[Two selected commercially based nucleic acid amplification tests for the diagnosis of tuberculosis]. / Zastosowanie wybranych komercyjnych testów molekularnych w mikrobiologicznej diagnostyce gruzlicy.

INTRODUCTION: This is the retrospective study on diagnostic effectiveness of two nucleic acid amplification tests: AMPLICOR Mycobacterium tuberculosis (MTB) and Xpert MTB/RIF. The first one was included into diagnostic procedure for tuberculosis from 1999 to 2009 and the second one has been used from November 2009. MATERIAL AND METHODS: Study groups comprised 1875 samples for AMPLICOR MTB (104 were inhibited), and 213 samples for Xpert MTB/RIF. RESULTS: The assay sensitivity was 81.9% for AMPLICOR MTB and 81.8% for Xpert MTB/RIF, and specificity was 97.2% and 99.5% respectively, as compared to the culture on Loewenstein-Jensen medium. However, sensitivity of each test correlated with Ziehl-Neelsen staining. For AFB+ samples, assay sensitivity was 97.8% for AMPLICOR MTB and 100% for Xpert MTB/RIF and for AFB- samples it was 58.1% and 50% respectively. CONCLUSIONS: The reported results show a high diagnostic usefulness of Xpert MTB/RIF test for samples, which are positive for Ziehl-Neelsen staining.

[The comparison between two methods for typing of nontuberculous mycobacteria: high pressure liquid chromatography and molecular assay GenoType Mycobacterium CM/AS]. / Porównanie dwóch technik typowania pratków niegruzliczych: cieczowej chromatografii wysokocisnieniowej i molekularnego systemu 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. 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 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.

[Diagnostic utility of the molecular assay GenoType MTBC (HAIN Lifesciences, Germany) for identification of tuberculous mycobacteria]. / Przydatnosc diagnostyczna molekularnego testu GenoType MTBC (HAIN Lifesciences, Niemcy) w identyfikacji pratków gruzlicy.

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.

[Pulmonary mycobacteriosis--the diagnostic challenge. The authors' experience]. / Mikobakteriozy pluc--trudnosci diagnostyczne doswiadczenia wlasne.

UNLABELLED: The diagnosis of NTM-related pulmonary disease is based on clinical symptoms, radiological features and several positive cultures of one and the same NTM species from samples obtained from the respiratory tract. Short hospitalization usually does not enable sufficient diagnostic procedures to meet the diagnostic criteria, and this may lead to the reduction of diagnostic sensitivity. The aim of the study was to draw attention to NTM-related pulmonary disease, to share the authors' experience in the diagnosing of pulmonary mycobacteriosis and to indicate the possibilities of improving the diagnostic accuracy in this disease. A group of 31 patients with sputum, bronchial washing and/or bronchoalveolar lavage fluid (BALF) NTM-positive cultures was selected from a cohort of 245 patients evaluated for tuberculous and nontuberculous mycobacterial diseases (total number of 1277 specimens were invastigated). In two of them NTM related pulmonary disease was diagnosed (caused by M. kansasii and M. avium) at the course of initial evaluation. In the remaining 29 patients the microbiological data did not allow to establish the diagnosis of mycobacterial lung disease mainly due to a small number of samples from the respiratory tract. From this group 13 patients were reevaluated within 3 - 6 months from the initial investigation. This allowed to identify two new cases of mycobacteriosis (M. kansasii and M. avium). Thus among 31 patients with NTM positive cultures from respiratory tract specimens 4 patients (4/31, 12,9%) met the diagnostic criteria for mycobacterial disaease. CONCLUSION: Microbiological analysis of an adequate number of samples in symptomatic patients with radiological features suggestive for NTM-related pulmonary disease increses the diagnostic sensitivity in pulmonary mycobacteriosis. Identification of the species in positive cultures is of great importance.

[Identification of Mycobacteriaceae species based on the hsp-65 gene polymorphism analysis by PCR - RFLP]. / Identyfikacja gatunkowa pratków z rodzaju Mycobacterium na podstawie analizy polimorfizmu genu hsp-65 z uzyciem techniki PCR-RFLP.

The polymorphism of the short fragment of the heat shock protein 65 encoding gene was evaluated by the PCR - RFLP technique described by Telenti and further developed by Devallois for identification of mycobacterial species in routine laboratory work. We analysed 58 strains representing 25 different mycobacterial species (24 reference strains and 34 clinical isolates). The results obtained by PCR-RFLP and HPLC identification techniques were highly concordant The results were compatible for 87,5% (21 / 24) reference strains and for 97,1% (33/34) clinical isolates. The PCR - RFLP method allowed for accurate identification mycobacterial species, especially pathogenic strains. Restriction patterns obtained for 25 species of Mycobacteriaceae genus could help in constructing the data base and algorithms used in routine laboratory practice.

[The mycolic acids analysis with HPLC technique in drug susceptibility testing of Mycobacterium tuberculosis strains]. / Analiza kwasów mikolowych technika HPLC w ocenie lekowrazliwosci Mycobacterium tuberculosis.

The aim of this study was to evaluate the utility of the quantitative analysis of mycolic acids by HPLC technique in drug susceptibility testing of the M. tuberculosis isolates to the first-line antituberculous drugs: isoniazid and rifampicin. Drug susceptibility of the 30 clinical M.tbc isolates was examined by the mycolic acids analysis with HPLC technique and results were compared to the proportion method on solid L-J medium and liquid medium in MGIT system. In HPLC method drug susceptibility of M. tuberculosis strains was described by TAMA index defined as the ratio of the total area under mycolic acids peaks (TAMA) from cultures with drug to the TAMA of control. At critical concentrations of drugs, TAMA indexes of resistant strains were >0.5, and TAMA indexes of susceptible strains were <0.05. The average error of the TAMA analysis was +/- 9.5% The quantitative analysis of mycolic acids by HPLC gives results compatible with standard proportion method and is a reliable method for determination of drug susceptibility of M. tuberculosis.

[Diagnosis of tuberculous lymphadenitis based on the fine needle aspiration samples analysis]. / Gruzlica wezlów chlonnych rozpoznana na podstawie badania materialu z biopsji cienkoiglowej.

Tuberculous lymphadenitis is one of the most common extrapulmonary manifestations of tuberculosis. The most common lymph nodes involved are in the cervical region. Lymphadenitis due to M. tuberculosis generally presents with enlarging neck lymph nodes over weeks or months associated with fever, weight loss and fatigue. Fine needle aspiration (FNA) of affected lymph nodes has been shown to yield a high sensitivity and specificity in the diagnosis of tuberculous lymphadenitis. Specimens should be examined cytologically, as well as by AFB smear and cultures. The time between the onset of symptoms, clinical presentation and final diagnosis is often too long. We present a case of 60 years old man with tuberculous lymphadenitis, initially suspected of lymphoproliferative disease.

[Modern etio-pathogenesis and diagnosis of mycobacterioses]. / Wspólczesna etiopatogeneza i rozpoznawanie mikobakterioz.

Over the last decades the incidence of infections with non-tuberculous mycobacteria (NTM) has increased. It has been noted in various regions of the world and it seems to concern also our country. The aim of this review is to call attention to clinical presentation and diagnostic criteria of mycobacterioses. The environmental sources of NTM, predispositions to dissemination of NTM infection and current epidemiological data on mycobacterioses are discussed. Since the accuracy of mycobacterial strain identification is most important for microbiological diagnosis of disease, it is suggested to perform it according to modern requirements, exclusively in selected, well-equipped laboratories.

[Mycolic acids analysis from various species mycobacterium by high pressure liquid chromatography (HPLC)]. / Analiza kwasów mikolowych róznych gatunków z rodzaju Mycobacterium metoda wysokocisnieniowej chromatografii cieczowej (HPLC).

HPLC is the most useful method to analyze various species of mycobacteria by using mycolic acids. The purpose was to prepare a library containing chromatographic patterns of mycolic acids derived from reference species Mycobacterium, which had been cultivated in standard conditions. 28 reference strains (27 ones from American Type Culture Collection and one cultivated from the vaccine M. bovis BCG) were used. The analysis of mycolic acids involved chromatographic separation of their bromophenacyl derivatives according to Centers for Disease Control recommendation. Mycolic acids profiles formed by HLPC were reproducible for all reference species in this study. Standard deviation of relative retention time of every peak did not exceed 2.5%. The species included into M. tuberculosis complex beyond M. bovis BCG shared the same mycolic acids pattern. HPLC is the only mean to distinguish M. tuberculosis from M. bovis BCG. The other studied stains had species specific patterns which differed from M. tuberculosis complex and M. bovis BCG. The prepared library comprising 28 reference elution profiles of mycolic acids from known mycobacteria species can be applied in diagnostic procedure of tuberculosis and mycobacteriosis.

[Use of mycolic acids analysis in diagnosis of tuberculosis and mycobacteriosis--three-year experience]. / Zastosowanie analizy kwasów mikolowych w diagnostyce gruzlicy i mikobakterioz--3 lata doswiadczen.

The aim of the study was to estimate the utility of the HPLC-based method of mycolic acids analysis to classify Mycobacterium species in routine diagnostic procedure on the basis of own three-year experience. 2142 patients' specimens were examined. 141 AFB were cultured. 36.2% strains were classified as M. tuberculosis complex by HPLC. The identification was confirmed by AMPLICOR MTB (Roche diagnostic, USA). M. xenopi (17.0%), M. kansasii (14.2%) and M. gordonae (14.2%) were the most frequent identified out of nontuberculous mycobacteria. Four mycobacteriosis cases were suspected because of repeated identification of the isolated strains. 136 strains on L-J slant shipped from other centres were identified. We confirm that the HPLC method is highly effective and specific for Mycobacterium species classification, which can be performed in no more than a couple of hours. In our opinion it is a very helpful tool, hard to replace in diagnostic procedure of tuberculosis and mycobacteriosis.