Characterization of Resistance Mutations Against First and Second Line Antituberculosis Drugs of Multi-Drug Resistant Mycobacterium tuberculosis Strains Isolated from Clinical Samples by in-House PCR Method and Comparison of Resistance Profiles

Introduction: Multi-drug resistant tuberculosis (MDR-TB) is still an important public health problem. Rapid agent diagnosis and resistance status determination are critical in establishing the correct treatment protocol. This study was conducted to determine resistance mutations against first- and second-line antituberculous drugs in the MDR-MTB strain isolated from respiratory tract specimens. Materials and Methods: After subculturing the isolated Mycobacterium tuberculosis strains on the Löwenstein-Jensen medium, DNA isolation was carried out using the boiling method. DNA isolates were kept at -40 °C until the day of analysis. Primer sequences specific to rpoB, InhA, katG, gyrA, eis, and rrs regions were used to determine isoniazid, rifampicin, quinolone, and aminoglycoside resistance. Results: The positivity rate of rpoB, InhA, katG, gyrA, eis, and rrs in 33 MDR-TB isolates was 27 (81.8%), 31 (93.9%), 25 (75.7%), 25 (75.7%), 20 (60.6%) and 14 (42.4%), respectively. Resistance mutations were not detected in susceptible isolates. Conclusion: According to the data obtained from the study, it was found that fluoroquinolone resistance mutations were higher in isolates defined as MDR-TB by conventional and molecular methods, and the in-house PCR method was a useful method for rapid resistance detection

Comparison and evaluation of Lowenstein-Jensen medium and 2% Ogawa medium for the diagnosis of tuberculosis

Tüberkülozun kesin tanısı basilin gösterilmesiyle konulur. Bakteriyolojik tekniklerle bakterinin kültürde üretilmesi altın standart olarak kabul edilmekte ve kültürde iki adet besiyeri kullanılması ve besiyerlerinden birinin sıvı olması önerilmektedir. Bununla birlikte farklı besiyerlerinin kullanılmasının tanıya önemli katkılar sağladığı da rapor edilmiştir. Bu çalışmada, bugün hala dünyada yaygın olarak kullanılmakta olan yumurta bazlı Löwenstein-Jensen (LJ) besiyeri ile Ogawa (%2) besiyerinin tanı değerinin karşılaştırılması amaçlanmıştır. Refik Saydam Hıfzıssıhha Merkezi Başkanlığı, Ulusal Tüberküloz Referans Laboratuvarına 2.5 yıl boyunca (Ocak 2006-Haziran 2008) başvuran 3311 tüberküloz şüpheli hastaya ait 7912 adet örnek (akciğer ve akciğer dışı örnekleri) toplanmıştır. Toplanan her bir örnek modifiye Petroff yöntemine (%4 NaOH) göre aynı gün içerisinde homojenizasyon-dekontaminasyon-konsantrasyon işlemine alınmıştır. Her bir örnek için iki adet Ogawa ve iki adet LJ besiyeri kullanılmıştır. İşlenmiş örneklerden bu besiyerlerine 150-200 ?l ekim yapılmış ve 35-37°C’de inkübe edilmiştir. Besiyerleri kontaminasyon yönünden inkübasyonun üçüncü gününde kontrol edilmiştir. Eğer kontaminasyon gözlenmemişse haftada bir kez olmak üzere üreme kontrolleri için sekiz hafta boyunca takip edilmiştir. Bu süre içerisinde üreme saptanan besiyerleri ayrılarak kültürlerin tanımlanması aşamasına geçilmiştir. Tür tayini için paranitrobenzoik asit (PNB), nitrat, niasin gibi biyokimyasal testler kullanılmıştır. Mycobacterium tuberculosis kompleksi olarak tanımlanan koloniler pozitif olarak rapor edilmiştir. Çalışma sonucunda toplam kontaminasyon oranı; Ogawa besiyeri için %3.1 ve LJ için %4 olarak bulunmuştur. Hastaların 248 (%7.5)’inde kültür pozitifliği (LJ + Ogawa ile) saptanmış; bunlardan 210’u her iki besiyeri ile de pozitif bulunurken, 20’si sadece LJ’de 18’i ise sadece Ogawa besiyerinde pozitif sonuç vermiştir. Her iki besiyerinin birlikte kullanılması dolayısıyla LJ ve Ogawa besiyerlerinin toplam pozitifliğe katkıları sırasıyla %8.1 (20/248) ve %7.3 (18/248); duyarlılıkları sırasıyla %92.7 (230/248) ve %91.9 (228/248) olarak bulunmuştur. LJ besiyeri referans yöntem olarak kabul edildiğinde, Ogawa besiyerinin duyarlılık, özgüllük, pozitif ve negatif prediktif değerleri sırasıyla %91.3, %99.4, %92.1 ve %99.4 olarak hesaplanmıştır. Sonuç olarak ucuz ve kontaminasyon oranının düşük olması göz önünde bulundurularak özellikle laboratuvara gönderilen tek örnekler için (beyin omurilik sıvısı, biyopsi, püy vb.) Ogawa besiyerinin, bir sıvı besiyerinin yanında, LJ ile birlikte ya da tek başına kullanılmasının mikobakterilerin üreme şansını artıracağı görüşüne varılmıştır.Accurate diagnosis of tuberculosis is based on the detection of the bacilli in the clinical specimen. The growth of mycobacteria in laboratory media is regarded as the gold standard for diagnosis and use of two different cultivation media, one of them being a liquid one, is recommended. In this study, the diagnostic values of egg-based Lowenstein-Jensen (LJ) medium used extensively all over the world and Ogawa (2%) medium were compared. A total of 7912 pulmonary and extrapulmonary clinical samples which belonged to 3311 patients with suspected tuberculosis, were enrolled in the National Tuberculosis Reference Laboratory in Refik Saydam National Public Health Agency during 2.5 years (January 2006- June 2008). The samples were processed by modified Petroff method (4% NaOH) for homogenizationdecontamination- concentration on the same day and inoculated on two Ogawa and two LJ media. The cultures were incubated at 35-37ºC for eight weeks and were controlled on the third day of incubation in terms of contamination. If no contamination were detected, the cultures were incubated for a total of eight weeks with regular weekly controls. The colonies detected in culture media were identified by biochemical tests, including paranitrobenzoic acid (PNB), niacin accumulation and nitrate reduction tests. Those indentified as Mycobacterium tuberculosis complex were reported as positive. The rates of contamination was 3.1% for Ogawa medium and 4% for LJ medium. Culture results were found positive for 248 patients (7.5%). While 210 of these were positive by both of the media, 20 (8.1%) patients were detected positive only by LJ and 18 (7.3%) only by Ogawa medium. The sensitivity of LJ medium was 92.7% (230/248) and of Ogawa medium was 91.9% (228/248). When LJ medium was taken as the reference method, the sensitivity, specificity, positive and negative predictive values of Ogawa medium were 91.3%, 99.4%, 92.1% and 99.4%, respectively. It was concluded that, when low price and low contamination rates were taken into consideration, Ogawa medium, used together with a liquid medium + LJ medium, would increase the yield of mycobacteria from single samples (cerebrospinal fluid, biopsy,pus, etc.) sent to the laboratories

Molecular epidemiological typing of M. tuberculosis isolates isolatedfrom Turkey's Eastern Anatolia with in house PCR method

Purpose: The aim of this study as to compare microscopy and culture results of samples, determine drug resistance rates of the isolates, evaluate epidemiological relationship between the strains with ERIC-PCR, RAPD-PCR, OUT-PCR based on in house PCR technique

Evaluation of quantiferon-TB gold and tuberculin skin test in patients with tuberculosis, close contact of patients, health care workers and tuberculosis laboratory personnel

Birengel, Serhat/0000-0002-5599-6488; Annakkaya, Ali Nihat N/0000-0002-7661-8830WOS: 000284385400003PubMed: 21063967Tüberkülin cilt testi (TCT), uzun bir süredir duyarlılık ve özgüllük değerlerindeki sınırlamalara rağmen etkin bir şekilde kullanılmaktadır. Aktif tüberkülozu olmaksızın TCT pozitif olan hastalar, latent tüberküloz enfeksiyonu (LTBE) olarak tanımlanır. LTBE olan hastaların TCT ile tanımlanması, hastalığın kontrolünde önemli bir aşamadır. Plazma interferon gamma (IFN-γ) düzeyini belirleyen Quantiferon-TB Gold Testi (QTG, Cellestis, Austuralya) latent tüberküloz (TB) enfeksiyonu tanısında TCT’ye göre avantajları olan in vitro tanısal bir testtir. Bu test Mycobacterium tuberculosis’e özgül ESAT-6 ve CFP-10 antijenlerini içerir. Bu çalışmanın amacı, TB’li hastalar, yakın temaslıları, sağlık çalışanları ve TB laboratuvarı personelinde QTG ve TCT testi sonuçlarının karşılaştırılmasıdır. Çalışmaya, 26 aktif akciğer TB’li hasta, 6 hasta yakını, 11 hasta teması olan sağlık çalışanı ve 8 TB laboratuvarı personeli dahil edilmiştir. TCT uygulanmadan önce QTG testi için kan örnekleri alınmıştır. Tüm bireyler BCG aşılaması yönünden sorgulanmış ve BCG skarı yönünden incelenmiştir. Çalışmaya katılanların tümünde BCG aşılaması öyküsü ve muayenede BCG skarı tespit edilmiştir. QTG testi plazma örneklerinde üretici firmanın önerileri doğrultusunda çalışılmıştır. TCT ile QTG testi arasındaki uyum kappa istatistiği ile ölçülmüştür. Aktif TB’li hastalarda (gerçek enfekte bireyler) TCT (PPD) pozitifliği %34.6 (9/26), QTG pozitifliği %65.3 (17/26) olarak belirlenmiş; pozitiflik oranı QTG testinde daha yüksek tespit edilmiş olmasına rağmen aradaki fark istatistiksel olarak anlamlı bulunmamıştır (p> 0.001). Diğer çalışma gruplarına bakıldığında; sağlık çalışanları, TB’li hastanın yakın temaslıları ve TB laboratuvarı çalışanlarında TCT ve QTG pozitiflik oranları sırasıyla; %36 (4/11) ve %27 (3/11); %16.6 (1/6) ve %83 (5/6); %37.5 (3/8) ve %75 (6/8) olarak tespit edilmiştir. PPD testi sonucu, QTG negatif grupta ortalama 11 mm iken, QTG pozitif grupta ortalama 14 mm olarak saptanmış ve aradaki fark istatistiksel olarak anlamlı bulunmuştur (p 0.001). TST and QTG positivity rates for health care workers, close house contact of TB patients and TB laboratory staff were as follows, respectively; 36% (4/11) and 27% (3/11); 16.6% (1/6) and 83% (5/6); 37.5% (3/8) and 75% (6/8). The mean PPD diameter was 11 mm in QTG negative group and 14 mm in QTG positive group with a statistically significant difference (p= 0.05) and gender (p< 0.001). In conclusion, QTG assay was superior to TST in its ability to detect LTBI and active TB infection, not to be affected with BCG vaccination, to discriminate responses due to non-tuberculous mycobacteria, and to avoid variability and subjectivity associated with application and reading the TST. Besides, QTG assay needs only one visit to the test unit. However, its being expensive than TST and requirement for special equipments and skilled laboratory personnel, are among the disadvantages of QTG assay

Serum Neopterin Level In Patients With Lung Cancer And Case Of Pneumoniae

Amaç: Neopterin, hücre aracılıklı immün yanıtı gösteren duyarlı bir belirteçtir. Bu nedenle T hücreleri ve makrofaj- ların rol oynadığı çeşitli infeksiyon hastalıklarında vücut sıvılarında neopterin düzeylerinin belirlenmesi hücre aracı- lıklı immün yanıt hakkında bilgi sağlar. Kanser ve pnömoni hastalarında serum neopterin düzeylerinin yüksek oldu- ğu bildirilmiştir. Bu çalışmanın amacı, akciğer kanseri olan hastalar ve pnömonili hastalarda serum neopterin düzey- lerinin tanısal değerinin araştırılmasıydı. Yöntem ve Gereçler: Bu çalışmada, 24 pnömonili hasta (10 K, 14 E) , 20 akciğer kanserli hasta (20 E) ve 16 sağ- lıklı gönüllüde (5 K, 11 E) serum neopterin düzeyleri istatistiksel olarak karşılaştırıldı. Neopterin düzeyleri üretici firmanın önerilerine göre ELISA yöntemiyle belirlendi. İstatistiksel değerlendirmede SPSS 11.5 programı kullanıldı. İstatistiksel analizlerde, univarite varyans analizi, kovaryans analizi ve Student T testler kullanıldı. p ? 0.05 değeri istatistiksel olarak anlamlı kabul edildi. Bulgular: Serum neopterin düzeyleri ortalaması sırasıyla; akciğer kanserli hastalarda 22.21 ± 8.60 nmol/l, pnömonili hastalarda 21.18 ± 8.76 nmol/l, kontrol grubunda ise 9.03 ± 4.15 nmol/l olarak belirlendi. Serum neopterin düzey- leri akciğer kanseri ve pnömoni hastalarında kontrol grubundan istatistiksel olarak anlamlı oranda yüksek saptandı. Serum neopterin düzeyleri kanserli hastalar ve pnömonili hastalar arasında istatistiksel olarak anlamlı farklılık gös- termedi. Sonuç: Serum neopterin düzeylerinin akciğer kanseri ve pnömonili hastalarda tanıda yardımcı test olarak kullanıla- bileceği, ancak; bu iki hastalığın birbirinden ayrımında kullanılamayacağı sonucuna varıldı.Aim:Neopterin is a sensitive marker for cell-mediated immune response. Because of this, the neopterin levels of body fluids show cell-mediated immune response in different infectious diseases which involve T cells and macrophages. It is reported that serum neopterin levels were found to be higher in patients with pneumoniae and patients with can- cer. The aim of this study was to investigate that the diagnostic value of neopterin in patients with lung cancer and pneumoniae. Material And Methods:In the present study, serum neopterin levels were compared between , 24 patients with pneu- moniae (10 female, 14 male) , 20 patients with lung cancer (20 male) and 16 healthy volunteers (5 female, 11 male). Neopterin concentrations were measured by ELISA method according to the protocol of manufacturer. In statistical evaluation, SPSS 11.5 programme was used.Univariate analysis of variance, covariance analysis and Student t tests were used in statistical analysis. p value &#8804;0.05 and p &#8804;0.001 was accepted as statistical significant. Results:Serum neopterin levels were found to be 22.21 ± 8.60 nmol/l, 21.18 ± 8.76 nmol/l, and 9.03 ± 4.15 nmol/l in patients with lung cancer, pneumoniae and control groups, respectively. Serum neopterin levels were established to be significantly higher in patients with lung cancer and patients with pneumoniae than in control groups. Serum neopterin levels were not significantly different between patients with lung cancer and patients with pneumoniae. Conclusion:It is our suggestion that serum neopterin levels may be used as a supplementtary test in diagnosis in pa- tients with lung cancer and patients with pneumoniae but it can not be used in in differential diagnosis of these two diseases

Comparison of the tuberculin skin test and the quantiferon test for latent Mycobacterium tuberculosis infections in health care workers in Turkey

Annakkaya, Ali Nihat N/0000-0002-7661-8830WOS: 000246892200007PubMed: 17515641The aim of this study was to compare the efficacy of the tuberculin skin test (TST) and the quantiferon test (QFT) for detecting latent tuberculosis infection (LTBI) in health care workers (HCWs). Seventy-six participants who were working in Duzce University Hospital, where tuberculosis patients were being treated, were included in the study. TST was performed according to the Mantoux technique. QFT was performed in accordance with the manufacturer's instructions. A positive TST result was defined as an induration diameter of >= 15 mm. TSTs were positive in 41 of 76 participants (53.9%) and QFT was positive in 65 of 76 participants (85.5%). There was a significant difference between the numbers of QFT-positive and TST-positive cases (P = 0.02). When the induration diameter of TST was >= 20 min, QFT positivity was 100%. Multivariate analysis revealed that there was a significant correlation between the percentage of patients with QFT positivity and the induration diameter of TST (P = 0.009). QFT thus seems to be more effective for LTBI diagnosis than TST. However, large-scale trials including quantitative measurement of QFT in subgroups taking into account the division where HCWs are employed and the different results of TST might clarify the usefulness of QFT in LTBI diagnosis

Clinical importance of serum neopterin level in patients with pulmonary tuberculosis

Objective: Neopterin is a sensitive marker for cell-mediated immune response. Because of this, the neopterin levels of body fluids show cell-mediated immune response in different infectious diseases which involve T cells and macrophages. The aim of this study was to determine the clinical importance of neopterin levels in patients with tuberculosis and compare with those levels of healthy subjects. Methods: Seventy patients with tuberculosis (46 newly diagnosed cases, 15 relapse cases, and 9 multidrug-resistant tuberculosis cases) and 18 healthy adult individuals were included in the study. Neopterin concentrations were measured by the ELISA method according to the protocol of the manufacturer. Chi-square test was used in statistical analysis; p≤0.05 was considered statistically significant. Results: Serum mean neopterin levels were 23.74±21.8nmol/L (median: 18.3) in newly diagnosed patients with pulmonary tuberculosis; 28.69±21.2nmol/L (median: 21.2) in relapse patients and 31.28±14nmol/L (median: 25.4) in multidrug-resistant tuberculosis cases, respectively. Serum mean neopterin levels were 4.03±5.12nmol/L (median: 5.1) in healthy subjects. The serum neopterin levels were found to be significantly higher in patients with tuberculosis than the control group. There was a statistically significant correlation between neopterin positivity (neopterin level ≥10nmol/L was accepted to be positive) and clinical symptoms of hemoptysis and weight loss. Besides statistically significant correlations between neopterin positivity and hemoglobin level, sedimentation rate, mean leukocyte count and radiological involvement (localized or diffuse) were determined. Conclusion: Serum neopterin levels can be used as a helper laboratory finding for the diagnosis of patients with tuberculosis. For this aim, further controlled studies are needed

Influence of sulfurization temperature on Cu2ZnSnS4 absorber layer on flexible titanium substrates for thin film solar cells

In this study, the effect of sulfurization temperature on the morphology, composition and structure of Cu2ZnSnS4 (CZTS) thin films grown on titanium (Ti) substrates has been investigated. Since Ti foils are flexible, they were preferred as a substrate. As a result of their flexibility, they allow large area manufacturing and roll-to-roll processes. To understand the effects of sulfurization temperature on the CZTS formation on Ti foils, CZTS films fabricated with various sulfurization temperatures were investigated with several analyses including x-ray diffraction (XRD), scanning electron microscopy (SEM), x-ray photoelectron spectroscopy and Raman scattering. XRD measurements showed a sharp and intense peak coming from the (112) planes of the kesterite type lattice structure (KS), which is strong evidence for good crystallinity. The surface morphologies of our thin films were investigated using SEM. Electron dispersive spectroscopy was also used for the compositional analysis of the thin films. According to these analysis, it is observed that Ti foils were suitable as substrates for the growth of CZTS thin films with desired properties and the sulfurization temperature plays a crucial role for producing good quality CZTS thin films on Ti foil substrates.TUBITAK (114F341