Journal of Clinical Mircobiology

Texto completo: acesso restrito. p. 2485-2493Improved diagnostics are needed for the detection ofMycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screenedM. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity withM. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing theesat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, includingMycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and theM. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen

Clinical and Diagnostic Laboratory Immunology

Texto completo: acesso restrito. p. 883-891Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ∼98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ∼93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection

EULAR/PReS endorsed consensus criteria* for the classification of childhood vasculitides

BACKGROUND: There has been a lack of appropriate classification criteria for vasculitis in children. OBJECTIVE: To develop a widely accepted general classification for the vasculitides observed in children and specific and realistic classification criteria for common childhood vasculitides (Henoch‐Schönlein purpura (HSP), Kawasaki disease (KD), childhood polyarteritis nodosa (PAN), Wegener's granulomatosis (WG), and Takayasu arteritis (TA)). METHODS: The project was divided into two phases: (1) the Delphi technique was used to gather opinions from a wide spectrum of paediatric rheumatologists and nephrologists; (2) a consensus conference using nominal group technique was held. Ten international experts, all paediatricians, met for the consensus conference. Agreement of at least 80% of the participants was defined as consensus. RESULTS: Consensus was reached to base the general working classification for childhood vasculitides on vessel size. The small vessel disease was further subcategorised into “granulomatous” and “non‐granulomatous.” Final criteria were developed to classify a child as HSP, KD, childhood PAN, WG, or TA, with changes introduced based on paediatric experience. Mandatory criteria were suggested for all diseases except WG. CONCLUSIONS: It is hoped that the suggested criteria will be widely accepted around the world because of the reliable techniques used and the international and multispecialist composition of the expert group involved

Molecular and Immunological Characterization of Mycobacterium tuberculosis CFP-10, an Immunodiagnostic Antigen Missing in Mycobacterium bovis BCG

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis

Journal of Clinical Mircobiology

p.3285–3290In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis

Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ∼98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ∼93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection