Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR.

Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients.

Rapid diagnosis of pulmonary tuberculosis.

INTRODUCTION: World Health Organization had estimated 9.4 million tuberculosis cases on 2009, with 1.7 million of deaths as consequence of treatment and diagnosis failures. Improving diagnostic methods for the rapid and timely detection of tuberculosis patients is critical to control the disease. The aim of this study was evaluating the accuracy of the cord factor detection on the solid medium Middlebrook 7H11 thin layer agar compared to the Lowenstein Jensen medium for the rapid tuberculosis diagnosis. METHODS: Patients with suspected tuberculosis were enrolled and their sputum samples were processed for direct smear and culture on Lowenstein Jensen and BACTEC MGIT 960, from which positive tubes were subcultured on Middlebrook 7H11 thin layer agar. Statistical analysis was performed comparing culture results from Lowenstein Jensen and the thin layer agar, and their corresponding average times for detecting Mycobacterium tuberculosis. The performance of cord factor detection was evaluated determining its sensitivity, specificity, positive and negative predictive value. RESULTS: 111 out of 260 patients were positive for M. tuberculosis by Lowenstein Jensen medium with an average time ± standard deviation for its detection of 22.3 ± 8.5 days. 115 patients were positive by the MGIT system identifying the cord factor by the Middlebrook 7H11 thin layer agar which average time ± standard deviation was 5.5 ± 2.6 days. CONCLUSION: The cord factor detection by Middlebrook 7H11 thin layer agar allows early and accurate tuberculosis diagnosis during an average time of 5 days, making this rapid diagnosis particularly important in patients with negative sputum smear.

Structural definition of trehalose 6-monomycolates and trehalose 6,6'-dimycolates from the pathogen Rhodococcus equi by multiple-stage linear ion-trap mass spectrometry with electrospray ionization.

The cell wall of the pathogenic bacterium Rhodococcus equi (R. equi) contains abundant trehalose monomycolate (TMM) and trehalose dimycolate (TDM), the glycolipids bearing mycolic acids. Here, we describe multiple-stage (MS(n)) linear ion-trap (LIT) mass spectrometric approaches toward structural characterization of TMM and TDM desorbed as [M + Alk](+) (Alk = Na, Li) and as [M + X](-) (X = CH(3)CO(2), HCO(2)) ions by electrospray ionization (ESI). Upon MS(n) (n=2, 3, 4) on the [M + Alk](+) or the [M + X](-) adduct ions of TMM and TDM, abundant structurally informative fragment ions are readily available, permitting fast assignment of the length of the meromycolate chain and of the α-branch on the mycolyl residues. In this way, structures of TMM and TDM isolated from pathogenic R. equi strain 103 can be determined. Our results indicate that the major TMM and TDM molecules possess 6, and/or 6'-mycolyl groups that consist of mainly C14 and C16 α-branches with meromycolate branches ranging from C18 to C28, similar to the structures of the unbound mycolic acids found in the cell envelope. Up to 60 isobaric isomers varying in chain length of the α-branch and of the meromycolate backbone were observed for some of the TDM species in the mixture. This mass spectrometric approach provides a direct method that affords identification of various TMM and TDM isomers in a mixture of which the complexity of this lipid class has not been previously reported using other analytical methods.

Role of lipid components in formation and reactivation of Mycobacterium smegmatis "nonculturable" cells.

We have found that transition of actively dividing Mycobacterium smegmatis cells into the dormant "nonculturable" state is accompanied by increase in the protein/lipid ratio and disappearance of one of the main lipid components of the mycobacterial cells, trehalose monomycolate. In this case, oleic acid is accumulated in the culture medium due to its secretion by the mycobacterial cells. Addition of lipids of different classes to "nonculturable" M. smegmatis cells induces their resuscitation. The lipid reactivating effect is evidently caused by the presence of fatty acids in their composition, because free fatty acids also exhibited reactivation effect. Oleic acid in concentration of 0.05-3 µg/ml exhibited maximal effect, and that allows us to draw a conclusion concerning its signal role in the transition of dormant cells into active state.

Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.

Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6'-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media / Detecção do fator corda e avaliação do aspecto macroscópico das colônias de micobactérias: um eficiente teste de triagem combinado para a identificação presuntiva do complexo Mycobacterium tuberculosis em meios só

OBJECTIVE: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. METHODS: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. RESULTS: The overall accuracy of cord factor detection alone was 95.4 percent (95 percent CI: 90.7-98.1 percent), and that of the combined screening test was 99.3 percent (95 percent CI: 96.4-100 percent). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. CONCLUSIONS: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

OBJETIVO: A diferenciação rápida entre Mycobacterium tuberculosis e micobactérias não-tuberculosas é fundamental para os pacientes coinfectados com tuberculose e HIV. Para tanto, utilizamos duas metodologias em nosso laboratório: detecção do fator corda e PCR-restriction enzyme analysis (PRA). O objetivo do estudo foi avaliar a acurácia desse teste de triagem em meio sólido como um método rápido para a identificação presuntiva do complexo M. tuberculosis, considerando custos e tempo de resultado. MÉTODOS: Foram processadas 152 cepas pelo teste de triagem combinado, que consistiu da detecção do fator corda por microscopia (esfregaço corado por Ziehl-Neelsen) e avaliação do aspecto macroscópico das colônias, e PRA (padrão ouro). Os custos foram estimados através da obtenção dos preços dos insumos necessários para a realização de cada teste. RESULTADOS: A acurácia da detecção do fator corda foi de 95,4 por cento (IC95 por cento: 90,7-98,1 por cento) e a do teste de triagem combinado foi de 99,3 por cento (IC95 por cento: 96,4-100 por cento). O custo da detecção do fator corda foi de R$ 0,60 e do PRA de R$ 16,00. Os resultados da detecção do fator corda estão prontos em 2 dias, ao passo que os de PRA necessitam de 4 dias. CONCLUSÕES: A identificação presuntiva de M. tuberculosis usando o aspecto macroscópico das colônias em conjunto com a detecção de fator corda por microscopia é um teste simples, rápido e de baixo custo. Recomendamos o teste de triagem combinado para rapidamente identificar M. tuberculosis em sítios com poucos recursos financeiros e em laboratórios menos equipados, enquanto se aguarda a identificação definitiva por métodos moleculares ou bioquímicos.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media.

OBJECTIVE: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. METHODS: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. RESULTS: The overall accuracy of cord factor detection alone was 95.4% (95% CI: 90.7-98.1%), and that of the combined screening test was 99.3% (95% CI: 96.4-100%). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. CONCLUSIONS: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

Avaliação do crescimento em cordas na identificação presuntiva do complexo Mycobacterium tuberculosis / Detection of cord factor for the presumptive identification of Mycobacterium tuberculosis complex

OBJETIVO: O Mycobacterium tuberculosis, sob certas condições apropriadas, cresce em cordões de serpentinas, denominados de fator corda, ou crescimento em cordas. O objetivo deste estudo é avaliar a detecção do fator corda como método de identificação presuntiva do complexo M. tuberculosis, comparando-o aos testes de tipificação (TIP) convencionais. MÉTODO: Foram analisadas 743 cepas, de janeiro de 2002 a dezembro de 2005, na Área de Micobactérias do Instituto Adolfo Lutz - Santos, obtidas de isolados clínicos coletados de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades Básicas de Saúde da Baixada Santista. Foram feitos esfregaços das cepas de micobactérias isoladas em meio líquido MB/BacT e meio sólido, Lowenstein-Jensen ou Ogawa-Kudoh, sendo 301 (40,5 por cento) cepas em meio líquido e 442 (59,5 por cento) em meio sólido. RESULTADOS: Os resultados de sensibilidade, especificidade e valores preditivos positivos e negativos, obtidos com a comparação do desempenho do método em ambos os meios de isolamento e TIP convencionais, foram respectivamente 98,5, 88, 97 e 93 por cento. Observou-se maior sensibilidade do método em meio sólido (100 por cento), com uma diferença de sensibilidade entre os meios analisados de apenas 2,7 por cento. CONCLUSÕES: Conclui-se, pelos resultados obtidos, que o fator corda é um critério real e rápido na identificação do complexo M. tuberculosis; além disso, em laboratórios com alta prevalência do complexo M. tuberculosis e que não dispõem de técnicas que permitam a precocidade de sua identificação, o fator corda possibilita o direcionamento aos testes conclusivos de identificação e adicionais de sensibilidade que se façam necessários.

OBJECTIVE: Virulent strains of the Mycobacterium tuberculosis complex, under certain appropriate conditions, grow as characteristic ropes, bundles or serpentine cords known as cord factor or growth in cords. The objective of the present study was to evaluate cord factor detection as a method of achieving presumptive identification of the M. tuberculosis complex, comparing it to conventional typing tests. METHODS: A total of 743 strains were analyzed from January of 2002 to December of 2005 in the Mycobacteria Sector of the Adolfo Lutz Institute, located in the city of Santos, Brazil. Samples were obtained from clinical specimens collected from patients with respiratory symptoms treated at basic health clinics in the greater metropolitan area of Santos. Ziehl-Neelsen-stained smears were prepared, 301 (40.5 percent) in MB/BacT broth and 442 (59.5 percent) on solid media, either Lowenstein-Jensen or Ogawa-Kudoh. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value obtained during the performance comparison of the two methods (cord factor detection and conventional typing) using both isolation media were, respectively, 98.5, 88, 97 and 93 percent. The method was more sensitive on solid medium (100 percent), and the difference in sensitivity between the two media types was only 2.7 percent. CONCLUSIONS: Taking into consideration the results obtained, we conclude that, in laboratories with a high incidence of M. tuberculosis complex isolation and limited economic resources, cord factor detection is a fast and valid criterion for identifying these mycobacteria using liquid or solid medium. It also enables subsequent conclusive identification tests, as well as additional sensitivity tests when necessary.

[Detection of cord factor for the presumptive identification of Mycobacterium tuberculosis complex]. / Avaliação do crescimento em cordas na identificação presuntiva do complexo Mycobacterium tuberculosis.

OBJECTIVE: Virulent strains of the Mycobacterium tuberculosis complex, under certain appropriate conditions, grow as characteristic ropes, bundles or serpentine cords known as cord factor or growth in cords. The objective of the present study was to evaluate cord factor detection as a method of achieving presumptive identification of the M. tuberculosis complex, comparing it to conventional typing tests. METHODS: A total of 743 strains were analyzed from January of 2002 to December of 2005 in the Mycobacteria Sector of the Adolfo Lutz Institute, located in the city of Santos, Brazil. Samples were obtained from clinical specimens collected from patients with respiratory symptoms treated at basic health clinics in the greater metropolitan area of Santos. Ziehl-Neelsen-stained smears were prepared, 301 (40.5%) in MB/BacT broth and 442 (59.5%) on solid media, either Lowenstein-Jensen or Ogawa-Kudoh. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value obtained during the performance comparison of the two methods (cord factor detection and conventional typing) using both isolation media were, respectively, 98.5, 88, 97 and 93%. The method was more sensitive on solid medium (100%), and the difference in sensitivity between the two media types was only 2.7%. CONCLUSIONS: Taking into consideration the results obtained, we conclude that, in laboratories with a high incidence of M. tuberculosis complex isolation and limited economic resources, cord factor detection is a fast and valid criterion for identifying these mycobacteria using liquid or solid medium. It also enables subsequent conclusive identification tests, as well as additional sensitivity tests when necessary.

Identification and structural characterisation of novel trehalose dinocardiomycolates from n-alkane-grown Rhodococcus opacus 1CP.

Rhodococcus opacus 1CP, a potent degrader of (chloro-) aromatic compounds was found to utilise C10-C16 n-alkanes as sole carbon sources. Highest conversion rates were observed with n-tetradecane and n-hexadecane, whereas the utilisation of n-dodecane and n-decane was considerably slower. Thin-layer chromatography of organic extracts of n-alkane-grown 1CP cultures indicated the growth-associated formation of a glycolipid which was characterised as a trehalose dimycolate by 1H-NMR spectroscopy and mass spectrometry. Total chain lengths between 48 and 54 carbons classify the fatty acid residues as nocardiomycolic acids. The presence of two double bonds in each mycolic acid is another feature that distinguishes the corresponding trehalose dinocardiomycolates from trehalose dicorynomycolates reported for Rhodococcus erythropolis DSM43215 and Rhodococcus ruber IEGM231. R. opacus 1CP was not found, even under nitrogen limitation, to produce anionic trehalose tetraesters which have previously been reported for R. erythropolis DSM43215.

Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry.

Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 microg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (alpha, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) alpha-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic alpha-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium avium-intracellulare group, M. phlei and M. flavescens. The M. avium-intracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester-mycoloyl TMM. This technique has marked advantages in the rapid analysis of not only intact glycolipid TMM, but also the mycolic acid composition of each mycobacterial species, since it does not require any degradation process.

[Biochemistry and bioactivities of mycobacterial components].

The most characteristic pathological change in mycobacterial infection is caseous necrosis followed by tuberculous cavity formation due to the cellular immunity induced by antigenic proteins and adjuvant active cell wall components. Mycobacterial cell well contains unique hydrophobic compounds possessing mycolic acids (a long branched-chain high molecular weight fatty acid) and shows distinctive properties such as acid-fastness and wax-like hydrophobicity. Mycobacteria do not produce exotoxin and the virulence cannot be determined by a single toxic substance, but the cell wall components to contact with the host cells are the most important surface molecule at the early stage of infection. Cord factor (trehalose 6,6'-dimycolate) is the most classical virulence factor which is lethally toxic for mice. However, cord factor exists among various species of mycobacteria and even in Nocardia and Rhodococcus. Furthermore, cord factor can produce granulomas without protein antigen in mice and it shows antitumor or non-specific prevention promotion of infection and induction promotion of various cytokines. Sulfolipid (tetracyl trehalose sulfate) also plays a role as a virulence factor by phagocytic process inhibition. Glycopeptidolipid (GPL) from M. avium and phenolglycolipid (PGL) from M. leprae also appeared to be immunomodulatory molecules which inhibit the developing of cellular immunity. Lipoarabinomannan (LAM) is also unique amphipatic molecule, like gram-negative endotoxin. Here, we discuss the involvement of various surface molecutes to contribute to pathogenesis in mycobacterial infection and immunity.

[Distribution of antigenic glycolipids among Mycobacterium tuberculosis strains and their contribution to virulence].

Tuberculosis is a chronic disease caused by Mycobacterium tuberculosis infection. The major pathological changes are immunologically hypersensitive granuloma formation due to the local proliferation or infiltration of immune cells. However, the mechanism for the development of the disease has not yet been fully understood. The first step of infection in intracellular survival in the phagocytic cells and this process has been reported to be regulated by cell surface glycolipid virulence factors. As genetical heterogeneity of M. tuberculosis among strains has been reported recently based on DNA fragmentation pattern, I have examined the distribution of cell surface glycolipids (cord factor, sulfolipids and penta acyl trehaloses) among the virulent (M. tuberculosis H37Rv and M. tuberculosis Aoyama B) and virulent (M. tuberculosis H37Ra) strains by two dimensional thin-layer chromatography of silica gel. Seven characteristic glycolipid components of the virulent strains were detected and separated by thin-layer chromatography of silica gel. Each glycolipid was identified by fast-atom-bombardment mass-spectrometry (FAB/MS) analysis of the intact lipid and gas-chromatography mass-spectrometry (GC/MS) analysis of the fatty acid or the carbohydrate moiety. As the result, molecular weight (m/z, 1,200-3,000) of each glycolipid was determined clearly by FAB/MS analysis. The structure of fatty acids (C16-C40) or mycolic acids (C76-C88) were determined by GC/MS analysis. Cord factor (TDM, trehalose 6,6'-dimycolate) and trehalose 6-monomycolate (TMM) showed strong granuloma forming activity, but other glycolipids practically did not. On the other hand, cord factor and trehalose 6-monomycolate showed phagocytosis inhibition (but showed promotion in the presence of complement) and marked inhibition of phagosome-lysosome fusion, while sulfolipids showed strong phagocytosis promotion and marked inhibition of phagosome-lysosome fusion. Penta acyl trehaloses showed phagocytosis promotion but no effect on phagosome-lysosome fusion. Cord factor and trehalose 6-monomycolate existed ubiquitously among virulent and avirulent strains, while sulfolipids and penta acyl trehaloses were detected in only virulent strains (M. tuberculosis H37Rv and M. tuberculosis Aoyama B). These results indicate that the existence of these toxic glycolipids contributes to the virulence of M. tuberculosis, profoundly. It is suggested that these glycolipids play an important role as virulence factors in the early stage of infection and expression of pathogenicity.

Studies of polymorphic DNA fingerprinting and lipid pattern of Mycobacterium tuberculosis patient isolates in Japan.

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L-rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.

Studies on lipids in mycobacterial cell wall: their important structure and function relating to pathogenicity and their biological activity.

The lipids cord-factor, mycosides and sulpholipids are supposed to be vitally linked with the pathogenecity of mycobacteria. In this paper an attempt has been made to clarify the understanding of the occurrence, organisation and possible interaction of the diverse lipids present in the mycobacterial cell wall and their possible structure and function.

Mechanisms of pathogenicity in mycobacteria.

The purpose of this article is to review current knowledge about the mechanisms of pathogenicity of mycobacteria. The following aspects of the problem are discussed: chemically-defined compounds implicated in the mechanisms of pathogenicity; location in the cell wall of these compounds and their biological activities; mechanisms of intracellular survival of pathogenic mycobacteria as compared to intracellular killing of non-pathogenic mycobacteria; and pathogenesis of mycobacterial infection. The future prospects in the elucidation of the mechanisms of pathogenicity and their possible application for a better control of mycobacterial diseases are briefly discussed.