The value of laboratory tests in the diagnosis of tuberculosis.

BACKGROUND AND AIM: In the last decades, the inhabitants of the Romanian region known as Jiu Valley underwent changes in their social and economic status which determined changes in behaviour and health, which influenced their general health condition. One of the consequences was the exacerbation of tuberculosis. In order to control this situation, there was a need to increase the efficiency of diagnosis. This optimization can be reached by a better detection of mycobacterium infection, optimal isolation of strains and identification of the resistance of strains to antituberculous drugs. METHODS: In order to identify the best diagnostic modality, we compared the efficacy of the classical bacteriological diagnosis, still performed in the field, to the modern methods of molecular biology. The study included two groups, one represented by 213 patients who were investigated using the classical bacteriological methods, and 49 who were diagnosed using the PCR method. RESULTS: The tuberculosis patients who have been evaluated only with the classical bacteriological methods where diagnosed as TB positive and treated according to the national guidelines, which are in agreement with the international guidelines. The PCR diagnostic methods had a superior diagnostic value compared to the traditional bacteriological method. CONCLUSIONS: The results revealed the superiority of the modern molecular biology methods based on PCR. However the bacteriological method remains useful in areas where PCR cannot be afforded.

MRI as indicator of pulmonary tuberculosis activity: from morphological to molecular level assessment - a case report.

We present the case of an adult patient with active post-primary pulmonary tuberculosis (TB) and discuss specific morphological and textural aspects found at high-field magnetic resonance imaging (MRI) of the lung. The 42-year-old man, heavy smoker, undertook a routine employment medical examination and was admitted to a referral pulmonology and TB center due to the abnormal findings seen on his chest radiography. The patient presented nonspecific symptoms, bilateral bronchial breath at pulmonary auscultation, inflammatory syndrome on the laboratory blood tests and positive sputum smears for acid-fast bacilli, which together with the typical aspect on the chest radiography lead to a diagnosis of post-primary pulmonary TB and administration of specific medication. To exclude a possible lung cancer and to reevaluate the extent of the disease, computed tomography and magnetic resonance imaging of the lung were performed. The magnetic resonance examination showed, with an accuracy similar to that of computed tomography, the morphology of active post-primary parenchymal TB lesions, as depicted on the T2-weighted acquisition. Moreover, the T1-weighted sequence using iterative decomposition allowed the assessment of both lipid and caseous pneumonia. To the best of our knowledge, this is the first reported case to assess post-primary pulmonary TB using high field MRI equipment, with an analysis from a morphological to a molecular level. By using a fast two-sequence protocol, both morphological, through T2-weighted acquisition, and textural information such as fat content, using dedicated T1-weighted sequence, can be obtained.

Morbidity by tuberculosis in the Jiu Valley: relation with diagnostic practice and the impact on therapeutic results.

BACKGROUND AND AIM: In the last two and a half decades the dynamics of tuberculosis has been modelled by social and economic conditions, with consequences on the life style, and effects on the onset and evolution of the disease. The Jiu Valley is an area with social problems: mining, mines closing down and ceasing activity, poverty. We looked for a relationship between changes of life style in the Jiu Valley and the dynamics of tuberculosis. METHODS: We studied 528 patients who asked for medical services in different hospitals in the Jiu Valley between 2010-2013. We structurally characterized this group, we identified the characteristics of life style, and we assessed the health state, in particular the relation with tuberculosis. RESULTS: We found out that the quality of life was influenced by the health state, especially by tuberculous disease. Quality of life was influenced by the life style, professional factors and their long term consequences. The study evidenced a strong relationship between apparently very different factors such as life style, professional factors on one side and those characterizing tuberculosis. CONCLUSIONS: We report the first detailed epidemiological data on tuberculosis in an economically poor area, the Jiu Valley.

A case control study of risk factors associated with pulmonary tuberculosis in romania: experience at a clinical hospital of pneumology.

BACKGROUND AND AIM: Tuberculosis (TB) remains a major public health issue in Romania. The aim of the present study was to evaluate the potential demographic, socioeconomic and behavioral risk factors for TB among hospitalized patients in Romania. METHODS: This is a case-control study conducted between March 1st 2014 and March 30th 2015 at Leon Daniello Clinical Hospital of Pneumology, Cluj Napoca. A total of 150 TB patients defined as "cases" were matched for age, sex and county of residence to 150 controls selected from patients attending the same hospital with respiratory diseases other than TB. Data collection was obtained through patient interviews using a structured questionnaire. Factors potentially associated with TB infection were analyzed using univariate and multivariate logistic regression. RESULTS: Factors independently associated with TB were illiteracy (OR=2.42, 95% CI 1.09-5.37), unemployment (OR=2.08, 95% CI 1.23-3.53), low household income (OR=4.12, 95% CI 2.53-6.71), smoking (more than 20 cigarettes per day) (OR=2.12, 95% CI 1.20-3.74), poor knowledge of TB (OR=3.46, 95% CI 1.97-6.07), presence of TB patient in household (OR=4.35, 95% CI 1.42-13.36), prior TB treatment (OR=2.2, 95% CI 1.93-2.5) and diabetes (OR=3.32, 95% CI 1.36-8.08). CONCLUSION: This study provided useful information that might help to develop and adapt effective policies for TB control in Romania.

A joint cross-border investigation of a cluster of multidrug-resistant tuberculosis in Austria, Romania and Germany in 2014 using classic, genotyping and whole genome sequencing methods: lessons learnt.

Molecular surveillance of multidrug-resistant tuberculosis (MDR-TB) using 24-loci MIRU-VNTR in the European Union suggests the occurrence of international transmission. In early 2014, Austria detected a molecular MDR-TB cluster of five isolates. Links to Romania and Germany prompted the three countries to investigate possible cross-border MDR-TB transmission jointly. We searched genotyping databases, genotyped additional isolates from Romania, used whole genome sequencing (WGS) to infer putative transmission links, and investigated pairwise epidemiological links and patient mobility. Ten isolates from 10 patients shared the same 24-loci MIRU-VNTR pattern. Within this cluster, WGS defined two subgroups of four patients each. The first comprised an MDR-TB patient from Romania who had sought medical care in Austria and two patients from Austria. The second comprised patients, two of them epidemiologically linked, who lived in three different countries but had the same city of provenance in Romania. Our findings strongly suggested that the two cases in Austrian citizens resulted from a newly introduced MDR-TB strain, followed by domestic transmission. For the other cases, transmission probably occurred in the same city of provenance. To prevent further MDR-TB transmission, we need to ensure universal access to early and adequate therapy and collaborate closely in tuberculosis care beyond administrative borders.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Evaluation of interleukin-10 levels in the plasma of patients with various stages of tuberculosis.

UNLABELLED: Mycobacterium tuberculosis (Mtb) infection remains one of the world's major causes of illness and mortality. A clear understanding of the host defense against Mtb is imperatively needed forthe control of this epidemic. When tuberculosis (TB) infection occurs, a variety of pro and anti-inflammatory cytokines play a vital role in the pathogenesis of this disease. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines reported to suppress the protective immune response against tuberculosis. AIM: The aim of the present study was to evaluate the association of plasma IL-10 levels with various disease stages of TB and the possible effects of treatment on these levels. MATERIALS AND METHODS: A group of 30 patients with active pulmonary TB and a control group of 21 healthy individuals were enrolled in this study. The levels of IL-10 were measured before, during, and after treatment using commercially available enzyme-linked immune-sorbent assay (ELISA). Data were analyzed using GraphPad Prism version 5.0. RESULTS: The results showed that the levels of IL-10 had significant differences between the TB and control groups (p<0.05). The patients with abnormal chest X-Ray findings had higher IL-10 levels when compared to patients with normal X-Rays (p=0.03). A subgroup of 18 patients were followed during the treatment and the mean plasma concentration of IL-10 in patients before therapy was higher than in patients at 3 months of therapy and in patients after 6 months of therapy (p=0.01). However, the IL-10 level remained significantly higher in patients at the end of treatment compared with controls. These findings could be used in follow-up as clinical biomarker of the success of tuberculosis therapy.

The concordance between phenotypic and genotypic M. tuberculosis drug susceptibility tests results: observational study.

UNLABELLED: Multi-drug resistant tuberculosis (MDR-TB) represents a major threat for TB control at the global level. Identification of mutations responsible for drug resistance by molecular methods can be used for rapid and specific detection of drug resistance. The aim of our study was to assess the concordance between phenotypic and genotypic tests results (GenoTypeMTBDRplus kit) for isoniazid and rifampicin resistance in M. tuberculosis isolated strains. MATERIAL AND METHODS: The specific zone mutations in rpoB, katG and inhA gene for rifampicin and isoniazid were investigated with molecular methods in 198 recently isolated unique strains from patients diagnosed with pulmonary tuberculosis. These results were compared with the absolute concentration drug susceptibility test results. RESULTS: Sensitivity, specificity, predictive positive value, predictive negative value, efficiency of genotypic method, calculated by comparing with conventional method for INH and RMP were 93.85%, 100.00%, 100.00%, 63.33%, 94.44%, and 99.26%, 82.25%, 92.46%, 98.07% and 93.93%, respectively). Cohen coefficient showed Kappa values = 0.746 (good strength of agreement) for INH, and Kappa value = 0.853 (very good strength of agreement) for RMP. CONCLUSION: The obtained results are consistent with those reported from other regions of the world. The use of rapid molecular assays reduces the time for drug resistance diagnostic to just a few days, and may help the control of the ongoing TB transmission.

[Tuberculosis in Romania. Problems and solutions]. / Tuberculoza in România. Probleme si solutii.

UNLABELLED: The analysis of the Management Unit of the National TB Programme (NTP) database, together with the reports of the TB county managers, allowed to the authors to identify some weaknesses of TB control in Romania in the recent years and to propose the appropriate measures. PROBLEMS: The marked decrease in the reduction of TB cases reported annually from 2,761 in 2005-2006, to 145 in 2007-2008 and the stagnation of mortality rate: 7.5 per ten thousand in 2007 and 7.6 per ten thousand in 2008. Deficiencies in data recording and reporting through informatic system of the NTP. Lack of financial resources for system maintenance and upgrade. Deficiencies in monitoring and control of mycobacterium resistance to antituberculous drugs phenomenon at national level. Sensitivity testing only for a small percentage of culture confirmed new TB cases (21%). Higher percentage of MDR in new TB cases compared to the results of national survey of mycobacterium drug resistance 2003-2004. Lack of personnel: 16 TB dispensaries without any pulmonologist, vacancies for 259 doctors, 436 nurses and 433 auxiliary personnel. Important deficiencies in the NTP network's infrastructure and logistics countrywide. Discontinuities in the supply with first and second line antituberculous drugs resulting in interruption of treatments. Lack of an officially endorsed protocol for the diagnosis, treatment and monitoring of cases with TB/HIV co-infection. Solutions: Revitalization of monitoring-supervision activities of the NTP running countrywide, provision with necessary financial resources to perform the scheduled visits in counties. Providing maintenance and upgrade of the informatic system for data collection. Implementation of the necessary measures in order to attract and maintain the personnel in the NTP network. Conduct the national survey of mycobacterium susceptibility to first and second line antituberculous drugs and drug susceptibility testing of the most culture confirmed TB cases. Restore the centralized procurement of TB drugs. Finalization and official endorsement of the protocol for TB/HIV co-infection initiated in 2004.

[Respiratory features of Aspergillus infection in eight years of pulmonology practice]. / Manifestari respiratorii ale infectiei cu Aspergillus intâlnite in 8 ani de practica pneumologia.

AIM: To evaluate clinical manifestations of Aspergillus infections in our pulmonology practice. METHODS: Between 2002-2009, we assessed retrospectively and prospectively, Aspergillus related diseases in patients admitted in Clinical Hospital of Pneumology from Cluj-Napoca, Romania. Positive diagnosis was based on identification of Aspergillus speciae (A spp) on Sabouraud medium, pathological, or immunological proves. RESULTS: 83 patients were included. A spp was isolated in 73 patients from: sputum 46, bronchial lavages (BL) 17, both sputum and BL 4, sputum and nasal secretion 1, or from surgical samples 5 cases. In those 10 cases without A spp positive cultures, diagnosis was confirmed by pathologic examination on surgical or bronchoscopical samples in 6 and 2 cases respectively. 76 patients had previous chronic respiratory diseases and 7 had nonrespiratory chronic diseases. We identified the following clinical forms: chronic cavitary aspergillosis 46 cases, aspergillus tracheobronchitis 19 cases, allergic aspergillosis 15 cases, invasive aspergillosis 3 cases. We observed a poor definition on nosological forms, diagnosis like "pulmonary aspergillosis" or "aspergilloma" were the most common. In 12 cases no clinical significance was attributed for detection A spp in bronchial smears. 60 patients supported specific management: antifungal therapy in 38 cases, surgical procedures in 14 cases and both methods in 8 cases, with many differences in treatment and follow-up. CONCLUSIONS: Chronic cavitary aspergillosis was the most frequent clinical form observed in our pulmonology practice. Using the clinical guidelines for fungal respiratory infections we can avoid the wrong diagnosis and then include a correct antifungal treatment in the complex management of our chronic pulmonary patients.

[Comparative study and sensitivity tests of Mycobacterium tuberculosis strains using nitrate reductase and absolute concentrations methods]. / Studiu comparativ al testarii sensibilitatii tulpinilor de Mycobacterium tuberculosis prin metoda nitrat reductazei si metoda concentratiilor absolute.

INTRODUCTION: The alarming increase of cases of tuberculosis with resistant germs renders compulsory the orientation towards rapid and cheap methods that would allow large-scale testing of mycobacterial strains. AIM: Evaluation of the performances of two phenotypic methods for mycobacteria sensitivity testing. nitrate reductase and absolute concentrations. MATERIAL AND METHOD: In two different laboratories we tested the same set of 20 mycobacterial strains from the 2008/2009 international panel and an additional 10 strains isolated in the second laboratory, using the absolute concentration method as standard and the nitrate reductase method, both using Lowenstein Jensen medium with rifampicine and isoniaside incorporated The results obtained in both laboratories are concordant for both methods and show sensitivity and specificity of 100% for nitrate reductase method compared to absolute concentration method The results obtained using the two methods in the two laboratories also showed a 100% reproducibility. CONCLUSIONS: Nitrate reductase method is easy to perform and to acknowledge. It does not require special equipment, besides what is usually found in the laboratory. It showed a 100% sensitivity and specificity. The 100% reproducibility and repetability of results can represent arguments for a possible use of the methods in all the laboratories belonging to the national network of mycobacteriology laboratories, in order to screen for multiresistant strains.

Romanian anti-TB drugs resistance surveillance 2003-2004.

UNLABELLED: Romania decided and initiated a DRS for anti-TB drugs at national level using the standardized methodology proposed by WHO and IUATLD. The DRS protocol was designed with technical assistance from WHO; the surveillance started in June 2003 and ended in June 2004. It was tested the susceptibility to the 4 first line anti-TB drugs: Isoniazide (H), Rifampicin (R), Streptomycin (S), Ethambutol (E). Drug susceptibility testing used: indirect absolute concentration method. There were included in the survey 1251 TB patients from the 60 clusters: 869 new cases and 382 previously treated. From the penitentiary system were included 85 TB patients, 47 new cases and 38 previously treated. RESULTS: [table: see text]. Estimations of the trend of anti-TB drug resistance in Romania for the next period was proposed.

[A simple enzyme test for proving the resistance to isoniazid of Mycobacterium tuberculosis strains]. / Test enzimatic simplu pentru semnalarea izoniazido-rezistentei tulpinilor de Mycobacterium tuberculosis.

Resistance to isoniazid is the consequence of mutation in some genes or genomic sequences. One of them is katG gene, which encode catalase-peroxidase. The activity of this enzyme can be revealed by simultaneous test catalase-peroxidase. The aim of the study was to assess the correlation between the results of catalase-peroxidase test and isoniazid- resistance of M. tuberculosis strains, in order to estimate the utility of this test in current practice. Material and method. We assessed the results of catalase- peroxidase test of 2268 identified M. tuberculosis strains (19 isolated from silicotuberculosis patients, 198 isolated from industrial dust and chemical polluted city patients, and 2051 strains isolated from other pulmonary tuberculosis patients), with known pattern of susceptibility/resistance. Six hundred twenty four strains (27.5%) had been INH mono-resistant or INH resistant associated with other resistance. It resulted a significant correlation between INH resistance and the negative result of catalase-peroxidase test (p <0.001). More than that, the negative result of peroxidase test was obtained in 63.5% of INH resistant strains. This simple and easy to do test, used for the strains after growth in primary culture, can he helpful in order to confirm the suspicion of INH resistance.

[Comparison of the results of M. tuberculosis cultures on liquid media in MB/BacT system and solid Löwenstein Jensen media in selected patients]. / Compararea rezultatelor culturilor obtinute în mediu lichid în sistemul MB/BacT si pe mediul Löwenstein Jensen de la pacienti selectati.

AIM: The study was performed to evaluate the performances of the MB/BacT System in comparison to solid media Löwenstein Jensen for the recovery and the time for the detection of mycobacteria from specimens of selected patients from a dust and chemical polluted area, where the incidence of tuberculosis is higher than the mean county incidence- as control- and silicotuberculosis patients. MATERIAL AND METHODS: We processed 193 selected specimens (156 as control and 37 from silicotuberculosis patients). All specimens have been processed for microscopic examination, culture on Löwenstein Jensen media and in Middlebrook7H9 liquid media in automatic system MB/BacT Sputum, bronchial aspirate and tongue swab had been previously decontaminated, but aseptically obtained specimens not, before inoculation onto the culture media. RESULTS: The culture positivity mean time for control was 14,56 days in MB/BacT system and 28.66 days on Löwenstein Jensen, and 17.7 days, and 77 days respectively for silicotuberculosis patients specimens, without significant difference between the two groups (chi2 = 0,33; p = 0,565; IC95). CONCLUSIONS: Simultaneously culture of selected specimens by using two different culture media for Mycobacterium strains isolation can contribute (it the improvement of the results by the shortage of diagnostic time. The precocity of the culture positive results in MB/BacTsystem (but also on LJ media), are directly related with the number of colonies forming units (CFU) in the processed specimen. The positive results on MB/BacT system in the first 14 days of incubation are related to rich cultures on LJ media, in the great majority of cases. Positive results on LJ media have been obtained after 21 days of incubation. All isolated strains have been M tuberculosis.

Abdominal miliary tuberculosis in a patient with AIDS: a case report.

We present a 34 year old patient, intravenous drug user, hospitalized with fever, distortion of general status, dry irritating cough, abdominal colicative pains, and we established the diagnosis of HIV infection advanced stage/AIDS; his antecedents revealed (August 2000) abdominal tuberculosis not treated during the last 3 months. He presented a pneumonia with Pneumocystis carinii during hospitalization. Death was due to a colon perforation with secundary peritonitis. Miliary tuberculous lesions in liver, spleen and colon were revealed at necropsy and cytomegalovirus was identified in necrotic samples also.