The use of selected commercial molecular assays for the microbiological diagnosis of tuberculosis

Introduction: We performed a retrospective assessment of the AMPLICOR Mycobacterium tuberculosis (MTB) assay for the molecular diagnosis of tuberculosis based on our own determinations between 1999 and 2009 and a preliminary assessment of the Xpert MTB/RIF system, which we are currently using. Materials and methods: The study groups comprised 1875 samples (including 104 inhibited samples) and 213 samples, respectively. Results: The sensitivities of the AMPLICOR MTB and the Xpert MTB/RIF assays were 81.9% and 81.8%, respectively, and their specificities were 97.2% and 99.5%, respectively, versus culture on Loewenstein-Jensen medium. Both assays showed a considerable difference in sensitivity depending on whether the test samples were smear-positive (AFB+) or smearnegative (AFB–). The sensitivities of the AMPLICOR MTB and the Xpert MTB/RIF assays were 97.8% and 100.0%, respectively, for AFB+ samples and 58.1% and 50.0%, respectively, for AFB– samples. Conclusions: Our results confirm full usefulness of the Xpert MTB/RIF assay for routine diagnosis in the case of smearpositive clinical samples.Wstęp: W pracy, retrospektywnie oceniono test AMPLICOR Mycobacterium tuberculosis (MTB) w molekularnej diagnostyce gruźlicy, na podstawie własnych oznaczeń w latach 1999–2009 oraz wstępnie oceniono system Xpert MTB/RIF, który jest na bieżąco w użyciu. Materiał i metody: Grupy badane obejmowały odpowiednio 1875 próbek (w tym 104 reakcje zahamowane) i 213 próbek. Wyniki: Czułość testów AMPLICOR MTB i Xpert MTB/RIF oceniono na 81,9% i 81,8%, a swoistość wynosiła odpowiednio 97,2% i 99,5%, wobec hodowli na pożywce Loewensteina-Jensena. Obydwa testy wykazywały znaczącą różnicę w czułości, w zależności od tego czy badane próbki były dodatnie (AFB+) czy ujemne (AFB–) w rozmazie. I tak dla próbek AFB+ czułość wynosiła 97,8% i 100%, a dla próbek AFB– czułość była 58,1% i 50%, odpowiednio dla testów AMPLICOR MTB i Xpert MTB/RIF. Wnioski: Przedstawione wyniki wykazują pełną przydatność testu Xpert MTB/RIF w rutynowej diagnostyce dla próbek klinicznych, dodatnich w mikroskopowym badaniu rozmazu. Pneumonol. Alergol. Pol. 2012; 80, 1: 6–1

Diagnostic utility of the molecular assay GenoType MTBC (HAIN Lifesciences, Germany) for identification of tuberculous mycobacteria

Wstęp: Nowy kompleksowy system GenoType firmy HAIN Lifescience (Niemcy) stwarza szerokie możliwości w diagnostyce gruźlicy i innych zakażeń prątkowych na poziomie molekularnym. Dzięki wprowadzeniu 5 wzajemnie uzupełniających się testów, system ten pozwala na wykrycie i typowanie mykobakterii oraz określenie lekowrażliwości na rifampicynę i izoniazyd na różnych etapach postępowania diagnostycznego, od badania materiałów bezpośrednich do wyizolowanych szczepów. Zaletą systemu jest dowolność w wyborze stosowanych testów (bez konieczności wykonywania wszystkich), w zależności od potrzeb i aktualnie stosowanych innych metod wykrywania prątków. System posiada certyfikat Unii Europejskiej do stosowania w rutynowej diagnostyce. Dotychczas żaden z testów systemu GenoType nie był stosowany w Polsce. Celem prezentowanej pracy była ocena precyzji typowania testem GenoType MTBC izolatów klinicznych zidentyfikowanych uprzednio jako M. tuberculosis complex metodą wysokociśnieniowej chromatografii cieczowej w ramach rutynowego postępowania diagnostycznego. Materiał i metody: Badanie miało charakter retrospektywny. Testem GenoType MTBC zbadano 161 izolatów klinicznych M. tuberculosis complex. Szczepy pochodziły od chorych na gruźlicę hospitalizowanych w Centralnym Szpitalu Klinicznym Warszawskiego Uniwersytetu Medycznego w latach 1999-2007. Wyniki: Typowanie testem GenoType MTBC okazało się w 100% zgodne z typowaniem metodą analizy kwasów mykolowych. Test GenoType MTBC wykazał ponadto, że wszystkie izolaty miały wzór hybrydyzacji charakterystyczny dla M. tuberculosis/M. canettii. Wnioski: 1. Test GenoType MTBC (HAIN Lifescience, Niemcy) prawidłowo rozpoznaje kliniczne szczepy M. tuberculosis complex i może w tym zakresie zastąpić wysokociśnieniową chromatografię cieczową w rutynowej diagnostyce gruźlicy. 2. W świetle pozytywnej oceny testu GenoType MTBC celowe wydaje się rozpatrzenie możliwości stosowania pozostałych testów systemu GenoType (HAIN Lifescience, Niemcy) w diagnostyce zakażeń prątkowych.Introduction: The GenoType system (HAIN Lifescience, Germany) offers new perspectives of detecting the tuberculous and non-tuberculous mycobacteria at the molecular level. The system compromises five independent tests that could be performed either on direct specimens or isolated strains, to identify the strains and test the resistance against rifampin and isoniazid. Up to now, non GenoType test was applied in Poland. The aim of the study was an evaluation the accuracy of GenoType MTBC test in speciation of the clinical isolates, previously classified as M. tuberculosis complex by HPLC analyze of mycolic acids. Material and methods: 161 clinical isolates, derived from the TB patients hospitalized in the Warsaw Medical University Hospital between 1999 and 2007 were assayed. Results: On the basis of the hybridization patterns, all 161 studied strains were identified as M. tuberculosis/M. canettii. Conclusions: 1. The GenoType MTBC test (HAIN Lifescience, Germany) precisely recognizes M. tuberculosis complex. The 100% accordance in speciation of M. tuberculosis by the GenoType MTBC test as compared to HPLC method was demonstrated. The GenoType MTBC test can replace HPLC in detection of tuberculous mycobacteria in clinical isolates. 2. As the GenoType MTBC test performs well, the other tests of GenoType system may be considered to be verified in diagnostic procedure of mycobacterial infection

Effect of phosphodiesterase 4 inhibitor (rolipram) on experimental allergic asthma-guinea pig model

Selective phosphodiesterases (PDE) inhibitors are the new group of antiasthmatic drugs, which integrate antiinflammatory activity with bronchoconstriction counteraction. Selective inhibitors of phosphodiesterase type 4 are used as alternative or assist drugs in treatment of respiratory system diseases. So far glucocorticosteroids remain the most efficient and widely used medicine in the treatment of asthma. However application of glucocorticosteroid is greatly limited because of numerous side effects, what induce to permanent search for new antiasthmatic drugs. Examination new substances are executed on animal models. Guinea pig model is widely used to research course of asthmatic reaction. This model is especially convenient on the ground of that: lung is major shock organ, airway respond to histamine, animals demonstrated early asthmatic reaction (EAR) and late asthmatic reaction (LAR), eosinophils flow in bronchoalveolar space during LAR. Inovalbumin(OA)sensitized guine a pigs hypersensitivity reaction breaks out as a result of OA provocation. Aims of our experiments, execute on guinea pig model were to determine the influence of rolipram (PDE 4 inhibitor) on modulation experimental asthmatic reaction and comparison activity of rolipram versus dexamethasone in attribution to chosen parameters of allergic reaction such as: lung resistance, influxofproteinandinflammatorycells in airways, and mastocytes degranulation. Experiments were made on guinea pigs sensitized and provoked with ovalbumin The obtain data indicate that rolipram was effective in reduction the rise of lung resistance during EAR, restricted influx of eosinophils to bronchoalveolar space between 1,5 and 24 hours after provocation, andreduced increase of histamine concentration in bronchoalveolar lavage fluid(BALf).Rolipramhadnoinfluenceon number of neutrophils present in BALf. Dexamethasone in double dose of 1,2mg/kg effectively bordered the growth of lung resistance during EAR, and broke influx of eosinophils and neutrophils to bronchoalveolar space

The comparison between two methods for typing of nontuberculous mycobacteria: high pressure liquid chromatography and molecular assay GenoType Mycobacterium CM/AS

Wstęp: W pracy oceniano przydatność diagnostyczną molekularnego systemu GenoType Mycobacterium CM/AS (HAIN Lifescience, Niemcy) w różnicowaniu izolatów klinicznych mykobakterii w porównaniu z analizą kwasów mykolowych z zastosowaniem cieczowej chromatografii wysokociśnieniowej. Zastosowanie obydwu testów umożliwia wyróżnienie 38 wzorów molekularnych, z których 24 można przypisać do poszczególnych gatunków mykobakterii, 10 wzorów odpowiada mykobaktedwóm, czasem kilku gatunkom, a 4 wzory odpowiadają Mycobacterium sp. lub Gram-dodatnim bakteriom z dużą zawartością par G-C w genomie. Materiał i metody: Badanie miało charakter retrospektywny i obejmowało 127 szczepów mykobakterii wyizolowanych na podłożu stałym Loewensteina-Jensena od pacjentów szpitala klinicznego Warszawskiego Uniwersytetu Medycznego w latach 1999-2007. Prątki identyfikowano metodą analizy kwasów mykolowych cieczową chromatografią wysokociśnieniową (HPLC) w ramach rutynowego postępowania diagnostycznego. Szczepy typowano powtórnie testami molekularnymi, najpierw GenoType Mycobacterium CM, a następnie - jeśli uzyskanego wzoru molekularnego nie można było przypisać do żadnego z wzorców - testem GenoType Mycobacterium AS. Wyniki: Spośród 127 badanych szczepów porównanie obu technik było możliwe dla 113 izolatów. Dla 105 ze 113 (93%) szczepów uzyskano wyniki zgodne. Należy podkreślić, że spośród 8 izolatów rozbieżnie typowanych w jednym przypadku w systemie GenoType Mycobacterium CM/AS stwierdzono obecność genomu M. tuberculosis complex. Szczep ten w typowaniu HPLC określono jako szybkorosnący gatunek M. abscessus/M. chelonae. Spośród pozostałych 14 ze 127 izolatów klinicznych 11 wytypowano tylko jedną z metod: 6 szczepów - techniką HPLC i 5 szczepów - w systemie molekularnym. W przypadku 3 szczepów identyfikacja nie powiodła się żadną z metod. Wnioski: System GenoType Mycobacterium CM/AS pozwala na szybką i rzetelną identyfikację gatunkową izolatów klinicznych mykobakterii. W porównaniu z obecnie stosowaną metodą HPLC system molekularny jest przyjazny dla środowiska i prostszy w wykonaniu. Pneumonol. Alergol. Pol. 2010; 78, 5: 363-368Introduction: The GenoType Mycobacterium CM and the GenoType Mycobacterium AS (HAIN Lifescience, Germany) were evaluated for the ability to differentiate mycobacterial species of clinical isolates. Serial use of the both assays is aimed to identify 38 different molecular patterns, of which 24 patterns can be assigned to single species, 10 patterns are allocated to two or more Mycobacterium species, and 4 patterns correspond to Mycobacterium species and gram-positive bacteria with a high G + C content. The analysis of mycolic acids by high pressure liquid chromatography (HPLC) was the reference method. Material and methods: A set of 127 nontuberculous mycobacterial isolates on Loewenstein-Jensen slants, derived from different patients between 1999 and 2007, was analyzed. The strains were primary classified by HPLC following the diagnostic procedure, and retyped by GenoType Mycobacterium CM/AS. Results: In total, results obtained by both methods were interpretable for 113 strains. Concordant results were obtained for 105 (93%) mycobacterial strains. One out of 8 inconcordant classified strains, which was classified a

Korelacje pomiędzy rozdęciem miąższu płucnego mierzonego wskaźnikiem RV%TLC i składem ciała oraz profilem cytokinowym u chorych na przewlekłą obturacyjną chorobę płuc

Introduction: Body composition is an important prognostic factor in patients with COPD. The decrease in fat free mass (FFM), muscle mass (MM) and increase in visceral fat is associated with an elevated secretion of cytokines which promote systemic inflammation. The aim of the study was to evaluate body composition and the cytokine profile in patients with COPD in relation with the presence of hyperinflation.Material and methods: The study group consisted of 149 patients (61F, 88M) with stable COPD in all stages of severity aged 68 ± 8.8 yrs. All the patients underwent spirometry and bodypletysmography with bronchial reversibility testing. Hyperinflation was defined as RV%TLC > 48% and > 126% predicted. Body composition was analyzed by bioimpedance. The following serum inflammatory markers were evaluated: C-reactive protein, IL-6, IL-8, TNF-a, CC16, adiponectin and resistin.Results: Hyperinflation was found in 96 patients (group A) and it was more frequent in women than men (49/61 vs. 47/88, p < 0.001). BMI and age in this group were comparable to those in patients without hyperinflation (group B). Patients with hyperinflation have lover FFM, FFM index, MM and MM index and total body water and higher fat mass and fat mass index. We found significantly higher serum concentrations of inflammatory markers in group A: IL-6 – 6.4 ± 10.9 vs. 3.6 ± 4.2 pg/ml, resistin – 9.3 ± 4.2 vs. 7.6 ± 2.4 ng/ml, CRP 4.1 ± 2.3 vs. 2.9±2.1 mg/l, respectively.Conclusions: Patients with hyperinflation have a lower FFMI, TBW and MMI and a higher proportion of fat tissue. Hyperinflation is associated with elevated concentrations of inflammatory markers what may be associated with more severe disease. Body compositions abnormality and higher activity of systemic inflammation could therefore be a negative prognostic factor in COPD patients.Wstęp: Skład ciała jest ważnym czynnikiem prognostycznym u chorych na POChP. Spadek beztłuszczowej masy ciała (FFM), masy mięśni (MM) i wzrost masy trzewnej tkanki tłuszczowej jest związane ze wzrostem wydzielania cytokin odpowiedzialnych za systemowe zapalenie. Celem pracy była ocena wpływu hiperinflacji układu oddechowego na stan odżywienia i profilu cytokin u chorych na POChP.Materiał i metody: Grupa badana składała się z 149 chorych (61K, 88M), w stabilnym okresie POChP, którzy reprezentowali wszystkie stopnie ciężkości choroby, w wieku 68 ± 8,8 lat. Wszyscy chorzy mieli wykonaną spirometrię i pletyzmografię z próbą rozkurczową. Rozdęcie było definiowane, jako zwiększenie RV%TLC > 48% and > 126% wn. Skład ciała był mierzony metodą bioimpendancji. Wykonano pomiar następujących cytokin w surowicy: białko C-reaktywne (CRP), IL-6, IL-8, TNF-a, CC16, adiponektyna i rezystyna.Wyniki: Rozdęcie stwierdzono u 96 chorych (grupa A), było ono częstsze u kobiet niż mężczyzn (49/61 v. 47/88, p < 0,001). Indeks masy ciała i wiek były podobne do grupy chorych bez rozdęcia (grupa B). Grupa A miała niższe FFM i FFMI, MM i MMI i całkowitą masę wody oraz wyższą masę tłuszczową i indeks masy tłuszczowej. W grupie A stwierdzono istotnie statystycznie wyższe stężenie w surowicy markerów zapalenia: IL-6 — 6,4 ± 10,9 v. 3,6 ± 4,2 pg/ml, resistin — 9,3 ± 4,2 v. 7,6 ± 2,4 ng/ml, CRP 4,1 ± 2,3 v. 2,9 ± 2,1 mg/l.Wnioski: Chorzy, u których stwierdza się rozdęcie płuc mają niższe FFMI, TBW, MMI i więcej tkanki tłuszczowej. U chorych z rozdęciem płuc stwierdza się podwyższone stężenie markerów stanu zapalnego w porównaniu z chorymi bez rozdęcia, co może świadczyć o bardziej zaawansowanym procesie chorobowym. Zarówno zaburzenia w składzie ciała jak i wyższa aktywność zapalenia systemowego w grupie chorych z rozdęciem płuc może wskazywać na ich gorsze rokowani

Periostin and Thymic Stromal Lymphopoietin—Potential Crosstalk in Obstructive Airway Diseases

Periostin and thymic stromal lymphopoietin (TSLP) are newly described markers of obstructive airway diseases and the mechanism by which both markers participate in immune response remains poorly understood. The aim of our study was to determine periostin and TSLP concentration in serum and induced sputum (IS) in patients with atopic asthma, chronic obstructive pulmonary disease (COPD), and controls, as well as to evaluate the potential link between periostin, TSLP, and Th2 immune response. Serum and IS levels of periostin, TSLP, IL-4, and IL-13 were determined in 12 atopic asthmatics, 16 COPD sufferers, and 10 controls. We noticed a significantly higher IS periostin and TSLP concentration at protein and mRNA level in asthmatics compared to the two other groups; additionally, periostin and TSLP were correlated positively with IS eosinophil count. A strong positive correlation between IS periostin and TSLP protein levels (r = 0.96) as well as mRNA expression level (r = 0.95) was found in patients with asthma. The results of our study show that periostin and TSLP are associated with eosinophilic airway inflammation and seem to be important drivers of atopic asthma but not COPD pathobiology. Very strong correlations between local periostin, TSLP, eosinophils, and IL-4 in asthma point to the link between periostin–TSLP and Th2 response

Effect of Phosphodiestrase 4 Inhibitor (Rolipram)on Experimental Allergic Asthma-Guinea Pig Model

Selective phosphodiesterases (PDE) inhibitors are the new group of antiasthmatic drugs, which integrate antiinflammatory activity with bronchoconstriction counteraction. Selective inhibitors of phosphodiesterase type 4 are used as alternative or assist drugs in treatment of respiratory system diseases. So far glucocorticosteroids remain the most efficient and widely used medicine in the treatment of asthma. However application of glucocorticosteroid is greatly limited because of numerous side effects, what induce to permanent search for new antiasthmatic drugs. Examination new substances are executed on animal models. Guinea pig model is widely used to research course of asthmatic reaction. This model is especially convenient on the ground of that: lung is major shock organ, airway respond to histamine, animals demonstrated early asthmatic reaction (EAR) and late asthmatic reaction (LAR), eosinophils flow in bronchoalveolar space during LAR. In ovalbumin (OA) sensitized guinea pigs hypersensitivity reaction breaks out as a result of OA provocation. Aims of our experiments, execute on guinea pig model were to determine the influence of rolipram (PDE 4 inhibitor) on modulation experimental asthmatic reaction and comparison activity of rolipram versus dexamethasone in attribution to chosen parameters of allergic reaction such as: lung resistance, influx of protein and inflammatory cells in airways, and mastocytes degranulation. Experiments were made on guinea pigs sensitized and provoked with ovalbumin The obtain data indicate that rolipram was effective in reduction the rise of lung resistance during EAR, restricted influx of eosinophils to bronchoalveolar space between 1.5 and 24 h after provocation, and reduced increase of histamine concentration in bronchoalveolar lavage fluid (BALf). Rolipram had no influence on number of neutrophils present in BALf. Dexamethasone in double dose of 1.2 mg/kg effectively bordered the growth of lung resistance during EAR, and broke influx of eosinophils and neutrophils to bronchoalveolar space

The Comparison between Two Methods for Typing of Nontuberculous Mycobacteria: High Pressure Liquid Chromatography and Molecular Assay Genotype Mycobacterium CM/AS

Introduction: The GenoType Mycobacterium CM and the GenoType Mycobacterium AS (HAIN Lifescience, Germany) were evaluated for the ability to differentiate mycobacterial species of clinical isolates. Serial use of the both assays is aimed to identify 38 different molecular patterns, of which 24 patterns can be assigned to single species, 10 patterns are allocated to two or more Mycobacterium species, and 4 patterns correspond to Mycobacterium species and gram-positive bacteria with a high G + C content. The analysis of mycolic acids by high pressure liquid chromatography (HPLC) was the reference method. Materials and methods: A set of 127 nontuberculous mycobacterial isolates on Loewenstein-Jensen media, derived from different patients between 1999 and 2007, was analyzed. The strains were primary classified by HPLC following the diagnostic procedure, and retyped by GenoType Mycobacterium CM/AS. Results: In total, results obtained by both methods were interpretable for 113 strains. Concordant results were obtained for 105 (93%) mycobacterial strains. One out of 8 incorcondantly classified strains, which was classified as M. abscessus/M. chelonae by HPLC, displayed a pattern of M. tuberculosis complex by a molecular method. Eleven clinical strains were differentiated only by one of used methods, either by HPLC (6 strains) or by GenoType CM/AS (5 strains). Three strains were not classified at all. Conclusions: Our results show that the GenoType Mycobacterium CM/AS system represents a useful tool to identify mycobacterial clinical isolates. The molecular system is as rapid and reliable as the HPLC, but much easier to perform and more friendly for the environment

Blood and Sputum Eosinophils of COPD Patients Are Differently Polarized than in Asthma

Different eosinophil subpopulations have been identified in asthma and other eosinophilic disorders. However, there is a paucity of data on eosinophil subpopulations in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to compare eosinophil phenotypes in blood and induced sputum in patients with COPD, asthma and controls. Stable patients with mild-to-moderate COPD (n = 15) and asthma (n = 14) with documented blood eosinophilia ≥100 cells/µL in the year prior to the study and the control group (n = 11) were included to the study. The blood and sputum eosinophil phenotypes were analyzed by flow cytometry. IL-5, IL-13, CCL5 and eotaxin-3 levels were measured in the induced sputum. The marker expression on blood eosinophils was similar among control, asthma and COPD groups. The expressions of CD125, CD193, CD14 and CD62L were higher on blood than on sputum eosinophils in all three groups. We found increased levels of CD193+ and CD66b+ sputum eosinophils from COPD patients, and an elevated level of CD11b+ sputum eosinophils in asthma compared to COPD patients. The results of our study suggest that the profile of marker expression on COPD sputum eosinophils differed from other groups, suggesting a distinct phenotype of eosinophils of COPD patients than in asthma or healthy subjects

Serum Amyloid A in Stable Patients with Chronic Obstructive Pulmonary Disease Does Not Reflect the Clinical Course of the Disease

Serum amyloid A (SAA) is a good systemic marker of the exacerbations of chronic obstructive pulmonary disease (COPD), but the significance of SAA in stable patients with COPD has not been widely investigated. We aimed to evaluate the SAA level in peripheral blood from stable patients with COPD and to search for correlations between SAA and other inflammatory markers and clinical characteristics of the disease. Serum SAA, IL-6, IL-8, TNF-alpha, basic blood investigations, pulmonary function testing and a 6-min walk test were performed. The correlations between SAA and other inflammatory markers, functional performance and the number of disease exacerbations were evaluated. A total of 100 consecutive patients with COPD were analyzed. No correlations between SAA and inflammatory markers as well as pulmonary function were found. Hierarchical clustering identified two clusters incorporating SAA: one comprised SAA, PaO2 and FEV1 and the second was formed of SAA and nine other disease markers. The SAA level was higher in patients with blood eosinophils < 2% when compared to those with blood eosinophils ≥ 2% (41.8 (19.5–69.7) ng/mL vs. 18.9 (1.0–54.5) ng/mL, respectively, p = 0.04). We conclude that, in combination with other important disease features, SAA may be useful for patient evaluation in stable COPD