The diagnostic value of urinary culture filtrate protein-10antigen in childhood tuberculosis

Introduction and Aim:Childhood tuberculosis (TB) remains a major problem worldwide.However, diagnosis of tuberculosis in children is often complicated by the difficulty in obtaining a proper sputum specimens and low sensitivity of the gold standard diagnostic test to confirm the presence of Mycobacterium tuberculosis(M.tb)in this age group. Recently, M.tbantigen detection in urinaryspecimenshas become a popular method. It is non-invasiveand handling of specimen is simple. It was reported that urinary CFP-10, a specific protein of M.tb, has emerged as a potential biomarker in the future. However, its diagnostic value as a new biomarker in childhood TB remains poorly understood.The aim of the study is to determine the diagnostic value of urinary CFP-10 in childhood TB. Methods: Seventy children with suspected pulmonary or extrapulmonary TB were enrolled. Tuberculosis was diagnosed by performingTuberculin skin test, chest x-ray, microscopic examination, and microbiological cultureobtainedfrom sputum or gastric lavage specimen. The level ofurinary CFP-10 antigen was analyzedbyELISA (Elabscience, China). Statistical analyseswereperformed using SPSS 21.0 and p-values of <0.05 were consideredstatistically significant. Results: The levels of urinary CFP-10 in subjects diagnosed with TB was higher than that of the non-TB subjects, 4.13(0.62) vs 0.43(0.14) pg/mL, p=0.005. The cut-off value forurinary CFP-10 level reached 0.39 pg/mL (sensitivity 65% and specificity 67%). This value became0.54 pg/mL (sensitivity 61% and specificity 62%)in microbiologically confirmed cases. Conclusion: The urinary CFP-10 level has moderate diagnostic value for diagnosing childhood TB.

Prime Vaccination with Chitosan-Coated Phipps BCG and Boosting with CFP-PLGA against Tuberculosis in a Goat Model

Attempts to improve the immune response and efficacy of vaccines against tuberculosis in cattle, goats, and other animal species have been the focus of research in this field during the last two decades. Improving the vaccine efficacy is essential prior to running long-lasting and expensive field trials. Studies have shown that vaccine protocols utilizing boosting with proteins improve the vaccine efficacy. The use of polymers such as chitosan and PolyLactic-co-Glycolic Acid (PLGA) improves the immune response against different diseases by improving the interaction of antigens with the cellular immune system and modulating the host immune response. This study shows that the prime BCG vaccination, boosted with a culture filtrate protein (CFP), alone or in combination with chitosan and PolyLactic-co-Glycolic Acid (PLGA), have the potential to reduce tuberculosis (TB) dissemination by reducing the number of animals with lesions, the number of lesions per animal, and the size of the lesions in vaccinated animals, compared with those not vaccinated or those vaccinated with BCG alone. The vaccinated groups showed significantly higher Interferon-γ levels in the blood compared to the control, nonvaccinated group after vaccination, after boosting, and after the challenge with the wild-type Mycobacterium bovis strain.

Early secretory antigenic target 6 and culture filtrate protein 10 as diagnostic indicators in IgA nephropathy associated with renal tuberculosis

Background: This study evaluated the diagnostic value of early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in immunoglobulin A nephropathy (IgAN) associated with renal tuberculosis (RT).Methods: Between January 2013 and January 2016, 40 patients with IgAN (IgAN group), 32 patients with RT (RT group), and 52 patients with IgAN associated with RT (IgAN + RT group) were selected for this study. A Tuberculin skin test (TST) was conducted, and serum Mycobacterium tuberculosis (MTB) antibody levels were measured. Urine samples were collected to culture MTB. Immunohistochemistry and western blotting were used to determine the expression of ESAT-6 and CFP-10 in renal tissues. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic values of ESAT-6 and CFP-10 in IgAN associated with RT.Results: TST, serum MTB antibody, and urine MTB assessments were negative in the IgAN group. The positive rates of the TST and serum MTB antibody and urine MTB testing were higher in the RT group than in the IgAN + RT group. Among the three groups, expression levels of ESAT-6 and CFP-10 were found to be the highest in the IgAN + RT group and were found to be the lowest in the IgAN group. The ROC curves indicated that the area under curve (AUC) value of ESAT-6 protein for IgAN + RT diagnosis was 0.907 with a cut-off of 26.72 as the critical value. Detection by ESAT-6 protein levels achieved 75.0% sensitivity and 94.2% specificity. The AUC value of the CFP-10 protein for diagnosis of IgAN + RT was 0.800, with a cut-off of 25.665 as the critical value. Detection by the protein levels of CFP-10 showed 63.9% sensitivity and 84.6% specificity.Conclusions: Our study provides evidence for the potential of the proteins ESAT-6 and CFP-10 as candidate markers for the diagnosis of IgAN associated with RT.

Sandwich Electrochemical Immunosensor for Early Detection of Tuberculosis Based on Graphene/Polyaniline-Modified Screen-Printed Gold Electrode

A rapid and sensitive sandwich electrochemical immunosensor was developed based on the fabrication of the graphene/polyaniline (GP/PANI) nanocomposite onto screen-printed gold electrode (SPGE) for detection of tuberculosis biomarker 10-kDa culture filtrate protein (CFP10). The prepared GP/PANI nanocomposite was characterized using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). The chemical bonding and morphology of GP/PANI-modified SPGE were studied by Raman spectroscopy and FESEM coupled with energy dispersive X-ray spectroscopy, respectively. From both studies, it clearly showed that GP/PANI was successfully coated onto SPGE through drop cast technique. Cyclic voltammetry was used to study the electrochemical properties of the modified electrode. The effective surface area for GP/PANI-modified SPGE was enhanced about five times compared with bare SPGE. Differential pulse voltammetry was used to detect the CFP10 antigen. The GP/PANI-modified SPGE that was fortified with sandwich type immunosensor exhibited a wide linear range (20–100 ng/mL) with a low detection limit of 15 ng/mL. This proposed electrochemical immunosensor is sensitive, low sample volume, rapid and disposable, which is suitable for tuberculosis detection in real samples.

Rapid diagnosis of pulmonary tuberculosis by combined molecular and immunological methods

Diagnosing pulmonary tuberculosis (TB) may be delayed until culture results become available.We ascertained the accuracy of a stepwise diagnostic algorithm for the rapid diagnosis of pulmonary TB by GeneXpert from sputum and/or bronchoalveolar lavage (BAL) followed by aMycobacterium tuberculosis-specific BAL ELISPOT assay in patients with a suspected diagnosis of pulmonary TB at a clinical referral centre in Germany.Among 166 patients with a presumptive diagnosis of pulmonary TB, 81 cases were confirmed byM. tuberculosisculture from sputum and/or BAL. In 66 out of 81 (81.5%) cases, patients initially hadM. tuberculosisdetected by GeneXpert from sputum; in addition, six out of 81 (7.4%) cases were diagnosed by GeneXpert on BAL fluid (together 72 out of 81 (88.9%) patients). Out of the remaining nine patients with negative GeneXpert results from sputum and BAL, BAL ELISPOT identified eight patients with culture-confirmed TB correctly (median time to culture positivity 26 days). At a cut-off of >4000 early secretory antigenic target-6- or culture filtrate protein-10-specific interferon-γ-producing lymphocytes per 1 000 0000 lymphocytes, the specificity of the BAL ELISPOT for active TB was 97%.In low TB incidence countries, nearly all patients with active pulmonary TB can be identified within the first few days of clinical presentation using a stepwise strategy with GeneXpert and BAL ELISPOT.

DIFFERENCES OF LYMPHOCYTE PROLIFERATION INDEX AFTER CULTURE FILTRATE PROTEIN 10 (CFP-10) STIMULATION IN PATIENTS WITH ACTIVE AND LATENT TUBERCULOSIS AND HEALTHY INDIVIDUALS

Angka kejadian tuberkulosis di dunia semakin meningkat, akibat rendahnya ketepatgunaan vaksin BCG untuk pencegahan infeksi TB.Saat ini telah dikembangkan vaksin DNA dari protein M.tuberculosis yaitu Culture filtrate protein-10 (CFP-10) yang dapat merangsangrespons imun seluler. Tujuan penelitian ini adalah mengetahui salah satu kemampuan antigenik antigen CFP-10 pasca stimulasi CFP-10 di pasien TB aktif, TB laten dan orang sehat. Penelitian bersifat eksperimen semu (quasi experimental). Sampel penelitian adalahPeripheral blood mononuclear cell (PBMC) dari 10 pasien TB paru aktif baru, 10 TB laten RS Khusus Paru Surabaya dan 10 orangsehat. Perlakuan kultur PBMC tanpa stimulasi (pembanding), dengan Mitogen (PHA) sebagai pembanding positif dan dengan stimulasiantigen CFP-10 diinkubasi pada CO2 5% selama 5 hari. Uji proliferasi limfosit dilakukan dengan menambahkan MTT. Indeks proliferasilimfosit adalah rasio antara absorban pembanding dan absorban dengan stimulasi pada λ 560 nm. Terdapat perbedaan bermakna indeksuji proliferasi limfosit pascastimulasi CFP-10 antara pasien TB paru aktif dan orang sehat (p=0,019) dan di pasien TB aktif, TB latendan orang sehat (p=0,0356). Antigen CFP 10 mampu merangsang respons imun protektif pada pasien TB aktif, TB laten dan orangsehat. Aktivitas imunogenik antigen CFP-10 dapat dipengaruhi oleh status imun seseorang dan kemampuan antigen CFP menginduksirespons imun protektif.

Humoral immune response to selective mycobacterial antigens and serodiagnosis of active tuberculosis in Bangladeshi patients

Background and objective: Serological test has become an important tool in the diagnosis of TB cases. This study focused on the analysis and comparison of antibody response to two Mycobacterium tuberculosis (MTB) antigens namely Ag85 complex and culture filtrate protein (CFP) in patients with tuberculosis. Antibody response to specific antigen was utilized as a diagnostic tool to detect active tuberculosis (TB) cases.Methods: Sera from 30 patients with active tuberculosis and 30 healthy individuals were tested by enzyme linked immunosorbent assay (ELISA) for the presence of immunoglobulin (Ig) G and IgM antibodies against Ag85 complex and culture filtrate protein (CFP) antigens of MTB.Results: The mean OD values of serum IgM and IgG antibodies against Ag85 and CFP were significantly (p<0.0001) higher in patients than that of healthy control individuals. Among the 30 tuberculosis patients, anti-Ag85 complex IgM and IgG was positive in 66.7% and 70.0% patients respectively. The seropositive rate of anti-CFP IgM and IgG was 33.3% and 56.7% respectively. The sensitivity and specificity of anti Ag85 and anti-CFP IgM and IgG ranged from 60.0% to 96.7%.Conclusion: The study demonstrated that determination of IgM and IgG antibodies against Ag85 complex and CFP could be used as a serological marker for diagnosis of active tuberculosis.IMC J Med Sci 2016; 10(2): 53-57