Incidence of active mycobacterial infections in Brazilian patients with chronic inflammatory arthritis and negative evaluation for latent tuberculosis infection at baseline - A longitudinal analysis after using TNFα blockers

Several studies point to the increased risk of reactivation of latent tuberculosisinfection (LTBI) in patients with chronic inflammatory arthritis (CIAs) after usingtumour necrosis factor (TNF)α blockers. To study the incidence ofactive mycobacterial infections (aMI) in patients starting TNF αblockers, 262 patients were included in this study: 109 with rheumatoid arthritis(RA), 93 with ankylosing spondylitis (AS), 44 with juvenile idiopathic arthritis(JIA) and 16 with psoriatic arthritis (PsA). All patients had indication for anti-TNFα therapy. Epidemiologic and clinical data were evaluated and asimple X-ray and tuberculin skin test (TST) were performed. The control groupincluded 215 healthy individuals. The follow-up was 48 months to identify cases ofaMI. TST positivity was higher in patients with AS (37.6%) than in RA (12.8%), PsA(18.8%) and JIA (6.8%) (p < 0.001). In the control group, TST positivity was32.7%. Nine (3.43%) patients were diagnosed with aMI. The overall incidence rate ofaMI was 86.93/100,000 person-years [95% confidence interval (CI) 23.6-217.9] forpatients and 35.79/100,000 person-years (95% CI 12.4-69.6) for control group (p <0.001). All patients who developed aMI had no evidence of LTBI at the baselineevaluation. Patients with CIA starting TNF α blockers and noevidence of LTBI at baseline, particularly with nonreactive TST, may have higher riskof aMI.     

Minimal contribution of Valpha14 natural killer T cells to Th1 response and host resistance against mycobacterial infection in mice

We elucidated the contribution of Valpha14 NKT cells to Th1 response and host resistance against mycobacterial infection. In Valpha14 NKT cell-deficient mice, host defense and DTH response to Mycobacterium bovis BCG were not different from wild-type mice after pulmonary infection. There was no significant difference in the lung concentrations of IFN-gamma between the two strains of mice. In addition, host defense to systemic infection with M. tuberculosis was similar to that of M. bovis. Our results indicate that Valpha14 NKT cells play only a marginal role, if any, in the Th1 response and host resistance to mycobacterial infection.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

The utility of fine needle aspiration in HIV positive children

Objective:  To determine the spectrum of disease, diagnostic accuracy and adequacy of fine needle aspirates (FNA) in human immunodeficiency virus (HIV) positive children who present with mass lesions.
Methods:  Between January 1997 and December 2002, 95 FNAs were performed in 91 children aged 15 years and younger who were known to be infected with the human immunodeficiency virus (HIV).
Results:  Head and neck masses including salivary gland swellings were the most common presentation (58.9%) followed by axillary masses (25.3%). Groin masses were aspirated in six children, flank and abdominal masses in four children, buttock masses in three children, a chest wall mass in one child and a sonar guided FNA of a lung mass in one child. Eight FNAs (8.4%) proved inadequate. Reactive lymphadenopathy was diagnosed in 42 cases, mycobacterial infection in 22, four children were diagnosed with abscess, one child had a fungal infection and five were found to have non-Hodgkin's lymphoma. There were four cases each of lymphoepithelial lesion and Kaposi sarcoma. There was one case each of nephroblastoma, rhabdomyosarcoma, myeloma, melanotic progonoma and spindle cells, not otherwise specified.
Conclusion:  Fine needle aspiration in HIV positive children is a worthwhile procedure and in most instances allows a rapid diagnosis obviating the need for surgery and enabling swift treatment to be undertaken where necessary. Ancillary studies form an important diagnostic component. Universal safety precautions must be strictly adhered to.     

Siderophilic bodies associated with hemosiderosis and atypical mycobacterial infection in an island siamang (Hylobates syndactylus)

Laminated iron concretions were noted in the liver of an aged siamang (Hylobates syndactylus) that had granulomatous enteritis and hepatitis due to Mycobacterium avium intracellulare infection. Preexisting hepatic siderosis, iron sequestration in macrophages, and compromised macrophage function due to mycobacterial infection are proposed as the basis for the abundance and size of the concretions. Similar siderophilic bodies and concomitant siderosis occurred in other siamangs. The concretions are similar to Schumann bodies and Michaelis-Gutmann bodies associated with granulomatous disease in other species.     

Unusual Diheme Conformation of the Heme-Degrading Protein from Mycobacterium tuberculosis

Heme degradation plays a pivotal role in the availability of the essential nutrient, iron, in pathogenic bacteria. A previously unannotated protein from Mycobacterium tuberculosis, Rv3592, which shares homology to heme-degrading enzymes, has been identified. Biochemical analyses confirm that Rv3592, which we have termed MhuD (mycobacterial heme utilization, degrader), is able to bind and degrade heme. Interestingly, contrary to previously reported stoichiometry for the Staphylococcus aureus heme degraders, iron-regulated surface determinant (Isd)G and IsdI, MhuD has the ability to bind heme in a 1:2 protein-to-heme ratio, although the MhuD-diheme complex is inactive. Furthermore, the 1.75-Å crystal structure of the MhuD-diheme complex reveals two stacked hemes forming extensive contacts with residues in the active site. In particular, the solvent-exposed heme is axially liganded by His75 and is stacked planar upon the solvent-protected heme. The solvent-protected heme is coordinated by a chloride ion, which is, in turn, stabilized by Asn7. Structural comparison between MhuD-diheme and inactive IsdG and IsdI bound to only one highly distorted metalloporphyrin ring reveals that several residues located in α-helix 2 and the subsequent loop appear to be responsible for heme stoichiometric differences and suggest open and closed conformations for substrate entry and product exit.