Mycobacterium tuberculosis Cell Wall Glycolipids Directly Inhibit CD4+ T-Cell Activation by Interfering with Proximal T-Cell-Receptor Signaling

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4⁺ T-cell responses. We have previously shown that M. tuberculosis can indirectly inhibit CD4⁺ T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine if M. tuberculosis could directly inhibit CD4⁺ T-cell activation. Murine CD4⁺ T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4⁺ T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence of M. tuberculosis and its subcellular fractions. CD4⁺ T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence of M. tuberculosis. Fractionation identified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose-capped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosis glycolipids directly inhibited CD4⁺ T-cell activation by interfering with proximal T-cell-receptor signaling.Aerosolized Mycobacterium tuberculosis infects alveolar and lung parenchymal macrophages, where it replicates unrestrained in the face of innate responses until T-cell immunity controls its growth. Despite robust activation of innate and adaptive immunity, M. tuberculosis survives and persists as a latent infection (15, 18). CD4⁺ T cells have a central role in controlling M. tuberculosis during acute and latent infections (53). Animal studies have shown that depletion or absence of CD4⁺ T cells during primary infection results in unchecked M. tuberculosis growth in the lung and decreased survival (37, 38, 41). Depletion of CD4⁺ T cells during latent infection also worsens disease and survival (46). In humans, loss of CD4⁺ T cells from progressive human immunodeficiency virus infection is directly responsible for the high rates of tuberculosis in human immunodeficiency virus-infected persons (48).Much is known about how M. tuberculosis manipulates macrophages for its survival (19, 29, 44). However, the way in which M. tuberculosis interferes with adaptive T-cell immunity is not well understood. Our recent studies have demonstrated that M. tuberculosis can modulate CD4⁺ T-cell function both indirectly and directly. M. tuberculosis, through Toll-like receptor 2 (TLR-2), inhibits gamma-interferon-regulated genes that result in decreased major histocompatibility complex class II (MHC-II) antigen processing by macrophages for effector and memory CD4⁺ T cells (22, 23, 40, 43). M. tuberculosis can also induce increased adhesion to fibronectin through α₅β₁ integrin on CD4⁺ T cells (45).M. tuberculosis molecules responsible for modulating CD4⁺ T-cell function reside in the mycobacterial cell wall and include the lipoproteins LpqH, LprG, and LprA as well as the glycolipid phosphatidylinositol mannoside (PIM). The lipoproteins bind to TLR-2 on macrophages, and PIM binds to VLA-5 (α₅β₁) on CD4⁺ T cells. The M. tuberculosis cell wall also contains lipoarabinomannan (LAM); complex lipids, such as phthiocerol dimycocerosate, and cord factor/dimycolytrehalose; and sulfolipids that are both targets of the immune response (e.g., glycolipids presented by CD1 to T cells) and agonists of host cell receptors (9, 10, 32). Although M. tuberculosis bacilli largely reside within macrophages, mycobacterial cell wall components, including glycolipids, can traffic outside infected macrophages through the production of exosomes. These exosomes can then deliver M. tuberculosis molecules to T cells and other host cells that are not directly interacting with M. tuberculosis-infected cells and thereby affect host immune responses (3, 4).The purpose of this study was to determine if M. tuberculosis can directly (i.e., independently from its effect on MHC-II antigen processing) interfere with CD4⁺ T-cell activation and, if so, what the M. tuberculosis molecules(s) and mechanism(s) are. Highly purified murine CD4⁺ T cells devoid of antigen-presenting cells (APCs) were activated by CD3 and CD28 monoclonal antibodies (MAbs) in the presence or absence of M. tuberculosis and biochemical fractions. We have found that M. tuberculosis bacilli directly inhibited CD4⁺ T-cell activation. Biochemical fractionation identified cell wall glycolipids as potent inhibitors of signaling through the T-cell receptor (TCR) complex by interfering with ZAP-70 phosphorylation.     

Development of a secondary immune response to Mycobacterium tuberculosis is independent of Toll-like receptor 2

Published work indicates that the contribution of Toll-like receptor 2 (TLR2) to host resistance during acute Mycobacterium tuberculosis infection is marginal. However, in these studies, TLR2 participation in the memory immune response to M. tuberculosis was not determined. The substantial in vitro evidence that M. tuberculosis strongly triggers TLR2 on dendritic cells and macrophages to bring about either activation or inhibition of antigen-presenting cell (APC) functions, along with accumulating evidence that memory T cell development can be calibrated by TLR signals, led us to question the role of TLR2 in host resistance to secondary challenge with M. tuberculosis. To address this question, a memory immunity model was employed, and the response of TLR2-deficient (TLR2 knockout [TLR2KO]) mice following a secondary exposure to M. tuberculosis was compared to that of wild-type (WT) mice based on assessment of the bacterial burden, recall response, phenotype of recruited T cells, and granulomatous response. We found that upon rechallenge with M. tuberculosis, both WT and TLR2KO immune mice displayed similarly enhanced resistance to infection in comparison to their naïve counterparts. The frequencies of M. tuberculosis-specific gamma interferon (IFN-γ)-producing T cells, the phenotypes of recruited T cells, and the granulomatous responses were also similar between WT and TLR2KO immune mice. Together, the findings from this study indicate that TLR2 signaling does not influence memory immunity to M. tuberculosis.Mycobacterium tuberculosis expresses a large repertoire of lipoproteins that can trigger signaling from Toll-like receptor 2 (TLR2) (13), including the 19-kDa lipoprotein (LpqH) (5), LprA (Rv1270) (29), and LprG (Rv1411c) (11). In addition to lipoproteins, lipomannan (31) and phosphatidyl-myo-inositol mannoside (PIM) (12, 16) also interact with TLR2 to initiate cellular activation (16). Despite this collection of TLR2 agonists on the tuberculosis (TB) bacillus, murine studies indicate that TLR2 is not essential for host resistance against acute M. tuberculosis infection (34, 37).It is well appreciated that memory immunity in tuberculosis does not provide long-term protective immunity, as evidenced in humans and experimental infections of mice. In a study performed in Cape Town, South Africa, it was determined that the incidence rate of TB attributable to reinfection after successful chemotherapy was four times that of new TB (40). In mouse models, immunological memory induced by M. tuberculosis infection can provide short-term protection, as evidenced by early reduction in the bacterial burden in the lungs following reexposure (6, 36). The memory immune mice exhibit a transient early induction of Th1 cells compared to naïve mice and concomitant early control of bacterial replication. However, despite the skewed kinetics, the memory mice do not achieve bacterial sterility in the lung, and bacteria continue to be maintained in a stable state. Clearly, this major gap in our understanding of how to induce sterilizing memory immunity in TB is an impediment to vaccine development.In vitro studies have documented opposing outcomes from antigen-presenting cells (APC) following interaction of their surface TLR2 with M. tuberculosis. For example, TLR2 signals upregulate B7 expression, induce interleukin 12 (IL-12) secretion (15), and initiate antimicrobial responses within M. tuberculosis-infected macrophages (22). TLR2 also initiates signaling that inhibits major histocompatibility complex (MHC) class II-dependent antigen presentation (24, 26) by macrophages and responsiveness to gamma interferon (IFN-γ) (2, 8, 18, 27). How these opposing changes to APC by TLR2 signals affect naïve T cell differentiation into effector and memory T cells following M. tuberculosis infection remains unclear. Furthermore, recent studies indicate that high expression levels of IL-12 in the environment promote effector T cell development while low expression levels (17) or even the absence (28, 41) of the cytokine is favorable for central memory T cell development. Together with the report that TLR2 regulates IL-10 production from macrophages and dendritic cells (DC) following M. tuberculosis infection (15, 30), these findings suggest that TLR2 signals may modulate the inflammatory milieu during T cell priming to influence effector versus memory T cell development.The literature on inhibition of APC function by TLR2 predicts that removal of TLR2 may improve APC functions and lead to better memory immunity. On the other hand, the finding that TLR2 signaling is anti-inflammatory and consequently conducive to memory T cell development predicts that absence of TLR2 may result in poorer memory immunity. Therefore, in this study, we examined whether the absence of TLR2 improved or worsened the capacity of the host to generate memory immunity upon rechallenge with M. tuberculosis.     

Immunostimulatory activity of major membrane protein II from Mycobacterium tuberculosis

Previously, we observed that both major membrane protein II of Mycobacterium leprae (MMP-ML) and its fusion with M. bovis BCG (BCG)-derived heat shock protein 70 (HSP70) (Fusion-ML) are immunogenic and that recombinant BCG secreting either of these proteins effectively inhibits the multiplication of M. leprae in mice. Here, we purified M. tuberculosis-derived major membrane protein II (MMP-MTB) and its fusion with HSP70 (Fusion-MTB) in a lipopolysaccharide-free condition and evaluated their immunostimulatory abilities. Both MMP-MTB and Fusion-MTB activated monocyte-derived dendritic cells (DC) in terms of phenotype and interleukin-12 (IL-12) production, but Fusion-MTB more efficiently activated them than MMP-MTB did. The IL-12 production was a consequence of the ligation of those recombinant proteins with Toll-like receptor 2. The M. tuberculosis-derived and M. leprae-derived recombinant proteins activated naïve T cells of both CD4 and CD8 subsets, but M. tuberculosis-derived proteins were superior to M. leprae-derived proteins and fusion proteins were superior to MMP, regardless of the origin of the protein. Memory-type CD4⁺ T cells obtained from BCG-vaccinated healthy individuals seem to be primed with MMP-MTB by the vaccination, and both M. tuberculosis-derived recombinant proteins produced perforin-producing CD8⁺ T cells from memory-type CD8⁺ T cells. Further, infection of DC and macrophages with M. tuberculosis H37Ra and H37Rv induced the expression of MMP on their surface. These results indicate that M. tuberculosis-derived MMP, as a sole protein or as part of a fusion protein, may be useful for developing new vaccinating agents against tuberculosis.Tuberculosis is a chronic infectious disease caused by intracellular infection with Mycobacterium tuberculosis (20). It is estimated that one-third of the global population is latently infected with this inhaled pathogen, which infects primarily macrophages and dendritic cells (DC), and tuberculosis is responsible for more than two million deaths yearly worldwide (11, 34, 36). The emergence of multidrug-resistant strains of M. tuberculosis mandates the development of more effective preventive and therapeutic strategies, including the development of improved vaccines (48). Protective immunity against M. tuberculosis is conducted chiefly by adaptive cellular immune responses, and gamma interferon (IFN-γ)-producing type 1 CD4⁺ T cells and CD8⁺ T cells are key components of this immunity (1, 12, 16). IFN-γ produced by activated T cells is believed to be an essential element of the host defense against M. tuberculosis (13). Further, the contribution of CD8⁺ T cells to protection by lysing infected cells is also important for bacterial killing (7, 19). CD8⁺ T cells can kill M. tuberculosis-infected host cells via a granule-dependent mechanism involving perforin and granulysin, which has a direct antimicrobial activity (42, 49).The only approved vaccine currently available against tuberculosis is M. bovis bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis. More than four billion doses of BCG have been administered so far, and is established as a safe vaccine (29). BCG appears to be effective at preventing diseases such as tuberculous meningitis and miliary tuberculosis in newborns and toddlers; however, it has no apparent effect on pulmonary tuberculosis in adults (3, 9). The reason why BCG cannot prevent disease development is not fully known, but one of the reasons is based on the fact that BCG has a capacity to block phagosome maturation to inhibit antigen (Ag) processing and presentation to type 1 T cells (14, 32, 38). Indeed, although M. tuberculosis directly delivers Ag to the major histocompatibility complex (MHC) class I processing pathway, BCG was less able to activate CD8⁺ T cells (35, 41). Further, BCG growing in human macrophages was not recognized by immune CD4⁺ T cells, although BCG-infected macrophages continued to express MHC class II molecules (35). These observations indicate the need for the development of a new vaccine against tuberculosis.Various new vaccine candidates which are based on Ags that are recognized in infected individuals are currently in clinical trials, including early secretory antigenic target 6 (ESAT-6), the Ag85 family, and a polyprotein Ag, designated Mtb72F, derived from M. tuberculosis proteins Mtb32 and Mtb59 (1, 2, 17, 18, 37, 39). However, a fully reliable new vaccine has not been established yet.A situation similar to that of tuberculosis can be found in leprosy, which is caused by infection with M. leprae, and the development of a new vaccine capable of inhibiting the multiplication of M. leprae is highly desirable. In both tuberculosis and leprosy, the activation of T cells is induced by DC loaded with bacilli or their components, which display various antigenic molecules on their surface, including the immunodominant Ags (15, 30), although there are conflicting results indicating that M. leprae inhibits the activation and maturation of DC (33). We are of the opinion that future vaccines, to be successful, must (i) be highly antigenic, (ii) have the capacity to activate both naïve CD4⁺ T cells and CD8⁺ T cells, and (iii) have the ability to be expressed on the surface of mycobacterium-infected Ag-presenting cells (APCs) such as macrophages and DC. Previously, we identified major membrane protein (MMP; gene name, bfrA or ML2038) as one of the immunodominant Ags of M. leprae (21). M. leprae-derived MMP (MMP-ML) ligates Toll-like receptor 2 (TLR2) and consequently activates the NF-κB pathway of host cells (21). DC pulsed with MMP-ML activate memory-type CD4⁺ and CD8⁺ T cells to produce IFN-γ in an Ag-specific fashion (21, 26). Further, MMP-ML is supposed to be recognized in vivo by T cells of M. leprae-infected individuals, including paucibacillary leprosy patients (26).Further, when we introduced MMP-ML with the Ag85A secretion signal of M. tuberculosis into BCG, the modified BCG, termed BCG-SM, secreted MMP-ML, enhanced the ability of BCG to activate naïve CD4⁺, and further, successfully activated naïve CD8⁺ T cells (25). Furthermore, BCG-SM at least partially inhibited the growth of M. leprae in C57BL/6 mice subsequently challenged by injection in the footpads (22). These observations indicate that MMP-ML could be a target molecule to be further analyzed as a vaccine candidate, and the fact that BCG-SM can activate both subsets of naïve T cells to produce IFN-γ indicates that secretion of MMP-ML, presumably in the phagosome of APCs, is a useful strategy to activate T cells (25). We sought another strategy to further enhance the T cell-stimulating activity of BCG, especially of the ability to activate IFN-γ-producing CD8⁺ T cells quickly and strongly. To this end, we used heat shock protein 70 (HSP70) as a fusion partner (6, 10, 44, 45). The gene encoding HSP70 of BCG was directly linked with that of MMP and extrachromosomally transformed into BCG (BCG-70 M) (31). BCG-70 M secreted the HSP70-MMP fusion protein (Fusion-ML) and activated not only Ag-specific naïve CD8⁺ T cells polyclonally but also naïve CD4⁺ T cells strongly (31). Further, the secreted Fusion-ML protein activated DC in terms of phenotype and the production of cytokines such as interleukin-12 (IL-12) (31). Thus, the production and secretion of HSP70 in phagosomes along with MMP-ML, using BCG as a vector, seem to be effective in activating human naïve CD8⁺ T cells. These observations led us to speculate that the use of MMP, which is commonly present in pathogenic mycobacteria, or of the HSP70-MMP fusion protein may be useful in inhibiting the multiplication of M. tuberculosis. However, the MMP homology between M. leprae and M. tuberculosis (MMP-MTB; gene name, bfrA or Rv1876) is 90.6% at the amino acid level. Therefore, in this study, we purified M. leprae- or M. tuberculosis-derived MMP and a fusion protein composed of HSP70 and M. leprae- or M. tuberculosis-derived MMP by using M. smegmatis and evaluated their immunostimulatory activities.     

Mycobacterium tuberculosis Cpn60.2 and DnaK Are Located on the Bacterial Surface,Where Cpn60.2 Facilitates Efficient Bacterial Association with Macrophages

Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ∼57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab′)₂ fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ∼34% was seen for anti-Cpn60.2 F(ab′)₂, while anti-DnaK F(ab′)₂ did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.The initiation of a tuberculous infection involves the adherence and phagocytosis of Mycobacterium tuberculosis bacilli by host cells. It is generally thought that the primary host niche of M. tuberculosis is the alveolar macrophage (Mφ). To access this cell, ligands on the outer surface of the M. tuberculosis bacillus must come in contact with surface receptors of the Mφ. Although a significant amount of information concerning the Mφ receptors involved in this interaction is available (15, 71), the identities of the mycobacterial cell surface components that mediate this binding are less well understood. However, evidence for the involvement of mycobacterial lipoarabinomannans (57), capsular polysaccharides (8), glycopeptidolipids (72), 19-kDa antigen (9), mycotin (21), and Apa glycoprotein (47) has been reported previously.For any of the aforementioned moieties to be involved in the binding of mycobacteria to host cells, they would have to be located on the surface of the bacterium. Early reports suggested that the outer surface of mycobacteria was composed of mycosides (10, 11). Later studies indicated the presence of an outer polysaccharide-rich layer (45, 50), which could explain the presence of the so-called electron-transparent zone often seen in electron micrographs of mycobacteria inside Mφ (13, 18) and more recently in axenically grown bacteria (17, 42, 43, 51). Support for this contention has come from studies describing the presence of an outer surface capsule on M. tuberculosis (12). Carbohydrates make up 85% of the capsule, and the predominant sugar is glucan (approximately 70% of all sugars present). Arabinomannan and mannan are also present in significant quantities, as are a number of proteins, some of which are glycosylated. While about 10% of the capsule is composed of proteins, there is very little lipid present (31, 37). No evidence for the presence of lipoarabinomannan in the capsule was found, though phosphatidylinositol mannoside (PIM) was identified (38). The presence of a glycan-rich capsule surrounding intracellular mycobacteria has been confirmed using specific monoclonal antibodies (MAbs) against arabinomannan and glucan (58, 59).We have shown previously that mechanical removal of capsular material from M. tuberculosis results in a 10-fold increase in bacterial binding to Mφ, suggesting that the capsule can act as an antiphagocytic barrier that limits the interaction of M. tuberculosis with Mφ (65). However, even though the capsule reduces binding of M. tuberculosis to Mφ, it does not eliminate it, and it is clear that at least some bacteria maintain the capacity to bind to Mφ. These observations, along with our earlier studies showing that only certain populations of Mφ efficiently bind M. tuberculosis (64, 67), suggest that the M. tuberculosis capsule modulates the interaction of bacteria with host cells, preventing uptake by some populations of Mφ and directing the bacteria to specific Mφ types or particular receptor-ligand interactions. In this study, we have evaluated the diversity of proteins present in the M. tuberculosis capsule and assessed the role of two bacterial molecular chaperones, Cpn60.2 and DnaK, in host cell binding. Using both competitive-inhibition and epitope-masking strategies, we have shown that while both Cpn60.2 and DnaK are present on the bacterial surface, only Cpn60.2 appears to be necessary to facilitate efficient bacterial association with Mφ.     

Cross-Reactive Immunity to Mycobacterium tuberculosis DosR Regulon-Encoded Antigens in Individuals Infected with Environmental,Nontuberculous Mycobacteria

Mycobacterium tuberculosis DosR regulon-encoded antigens are highly immunogenic in M. tuberculosis-infected humans and are associated with latent tuberculosis infection. We have investigated the hypothesis that infection with or exposure to nontuberculous mycobacteria (NTM) can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens since responsiveness has been observed in non-M. tuberculosis-exposed but purified protein derivative-responsive individuals. M. tuberculosis DosR regulon-encoded antigen-specific T-cell responses were studied in peripheral blood mononuclear cells (PBMCs) of NTM-infected/exposed individuals. BLASTP was used to determine the presence of M. tuberculosis DosR regulon-encoded protein orthologs among environmental mycobacteria and nonmycobacteria. Significant gamma interferon production was observed in PBMCs from NTM-infected/exposed individuals in response to M. tuberculosis DosR regulon-encoded antigens. DosR regulon-encoded protein orthologs were prominently present in tuberculous and environmental mycobacteria and surprisingly also in nonmycobacteria. The ubiquitous presence of the highly conserved DosR master regulator protein Rv3133c suggests that this is a general adaptive bacterial response regulator. We report a first series of M. tuberculosis antigens to which cross-reactive immunity is induced by NTM infection/exposure. The high conservation of M. tuberculosis DosR regulon-encoded antigens most likely enables them to induce cross-reactive T-cell responses.Yearly, tuberculosis (TB) claims 1.7 million lives. Its global incidence exceeds 9 million new cases. TB morbidity and mortality are likely to increase further as a result of TB reactivation in human immunodeficiency virus type 1-Mycobacterium tuberculosis-coinfected individuals, as well as the rising frequencies of multidrug-resistant and extensively drug-resistant M. tuberculosis strains (41). It is estimated that 2 billion people carry a latent TB infection. This vast reservoir forms a major source of new TB cases: 1 out of every 10 M. tuberculosis-infected individuals will develop active TB disease at one point in their lifetime, while the remainder are able to contain the bacilli without any clinical symptoms.In a series of recent M. tuberculosis antigen discovery studies, aiming at identifying new M. tuberculosis biomarker and vaccine antigens, we found that genes from the M. tuberculosis DosR (Rv3133c) regulon encode antigens that can induce specific T-cell immunity in M. tuberculosis-infected individuals (20, 32). Tubercle bacilli express the DosR regulon under in vitro conditions of hypoxia, low-dose nitric oxide (40), and carbon monoxide, (19, 37), conditions thought to be encountered by persisting intracellular bacilli in the immunocompetent host. Studies have also reported upregulated expression of DosR regulon genes in infected murine macrophages and infected murine lung tissues (19, 35-37). Importantly, immunity to M. tuberculosis DosR regulon-encoded antigens might contribute to the control of persistent M. tuberculosis infection since several DosR regulon-encoded antigens were preferentially recognized by subjects with latent TB infection (20, 32). Finally, we also found that Mycobacterium bovis BCG vaccination does not induce immune responses to M. tuberculosis DosR regulon-encoded antigens, which might partly underlie its inefficacy against late M. tuberculosis infection (16, 21).During these studies, we unexpectedly observed that some M. tuberculosis DosR regulon-encoded antigens were also recognized by healthy controls (HCs) who had a positive in vitro gamma interferon (IFN-γ) response to purified protein derivative (PPD) or M. tuberculosis lysate. However, none of these tuberculin skin test (TST)-negative HCs had had any known exposure to or infection with M. tuberculosis or had received BCG vaccination (20). We therefore hypothesized that these responses could be due to exposure to environmental, nontuberculous mycobacteria (NTM).NTM comprise all mycobacterial species that are not included within the M. tuberculosis complex (M. tuberculosis, M. bovis, M. bovis BCG, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canetti) or Mycobacterium leprae (8, 28). NTM are facultative intracellular bacteria with specific niches in the environment and are ubiquitously present and opportunistic. Diagnosis and treatment of lung infections caused by NTM involve careful evaluation of the infection since no reference standard or parameter exists by which the different NTM can be characterized (5). It has long been suspected that exposure to NTM can shape host immunity. For example, neonatal BCG vaccination results in significant protection against pediatric TB but generally fails to protect adequately against pulmonary TB in adults; this latter failure has been attributed in part to the immunomodulatory effects of NTM exposure/infection (2, 7, 10, 26), although the kinetics of the latter as well as the underlying mechanisms involved remain undefined.Here, we have investigated whether infection with or exposure to NTM can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens. We also studied the presence of M. tuberculosis DosR regulon protein orthologs and homologs among mycobacteria and several nonmycobacterial species.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Protection of Mycobacterium tuberculosis from Reactive Oxygen Species Conferred by the mel2 Locus Impacts Persistence and Dissemination

Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox^−/− and iNOS^−/− mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.Despite extensive efforts to eradicate tuberculosis, caused by Mycobacterium tuberculosis, worldwide and prevent the spread of antibiotic-resistant strains, tuberculosis remains one of the most frequent causes of death in humans. Currently, one-third of the world''s population is thought to be persistently infected with tuberculosis (6, 19, 20). A better understanding of the mechanisms that lead to persistence in humans is needed before it will be possible to develop rational strategies to prevent establishment of latency and block reactivation from it. Although the mouse model, even when infected by the natural low-dose aerosol route, does not replicate all aspects of pathogenesis by tuberculosis, its cost-effectiveness and the presence of numerous reagents make it an important tool for examination of the acute and persistent stages of infection (26, 50, 51). As a result, much of our knowledge regarding the role of the host in controlling infections as well as the bacterial factors involved has been obtained using the mouse model (26, 31, 34).Initially, tubercle bacilli encounter naïve alveolar macrophages in the mouse lung that produce low levels of reactive oxygen species (ROS) and undergo an oxidative burst in response to infection (8, 52). In the absence of ROS production, there is a modest transient advantage for tuberculosis in the lung, suggesting that ROS initially assist in the control of bacterial growth (16). This innate immune response does little to prevent the growth of the bacteria in mouse lungs, since for the first month after infection, bacterial numbers increase to a million or more. This time point corresponds to the peaks in the numbers of CD4 and CD8 T cells in the lung (38), maximal tumor necrosis factor alpha (TNF-α) levels (5, 32), and the initiation of a strong cell-mediated immune response. The cell-mediated immune response leads to control of bacterial growth, higher gamma interferon (IFN-γ) levels and tuberculosis persistence at stable numbers for between 1 and 2 years (38) before reactivation, most likely the result of a weakened immune system due to the age of the mice (49). TNF-α (44, 57) and IFN-γ (15, 27) levels are important for controlling tuberculosis growth and maintenance of the persistent state in the mouse model of infection. One of the reasons that TNF-α and IFN-γ help to control tuberculosis is that they provide signals for the production of both ROS and reactive nitrogen species (RNS) by macrophages (8, 25, 28, 32, 40, 65). Although M. tuberculosis is considered relatively resistant to ROS, mutations that impact those bacterial pathways involved in resistance can affect virulence (21, 33, 39, 47, 53). In contrast to ROS, RNS, produced by the nitric oxide synthase (iNOS) that is induced by IFN-γ, inhibit the growth of mycobacteria and are critical to maintenance of the persistent state in mice (7, 8, 22, 27, 29, 42). Although the role of RNS in the control of M. tuberculosis growth in mice seems well proven, the involvement of RNS in human (58) and in other animal, including guinea pig (36, 68), tuberculosis models is less clear, possibly because the presence of NO has been difficult to demonstrate (9, 24, 46, 54, 67). These observations suggest that M. tuberculosis genes involved in resistance to either ROS or RNS will play a role in persistence.We recently identified a locus in Mycobacterium marinum, designated mel2, that affects resistance to ROS and RNS (61, 62). Interestingly, this locus has three genes, melF, melG, and melH, with similarity to the luxA, luxG, and luxH bioluminescence genes, respectively, in other bacterial species (23). The luciferase (luxA) genes in other organisms can affect resistance to ROS (41, 55, 66) in addition to their role in bioluminescence. Bacterial luciferases are thought to scavenge H₂O₂ in a catalase-like reaction in which the enzyme-bound flavin mononucleotide (FMN) hydroperoxide and H₂O₂ decompose, releasing water and oxygen, to the enzyme-bound hydroxy-FMN that spontaneously decomposes to water, light, and enzyme-bound FMN (66). These observations suggest the intriguing possibility that the mel2 locus will be important for tuberculosis pathogenesis, since M. tuberculosis must resist ROS produced by the host immune response during infections. In order to test this hypothesis, we isolated an M. tuberculosis mutant in the mel2 locus and confirmed the loss of mel2 activity by an increase in ROS susceptibility. The mel2 mutant was evaluated for its role in virulence using the mouse model and macrophage infection in vitro. Mice and macrophages deficient in ROS and RNS were used to probe the primary host defense mechanisms that the mel2 locus is necessary to protect tuberculosis against. We found that the mel2 mutant displays increased susceptibility to ROS and is defective for growth in activated macrophages. Our data also suggest that the mel2 locus plays an important role during the persistence and dissemination of M. tuberculosis. The primary mechanism by which mel2 affects these events appears to be resistance to ROS. Overall, these studies provide evidence for the role of bioluminescence-related genes, such as mel2, in protecting M. tuberculosis, and most likely other bacterial species that carry similar genes, against ROS.     

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

A multiplex method using the SNaPshot technique was developed to screen for six common mycobacterial species: Mycobacteria tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae. A total of 468 mycobacterial clinical isolates were subjected to analysis for the presence of the six mycobacterial species by the multiplex SNaPshot method. Of the 468 mycobacterial isolates, 464 (99.15%) could be correctly identified by this assay. The multiplex SNaPshot technique is a promising discriminatory tool for rapid and accurate identification of frequently encountered clinical mycobacterial species.Even though Mycobacterium tuberculosis continues to be a serious health concern worldwide, it has been increasingly recognized that nontuberculous mycobacteria (NTM) are important human pathogens (4, 16, 23). NTM are ubiquitous organisms, with nearly 100 different species found in soil and water that can act as opportunistic pathogens in humans, causing a wide variety of skin and soft tissue infections, lymphadenitis, and lung disease (6, 15). The early differentiation of M. tuberculosis from NTM and the identification of species among NTM are crucial for immediate implementation of the appropriate therapy because susceptible drugs vary widely among different species (9).Conventionally, identification of mycobacteria is carried out by time-consuming biochemical tests that are not always accurate (5, 17, 25). Chromatographic techniques such as high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and thin-layer chromatography (TLC) are labor-intensive, difficult, or expensive (21, 26). DNA sequence analysis of the 16S rRNA gene region is now regarded as the gold standard for the identification of mycobacteria (13, 22, 25, 27). However, equipment and running costs are high. Simple genotypic assays for the identification of mycobacteria, such as Accuprobe (Gen-Probe Inc., San Diego, CA) (1), INNO-LiPA (27), and Genotype Mycobacterium (Hain Diagnostika) (19) are available commercially. Even though these tests are simple, they are often suited for small test volumes and are too expensive for high-throughput laboratories to use in a routine clinical diagnostic setting.In this study, we developed a novel multiplex SNaPshot method using fluorescently labeled terminators and capillary electrophoresis to screen for six common clinically encountered mycobacterial species (M. tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae) based on eight single nucleotide polymorphisms (SNPs) located in conserved regions of the 16S rRNA and Hsp65 genes.     

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.Of the known species in the genus Mycobacterium, Mycobacterium tuberculosis is the most common and most important pathogen, causing 2 million deaths and over 8 million cases of tuberculosis worldwide annually (2, 3, 4, 7). In addition to M. tuberculosis, infections with nontuberculosis mycobacteria (NTM) can also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of different mycobacterial species, the treatments of mycobacterial infections are different (13, 30, 33, 34). Thus, it is very important to differentiate between mycobacteria at the species level during early-stage diagnostics.Instead of a culture-based identification scheme, which may take 4 to 6 weeks or longer to identify slowly growing mycobacteria, PCR-based protocols (sequencing or PCR-restriction analysis [PRA]) targeting chronometer molecules, such as 16S rRNA (5, 6, 28), hsp65 (17, 19, 25), and rpoB (1, 16, 21), have been widely used to identify mycobacteria. However, in spite of the successful application of these conventional PCR-based methods to culture isolates, there are some drawbacks in their direct application to clinical specimens. This is especially true for sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, producing confusing results by the simultaneous amplification of both commensals and mycobacterial strains. We have recently developed several methods for mycobacterial species identification based on amplification of hsp65 gene sequences directly from sputum samples (15, 27). Limitations due to the intrinsic features of conventional PCR prevented feasible identification of mycobacterial species from sputum samples using this method.The use of the real-time PCR assay in the diagnosis of many infectious diseases has been increasing, as it represents an appealing alternative to conventional PCR. It is an improvement over conventional methods because of its increased sensitivity and specificity, low contamination risk, and ease of performance and speed (8). In particular, fluorescence resonance energy transfer (FRET)-based real-time PCR permits not only the simultaneous identification of multiple target species but also the direct identification of target species from primary specimens such as sputum specimens through melting curve analysis of the amplification product (8). These characteristics of FRET-based real-time PCR provide a useful advantage for the identification of mycobacteria from sputum samples. Recently, several real-time PCR-based methods for mycobacterial detection and identification have been developed and evaluated (9, 22, 23, 26, 29). However, direct application of the real-time PCR-based method to primary specimens was generally limited to M. tuberculosis alone (11, 26). So far, a method which can simultaneously identify several pathogenic NTM as well as M. tuberculosis from primary sputum samples in a single reaction has not been developed.In the present study, we sought to develop a multiprobe real-time PCR targeting the hsp65 gene (HMPRT-PCR) based on melting curve analysis (HybProbes). This enabled the simultaneous identification of several pathogenic mycobacteria, including M. tuberculosis, in a single PCR performed on cultures and sputum samples. The usefulness of these methods was evaluated by blindly applying them to cultured and sputum samples.     

Acute Immune Response to Mycobacterium massiliense in C57BL/6 and BALB/c Mice

Mycobacterium massiliense is an environmental opportunistic pathogen that has been associated with soft tissue infection after minor surgery. We studied the acute immune response of C57BL/6 and BALB/c mice infected intravenously with 10⁶ CFU of an M. massiliense strain isolated from a nosocomial infection in Brazil. The results presented here show that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells, and natural killer cells induced by gamma interferon and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.Mycobacteria that do not belong to the complex Mycobacterium tuberculosis are known as nontuberculous mycobacteria (NTM) or atypical mycobacteria. NTM are ubiquitous microorganisms found worldwide in soil and water (3, 23, 38). These environmental mycobacteria are considered emerging and environmental opportunistic pathogens (6, 23).Mycobacterium massiliense is an environmental nonphotochromogenic, rapidly growing Mycobacterium strain that has been associated with soft-tissue infection after minor surgery or intramuscular injection (3, 5, 17, 22, 26, 46) and with pulmonary infection due to diseases, such as cystic fibrosis (29, 41). This species differs only slightly from Mycobacterium abscessus, sharing a 99.6% sequence identity of their 16S rRNA genes; genetic differences can be observed by comparative sequence analysis of the rpoB and hsp65 genes (1, 25, 42). Infections with these agents tend to respond poorly to macrolide-based chemotherapy (3), even though the organisms are susceptible to clarithromycin (15, 44, 47).M. massiliense infection mainly affects immunocompetent individuals and occasionally is associated with disseminated disease (8). An outbreak of M. massiliense occurred in Goiania, Brazil, where 30 individuals were infected after undergoing knee joint and laparoscopic surgery (5). Despite the fact that the infected individuals were from different hospitals, a unique M. massiliense strain was identified and characterized by pulsed-field gel electrophoresis.Disease pathogenesis involves host-pathogen interactions that directly affect parasite clearance. Typically, when environmental bacteria are passively introduced into the host, rapid bacterial clearance occurs due to an efficient innate immune response (30). Nonetheless, accidental infections with M. massiliense have been described as having a chronic evolution and, in some cases, the disease is disseminated irrespective of the host''s immune status. Such findings raise the possibility that this species is more virulent and/or pathogenic than other environmental mycobacteria, such as M. chelonae and M. abscessus.Recently, a murine model of M. abscessus infection was described, and isogenic mice were shown to be good models to address the immune response of the host (34, 39). In the present study, we analyzed the immune response of C57BL/6 and BALB/c mice infected with a clinical isolate of M. massiliense obtained from the recent outbreak in Goiania, Brazil. We show here that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells (DCs), and natural killer (NK) cells induced mainly by gamma interferon (IFN-γ) and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.     

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand,Near-Patient Technology

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System''s MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay''s buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.An alarming increase in the global incidence of drug-resistant Mycobacterium tuberculosis infection has created a critical need for methods that can rapidly detect M. tuberculosis and identify drug-resistant cases (53). Failure to quickly and effectively recognize and treat patients with drug-resistant tuberculosis (TB), particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, leads to increased mortality, nosocomial outbreaks, and resistance to additional antituberculosis drugs (14, 37). However, MDR and XDR tuberculosis can be effectively treated if properly identified (35). A number of new diagnostic approaches have brought incremental improvements in detection and drug susceptibility testing (2, 9, 19, 24, 37, 41, 46); however, none can realistically provide actionable information within the time frame of a single office or clinic visit. Thus, despite technical advances, rapid diagnostics have not yet been able to have an impact on critical initial decisions regarding hospitalization, isolation, and the choice of treatment regimens for suspected tuberculosis patients.Previously, we showed that direct molecular detection of M. tuberculosis and rifampin resistance could be accomplished simultaneously (27); more recently, our group developed a single-tube, molecular beacon-based real-time PCR assay for the detection of rifampin-resistant M. tuberculosis (42, 43). Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene, which occur in 95 to 98% of all rifampin-resistant strains (and which are almost invariably absent in rifampin-susceptible strains), were detected by five overlapping molecular beacons (34). The assay proved to be simple, rapid, specific, and highly sensitive in tests on isolates of M. tuberculosis from New York City, Madrid (42), India, and Mexico (51). As most rifampin-resistant isolates are also resistant to isoniazid, rifampin resistance can be used as a marker for MDR M. tuberculosis (36, 44, 49). However, like all nucleic acid amplification-based assays for M. tuberculosis detection (15), this assay was too complex and too prone to operator errors, sample cross-contamination, and biohazards for rapid near-patient use.The Cepheid GeneXpert System (Sunnyvale, CA), a single-use sample-processing cartridge system with integrated multicolor real-time PCR capacity (45), has the potential to greatly simplify nucleic acid amplification tests. Here, we utilized this new technology to develop an on-demand, near-patient PCR assay that employs a novel six-color dye set to detect M. tuberculosis and identify rifampin resistance as a surrogate for MDR directly from a patient''s sputum in less than 2 h. The many features of this system, including sample decontamination, hands-free operation, on-board sample processing, and ultrasensitive hemi-nested PCR, enabled us to create a low-complexity assay with a sensitivity that approached certain culture methods. This type of assay may prove to be useful in the initial management of suspected tuberculosis cases in both the United States and the world at large.     

Mycobacterium tuberculosis Transmission Is Not Related to Household Genotype in a Setting of High Endemicity

Among the different strains of Mycobacterium tuberculosis, Beijing has been identified as an emerging genotype. Enhanced transmissibility provides a potential mechanism for genotype selection. This study evaluated whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in contact with an adult tuberculosis (TB) index case in the child''s household. We conducted a prospective, community-based study at two primary health care clinics in Cape Town, South Africa, from January 2003 through December 2004. Bacteriologically confirmed new adult pulmonary TB cases were genotyped by IS6110 DNA fingerprinting; household contacts less than 5 years were traced and screened for M. tuberculosis infection and/or disease. A total of 187 adult index cases were identified from 174 households with children aged less than 5 years. Of 261 child contacts aged 0 to 5 years, 219 (83.9%) were completely evaluated and the isolate from the index case was successfully genotyped. M. tuberculosis infection (induration of ≥10 mm by Mantoux tuberculin skin test) was documented in 118/219 (53.9%) children; 34 (15.5%) had radiographic signs suggestive of active TB. There was no significant difference in the ratio of infected children among those exposed to the Beijing genotype (51/89; 57.3%) and those exposed to non-Beijing genotypes (55/115; 47.8%) (odds ratio, 1.5; 95% confidence interval, 0.8 to 2.7). Genotyping was successful for six children diagnosed with active TB; the isolates from only two children had IS6110 fingerprints that were identical to the IS6110 fingerprint of the isolate from the presumed index case. We found no significant association between the M. tuberculosis genotype and transmissibility within the household. However, undocumented M. tuberculosis exposure may have been a major confounding factor in this setting with a high burden of TB.From an evolutionary perspective, the global tuberculosis (TB) epidemic presents a dynamic picture. Mycobacterium tuberculosis generates significant genetic diversity through deletion, duplication, and recombination events; but unlike most other bacterial pathogens, gene exchange is rare (31, 33). The absence of horizontal gene transfer results in strict clonality with distinct genetic lineages that permit accurate phylogenetic reconstruction. Selection of the most successful genotypes is mediated by genotype-specific differences in host-pathogen interactions (15, 18), some of which have been well characterized in animal models (11, 24, 25). Pathogen-related factors that may contribute to M. tuberculosis genotype selection include variability in transmissibility (the ability to spread from person to person), pathogenicity (the ability to cause clinical disease), the level of protection afforded by Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccination, and the acquisition of drug resistance.The Beijing genotype predominates in parts of East Asia (17, 23, 38, 41), northern Eurasia (12, 31), and southern Africa (8, 39). Beijing has been regarded as an emerging genotype on the basis of its global distribution, its association with young age (4), and its proportional increase in prevalence over time (8, 39). An increased ability to circumvent the protection afforded by BCG vaccination is suggested by the positive association (of the Beijing genotype) with the presence of a BCG scar in human populations (4) and has been observed in mice (24), although more recent findings challenge this observation (20). Multiple mechanisms have been explored to explain the potential link between the emergence of the Beijing genotype and low-level BCG protection (1), which may provide the Beijing genotype with a selective advantage in populations in which universal BCG vaccination is practiced.The association between the Beijing genotype and drug-resistant TB is well documented in multiple settings (2, 9, 31, 32, 37). Although it has been demonstrated that the acquisition of drug resistance is usually associated with a fitness cost, this finding seems variable and strain dependent (14) and may be insufficient to prevent transmission (16). Some Beijing genotypes retain their fitness in vitro, despite the acquisition of drug resistance (36), while compensatory evolution may account for significantly higher levels of fitness in clinical strains than in their progenitors (16). The geographic clustering of drug-resistant cases with evidence of clonal expansion suggests the successful transmission of drug-resistant Beijing genotypes (37). This is supported by the frequency with which isolates of the Beijing genotype are identified among children with drug-resistant TB (30), which indicates successful transmission within the community (34).Variable transmissibility, irrespective of drug resistance, represents a relatively unexplored potential mechanism for the emergence of an M. tuberculosis genotype. Conventional molecular tools are limited by an inability to distinguish factors related to transmissibility from those related to pathogenicity, since only patients with active disease can be evaluated. The value of experimental animal models is equally limited, since artificially induced infection does not allow simulation of the natural airborne transmission of M. tuberculosis. The household provides an appropriate setting in which variables related to recent M. tuberculosis transmission in the human host may be studied and allows the evaluation of young children likely to have been infected through contact with others at the household level.The study described here aimed to determine whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in household contact with an adult TB index case.     

Dendritic Cells Inhibit the Progression of Listeria monocytogenes Intracellular Infection by Retaining Bacteria in Major Histocompatibility Complex Class II-Rich Phagosomes and by Limiting Cytosolic Growth

Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2⁺ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2⁺, major histocompatibility complex class II⁺ (MHC-II⁺) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response.Dendritic cells (DC) comprise a heterogeneous group of antigen-presenting cells (APC) that have the unique capacity to activate naïve T cells, and thus they are required for the initiation of an adaptive immune response (4). Immature DC link the innate to the adaptive immune response by sampling material from the local environment and differentiating in a manner appropriate to the ingested cargo. Maturation into APC capable of presenting antigens for the initiation of an adaptive immune response results when DC pattern recognition receptors encounter molecular signatures associated with pathogens (4, 25). In recent years, studies using predominantly bone marrow-derived myeloid DC and model antigens have indicated that the unique ability of DC to induce primary T-cell responses is a function of endosomal/lysosomal specializations that effectively preserve immunogenic peptides (26, 34, 43). Unlike other professional APC, antigen processing and presentation in DC is coupled to the receipt of appropriate maturation signals (25, 26). Immature DC have a high capacity to ingest soluble and particulate material and sequester it in major histocompatibility complex class II (MHC-II)-rich compartments (MIIC) (19, 30). The degradation and loading of peptides onto MHC-II, as well as the delivery to and retention of peptide-MHC complexes on the cell surface, is enhanced after the receipt and assimilation of inflammatory signals (11, 19, 30, 44). Recent evidence suggests that antigen degradation is limited in immature DC endosomes and lysosomes relative to that of macrophages by lower protease content as well as by an acidification response that controls the rate and extent of proteolysis (14, 26, 42). These adaptations have been proposed to prevent the destruction of immunogenic epitopes destined for presentation to T cells (14, 26, 35).Listeria monocytogenes belongs to a class of intracellular pathogens that must escape the host cell endosomal/lysosomal system in order to grow. L. monocytogenes infects a broad range of cell types, including DC, and the intracellular life cycle has been characterized in detail (31). The growth stage of the L. monocytogenes life cycle proceeds after the rupture of the phagosomal membrane by a pH- and cholesterol-dependent pore-forming cytolysin, listeriolysin O (LLO), with the assistance of a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Within 30 min of uptake by macrophages, L. monocytogenes escapes from LAMP1-negative, late endosomal vesicles, which acidify to pH 5.5, providing an optimal environment for LLO-mediated pore formation (5, 18, 28). Consequently, the majority of L. monocytogenes can be found free and replicating in the macrophage cytosol in the first hour of infection. Bacterial escape is inefficient after phagosomal fusion with lysosomes (18), presumably due to a harsh environment (low pH, high proteolysis) that does not support LLO activity. L. monocytogenes thus takes advantage of a small window of opportunity within minutes after ingestion by macrophages, during which the phagosomal conditions are favorable for LLO to mediate bacterial escape to the cytosol. Once in the cytosol, L. monocytogenes recruits the host cell actin machinery to spread to adjacent cells by a process of intercellular spread. Escape from secondary spreading vacuoles, mediated by LLO in combination with two bacterial phospholipases (PI-PLC and a broad-range PLC, PC-PLC) (1), allows the infection to progress while shielding the bacteria from extracellular immune defenses of the host. The intracellular lifestyle dictates that clearance of the pathogen requires the processing and presentation of bacterial antigens via the MHC-I pathway and the activation of a CD8⁺ T-cell response (22). The presentation of antigens via the MHC-II pathway and activation of a CD4⁺ T-cell response is necessary for the maintenance of long-term CD8⁺ T-cell memory (41).DC mature in response to infection with wild-type (WT) L. monocytogenes (3, 9, 27) and are required for the initiation of an adaptive immune response in the murine intravenous model (21). We have previously reported that bone marrow-derived DC have the capacity to restrict large numbers of L. monocytogenes to a membrane-bound compartment and, consequently, to limit their intracellular growth (50). This is in contrast to bone marrow-derived macrophages (BMM), which support relatively unrestricted bacterial growth in the cytosol. Consistent with these findings, DC become infected with L. monocytogenes in vivo, but unlike macrophages, they do not appear to provide a significant niche for bacterial replication (2, 27). Given the critical role of DC in the immune response, the limitation of intracellular bacterial infection may preserve and enhance their specialized antigen-presenting function. Therefore, to begin to address how DC control L. monocytogenes growth, we examined the characteristics of DC phagosomes early after infection. Evidence is provided that phagosomal maturation events associated with the acquisition of MHC-II molecules reduce the efficiency of L. monocytogenes escape to the DC cytosol, possibly by presenting suboptimal environmental conditions for LLO. Along with an additional as-yet undefined mechanism that controls the extent of bacterial growth in the cytosol, these DC-specific responses to L. monocytogenes infection preserve cellular integrity during the course of the maturation response.     

High-Level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).In bacterial populations, the generation of antibiotic resistance depends on the rate of emergence of resistant mutants (1, 19, 23). Correlations between high mutation rates, the geographic distribution of mutations, antibiotic resistance, and virulence in bacteria have been reported in several studies (9, 20, 33, 37). Knowledge of geographic variations is important for monitoring rifampin (rifampicin) resistance within a defined population of patients infected with Mycobacterium tuberculosis, as the prevalence of the mutations studied so far varies for M. tuberculosis strains isolated from different countries (24, 26, 29, 33, 36). In 2004, the prevalence of tuberculosis in Iran was reported to be 17 per 100,000, and at the southern border of Iran (Zabol province) where tuberculosis is endemic, the prevalence was 141 per 100,000 (20). Rifampin resistance is of particular epidemiologic importance, since it represents a valuable surrogate marker for multidrug-resistant (MDR) tuberculosis strains, and the prevalence of MDR strains is a significant obstacle to tuberculosis therapy (4, 21, 26). DNA sequencing studies indicate that more than 95% of rifampin-resistant M. tuberculosis strains have mutations within the 81-bp hot-spot region (codons 507 to 533) of the RNA polymerase β-subunit (rpoB) gene (4, 19, 32). Over the last 15 years, Kapur et al. and Telenti et al. have identified the molecular basis of rifampin resistance in M. tuberculosis (9, 29). Thus, it is important to determine the molecular bases of mutations and their distribution at the level of each country prior to molecular testing introduction for routine diagnostics (9, 11, 13, 15, 16, 23).In this study, we investigated the significance of multiple mutations in the rpoB gene and their correlation with highly prevalent nucleotide changes in codons 523, 531, and 526 and also demonstrated the highly prevalent nucleotide changes observed in the last nine codons of the β-subunit (523 to 531) that are associated with higher levels of resistance to rifampin (MIC, ≥100 μg/ml) in patients bearing secondary M. tuberculosis infection.     

Patients with Multidrug-Resistant Tuberculosis Display Impaired Th1 Responses and Enhanced Regulatory T-Cell Levels in Response to an Outbreak of Multidrug-Resistant Mycobacterium tuberculosis M and Ra Strains

In Argentina, multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 1990s and thereafter disseminated to the immunocompetent community. Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR Mycobacterium tuberculosis outbreak strains, such as the so-called strain M of the Haarlem lineage and strain Ra of the Latin America and Mediterranean lineage. In the current study, we evaluated the immune responses induced by strains M and Ra in peripheral blood mononuclear cells from patients with active MDR-TB or fully drug-susceptible tuberculosis (S-TB) and in purified protein derivative-positive healthy controls (group N). Our results demonstrated that strain M was a weaker gamma interferon (IFN-γ) inducer than H37Rv for group N. Strain M induced the highest interleukin-4 expression in CD4⁺ and CD8⁺ T cells from MDR- and S-TB patients, along with the lowest cytotoxic T-lymphocyte (CTL) activity in patients and controls. Hence, impairment of CTL activity is a hallmark of strain M and could be an evasion mechanism employed by this strain to avoid the killing of macrophages by M-specific CTL effectors. In addition, MDR-TB patients had an increased proportion of circulating regulatory T cells (Treg cells), and these cells were further expanded upon in vitro M. tuberculosis stimulation. Experimental Treg cell depletion increased IFN-γ expression and CTL activity in TB patients, with M- and Ra-induced CTL responses remaining low in MDR-TB patients. Altogether, these results suggest that immunity to MDR strains might depend upon a balance between the individual host response and the ability of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.Human interventions, namely, mistreatment of tuberculosis (TB) and poor patient compliance, selectively favor the multiplication of drug-resistant Mycobacterium tuberculosis mutants over drug-susceptible bacilli in tuberculous lesions. M. tuberculosis isolates are considered to be multidrug resistant (MDR) when showing resistance to isoniazid and rifampin (rifampicin), the most effective drugs for TB treatment; they become extensively drug resistant when showing additional resistance to key second-line drugs (32, 36). MDR-TB and extensively drug-resistant TB are very difficult to treat, their prognosis is somber, and mortality is high (14, 27).In Argentina, a total of 11,464 new cases of TB were reported in 2006, with an incidence of 29.1 per 100,000 inhabitants. MDR-TB occurred in 4.5% of the cases. MDR-TB outbreaks emerged in Argentina among hospitalized patients with AIDS in the early 1990s (1, 45) and thereafter disseminated to immunocompetent individuals (37-39). Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR M. tuberculosis outbreak strains, such as the so-called strain M of the Haarlem family and strain Ra of the Latin America and Mediterranean (LAM) family. Each of these two strains managed to perpetuate in its geographical niche, i.e., Buenos Aires and the Rosario City area, respectively. In particular, strain M appears to be highly prosperous in the country and is able to build up further drug resistance without impairing its ability to spread (29).TB development depends not only on the host immune response and on its natural resistance/susceptibility to M. tuberculosis infection but also on differences in transmissibility, virulence, and immunogenicity among M. tuberculosis strains, determined by the genetic background of the organisms. It is becoming evident that certain strains of M. tuberculosis with special transmission potential are able to manipulate host immunity by inducing Th1 or Th2 responses which could impact disease outcome and/or evolution (5, 28, 30, 31, 40-42, 51). Protective immunity against TB is mediated by a Th1-type immune response characterized by high levels of interleukin-12 (IL-12) from infected macrophages and gamma interferon (IFN-γ) from antigen-specific T cells, which control and contain infection in the lungs (24). Peripheral blood mononuclear cells (PBMC) from MDR-TB patients have been shown to poorly respond in vitro to H37Ra whole bacilli, purified protein derivative (PPD), and specific antigens, such as ESAT-6 and the 30-kDa protein (16, 25, 26, 33). Furthermore, increased IL-4 secretion by CD4⁺ T cells after H37Rv total lipid stimulation was observed in MDR-TB patients (50).Although a Th1 profile is necessary for a protective response, it may also cause immunopathologic damage; for this reason, either regulatory T cells (Treg cells) or a Th2 response might play important regulatory functions protecting the patient from collateral host tissue damage. Nevertheless, an excessive Th1 downregulation might favor disease progression. In this context, increased levels of CD4⁺ CD25^high Foxp3⁺ Treg cells have been detected in PBMC from drug-susceptible tuberculosis (S-TB) patients compared to those from PPD-positive (PPD⁺) healthy donors (18-20, 46). In addition, the results of studies involving in vitro depletion of CD4⁺ CD25^high T lymphocytes suggest a role of Treg cells in TB pathogenesis (18-20, 43).In the current study, we examined immune profiles induced by two MDR M. tuberculosis strains disseminated in Argentina, namely, strains M and Ra. Our results demonstrate that strain M is a weak inducer of IFN-γ and elicits a remarkably low cytotoxic T-cell (CTL) activity. Also, in vitro expansion of Treg cells in PBMC from TB patients is not M. tuberculosis strain dependent and efficiently suppresses antigen-induced IFN-γ and CTL responses.     

Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

A multiplex PCR assay for the simultaneous detection of Mycobacterium tuberculosis and Pneumocystis jirovecii was developed using IS6110-based detection for M. tuberculosis and mitochondrial large-subunit (mtLSU) rRNA gene detection for P. jirovecii. Ninety-five pulmonary blinded samples were examined using the developed multiplex PCR assay, and the results were compared with those obtained by the single nested PCRs targeting IS6110 for M. tuberculosis and mtLSU rRNA for P. jirovecii. Of the 95 pulmonary samples tested, the multiplex nested PCR developed here could detect 36 cases of M. tuberculosis infection, 35 cases of P. jirovecii infection, and 17 cases of M. tuberculosis and P. jirovecii coinfections. The sensitivities of the multiplex nested PCR in detecting M. tuberculosis and P. jirovecii were 92.1% and 81.4%, respectively, whereas the specificities in detecting M. tuberculosis and P. jirovecii were 98.2% and 100%, respectively.Pulmonary tuberculosis (TB) and Pneumocystis jirovecii pneumonia are two of the most common opportunistic infections found in association with AIDS worldwide (4), including Thailand (2, 3, 6, 10). About one-third of the world''s population and one-third of people infected with HIV are infected with Mycobacterium tuberculosis. The World Health Organization (WHO) reported that globally 9.2 million new cases of TB and 1.7 million deaths from TB occurred in 2006, and of these, 0.7 million cases and 0.2 million deaths, respectively, were in HIV-positive people (21). At present, Pneumocystis pneumonia, caused by Pneumocystis jirovecii (previously known as P. carinii f. sp. hominis), remains one of the most common AIDS-defining illnesses and is a frequent cause of morbidity and mortality in HIV-infected patients (7). Geographically, TB is the most common respiratory opportunistic infection in people infected with HIV worldwide, especially in the developing world (1, 4, 6, 8, 9, 12), whereas P. jirovecii pneumonia is more prevalent in industrialized countries (4, 5, 13, 16). In Thailand, TB has been the most common opportunistic infection in people with AIDS, whereas P. jirovecii pneumonia has been the second most common (12, 18). The total number of the two infections represents one-half of opportunistic infections in AIDS cases.TB and P. jirovecii pneumonia can clinically and radiologically mimic each other, including having similar presentations in patients, and they cannot always be diagnosed by clinical presentation or sputum examination. In addition, coinfection in individuals may also occur. Therefore, accurate and rapid diagnosis is required. The molecular means of diagnosis is considered to be a reliable technique, and it is essential that it be developed or improved to simultaneously diagnose TB and P. jirovecii pneumonia. Having a technique for differential diagnosis of the two infections would contribute to the ability to provide immediate treatment, controlling the diseases and decreasing the rates of transmission. The aim of the present study was to develop a multiplex PCR technique for the detection of M. tuberculosis and P. jirovecii simultaneously in clinical samples. In the present study, the development of a multiplex PCR involved selection of the appropriate genes, as well as the optimum PCR mixture and PCR thermal profile. The multiplex PCR was applied to test its sensitivity and specificity with clinical specimens.