh.

Morphology of peripheral blood red cells was investigated in the course of enterosorption in patients with anthracotic and lung tuberculosis by means of scanning electron microscopy. Application of enterosorbent noticeably reduced the number of normocytes, this evidencing stimulating effect of detoxication. The proportion of deformed red cells lowered in parallel to inhibiting activity of tuberculous process activity in the lungs as a result of detoxicating effect of the treatment.

[Assessment of the activity of pulmonary tuberculosis detected in a migrating rural population]. / Otsenka aktivnosti tuberkuleza legkikh, vyiavlennogo u migriruiushchego sel'skogo naseleniia.

The author established that the activity of freshly detected pulmonary tuberculosis was confirmed by a reduction of the blood serum content of DNA, increase of DNA, nucleotides and ceruloplasmin. One could not find any essential differences in disorders of the above values in seasonal workers and in the group of "nonemigrant" population.