Coexistence of Cryptococcal Fungemia and Pneumocystis jirovecii Pneumonia in an HIV-Infected Patient: A Case Report.

INTRODUCTION: Opportunistic infections caused by bacteria and fungi are common in human immunodeficiency virus (HIV)-infected patients. Cryptococcus neoformans and Pneumocystis jirovecii are the most common opportunistic infections in immunosuppressed individuals, but their coexistence is rare. To our knowledge, this is the first case presented in Turkey involving the coexistence of C.neoformans fungemia and P.jirovecii pneumonia. CASE PRESENTATION: A 26-year-old male patient presented with a cachectic appearance, cough, sputum, weakness, shortness of breath, and a weight loss of 15 kg in the last three months. It was learned that the patient was diagnosed with HIV three years ago, did not go to follow-ups, and did not use the treatments. CD4 cell count was 7/mm3 (3.4%), CD8 cell count was 100 (54%) mm3, and HIV viral load was 5670 copies/mL. In thorax computed tomography (CT), increases in opacity in diffuse ground glass density in both lungs and fibroatelectasis in lower lobes were observed. With the prediagnosis of P. jiroveci pneumonia, the HIV-infected patient was given trimethoprim-- sulfamethoxazole 15 mg/kg/day intravenously (i.v.). On the 4th day of the patient's hospitalization, mutiplex PCR-based rapid syndromic Biofire (Film Array) blood culture identification 2 (BCID2) test (Biomerieux, France) was applied for rapid identification from blood culture. C. neoformans was detected in the blood culture panel. The treatment that the patient was taking with the diagnosis of C. neoformans fungemia was started at a dose of liposomal amphotericin B 5 mg/kg/- day + fluconazole 800 mg/day. CONCLUSION: While the incidence of opportunistic infections has decreased with antiretroviral therapy (ART), it remains a problem in patients who are unaware of being infected with HIV or who fail ART or refuse treatment. High fungal burden, advanced age, low CD4+ cell count, and being underweight are risk factors for mortality in HIV-positive patients. Our case was a cachectic patient with a CD4 count of 7 cells/mm3. Despite the early and effective treatment, the course was fatal.

Treatment Results of Multidrug-Resistant Tuberculosis Patients in the Aegean Region.

OBJECTIVE: We aimed to evaluate 109 rifampicin-resistant or multidrug-resistant tuberculosis patients who are treated in Izmir Chest Diseases MDR Tuberculosis Centre. MATERIAL AND METHODS: The patient profile, side effects, treatment success, and mortality of rifampicin-resistant or multidrugresistant tuberculosis patients who were followed up and treated in our hospital's tuberculosis service between 2010 and 2018 were analyzed retrospectively. RESULTS: Of the rifampicin-resistant or multidrug-resistant tuberculosis patients, 83 (76.1%) were male and the mean age was 46.3 ± 16.3 years. Of the cases 13 (11.9%) had rifampicin resistance without isoniazid. Since 5 out of 109 patients diagnosed with multidrugresistant tuberculosis emigrated to other countries, the treatment results of 104 patients were evaluated. As a result of the treatment, the cure was achieved in 81 (77.9%) patients and treatment was completed in 13 (12.5%). Treatment success was found as 90.4%. No patient experienced recurrence. The mortality rate was determined as 9.6%. The cure rate of patients treated with ≥6 drugs (90.9%) was statistically significantly (P = .029) higher than the group treated with ≤5 drugs (71.8%). CONCLUSIONS: Multidrug-resistant tuberculosis treatment is a long-term, financially burdensome practice that may cause serious side effects and complications, and it requires strict discipline. The fight against tuberculosis can be successful with tuberculosis control programs that are pursued with determination.

[An Infant with Zoonotic Pulmonary Tuberculosis due to Mycobacterium bovis]. / Mycobacterium bovis'e Bagli Zoonotik Pulmoner Tüberküloz Tanili Bir Infant.

Bovine tuberculosis might be seen in low-income countries, especially in children fed with raw milk. The most common transmission route is fecal-oral way, and it is most likely through unpasteurized dairy products. Although clinical and radiological findings are like non-zoonotic tuberculosis, treatment approaches may differ in individuals with zoonotic tuberculosis. Prevention of zoonotic diseases requires multidisciplinary approaches. These approaches include the development of veterinary and surveillance studies for the detection of communicable diseases in farm animals, as well as informing the public about raw milk consumption. In this case report, a patient with zoonotic pulmonary tuberculosis related to Mycobacterium bovis because of consumption of raw milk was presented. A five-month-old male was admitted to the hospital due to a persistent, feverless, non-productive cough since birth. Empirical antibiotic treatment was started with a preliminary diagnosis of pneumonia because of left upper lobe and right pericardial infiltration on chest X-ray. However, after two weeks of antimicrobial therapy, the patient's clinical and laboratory findings did not improve. This led to the referral for a computed tomography imaging, which revealed tracheomalacia, consolidation on the right upper lobe, an indistinguishable mass or consolidation on the left middle lobe of the lung, peribronchial thickening on the basal segment of the lower lobe, and mediastinal lymphadenopathy. Three consecutive days of fasting gastric lavage fluid was sent to the reference laboratory for acid-resistant bacillus examination, polymerase chain reaction (PCR) and culture studies. As the clinical findings were compatible and PCR was positive, the patient was started on quadruple antituberculous therapy. After initiation of anti-tuberculosis drugs, the patient's findings radiologically and clinically were improved. Mycobacterium bovis was grown in the culture. In the meantime, it was discovered that the patient was fed with raw milk. Due to the patient's clinical symptoms and the growth of Mycobacterium bovis in the gastric lavage fluid culture, the diagnosis of bovine tuberculosis was made. The culprit was that the milk of the cow belonging to the patient's family, which was later found to be infected with M.bovis, was milked and given to the patient without boiling. Today, unpasteurized dairy products continue to be consumed, especially in rural areas. One of the most important steps to prevent zoonotic diseases is to raise awareness about not consuming raw milk and undercooked meat. To elucidate the epidemiological link in childhood, taking a good anamnesis, including questioning raw milk consumption, is essential in the diagnosis of tuberculosis.

Investigation of miRNA and cytokine expressions in latent tuberculosis infection and active tuberculosis.

BACKGROUND: In tuberculsosis (TB), miRNA has been used as a biomarker to distinguish between healthy individuals and TB patients. The aim of this study was to investigate (i) the association of the miRNA and cytokine expression levels, the course of tuberculosis infection, clinical forms and response to treatment, and (ii) the effects of genotypic features of bacteria on the course of tuberculosis and the relationship between miRNA and cytokine expressions and bacterial genotypes. METHODS: A total of 200 cases (100: culture positive active tuberculosis, 50: quantiferon positive latent tuberculosis infection and 50: quantiferon negative healthy controls) were included in the study. For the tuberculosis group at the time of admission and after treatment, for the latent tuberculosis infection and healthy control groups at the time of admission, miRNA and cytokine expressions were determined. Genotyping of M.tuberculosis isolates was performed by spoligotyping method. RESULTS: While, in the comparison of miRNA expressions between the pretreatment patient group and the healthy control group, there was a statistically significant decrease in the expression of miR-454-3p, miR-15a-5p, miR-590-5p, miR-381, and miR-449a in the Pulmonary TB group, there was no significant change in miRNA expression in extrapulmonary TB patients. When the cytokine expressions of the patient group and the healthy control group were compared before treatment, the expressions of all cytokines in the patient group decreased. However, the only cytokine that showed a significantly lower expression was IL12A in PTB patients. DISCUSSION: There is no significant relationship between the clinical course of the disease, cytokine and miRNA expression, and the genotype of the bacteria.

Antibiotic resistance rates and penicillin MIC distribution in patients with streptococcal pneumonia between 2013-2019, and use of antibiotics in clinical practice.

PURPOSE: The purpose of the present study is to investigate the antibiotic resistance rates and use of antibiotics in patients with streptococcal pneumonia in a reference tertiary care hospital for pulmonary diseases in Izmir, Turkey. METHODS: A total of 1224 cases with streptococcal pneumonia between 2013 and 2019 were included in the study, retrospectively. Drug susceptibility testing for penicillin and other antibiotics were performed according to the recommendations of EUCAST criteria. Clinical data and general characteristics were collected and evaluated for each patient in accordance with the susceptibility testing report. RESULTS: Totally, resistance rates for trimethophrim-sulfamethoxazole, penicillin (oxacillin), erythromycin, tetracycline, clindamycin and levofloxacin resistance were 63.5%, 39.8%, 37.7%, 37.6%, 28.8%, and 4.8%, respectively. Antibiotic resistance was not detected against vancomycin,teicoplanin and linezolid. Multidrug resistance rate was found to be 27.1%. It was observed that there was a statistically significant decrease in trimethophrim-sulfamethoxazole, penicillin (oxacillin), erythromycin, clindamycin and levofloxacin resistance rates by years (p: 0.000, 0.004, 0.000, 0.001, 0.010, respectively). The penicillin MIC distribution was higher at the range of 0.12-2 â€‹µg/mL and there was statistical difference among the ranges of MIC values for the representative years (p:0.033). Among the antibiotics investigated, the most commonly used antibiotic was moxifloxacin. CONCLUSIONS: Trimethophrim-sulfamethoxazole resistance rate has been found higher than other antibiotics. As penicillin MIC values were at the range of 0.12-2 â€‹µg/mL frequently, high doses of penicillin treatment might be required in some patients. It is noteworthy that significant decrease in resistance rates in penicillin, erythromycin, clindamycin and tetracycline could be due to the vaccination programme carried out since 2008 in Turkey. As the empiric use of quinolones is high it would be more appropriate to use it according to the susceptibility testing. It is important to determine the regional antimicrobial susceptibility for Streptococcus pneumoniae to select appropriate empirical antimicrobials in the clinical practice.

Evaluation of antimicrobial susceptibilities of non-tuberculous mycobacteria against linezolid and tigecycline.

Mycobacterial susceptibility testing is important for the management of nontuberculous mycobacteria (NTM) infections. The aim of the study is to determine the susceptibilities of tigecycline (TGC) and linezolid (LZD) against NTM. The study was carried out using stocks of NTM strains in the tuberculosis department of the microbiology laboratory. It was designed a retrospective study. LZD and TGC sensitivities of study isolates were analyzed by microdilution. Forty NTM isolates have been studied. LZD and TGC sensitivities varied according to the NTM type. It is concluded that each isolate should be individually evaluated due to variable susceptibilities to LZD and TGC.

[Investigation of pncA, rpsA and panD Gene Mutations Associated with Resistance in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates and Spoligotyping]. / Pirazinamid Dirençli Mycobacterium tuberculosis Izolatlarinda Direnç ile Iliskili pncA, rpsA ve panD Gen Mutasyonlari ve Spoligotiplerin Arastirilmasi.

Pyrazinamide (PZA) is one of the first-line anti-tuberculous drugs used in the treatment of tuberculosis (TB). Considering the ability of PZA to shorten the treatment period from 9-12 months to six months by eliminating persistent bacilli, it appears to be an important cornerstone of TB therapy. While the main mechanism causing the PZA resistance is pncA mutations at a rate of 70-97%, it has been determined that rpsA and panD mutations can also cause resistance. In this study, we aimed to investigate the pncA, rpsA and panD gene mutations, the efficiency of the pyrazinamidase (PZAse) enzyme test in determining PZA resistance, the drug susceptibility and their families in PZA-resistant Mycobacterium tuberculosis isolates. Totally 46 PZA resistant M.tuberculosis isolates were included in the study. The pncA, rpsA and panD mutations caused by PZA resistance were investigated by in-house PCR followed by DNA sequencing method. Drug susceptibility was determined with Bactec MGIT 960 (Becton Dickinson, USA) system, the presence of PZAse was evaluated by colorimetric PZAse enzyme assay and the families were determined by the spoligotyping method. Of the 46 PZA-resistant isolates, 24 (52.2%) were identified as PZA monoresistant, 11 (23.9%) multidrug resistant (MDR)-TB and 11 (23.9%) poly drug resistant (PDR)-TB. Gene mutations associated with resistance were detected in 73.9% (34) of PZA-resistant M.tuberculosis isolates. The pncA, rpsA and panD mutations were found in 71.7% (33), 28.2% (12) and 4.3% (2) of the isolates, respectively. The coexistence of pncA/rpsA and pncA/panD gene mutations were determined in 12 and two isolates, respectively. The pncA gene mutations were observed in 3 (33.3%) of 9 (19.6%) isolates whose enzyme presence was detected by the colorimetric PZAse test. In the pncA gene, eight different point mutations in the form of missense mutation;A226C (27.3%), A152C (24.2%), C169G (21.2%) A422C (9.1%), G145A (6.1%), A29G (6.1%), A424G (3%) and T464G (3%) were detected. In the rpsA gene, A636C (42.9%) silent and G1318A (42.9%) missense mutations and in the panD gene, C66G (50%) nonsense and A145G (50%) missense mutations were the most common mutations detected. As a result of genotyping of PZA resistant isolates, the most common genotypes were found in T1 cluster with 17 (36.9%) isolates; followed by the families of Beijing with 7 (15.2%) isolates, H3 with 6 (13%) isolates, TUR with 5 (10.9%) isolates, and LAM 9 with 4 (8.7%) isolates, respectively. In addition, 2 (4.3%) isolates belonging to the ORPHAN family and one isolate belonging to each of LAM TUR, LAM 2, LAM 7, T2, T5-RUS1 families were identified. Our study is the first to investigate all pncA, rpsA and panD gene mutations that have been found to cause PZA resistance in Turkey. Epidemiological studies on PZA resistance will make important contributions to the determination of resistance mechanism and the development of methods that will provide rapid diagnosis for the detection of resistance.

Multicenter evaluation of AYC.2.2 agar for the isolation of mycobacteria from clinical samples.

PURPOSE: The aim of this multicenter study is to evaluate AYC.2.2 agar for the isolation of mycobacteria from clinical samples. METHODS: Totally 5559 media were tested in 7 centers. AYC.2.2 agar media for the study were prepared by C1 and sent to other centers under appropriate conditions. Other media except AYC.2.2 agar were purchased commercially. The media were subjected to routine laboratory operations in the center where they were sent. After the samples received for routine processing (in all centers, samples were processed with the same method (NALC-NaOH)), they were cultivated on routine media and AYC.2.2 agar afterward. RESULTS: C1: Average growth time was determined as 12.74±3.74 days with MGIT 960 system; 24.42±4.75 days with LJ and 24.37±4.96 days with AYC.2.2 agar. C2: Average growth time was determined as 18.25±9.32 days with TK-Medium, 28.73±7.44 days with LJ, and 31.72±6.35 days with AYC.2.2 agar. C3: Average growth time was determined as 20.48±7.24 days with Ogawa medium, 20.74±7.12 days with LJ, and 20.26±7.43 days with AYC.2.2 agar. C4: Average growth time was determined as 15.27±6.37 days with MGIT 960 system, 22.14±9.1 days with LJ, and 22±8.45 days with AYC.2.2 agar. C5: Average growth time was determined as 13±4.24 days with MGIT 960 system, 32.16±6.23 days with LJ, and 33±5.73 days with AYC.2.2 agar. C6: Average growth time was determined as 9±3.11 days with MGIT 960 system, 18.68±5.32 days with LJ, and 18.34±4.63 days AYC.2.2 agar. C7: Average growth time was determined as 14.74±7.65 with MGIT 960 system, 26.01±8.21 days with LJ, and 26.24±7.88 days with AYC.2.2 agar. CONCLUSIONS: In conclusion, similar results were obtained with LJ and Ogawa media and AYC.2.2 agar. Furthermore, more studies should be conducted for isolation of M. tuberculosis and performing antibiotic susceptibility tests using AYC.2.2 agar before it can be used as a routine media in the laboratories.

The Relation Between the Emergence of Fluoroquinolone Resistance and Fluoroquinolone Exposure in New Cases of Active Pulmonary Tuberculosis.

OBJECTIVE: This study aimed to determine the ratio of fluoroquinolone (FQ) exposure before the diagnosis of patients with a new case of active pulmonary tuberculosis (TB) and to investigate the correlation of this treatment with the emergence of FQ-resistant strains. MATERIAL AND METHODS: In this retrospective comparative case series study, a total of 132 patients, who had been diagnosed with adult, culture-positive, active pulmonary TB were reviewed. The FQ group had 30 patients who had had ≥1 time and ≥7 days of FQ exposure within 1 year before the diagnoses. The control group included an equal number of patients with TB with similar demographic characteristics (non-FQ group). Ofloxacin (OFX) and moxifloxacin (MFX) resistance were examined at 2 different concentrations (2 and 4 mg/L for OFX; 0.25 and 0.5 mg/L for MFX). RESULTS: Of the 132 patients, 30 (22%) had 7 days or longer of FQ monotherapy within 1 year of initiation of anti-TB treatment. FQ resistance was detected in 2 (3.3%) patients. In the FQ group, MFX resistance at 0.25 mg/L concentration was observed in 1 patient, whereas another patient had OFX and MFX resistance at 4 mg/L and 0.5 mg/L concentrations, respectively. In the non-FQ group, no FQ resistance was detected in any of the patients. No statistically significant difference in terms of development of FQ resistance was found between the ratios of FQ and non-FQ groups (p=0.492). Although there was no statistically significant difference, 2 patients, in whom resistance was detected, had FQ exposure before their diagnosis. CONCLUSION: The FQ exposure ratio before the diagnosis is high (22%) in this cohort that includes patients with new active pulmonary TB, and the presence of patients with FQ resistance (even if only a few) should be a noteworthy and cautionary result in terms of FQ exposure and resistance development.

A new colorimetric method for rapid detection of ethambutol and streptomycin resistance in Mycobacterium tuberculosis: crystal violet decolorization assay (CVDA).

Streptomycin (STR) and ethambutol (EMB) are important drugs used for the treatment of tuberculosis. There is a need for fast, reliable and inexpensive methods for detecting resistance to these drugs. The aim of this study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for the detection of STR and EMB resistance that is important drugs in tuberculosis treatment. In this study, drug susceptibility testing was performed on 140 Mycobacterium tuberculosis isolates provided from nine centers. Three tubes were used for each isolate. One of the tubes had a concentration of 2 mg/L STR and the other 5 mg/L EMB. The third was drug-free control tube. Sensitivity, specificity, positive predictive value (PPD), negative predictive value (NPD) and agreement for STR were found to be 81.8%, 94.6%, 87.8%, 91.5% and 90.57%, respectively. For EMB, sensitivity, specificity, PPD, NPD, and agreement were found to be 76%, 98.23%, 90.47%, 94.87% and 94.2%, respectively. The results were obtained in 11.3 ± 2.7 days (8-21 days). CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of STR and EMB resistance, and it could be adapted for drug susceptibility testing.

In vitro effects of ciprofloxacin, levofloxacin and moxifloxacin on Mycobacterium tuberculosis isolates.

INTRODUCTION: Increased tuberculosis prevalence, and isolation of multidrug resistant (MDR) Mycobacterium tuberculosis strains frequently as causative organisms from tuberculosis infections are resulted in increasing need of new anti-tuberculosis drugs. Nowadays, fluoroquinolones known to have fewer side effects than the other drugs used in treatment of tuberculosis are sometimes assessed even as first-line anti-tuberculosis drugs due to their in vitro and in vivo strong activity. It was aimed in this study to investigate phenotypically the fluoroquinolone susceptibility in MDR and non-MDR M. tuberculosis isolates. MATERIALS AND METHODS: A total of 126 MDR and non-MDR M. tuberculosis isolates from mycobacteriology laboratory of two hospitals in the Aegean Region of Turkey were included in the study. Ciprofloxacin (CIP), levofloxacin (LEV) and moxifloxacin (MXF) susceptibilities were assessed by agar proportion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. RESULT: Twelve (15.2%), 5 (6.3%) and 4 (5.1%) of the MDR M. tuberculosis strains were resistant to CIP, LEV, MXF, respectively [resistance breakpoints (µg/mL); CIP (> 2), LEV (> 1), MXF (> 0.5)] while non-MDR strains were susceptible to CIP, LEV, MXF. CONCLUSIONS: Consequently, although high fluoroquinolone susceptibilities were evaluated as a pleasing data in this study, to preserve their efficiency for many years steadily, quinolone usage and resistance increment in MDR M. tuberculosis isolates should be monitored elaborately.

The Contribution of Ultrasonographic Characteristics of Mediastinal Lymph Nodes on Differential Diagnosis of Tuberculous Lymphadenitis from Sarcoidosis.

BACKGROUND: Sarcoidosis and Mediastinal Tuberculous Lymphadenitis (MTLA) are two granulomatous diseases. Differentiation between these two diseases is dependent on clinical presentation, microbiological investigation, and cytopathological examination. In endemic regions, differential diagnosis of MTLA and sarcoidosis might be difficult. Endobronchial ultrasound guided Transbronchial Needle Aspiration (EBUS-TBNA) is a new diagnostic procedure for the diagnosis of mediastinal lymphadenopathy. EBUS not only enables the sampling of Lymph Nodes (LN), but also visualization of sonographic features of them. We hypothesized that the sonographic features of LN may help to differentiate MTLA from sarcoidosis. MATERIALS AND METHODS: This is a retrospective analysis of patients with intrathoracic lymphadenopathy who underwent EBUS-TBNA and were finally diagnosed as sarcoidosis or MTLA. Size, shape, margin, echogenicity, and coagulation necrosis were compared between the groups. RESULTS: A total of 257 LNs (215 sarcoidosis, 42 MTLA) were examined in 101 patients. A heterogeneous echotexture of lymph nodes was significantly more common (P <0.0001) in MTLA (69%) than sarcoidosis (36.2%). Also, necrosis was statistically significantly higher in MTLA compared to sarcoidosis (P<0.0001). The vascular pattern was similar in both groups (P=0.9050). Nearly half of the patients had grade 1 vascular pattern in both groups. The odds for diagnosis of MTLA were significantly higher in the presence of heterogeneous echotexture (odds ratio [OR], 7,00) or necrosis sign (OR, 131,2). CONCLUSION: Vascular patterns of two diseases were similar. Heterogeneous echotexture and necrosis sign in the LNs on EBUS are specific for MTLA. Combination of these findings with a positive tuberculin skin test, favors the diagnosis of MTLA over sarcoidosis.

Pulmonary tuberculosis due to Mycobacterium bovis revealed by skin lesion in slaughterhouse worker.

A 46-year-old male patient who has worked as a butcher was admitted to the hospital with an unhealing wound on the dorsal side of his hand. Incisional biopsy was performed from the lesion and histopathological diagnosis revealed a granulomatous inflammatory process, compatible with tuberculosis. The patient was directed to the department of chest diseases for further investigation in terms of pulmonary Tuberculosis (TB) infection. On the chest X-ray and thoracic CT scan, a minimal infiltration was observed in the left upper lobe. In two respiratory samples obtained by fiberoptic bronchoscopy, Mycobacterium tuberculosis complex was isolated and identified as M. bovis in subspecies level. After overall clinical evaluation, anti-TB treatment was initialized and a radiologic/clinical regression was observed during the follow-up procedure. This case has been reported as a rare and noteworthy pulmonary TB disease due to M. bovis in a slaughterhouse worker with a cutaneous granulomatous inflammatory reaction.

The diagnostic accuracy of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in mediastinal tuberculous lymphadenitis

Background/aim: Mediastinal lymph nodes are the second most commonly affected lymph nodes in tuberculous lymphadenitis. It is often difficult to diagnose tuberculosis in patients with lymphadenopathy without parenchymal lesions. The aim of this study was to describe the diagnostic utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with isolated mediastinal tuberculous lymphadenitis (MTLA). Materials and methods: This study included 527 patients who had undergone EBUS-TBNA between December 2012 and December 2014. Patients with the final diagnosis of MTLA were evaluated. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of EBUS-TBNA were calculated.Results: The prevalence of MTLA in all patients who had undergone EBUS-TBNA for mediastinal lymphadenopathy of unknown etiology was 5.2% (28/527). EBUS-TBNA was diagnostic in 21/28 (75%) patients, and the remaining 7 patients required additional procedures for confirmation of diagnosis. Sensitivity, specificity, PPV, NPV, and accuracy of combined cytopathological and microbiological examinations of EBUS-TBNA in the diagnosis of MTLA were 87.5%, 98.5%, 91.4%, 98%, and 94.4%, respectively. There were no major complications.Conclusion: EBUS-TBNA is a safe and effective procedure for the diagnosis of MTLA. When microbiological and cytopathological examinations of samples are combined, EBUS-TBNA demonstrates good diagnostic accuracy and NPV for the diagnosis of MTLA.

Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

[Multicenter evaluation of the indirect nitrate reductase assay for the rapid detection of multidrug-resistant tuberculosis]. / Çok ilaca dirençli tüberkülozun hizli tespiti için dolayli nitrat redüktaz testinin çok merkezli degerlendirilmesi.

Multidrug-resistant tuberculosis (MDR-TB) is defined as resistance to at least isoniazid (INH) and rifampicin (RIF), and it complicates the implementation of tuberculosis control programmes. The rapid detection of MDR-TB is crucial to reduce the transmission of disease. The nitrate reductase assay (NRA) is one of the colorimetric susceptibility test methods for rapid detection of MDR-TB and based on the ability of reduction of nitrate to nitrite by Mycobacterium tuberculosis. The aim of this study was to evaluate the performance of the NRA for the rapid detection of MDR-TB. A total of 237 M.tuberculosis complex (MTC) isolates that were identified by the same method (BD MGIT(TM) TBc Identification Test, USA) from nine different medical centers in Turkey were included in the study. The susceptibility results of the isolates against INH and RIF obtained by reference test (Bactec MGIT(TM) 960, BD, USA) were then compared with NRA. In order to ensure consistency between centers, Löwenstein-Jensen (LJ) medium with antibiotics and without antibiotics (growth control) and Griess reagent solution were prepared in a single center (Ondokuz Mayis University School of Medicine, Medical Microbiology Department) and sent to all participant centers with the standardized test procedure. After the inoculation of bacteria into the test tubes, the tubes were incubated at 37°C, and after seven days of incubation, 500 µl Griess reagent was added to the LJ medium without antibiotics. If a color change was observed, an equal volume of Griess reagent was added to test LJ media with antibiotics. When a color change was observed in LJ media with antibiotics, it was considered that the isolate was resistant to tested antibiotics. Among 237 MTC isolates, 16 were resistant only to INH and nine were resistant only to RIF; 93 isolates (39.2%) were resistant (MDR) and 119 isolates (50.2%) were susceptible to both of the drugs determined with the reference susceptibility test. In the study, five INH-resistant isolates determined with reference method were found susceptible with NRT and eight INH-susceptible isolates determined with reference method were found resistant with NRT. In contrast, one RIF-resistant isolate determined with reference method was found susceptible with NRT and three RIF-susceptible determined isolates were found resistant with NRT. Accordingly, the concordance rate between the reference method and NRA were estimated as 94.5% for INH and 98.3% for RIF. The sensitivity, specificity, positive and negative predictive values of NRA were detected as 95.4%, 93.7%, 92.8% and 96% for INH, and 99%, 97.8%, 97.1% and 99.2% for RIF, respectively. The results of the 111 isolates were obtained on the seventh day, while the rest of the results were obtained between 10-14 days. In conclusion, the data of this multicenter study showed that NRA is a reliable, relatively inexpensive and practical method to perform for the rapid detection of MDR-TB.

Five Years' Evaluation of the BD ProbeTec System for the Direct Molecular Detection of Mycobacterium tuberculosis Complex in Respiratory and Nonrespiratory Clinical Samples.

In this study, Mycobacterium tuberculosis complex was detected by BD ProbeTec ET system in 4716 respiratory and 167 nonrespiratory samples [mostly (98%) smear negative). Sensitivity, specificity, positive and negative predictive values were 81.8%, 98.3, 85.1 and 97.9 for respiratory and 100%, 96.2, 64.7 and 100, for nonrespiratory samples, respectively. Among 149 (3.1%) ProbeTec DTB positive and culture negative samples, 72 (65 respiratory and seven nonrespiratory) (48.3%) were recovered from the patients who were evaluated as having TB infection. The system has been found as useful in early diagnosis of tuberculosis infection in association with the clinical, radiological and histopathological findings.

[Comparison of the performances of MTD Gene-Probe® test, BACTEC 960™ system and Löwenstein-Jensen culture methods in the diagnosis of smear-negative tuberculosis cases]. / Yayma negatif tüberküloz olgularinin tanisinda MTD Gen-Probe® testi, BACTEC 960™ sistemi ve Löwenstein-Jensen kültür yöntemlerinin performansinin karsilastirilmasi

The aim of this study was to compare the results of nucleic acid amplification-based MTD (Mycobacterium tuberculosis direct test) Gene-Probe® method in samples obtained from acid-fast bacilli (ARB) smear-negative patients with suspected tuberculosis (TB), with the culture results obtained from automated BACTEC 960™ (MGIT) system and Löwenstein-Jensen (LJ) medium. In addition, the contribution of molecular methods in early diagnosis of pulmonary TB and the effect of radiological prevalence of the disease associated with or without cavity to the molecular diagnosis and/or growth time in culture media have been evaluated. A total of 107 patients (86 male, 21 female; mean age: 49.89 ± 17.1 years, age range: 18-81 years) who were clinically and radiologically suspected of having pulmonary TB and/or TB pleurisy, were included in the study. Of the samples 65 (60.7%) were sputum, 32 (29.9%) were bronchial aspiration, 5 (4.7%) were pleural fluid, and 5 (4.7%) were transthoracic fine needle aspiration biopsy materials. Patient samples were cultured in solid LJ media and liquid-based BACTEC 960 system (Becton Dickinson Co., USA) in the same working day. Meanwhile, MTD Gen-Probe test (Gen-Probe Inc., USA) was studied in two separate working days of the week as specified by the laboratory. The samples were incubated until positivity was determined in BACTEC 960 system and/or growth was detected in LJ medium. Negative cultures were incubated for 42 days and were finalized. When mycobacterial growth was determined in the culture, identification of M.tuberculosis complex (MTBC) and differentiation from nontuberculous mycobacteria were performed by conventional methods and BACTEC 460 NAP test. Forty five (42%) patients were diagnosed as pulmonary paranchimal TB (40 were active pulmonary TB, 1 was miliary TB and 4 were culture-negative pulmonary TB), while 4 (3.7%) patients diagnosed as extrapulmonary TB and 58 (57.9%) patients were diagnosed as other pulmonary diseases unrelated with TB. LJ cultures yielded positive results in 32 of 45 (71%) pulmonary TB patients, and BACTEC 960 were found positive in 84.4% (38/45) of those patients. On the other hand the positivity rate of MTD Gen-Probe test was detected as 37.4% (40/107). The sensitivity, specificity, positive and negative predictive values for MTD Gen-Probe test were estimated as 89%, 100%, 100% and 93%, respectively. Those values for BACTEC 960 system were found as 82%, 98%, 97% and 88%, and for LJ culture method as 71%, 100%, 100% and 83%, respectively. Average periods to make a decision for diagnosis of TB by MTD Gen-Probe, BACTEC 960 (MGIT) and LJ culture methods were calculated as 2.36 days, 20.11 days and 32.49 days, respectively. In comparison of the methods in terms of turnaround times, MTD Gen-Probe test was found superior to LJ culture method, however the turnaround times for BACTEC 960 and LJ culture methods were similar. When the clinical data were evaluated, no effect of radiological density of lesion was identified on the diagnosis time of molecular test and time of growth in liquid based automated BACTEC system and/or LJ culture method. However, LJ culture demonstrated earlier reactivity in patients with cavitary lesions. As a result, MTD Gene-Probe test was observed as a reliable and rapid method for the early diagnosis of pulmonary TB patients, early initiation of therapy, prevention of disease progression and transmission.

[Investigation of bacterial etiology with conventional and multiplex PCR methods in adult patients with community-acquired pneumonia]. / Toplum Kökenli Pnömonisi Olan Eriskin Hastalarda Konvansiyonel ve Multipleks PCR Yöntemleriyle Bakteriyel Etiyolojinin Arastirilmasi

Community-acquired pneumonia (CAP) is still a serious life-threatening disease, in which the etiologic agent cannot be identified in more than 50% of patients despite advanced diagnostic methods. The most commonly used methods in the determination of CAP etiology are culture and serological tests. Since early and accurate therapy reduces the mortality in CAP cases, rapid and reliable diagnostic methods are needed. The aim of this study was to determine the bacterial etiology in adult patients with CAP by implementing multiplex polymerase chain reaction/reverse line blot hybridization (M-PCR/RLBH) assay combined with conventional methods. A total of 128 cases (94 were male; age range: 19-81 years, mean age: 58) who were admitted to our hospital and clinically diagnosed as CAP between November 2008 - November 2010, were included in the study. Respiratory samples (sputum and/or bronchoalveolar lavage) obtained from patients were searched by M-PCR/RLBH method (Gen ID®, Autoimmun Diagnostika GmbH, Germany) in terms of the presence of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila nucleic acids. The samples were simultaneously inoculated onto 5% sheep blood agar, chocolate agar, haemophilus isolation agar, buffered charcoal yeast extract-selective agar and EMB agar media for cultivation. Serum samples obtained from the cases were tested for IgM and IgG antibodies against C.pneumoniae by microimmunofluorescence (Focus Diagnostic, USA) and against L.pneumophila and M.pneumoniae by indirect immunofluorescence (Euroimmun, Germany) methods. The bacterial etiology was identified in 59 (46.1%) of 128 patients with CAP and a total of 73 pathogens were detected. The leading organism was S.pneumoniae (n= 32, 25%), followed by H.influenzae and M.pneumoniae (n= 9, 7%), gram-negative bacilli (n= 10, 7.8%), M.catarrhalis (n= 6, 4.7%), C.pneumoniae (n= 4, 3.2%), L.pneumophila (n= 2, 1.6%) and Staphylococcus aureus (n= 1, 1.4%). Infection with atypical pathogens were detected in 15 (11.7%), and mixed infections in 14 (10.9%) patients. The detection rate of microorganisms (S.pneumoniae, H.influenzae, M.catarrhalis, C.pneumoniae, L.pneumophilia, M.pneumoniae) searched by M-PCR/RLBH method was 41.4% (53/128), while those microorganisms were detected in 23.4% (30/128) of the patients by conventional methods, representing a significant difference (p< 0.05). It was concluded that M-PCR/RLBH method supplemented the determination of bacterial etiology in CAP cases by increasing the rate of detection from 23.4% to 41.4%. The results indicated that empirical treatment of CAP should primarily include antibiotics against S.pneumoniae, M.pneumoniae and H.influenzae in our region.