Métodos convencionais e moleculares para o diagnóstico da tuberculose pulmonar: um estudo comparativo / Conventional and molecular techniques in the diagnosis of pulmonary tuberculosis: a comparative study

OBJETIVO: Comparar quatro métodos laboratoriais no diagnóstico de tuberculose pulmonar. MÉTODOS: Foram realizadas pesquisa direta pelas colorações de Ziehl-Neelsen e auramina, cultura para micobactérias em meio Lõwenstein-Jensen (LJ) e polymerase chain reaction (PCR, reação em cadeia da polimerase) para Mycobacterium tuberculosis em 160 amostras de secreção respiratória de pacientes com suspeita de tuberculose pulmonar. As cepas isoladas foram identificadas por método radiométrico utilizando-se p-nitro-alfa-acetilamino-beta-hidroxipropiofenona (NAP) e métodos clássicos. A sensibilidade dos métodos foi comparada com o padrão ouro para o diagnóstico da tuberculose pulmonar, definido por critérios clínicos, radiológicos e microbiológicos. RESULTADOS: Dos 160 pacientes, 142 foram diagnosticados com tuberculose pulmonar de acordo com o padrão ouro. As técnicas de Ziehl-Neelsen e auramina, cultura em meio LJ e PCR apresentaram sensibilidade de 54,2 por cento, 58,4 por cento, 67,6 por cento e 77,5 por cento, respectivamente, quando comparados ao critério diagnóstico adotado. A especificidade dos quatro métodos foi de 100 por cento. A concordância na identificação da micobactéria entre PCR e o método radiométrico utilizando NAP foi alta (96,8 por cento). A sensibilidade da PCR foi de 50,8 por cento nas amostras com baciloscopia negativa e de 98,8 por cento naquelas com baciloscopia positiva. Nas amostras com resultados negativos na baciloscopia e cultura, a sensibilidade da PCR foi menor que nas com resultados positivos (25,6 por cento e 99,0 por cento, respectivamente). CONCLUSÕES: A PCR é método promissor no diagnóstico da tuberculose pulmonar, mesmo em amostras paucibacilares. Além disso, apresenta a vantagem da identificação simultânea e rapidez do resultado.

OBJECTIVE: To compare four laboratory methods in the diagnosis of pulmonary tuberculosis. METHODS: Respiratory secretion specimens were collected from 160 patients suspected of having pulmonary tuberculosis. Direct testing for Mycobacterium tuberculosis was carried out using Ziehl-Neelsen and auramine staining. In addition, culture in Lõwenstein-Jensen (LJ) medium and polymerase chain reaction (PCR) were used. The strains isolated were identified by means of a radiometric method using p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) and classical methods. The sensitivity of the methods was compared to the gold standard for the diagnosis of pulmonary tuberculosis, based on clinical, radiological and microbiological criteria. RESULTS: Of the 160 patients, 142 were diagnosed with pulmonary tuberculosis according to the gold standard. The sensitivity of Ziehl-Neelsen staining, auramine staining, culture in LJ medium and PCR was 54.2 percent, 58.4 percent, 67.6 percent and 77.5 percent, respectively, when compared with the diagnostic criterion adopted. All four methods presented 100 percent specificity. In the identification of mycobacteria, there was high (96.8 percent) concordance between PCR and the radiometric method using NAP. The sensitivity of PCR was 50.8 percent in samples with negative sputum smear microscopy results and 98.8 percent in those with positive results. The sensitivity of PCR was lower in specimens with negative results in sputum smear microscopy and culture than in those with positive results (25.6 percent and 99.0 percent, respectively). CONCLUSIONS: We found PCR to be a promising method for the diagnosis of pulmonary tuberculosis, even in paucibacillary specimens. Simultaneous identification and faster results are additional advantages of this method.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens.

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC 12B vials. At a growth index (GI) > or=30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC® 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC® 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC® NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC® 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC® 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.

Conventional and molecular techniques in the diagnosis of pulmonary tuberculosis: a comparative study.

OBJECTIVE: To compare four laboratory methods in the diagnosis of pulmonary tuberculosis. METHODS: Respiratory secretion specimens were collected from 160 patients suspected of having pulmonary tuberculosis. Direct testing for Mycobacterium tuberculosis was carried out using Ziehl-Neelsen and auramine staining. In addition, culture in Löwenstein-Jensen (LJ) medium and polymerase chain reaction (PCR) were used. The strains isolated were identified by means of a radiometric method using p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) and classical methods. The sensitivity of the methods was compared to the gold standard for the diagnosis of pulmonary tuberculosis, based on clinical, radiological and microbiological criteria. RESULTS: Of the 160 patients, 142 were diagnosed with pulmonary tuberculosis according to the gold standard. The sensitivity of Ziehl-Neelsen staining, auramine staining, culture in LJ medium and PCR was 54.2%, 58.4%, 67.6% and 77.5%, respectively, when compared with the diagnostic criterion adopted. All four methods presented 100% specificity. In the identification of mycobacteria, there was high (96.8%) concordance between PCR and the radiometric method using NAP. The sensitivity of PCR was 50.8% in samples with negative sputum smear microscopy results and 98.8% in those with positive results. The sensitivity of PCR was lower in specimens with negative results in sputum smear microscopy and culture than in those with positive results (25.6% and 99.0%, respectively). CONCLUSIONS: We found PCR to be a promising method for the diagnosis of pulmonary tuberculosis, even in paucibacillary specimens. Simultaneous identification and faster results are additional advantages of this method.

Lipase-catalyzed chemo- and enantioselective acetylation of 2-alkyl/aryl-3-hydroxypropiophenones.

The chemo- and enantioselective capabilities of porcine pancreatic lipase (PPL) in tetrahydrofuran, and Candida rugosa lipase (CRL) in diisopropyl ether have been investigated for the acetylation of racemic 2-alkyl/aryl-3-hydroxypropiophenones, which are important precursors in the synthesis of biologically active chromanones and isoflavanones. A highly chemoselective acetylation of primary hydroxy group in preference to phenolic hydroxy group leading to the formation of enantiomerically enriched monoacetates has been observed.

Methods for the rapid identification of mycobacterial species.

The use of rapid tests for the identification of mycobacteria has been advocated primarily for Mycobacterium tuberculosis; however, they have been accepted less widely than expected. Chromatographic methods are best suited for larger laboratories, whereas nucleic acid probes may be used by laboratories that can justify the cost based on volume and pricing. Nucleic acid sequencing offers the possibility of providing the most exact identification of species of mycobacteria, but its use is limited to reference laboratories that have the applicable resources. Because of their clinical importance, all mycobacteria should be identified using the most rapid methods available.

Alternative use of polymerase chain reaction instead of rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone test for the early detection of Mycobacterium tuberculosis in BACTEC 12B cultures.

Compared with conventional culture media, the TB BACTEC system has demonstrated improved isolation rates as well as an earlier detection time for mycobacterial species. However, the identification of M. tuberculosis by the rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test in the TB BACTEC 460 system may require 6 days for interpretable results. We evaluated the usefulness of a polymerase chain reaction (PCR) assay for earlier identification of M. tuberculosis in positive BACTEC 12B cultures. A total of 262 TB BACTEC culture specimens with GIs > or = 10 were assayed by PCR, and the results were compared with those of the NAP test. The aliquot from BACTEC 12B vials was boiled for 10 min, and 2 microliters of the boiled suspension was used for the PCR assay. One set of primers based on the IS 6110 sequence of M. tuberculosis was used to amplify a 457 bp fragment of DNA. Of the 173 TB BACTEC culture specimens which were identified as M. tuberculosis by the NAP test. 171 were PCR positive. Of the 21 TB BACTEC cultures identified as MOTT by the NAP test. 19 were PCR negative, but 2 were PCR positive: these two cultures were shown to grow both M. tuberculosis and MOTT in BACTEC 12B vials. Of the remaining 68 cultures which were contaminated with AFB-negative bacteria, the PCR identified M. tuberculosis in 13, in agreement with the NAP results in the reprocessed specimens. Overall, the PCR results in the 262 BACTEC culture specimens with GIs > or = 10 were sensitive in 99.5% (186/187) and specific in 100% (68/68). The mean time for the identification of M. tuberculosis in TB BACTEC cultures with GIs > or = 10 was 7 h by the PCR compared to 5.9 days by the NAP test. These results suggest that the PCR could be used as an alternative to the NAP test for the rapid identification of M. tuberculosis in BACTEC 12B cultures, particularly in those which contained both M. tuberculosis and MOTT or M. tuberculosis and AFB-negative bacteria.

[Disseminated Mycobacterium genavense infection in patients with HIV infection. Description of 5 cases and review of the literature]. / Infección diseminada por Mycobacterium genavense en pacientes con infección por HIV. Descripción de 5 casos y revisión de la literatura.

BACKGROUND: Five cases of disseminated infection by Mycobacterium genavense in patients with HIV infection are reported with a review of the literature. MATERIAL AND METHODS: A description of the clinical, epidemiologic and therapeutic characteristics of five cases are presented. The initial isolation of the microorganism was performed in Bactec 13A from blood and bone marrow aspirates. Subcultures were made in different media and the growth characteristics evaluated. Sensitivity to NAP was determined by radiometric techniques and gas chromatography allowed a possible identification. Definitive identification was based on PCR amplification of the gene which codifies the 65kDa protein and the posterior restriction of the amplified fragments by using BstEII and HaeIII. RESULTS: All five patients were males with HIV infection and a lymphocyte count of less than 25 cells/mm3 with an non-specific clinical picture similar to that produced by M. avium complex (MAC). Empiric antiMAC treatment was administered in four of the patients with good clinical response. All five strains were sensitive to NAP. The greatest growth rate was obtained in the subcultures with acid pH in liquid medium. Gas chromatography was very similar to that of M. simiae. Amplification of the gene which codifies the 65 kDa protein and posterior restriction with BstEII resulted in 2 fragments of 325 and 125 bp, while restriction with HaeIII resulted in two fragments of 140 and 105 bp. CONCLUSIONS: Mycobacterium genavense represents 9% of the disseminated infections by mycobacteria in AIDS patients. The clinical manifestations, empiric treatment and response is similar to that of infection by M. avium complex. Growth is favored by acid pH in liquid medium. Susceptibility to NAP leads to possible identification which should be confirmed by molecular techniques.

Rapid preliminary differentiation of species within the Mycobacterium tuberculosis complex: proposition of a radiometric method.

A radiometric method using the "Bactec 460-TB" apparatus now enables rapid obtaining of drug-susceptibility data and differentiation of clinical mycobacterial specimens into those belonging to the Mycobacterium tuberculosis complex (comprised of M. tuberculosis, M. bovis, M. bovis BCG and M. africanum) and non-tuberculous mycobacteria, using a specific inhibitor (p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone). In the present work, further differentiation of the M. tuberculosis complex into 4 individual species was achieved using the susceptibility of strains to 1.25 micrograms/ml of thiophene-2-carboxylic acid hydrazide, 40 micrograms/ml of D-cycloserine, and 100 micrograms/ml of pyrazinamide.

Selective inhibition of the Mycobacterium tuberculosis complex by p-nitro-alpha-acetylamino-beta-hydroxypropio phenone (NAP) and p-nitrobenzoic acid (PNB) used in 7H11 agar medium.

The potential of p-nitrobenzoic acid (PNB) and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) for discriminating the Mycobacterium tuberculosis (M.tb) complex from other mycobacteria was evaluated in cases of clinical isolates. For this purpose, 500 micrograms/ml of PNB and 5 or 10 micrograms/ml of NAP were incorporated in 7H11 agar medium, and appropriate dilutions corresponding to about 10(6) viable units were then plated on PNB- or NAP-containing media. Results were reported as growth or no growth as compared to a parallel control. Our data reconfirmed the potential of PNB to discriminate M.tb complex bacteria. This study also showed for the first time that the NAP test (used until now in 7H12a broth only) can also be successfully applied using 7H11 agar. Also, preliminary data with 5 micrograms/ml of NAP on a limited number of strains suggested that it may discriminate the M.tb complex bacilli from M. xenopi and M. gastri better than PNB in 7H11 agar.

Radiometric diagnosis of mycobacteria.

The aim of this study was to determine the impact of recently developed rapid radiometric techniques on the clinical diagnostic operations of a reference laboratory for mycobacteria. Selective inhibition by rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone was used to rapidly screen submitted cultures for identification of mycobacterial species other than Mycobacterium tuberculosis. The radiometric drug susceptibility test was applied only to those cultures presumptively identified as belonging to the Mycobacterium tuberculosis complex. All referred cultures were tested without additional subculture. The results showed that non-pigmented mycobacteria other than Mycobacterium tuberculosis can be screened with about 99% reliability, most of them within 24 hours. Unnecessary drug susceptibility testing of mycobacteria other than tubercle bacilli can be avoided at an early stage, thus shortening the average reporting time of the Mycobacterium tuberculosis complex to nine days following the receipt of the cultures. Ways of limiting erroneous reporting are discussed.

Evaluation of the p-nitro-alpha-acetylamino-beta-hydroxypropiophenone differential test for identification of Mycobacterium tuberculosis complex.

The p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) differential test for the identification of Mycobacterium tuberculosis recovered from clinical specimens was evaluated by two laboratories and found to be a rapid and accurate procedure with a specificity exceeding 99%.

Evaluation of a rapid radiometric differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone.

This study is an evaluation of a rapid technique for the differentiation of the Mycobacterium tuberculosis complex from other mycobacteria, using p-nitro-alpha-acetylamino-beta- hydroxypropiophenone (NAP) as a selective inhibitory agent. A total of 416 coded cultures, 234 cultures belonging to the M. tuberculosis complex and 182 cultures belonging to 35 other mycobacterial species, were tested in two laboratories for p-nitro-alpha-acetylamino-beta- hydroxypropiophenone inhibition to concentrations of 5 and 10 micrograms of NAP per ml in Middlebrook 7H12 liquid medium. Two testing modes were compared: the indirect, in which a large bacterial inoculum was used from an isolated culture on a solid medium, and the direct, which used a small inoculum from 7H12 medium. A decrease or no increase in daily 14CO2 output as measured by a BACTEC system was considered evidence of inhibition. The data presented show that a concentration of 5 micrograms of NAP per ml can effectively separate the M. tuberculosis complex from other mycobacterial species in 4 to 6 days. The direct test data show that, unlike other conventional biochemical tests, it does not require a heavy inoculum of mycobacteria and can therefore be performed soon after growth is detected by the radiometric method.

Structure-activity relationship of 5-hydroxykynurenamine analogues in isolated dog cerebral arteries.

In helically-cut strips of cerebral arteries isolated from dogs, analogues of 5-hydroxykynurenamine (5-HK), including 2-(3'-aminopropyl)-aniline (Cpd. I), 2'-amino-3-dimethylamino-3'-hydroxypropiophenone(CPD. II), 2'-amino-3-dimethylamino-5'-hydroxypropiophenone (Cpd. III) and 2',3-diamino-propiophenone (kynurenamine), caused a dose-related contraction which was antagonized by treatment with methysergide. The potency for inducing contractions was in the order of 5-hydroxytryptamine greater than 5-HK greater than Cpd. III greater than kynurenamine, Cpd. I and Cpd. II. Treatment with the 5-HK analogues antagonized the contractile response to 5-hydroxytryptamine in a dose-dependent manner, the antagonistic potency being in the order of 5-HK greater than Cpd. III greater than kynurenamine, Cpd. II greater than Cpd. I. Alterations in the hydroxy group on the benzene ring and/or radicals of long side chain of 5-HK attenuated the agonistic and antagonistic actions of 5-HK; however, the attenuation of these actions differed. Thus, the radicals appear to be involved in the agonistic and antagonistic actions to a different extent.