Novel health system strategies for tuberculin skin testing at primary care clinics: Performance assessment and health economic evaluation.

BACKGROUND: Tuberculin skin test (TST) for guiding initiation of tuberculosis preventive therapy poses major challenges in high tuberculosis burden settings. METHODS: At a primary care clinic in Johannesburg, South Africa, 278 HIV-positive adults self-read their TST by reporting if they felt a bump (any induration) at the TST placement site. TST reading (in mm) was fast-tracked to reduce patient wait time and task-shifted to delegate tasks to lower cadre healthcare workers, and result was compared to TST reading by high cadre research staff. TST reading and placement cost to the health system and patients were estimated. Simulations of health system costs were performed for 5 countries (USA, Germany, Brazil, India, Russia) to evaluate generalizability. RESULTS: Almost all participants (269 of 278, 97%) correctly self-identified the presence or absence of any induration [sensitivity 89% (95% CI 80,95) and specificity 99.5% (95% CI 97,100)]. For detection of a positive TST (induration ≥ 5mm), sensitivity was 90% (95% CI 81,96) and specificity 99% (95% CI 97,100). TST reading agreement between low and high cadre staff was high (kappa 0.97, 95% CI 0.94, 1.00). Total TST cost was 2066 I$ (95% UI 594, 5243) per 100 patients, 87% (95% UI 53, 95) of which were patient costs. Combining fast-track and task-shifting, reduced total costs to 1736 I$ (95% UI 497, 4300) per 100 patients, with 31% (95% UI 15, 42) saving in health system costs. Combining fast-tracking, task-shifting and self-reading, lowered the TST health system costs from 16% (95% UI 8, 26) in Russia to 40% (95% UI 18, 54) in the USA. CONCLUSION: A TST strategy where only patients with any self-read induration are asked to return for fast-tracked TST reading by lower cadre healthcare workers is a promising strategy that could be effective and cost-saving, but real-life cost-effectiveness should be further examined.

Antibody Fc Glycosylation Discriminates Between Latent and Active Tuberculosis.

BACKGROUND: Mycobacterium tuberculosis remains a global health problem and clinical management is complicated by difficulty in discriminating between latent infection and active disease. While M. tuberculosis-reactive antibody levels are heterogeneous, studies suggest that levels of IgG glycosylation differ between disease states. Here we extend this observation across antibody domains and M. tuberculosis specificities to define changes with the greatest resolving power. METHODS: Capillary electrophoretic glycan analysis was performed on bulk non-antigen-specific IgG, bulk Fc domain, bulk Fab domain, and purified protein derivative (PPD)- and Ag85A-specific IgG from subjects with latent (n = 10) and active (n = 20) tuberculosis. PPD-specific isotype/subclass, PPD-specific antibody-dependent phagocytosis, cellular cytotoxicity, and natural killer cell activation were assessed. Discriminatory potentials of antibody features were evaluated individually and by multivariate analysis. RESULTS: Parallel profiling of whole, Fc, and Fab domain-specific IgG glycosylation pointed to enhanced differential glycosylation on the Fc domain. Differential glycosylation was observed across antigen-specific antibody populations. Multivariate modeling highlighted Fc domain glycan species as the top discriminatory features, with combined PPD IgG titers and Fc domain glycans providing the highest classification accuracy. CONCLUSIONS: Differential glycosylation occurs preferentially on the Fc domain, providing significant discriminatory power between different states of M. tuberculosis infection and disease.

Simple and rapid liquid chromatographic and electrophoretic methods for phenol quantification and its stability in tuberculin purified protein derivative preparations.

Phenol is commonly used as an antimicrobial agent with an initial concentration of 0.35% (w/v) in injectable diluted tuberculin purified protein derivative (TPPD) solution. The anti-microbial action of phenol in TPPD is directly concentration dependent. Furthermore, high phenol content (>0.5%) may have a negative effect on the stability and clinical effectiveness of TPPD solution. Therefore, simple, rapid and reliable reversed phase liquid chromatographic (RPLC) and capillary zone electrophoretic (CZE) methods were firstly developed and validated for phenol quantification in Connaught tuberculin (CT68) PPD diluted preparations at 5 TU per test dose of 0.1 mL. In RPLC, the elution was carried out by 80% (v/v) ACN mixed with 20% (v/v) phosphate buffer containing 0.05% (v/v) triflouroacetic acid (pH 3.2) at 0.2 mL min-1 flow rate and 20.0 °C column temperature. In addition, phenol was separated from tuberculin (CT68) protein with a resolution of (R = 2.81) and was quantified within 3 min. In CZE, the migration of phenol was performed by 50 mmol L-1 borate buffer (pH 9.8) at -20 kV applied voltage and 25.0 °C capillary temperature. Furthermore, excellent linearity was achieved within 0.17-0.53% (w/v) for the phenol content with coefficients of determination (r2) higher than 0.9995. Moreover, the detection and quantification limits were found to be 0.046 & 0.153% and 0.051 & 0.171% (w/v) with RPLC and CZE respectively. Additionally, the intraday precision (RSD%, n = 9) was ranged between 0.18 and 0.39 and 0.33-54 with RPLC and CZE respectively. Moreover, the interday precision (RSD%, n = 27) was varied between 2.06 and 2.99 and 2.25-3.40 by RPLC and CZE, respectively. Furthermore, the obtained mean recoveries were ranged between 91.32 and 107.51% with RPLC and 90.71-108.92% with CZE. In addition, the effect of different storage temperatures at 4, 25 and 37 °C over storage periods of 2, 7, 14, 21 and 30 days was also studied on the TPPD product. The obtained results have revealed that the phenol content was effectively decreased about 37% of its original content after 30 days at storage temperatures of 25 and 37 °C. However, the phenol content did not change and was stable up to 21 days at storage temperature of 4 °C. Therefore, the simple and rapid proposed analytical methods could be used for a rapid expiry investigation of TPPD products based on phenol quantification, as a marker.

Potencial do teste IGRA (Interferon Gama Release Assay) para o diagnóstico de tuberculose ocular. Revisão e análise comparativa com o teste tuberculínico cutâneo (PPD) / The potential of the IGRA (Interferon Gamma Release Assay) test for the diagnosis of ocular tuberculosis. Review and comparative analysis with the tuberculosis skin test

Resumo A detecção precisa da infecção latente por tuberculose está se tornando cada vez mais importante devido ao aumento do uso de medicamentos imunossupressores e da epidemia do vírus da imunodeficiência humana, o que aumentou o risco de reativação à tuberculose ativa (TB). O Teste IGRA QuantiFERON® TB Gold apresenta vantagens frente ao teste de PPD como por exemplo, requer somente uma coleta de amostra sanguínea ; não há necessidade que o paciente retorne ao laboratório para leitura e interpretação dos resultados; Os resultados são objetivos, não requerem interpretação do leitor ou interferência de critérios subjetivos; trata-se de um teste in vitro, portanto não há "efeito booster" (potenciação da reação tuberculínica); o teste não é afetado por vacinação prévia por BCG ou infecção por outras espécies de micobactérias. Limitações são descritas, apesar de raras, como reações cruzadas deste método com infecções por algumas espécies de micobactérias não-tuberculosis (incluindo Mycobacterium kansasii, Mycobacterium szulgai e Mycobacterium marinum). Ainda há poucos dados sobre o teste IGRA em certas populações, como por exemplo, em crianças, pacientes imunocomprometidos e mulheres grávidas. Nestes grupos, a interpretação do teste pode ser difícil e mais estudos se fazem necessários.

Abstract Precise detection of latent tuberculosis infection is becoming increasingly important due to increased use of immunosuppressive drugs and the human immunodeficiency virus epidemic , which increased the risk of reactivation to active tuberculosis (TB).The QuantiFERON® TB Gold IGRA Test has advantages over the skin test for TB, otherwise known as a Mantoux tuberculin test, for example, requires only a blood sample collection; there is no need for the patient to return to the laboratory for reading and interpretation of the results; The results are objective, do not require interpretation of the reader or interference of subjective criteria; it is an in vitro test, so there is no "booster effect" (potentiation of the tuberculin reaction); the test is not affected by prior BCG vaccination or infection with other species of mycobacteria. Limitations are described, although rare, as cross-reactions of this method with infections by some species of non-tuberculosis mycobacteria (including Mycobacterium kansasii, Mycobacterium szulgai and Mycobacterium marinum). There is still little data on the IGRA test in certain populations, such as in children, immunocompromised patients and pregnant women. In these groups, the interpretation of the test can be difficult and more studies are needed.

Ojo rojo en tatuaje: Un curioso ojo rojo de etiología tuberculosa / Red-eyed-tatoo: An unusual red eye of tuberculous etiology

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Proteomic analysis of protein purified derivative of Mycobacterium bovis.

BACKGROUND: Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). METHODS: Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. RESULTS: Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. CONCLUSIONS: This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Linfadenitis por micobacterias no tuberculosas: experiencia de 15 años / Lymphadenitis due to non-tuberculous mycobacteria: experience over 15 years

OBJETIVO: Estudiar la epidemiología, las manifestaciones clínicas, el manejo diagnóstico-terapéutico y la evolución de las linfadenitis por micobacterias no tuberculosas en la población pediátrica de Aragón. MATERIAL Y MÉTODOS: Estudio retrospectivo de pacientes menores de 15 años diagnosticados de linfadenitis por micobacteria no tuberculosa entre 2000 y 2015. CRITERIOS DE INCLUSIÓN: pacientes con linfadenitis y cultivo positivo. Los resultados se expresan como medias, rango y desviación típica para las variables cuantitativas, y porcentajes para las cualitativas. RESULTADOS: Se detectan 27 casos, edad media de presentación 39,9 meses (rango 10 meses-8 años). El tiempo desde inicio de los síntomas hasta la primera consulta especializada es 1,7 ± 1,1 meses. La localización más frecuente es submaxilar en 17/27 casos (63%), lado derecho en el 59,3%, con tamaño de 2,96 ± 1,26 cm. Solo 16/27 presentan fistulización. Prueba de tuberculina superior a 10 mm en 7/24 (29,1%). El cultivo es positivo para Mycobacterium avium en 14/27 (51,9%), Mycobacterium intracellulare 3/27 (11,1%), Mycobacterium lentiflavum 3/27 (11,1%). El 92,6% (23/27) es tratado inicialmente con amoxicilina-clavulánico. La combinación de antibióticos y cirugía se aplica en 16/27 casos (59,3%), solo antibioterapia 7/27 (25,9%) y únicamente exéresis 4/27 (14,8%). Dos pacientes precisan reintervención y un caso desarrolla neutropenia grave secundaria a rifabutina. Solo un caso (3,7%) presenta parálisis facial transitoria como secuela. CONCLUSIONES: La combinación de antibioterapia y cirugía es el tratamiento más frecuente. El retraso en el diagnóstico hace que la exéresis como primera opción terapéutica se realice únicamente en uno de cada 7 pacientes

OBJECTIVE: To study the epidemiology, clinical features, diagnosis, therapeutic management, and outcome of non-tuberculous mycobacterial lymphadenitis in a paediatric population of Aragón (Spain). MATERIAL AND METHODS: A retrospective study was conducted on patients under 15 years-old diagnosed with non-tuberculous mycobacterial lymphadenitis between the years 2000 and 2015. Inclusion criteria: patients with lymphadenitis and positive culture. Quantitative values are shown as mean, rank, and standard deviation, and qualitative data as frequencies. RESULTS: Twenty-seven cases were registered, with a mean age of presentation of 39.9 months (range 10 months-8 years). The mean time between the symptoms onset and first consultation was 1.7 ± 1.1 months. The most frequent location was sub-maxilar in 17/27 cases (63%), on the right side in 59.3%, and size 2.96 ± 1.26 cm. Fistulae were observed in 16/27 cases. Tuberculin test was greater than 10 mm in 7/24 (29.1%). Microbiological cultures were positive for Mycobacterium avium in 14/27 (51.9%), Mycobacterium intracellulare 3/27 (11.1%), and Mycobacterium lentiflavum 3/27 (11.1%). Combined treatment of antibiotics and surgery was given in 16/27 cases (59.8%), medical treatment only in7/27 (25.9%), and surgical exeresis alone in 4/27 (14.8%). Two patients required a new surgery, and one showed severe neutropenia secondary to rifabutin. Only one case (3.7%) suffered from temporary facial palsy as sequel. CONCLUSIONS: The most frequent treatment was the combination of antibiotics and surgery. Delay in diagnosis seemed to be responsible for the limited number of exeresis as first option, only one for every seven patients

Investigations and implications of associations between mycobacterial purified protein derivative hypersensitivity and MAP-antibody ELISA in Irish dairy cows.

Intradermal testing, involving administration of purified protein derivative (PPD), to elicit a delayed hypersensitivity (DTH) response, is used as a diagnostic tool for bovine tuberculosis (bTB) and to aid in the identification of exposure to Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD). Further research is required to increase the diagnostic value of skin testing for MAP. The aim of this study was to investigate if animals showing DTH reactions to PPD had an associated increase in MAP ELISA response, thereby identifying potential cases of sub-clinical JD. A 139-cow dairy herd was recruited to the study. During the mandatory annual bTB test, skin thickness measurements (mm) were recorded at the site of avian and bovine PPD administration. Cows were categorised based on recording no DTH, DTH at both PPD administration sites and DTH at one PPD site only. Blood samples were collected pre and post bTB testing, and ELISA tested. Generalised estimating equations were performed to identify associations between DTH responses and MAP ELISA results. Significant associations were identified between PPD DTH responses and MAP ELISA readings. Animals with DTH at both avian and bovine PPD sites were most likely to test ELISA positive in the post-PPD period relative to other categories. Further research is required to identify whether skin thickness increases post-PPD and associated increase in ELISA response, identifies animals previously exposed to MAP, or if results are due to cross reactivity.

Un caso de linfadenitis granulomatosa por Mycobacterium tuberculosis en nuestro medio / Granulomatous lymphadenitis by Mycobacterium tuberculosis in our midst

La afectación ganglionar por Mycobacterium tuberculosis puede ocurrir tras una diseminación linfohemática a partir de una afectación primaria pulmonar, o por primoinfección extrapulmonar, cuya puerta de entrada son las mucosas o contacto con objetos contaminados. Se presenta el caso de un niño de diez años, nacido en España, afecto de adenitis tuberculosa en la región inguinal, cuya infección se produjo tras una herida en el pie ocurrida en una playa de Brasil. Tras el inicio de la terapia antituberculosa desarrolló una escrófula que requirió desbridamiento quirúrgico, con buena evolución posterior (AU)

Tuberculous lymphadenitis can be caused either by an haematogenous spread from a pulmonary primary form or by an extrapulmonary infection due to cutaneous lesions or contact with contaminated items. We present a 10-year-old spanish boy, who has an inguinal tuberculous lymphadenitis. Infection was secondary to a foot injury caused in a Brazilian beach. A scrofula appeared after triple therapy was instaured, needing surgical debridement with a favourable clinical evolution (AU)

Tuberculosis infantil: presentación de 3 casos y discusión / Pediatric tuberculosis: case report and discussion

Se presentan tres casos de tuberculosis infantil. Los dos primeros casos son dos hermanos de 5 años y 20 meses de edad con tuberculosis pulmonar, contactos intradomiciliarios del caso índice, la madre de ambos afecta de tuberculosis pulmonar bacilífera, con antecedente de viaje hace 4 meses a su pais de origen (Marruecos); ambos diagnosticados por positividad de la prueba de tuberculina y estudio radiográfico. El tercero es una niña de 9 años afecta de artritis idiopática juvenil en tratamiento inmunosupresor que desarrolló tuberculosis miliar con afectación encefálica, diagnosticada inicialmente en contexto de estudio de adenopatía (AU)

Three cases of childhood tuberculosis are presented: first and second cases are two brothers of 5 years and 2.0 months old respectively with pulmonary tuberculosis, household contacts of the index case: their mother, who was affected by smear-positive pulmonary tuberculosis, four months after visiting their country of origin (Morocco); both brethren were diagnosed by positivity of the tuberculin test and radiographic study. The third case is a 9 ycar old girl, juvenile idiopathic arthritis patient treated With immunosuppressive therapy, who has developed miliary tuberculosis with meningoencephalic involvement and she was initially diagnosed by the study of an adenopathy (AU)

Mycobacterium bovis en fauna silvestre de la cuenca lechera de Santa Fe, Argentina / Mycobacterium bovis in wildlife of the dairy regions of Santa Fe (Argentina)

El control y la erradicación de la tuberculosis bovina basados en la detección de los animales infectados y su inmediata faena permitió lograr progresos satisfactorios en varios países y regiones, pero no todos pudieron lograrlo debido principalmente a la presencia de fauna silvestre infectada con Mycobacterium bovis. La Argentina aplica desde 1999 estas mismas premisas y ha logrado avances en los rodeos lecheros, aunque no se ha evaluado el factor ambiental como la fauna silvestre. El objetivo de este trabajo fue determinar si la fauna silvestre de la cuenca lechera de Santa Fe está infectada con M. bovis. Se realizó la captura/sacrificio de fauna silvestre presente en 5 rodeos lecheros con altos niveles de reaccionantes positivos a la prueba de tuberculina. Sobre 95 mamíferos silvestres examinados, se aisló M. bovis de 7 individuos de comadreja overa (Didelphis albiventris), de uno de zorro gris (Lycolapex gimnocercus) y de uno de rata (Rattus norvegicus). Los sitios anatómicos que produjeron estos aislamientos variaron de acuerdo con las especies; en ninguno de los ejemplares evaluados se observaron lesiones macroscópicas de tuberculosis. Los espoligotipos de M. bovis aislados con mayor frecuencia de los animales silvestres correspondieron a los tipos 34 (4 aislamientos) y 12 (3 aislamientos); el primero es el más corrientemente aislado del ganado en Argentina. Se discute en este estudio el papel de la comadreja overa (D. albiventris) como hospedador circunstancial de M. bovis

Control eradication campaigns of bovine tuberculosis based on the «test and slaughter¼ approach were successful in many countries and regions; however, in some areas the infection persists and one of the main reasons is Mycobacterium bovis infection in wild life species. Argentina has applied the same approach since 1999, achieving progress in dairy cattle herds. Nonetheless, the wildlife role has never been investigated. The objective of this study was to determine if wildlife from the Santa Fe dairy area is infected with M. bovis. Wildlife species having a positive tuberculin skin test were captured in five dairy farms. Ninety five wildlife mammals were captured; M. bovis was recovered from 7 possums (Didelphys albiventris), from one fox (Lycolapex gimnocercus) and from one rat (Rattus norvegicus). None of the animals exhibited macroscopic lesions. The most frequently isolated M. bovis spoligotypes were types 34 (4 isolates) and 12 (3 isolates). Spoligotype 34 is the most frequently isolated type in Argentine cattle. The role of D. albiventris as spillover host of M. bovis is discussed in this study

Does an Electronic Health Record Improve Completeness of Prenatal Studies?

OBJECTIVE: To determine whether implementation of an electronic health record (EHR) would increase the rate of prenatal Human Immunodeficiency Virus (HIV) and purified protein derivative (PPD) testing. METHODS: Eligible participants received prenatal care and delivered at term at a single academic institution in March-April 2011, March-April 2012, and March-April 2013. As part of routine prenatal care, all women were tested for HIV and tuberculosis (via a PPD test) during each pregnancy. The 2011 cohort was charted on paper. The 2012 and 2013 cohorts were charted via EHR. To appear in the prenatal labs display in EHR, PPD results must be manually documented, while HIV results are uploaded automatically. Documentation of PPD and HIV tests were analyzed. RESULTS: The 2011, 2012, and 2013 cohorts had 249, 208, and 190 patients, respectively. Complete PPD and HIV results were less likely to be charted in the 2012 EHR cohort compared to the paper chart cohort (72.1% vs. 80.1%; p=0.03). This was driven by fewer documented completed PPD tests (2011 83.9% vs. 2012 72.6%; p=0.003). PPD test documentation improved non-significantly to 86.2% in the 2013 EHR cohort (p=0.5). HIV documentation rates increased from 95.2% in the paper chart cohort to 98.6% in the 2012 EHR cohort (p=0.04), and to 98.9% in the 2013 EHR cohort (p=0.03). CONCLUSIONS: EHR implementation corresponded with a marked decrease in documentation of PPD test completion. HIV documentation rates improved. PPD results were likely charted incorrectly in provider notes due to training deficiencies and lack of standardization, which did not improve significantly after retraining.

Increase in the number of tuberculosis cases treated following tuberculin skin testing in first-year schoolchildren in Madagascar.

BACKGROUND: Tuberculosis continues to cause unacceptably high levels of disease and death worldwide. Active preventive strategies are required to improve tuberculosis control and to increase the number of cases treated in developing countries. The aim of this study was to evaluate the utility of the tuberculin skin test (TST) in first-year schoolchildren as a means of increasing the number of tuberculosis cases detected through the screening of close contacts. METHODS: All members of the households of 90 schoolchildren assigned to three groups on the basis of TST category (≤ 5 mm, [5-15)mm, ≥ 15 mm) were screened for sputum smear-positive pulmonary tuberculosis. The percentage detection of tuberculosis in close contacts was compared between TST categories. RESULTS: We identified 433 close contacts of the 90 schoolchildren, who were then evaluated for tuberculosis. We identified 11 cases of pulmonary tuberculosis among the close contacts (7 already on treatment and 4 previously undiagnosed): 0 in TST category ≤ 5 mm, 3 in TST category [5-15) mm and 8 in TST category ≥ 15 mm). This approach increased the detection of tuberculosis cases by a factor of 1.6 in first-year schoolchildren of the TST ≥ 5 mm group. CONCLUSION: TST in first-year schoolchildren is a potentially effective method for improving the detection of tuberculosis in close contacts.

Long-incubation-time gamma interferon release assays in response to purified protein derivative, ESAT-6, and/or CFP-10 for the diagnosis of Mycobacterium tuberculosis infection in children.

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.

Screening for latent tuberculosis in Norwegian health care workers: high frequency of discordant tuberculin skin test positive and interferon-gamma release assay negative results.

BACKGROUND: Tuberculosis (TB) presents globally a significant health problem and health care workers (HCW) are at increased risk of contracting TB infection. There is no diagnostic gold standard for latent TB infection (LTBI), but both blood based interferon-gamma release assays (IGRA) and the tuberculin skin test (TST) are used. According to the national guidelines, HCW who have been exposed for TB should be screened and offered preventive anti-TB chemotherapy, but the role of IGRA in HCW screening is still unclear. METHODS: A total of 387 HCW working in clinical and laboratory departments in three major hospitals in the Western region of Norway with possible exposure to TB were included in a cross-sectional study. The HCW were asked for risk factors for TB and tested with TST and the QuantiFERON®TB Gold In-Tube test (QFT). A logistic regression model analyzed the associations between risk factors for TB and positive QFT or TST. RESULTS: A total of 13 (3.4%) demonstrated a persistent positive QFT, whereas 214 (55.3%) had a positive TST (≥ 6 mm) and 53 (13.7%) a TST ≥ 15 mm. Only ten (4.7%) of the HCW with a positive TST were QFT positive. Origin from a TB-endemic country was the only risk factor associated with a positive QFT (OR 14.13, 95% CI 1.37 - 145.38, p=0.026), whereas there was no significant association between risk factors for TB and TST ≥ 15 mm. The five HCW with an initial positive QFT that retested negative all had low interferon-gamma (IFN-γ) responses below 0.70 IU/ml when first tested. CONCLUSIONS: We demonstrate a low prevalence of LTBI in HCW working in hospitals with TB patients in our region. The "IGRA-only" seems like a desirable screening strategy despite its limitations in serial testing, due to the high numbers of discordant TST positive/IGRA negative results in HCW, probably caused by BCG vaccination or boosting due to repetitive TST testing. Thus, guidelines for TB screening in HCW should be updated in order to secure accurate diagnosis of LTBI and offer proper treatment and follow-up.

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis.

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.

Reduced CD27 expression on antigen-specific CD4+ T cells correlates with persistent active tuberculosis.

OBJECTIVE: CD27, a member of the tumor necrosis factor receptor family, has important role in generation of T cell immunity. In this study, association of CD27 expression on mycobacterial antigen-specific CD4+ T cells with pulmonary tuberculosis (TB) was investigated. METHODS: Mycobacterial antigen-specific CD4+ T cells were identified based on CD154 expression and CD27 expression on antigen-specific CD4 T cells was analyzed by flow cytometry. RESULTS: Compared with tuberculin-positive controls, patients with bacterial culture-positive pulmonary TB had significantly reduced CD27 expression on antigen-specific CD4 T cells. The persistent active TB patients had much lower percentages of CD27+ antigen-specific CD4 T cells than culture-positive new TB patients (P=0.008) and healthy controls (P=0.005). Logistic regression analysis on frequencies of CD27-expressing antigen-specific CD4 T cells and TB patients' clinical characteristics indicated that low percentage of CD27+ antigen-specific CD4 T cells correlated significantly with persistent active tuberculosis (P=0.002, odds ratio=19.6). CONCLUSION: It is concluded that frequency of CD27+ antigen-specific CD4 T cells could be used as an immunological marker for persistent active TB.

Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins.

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens. One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N=20) were significantly higher (p<0.01) than in control animals (N=20), despite the conserved nature of this protein. In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.

Diagnóstico y tratamiento de la tuberculosis / No disponible

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Prueba de la tuberculina y riesgo de tuberculosis. No sólo el tamaño es importante / The tuberculin skin test and risk of tuberculosis: size is not the only issue

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No disponible