Performance of QuantiFERON-TB Gold Plus assays in children and adolescents at risk of tuberculosis: a cross-sectional multicentre study.

INTRODUCTION: The QuantiFERON-TB Gold Plus (QFT-Plus) assay, which features two antigen-stimulated tubes (TB1 and TB2) instead of a single tube used in previous-generation interferon-gamma release assays (IGRAs), was launched in 2016. Despite this, data regarding the assay's performance in the paediatric setting remain scarce. This study aimed to determine the performance of QFT-Plus in a large cohort of children and adolescents at risk of tuberculosis (TB) in a low-burden setting. METHODS: Cross-sectional, multicentre study at healthcare institutions participating in the Spanish Paediatric TB Research Network, including patients <18 years who had a QFT-Plus performed between September 2016 and June 2020. RESULTS: Of 1726 patients (52.8% male, median age: 8.4 years), 260 (15.1%) underwent testing during contact tracing, 288 (16.7%) on clinical/radiological suspicion of tuberculosis disease (TBD), 649 (37.6%) during new-entrant migrant screening and 529 (30.6%) prior to initiation of immunosuppressive treatment. Overall, the sensitivity of QFT-Plus for TBD (n=189) and for latent tuberculosis infection (LTBI, n=195) was 83.6% and 68.2%, respectively. The agreement between QFT-Plus TB1 and TB2 antigen tubes was excellent (98.9%, κ=0.961). Only five (2.5%) patients with TBD had discordance between TB1 and TB2 results (TB1+/TB2-, n=2; TB1-/TB2+, n=3). Indeterminate assay results (n=54, 3.1%) were associated with young age, lymphopenia and elevated C reactive protein concentrations. CONCLUSIONS: Our non-comparative study indicates that QFT-Plus does not have greater sensitivity than previous-generation IGRAs in children in both TBD and LTBI. In TBD, the addition of the second antigen tube, TB2, does not enhance the assay's performance substantially.

Dynamics of Reverse Transcription-Polymerase Chain Reaction and Serologic Test Results in Children with SARS-CoV-2 Infection.

OBJECTIVES: To determine the time to reverse transcription-polymerase chain reaction (RT-PCR) negativity after the first positive RT-PCR test, factors associated with longer time to RT-PCR negativity, proportion of children seroconverting after proven severe acute respiratory syndrome coronavirus 2 infection, and factors associated with the lack of seroconversion. STUDY DESIGN: The Epidemiological Study of Coronavirus in Children of the Spanish Society of Pediatrics is a multicenter study conducted in Spanish children to assess the characteristics of coronavirus disease 2019. In a subset of patients, 3 serial RT-PCR tests on nasopharyngeal swab specimens were performed after the first RT-PCR test, and immunoglobulin G serology for severe acute respiratory syndrome coronavirus 2 antibodies was performed in the acute and follow-up (<14 and ≥14 days after diagnosis) phase. RESULTS: In total, 324 patients were included in the study. The median time to RT-PCR negativity was 17 days (IQR, 8-29 days), and 35% of patients remained positive more than 4 weeks after the first RT-PCR test. The probability of RT-PCR negativity did not differ across groups defined by sex, disease severity, immunosuppressive drugs, or clinical phenotype. Globally, 24% of children failed to seroconvert after infection. Seroconversion was associated with hospitalization, persistence of RT-PCR positivity, and days of fever. CONCLUSIONS: Time to RT-PCR negativity was long, regardless of the severity of symptoms or other patient features. This finding should be considered when interpreting RT-PCR results in a child with symptoms, especially those with mild symptoms. Seroprevalence and postimmunization studies should consider that 11 in 4 infected children fail to seroconvert.

Interferon-Gamma Release Assays Differentiate between Mycobacterium avium Complex and Tuberculous Lymphadenitis in Children.

OBJECTIVES: To assess the performance of interferon-gamma release assays (IGRAs) in the differential diagnosis between Mycobacterium avium complex (MAC) and tuberculosis (TB) in children affected with subacute/chronic submandibular/cervical lymphadenitis. STUDY DESIGN: Multicenter observational study comparing children with microbiologically confirmed MAC lymphadenitis from the European NontuberculouS MycoBacterial Lymphadenitis in childrEn study with children with TB lymphadenitis from the Spanish Network for the Study of Pediatric TB database. RESULTS: Overall, 78 patients with MAC and 34 with TB lymphadenitis were included. Among MAC cases, 44 out of 74 (59.5%) had positive tuberculin skin test (TST) results at the 5-mm cut-off, compared with 32 out of 33 (97%) TB cases (P < .001); at the 10-mm cut-off TST results were positive in 23 out of 74 (31.1%) vs 26 out of 31 (83.9%), respectively (P < .001). IGRA results were positive in only 1 out of 32 (3.1%) patients with MAC who had undergone IGRA testing, compared with 21 out of 23 (91.3%) TB cases (P < .001). Agreement between TST and IGRA results was poor in MAC (23.3%; κ = 0.017), but good in TB cases (95.6%; κ = 0.646). IGRAs had a specificity of 96.9% (95% CI 84.3%-99.8%), positive predictive value of 95.4% (95% CI 78.2%-99.8%), and negative predictive value of 93.9% (95% CI 80.4%-98.9%) for TB lymphadenitis. CONCLUSIONS: In contrast to TST, IGRAs have high specificity, negative predictive value, and positive predictive value for TB lymphadenitis in children with subacute/chronic lymphadenopathy, and consequently can help to discriminate between TB and MAC disease. Therefore, IGRAs are useful tools in the diagnostic work-up of children with lymphadenopathy, particularly when culture and polymerase chain reaction results are negative.

The clinical relevance of the microbiome when managing paediatric infectious diseases-Narrative review.

In recent years, the field of infectious diseases has been hit by the overwhelming amount of information generated while the human microbiome is being disentangled. Based on the interaction between the microbiota and the immune system, the implications regarding infectious diseases are probably major and remain a challenge. AIMS: This review was conceived as a comprehensive tool to provide an overview of the available evidence regarding the influence of the microbiome on infectious diseases in children. METHODS: We present the main findings aroused from microbiome research in prevention, diagnosis and treatment of infectious disease under a paediatric perspective, to inform clinicians of the potential relevance of microbiome-related knowledge for translation to clinical practice. RESULTS AND CONCLUSION: The evidence shown in this review highlights the numerous research gaps ahead and supports the need to move forward to integrating the so-called microbiome thinking into our routine clinical practice.

Diagnostic Accuracy of QuantiFERON-TB Gold Plus Assays in Children and Adolescents with Tuberculosis Disease.

In 2016, a new interferon-gamma release assay, QuantiFERON-TB Gold Plus, was introduced. We conducted a cross-sectional multicenter study, involving 158 children and adolescents with tuberculosis disease. The overall sensitivity of the assay was 82.9% (IQR 77.0%-88.8%), indicating that in children this test does not have higher sensitivity than previous generation interferon-gamma release assays.

Semen searching when sperm is absent.

Sexual assault cases have varying factors that may mask semen findings when analysing evidence at the forensic laboratory. Semenogelin (Sg) is a potential marker for the identification of semen even at azoospermy or when few sperm cells are found. The current study examined Sg in normospermic and azoospermic donors as an internal evaluation of sensitivity, specificity and interference. The impact of a historical review of 53 judicial sexual assault cases over a five-year period was also analysed. The use of varying tests was of importance to prioritize certain samples within cases. Semen findings by Sg were then compared to prostate-specific antigen (PSA), phosphatase enzyme (AP) and Y-chromosome presence, the latter being used in an attempt to link semen fluid identification with obtaining a male DNA profile. Test findings were the highest ever registered for Sg (1:400,000), PSA (1:800,000), AP (1:25,000) and sperm cytology (SC) (1:50,000). Our results demonstrated the usefulness of using the Sg marker to avoid a false semen-negative result (6% cases), particularly in cases where sperm was absent or scarce (11% spermatozoa positive cases). Results were expressed in categories according to the set: Sg-PSA-AP. Thus, categories I (full positive, 46%), VI (full negative, 27%) and III (Sg/PSA positive; 11%) were the most frequent and Y-chromosome was obtained in 59%, 12% and 12% ratios, respectively. In conclusion, Sg was recommended for the workflow procedure of semen investigation when sperm absence is expected either from azoospermic/oligospermic or normospermic semen, especially before/after ejaculation.

Evaluation of radiological capacity and usage in paediatric TB diagnosis: A mixed-method protocol of a comparative study in Mozambique, South Africa and Spain.

INTRODUCTION: Tuberculosis remains one of the top ten causes of mortality globally. Children accounted for 12% of all TB cases and 18% of all TB deaths in 2022. Paediatric TB is difficult to diagnose with conventional laboratory tests, and chest radiographs remain crucial. However, in low-and middle-income countries with high TB burden, the capacity for radiological diagnosis of paediatric TB is rarely documented and data on the associated radiation exposure limited. METHODS: A multicentre, mixed-methods study is proposed in three countries, Mozambique, South Africa and Spain. At the national level, official registry databases will be utilised to retrospectively compile an inventory of licensed imaging resources (mainly X-ray and Computed Tomography (CT) scan equipment) for the year 2021. At the selected health facility level, three descriptive cross-sectional standardised surveys will be conducted to assess radiology capacity, radiological imaging diagnostic use for paediatric TB diagnosis, and radiation protection optimization: a site survey, a clinician-targeted survey, and a radiology staff-targeted survey, respectively. At the patient level, potential dose optimisation will be assessed for children under 16 years of age who were diagnosed and treated for TB in selected sites in each country. For this component, a retrospective analysis of dosimetry will be performed on TB and radiology data routinely collected at the respective sites. National inventory data will be presented as the number of units per million people by modality, region and country. Descriptive analyses will be conducted on survey data, including the demographic, clinical and programmatic characteristics of children treated for TB who had imaging examinations (chest X-ray (CXR) and/or CT scan). Dose exposure analysis will be performed by children's age, gender and disease spectrum. DISCUSSION: As far as we know, this is the first multicentre and multi-national study to compare radiological capacity, radiation protection optimization and practices between high and low TB burden settings in the context of childhood TB management. The planned comparative analyses will inform policy-makers of existing radiological capacity and deficiencies, allowing better resource prioritisation. It will inform clinicians and radiologists on best practices and means to optimise the use of radiological technology in paediatric TB management.

CXCL12γ isoform is expressed on endothelial and dendritic cells in rheumatoid arthritis synovium and regulates T cell activation.

OBJECTIVE: CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation. METHODS: Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti-pan-CXCL12 or anti-CXCL12γ-specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding-defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures. RESULTS: CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN-positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell-stimulatory effects of soluble CXCL12 proteins. CONCLUSION: CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.

The Value of the Second QuantiFERON-TB Gold-Plus Antigen Tube at Diagnosis and at Treatment Completion in Spanish Children With Tuberculosis.

We studied 295 children (tuberculosis disease, n = 159; latent tuberculosis infection, n = 136) with positive QuantiFERON-TB Gold-Plus assay results. No significant differences between first and second antigen tube interferon-gamma responses were detected, irrespective of patient and disease characteristics at diagnosis. Of patients with a repeat assay after treatment completion (n = 65), only 16.9% converted to negative results.

Adequacy of the 10 mg/kg Daily Dose of Antituberculosis Drug Isoniazid in Infants under 6 Months of Age.

In 2010, the WHO recommended an increase in the daily doses of first-line anti-tuberculosis medicines in children. We aim to characterize the pharmacokinetics of the once-daily isoniazid (INH) dose at 10 mg/kg of body weight in infants <6 months of age. We performed a multicenter pharmacokinetic study in Spain. The N-acetyltransferase 2 gene was analyzed to determine the acetylation status. Samples were analyzed using a validated UPLC-UV assay. A non-compartmental pharmacokinetic analysis was performed. Twenty-three pharmacokinetic profiles were performed in 20 infants (8 females) at a median (IQR) age of 19.0 (12.6-23.3) weeks. The acetylator statuses were homozygous fast (n = 1), heterozygous intermediate (n = 12), and homozygous slow (n = 7). INH median (IQR) Cmax and AUC0-24h values were 4.8 (3.7-6.7) mg/L and 23.5 (13.4-36.7) h*mg/L and the adult targets (>3 mg/L and 11.6-26.3 h*mg/L) were not reached in three and five cases, respectively. The age at assessment or acetylator status had no impact on Cmax values, but a larger INH AUC0-24h (p = 0.025) and trends towards a longer half-life (p = 0.055) and slower clearance (p = 0.070) were observed in homozygous slow acetylators. Treatment was well tolerated; mildly elevated alanine aminotransferase levels were observed in three cases. In our series of young infants receiving isoniazid, no major safety concerns were raised, and the target adult levels were reached in most patients.

[Serological profile of immigrant pregnant women against HIV, HBV, HCV, rubella, Toxoplasma gondii, Treponema pallidum, and Trypanosoma cruzi]. / Perfil serológico en gestantes extranjeras frente a VIH, VHB, VHC, virus de la rubéola, Toxoplasma gondii, Treponema pallidum, y Trypanosoma cruzi.

INTRODUCTION: The increase in immigration is changing the prevalence of mother to child infectious diseases. Our aim is to determine the serological profile of foreign pregnant women against these infections. METHODS: A retrospective cross sectional study was performed in a tertiary hospital from Madrid between August 2007 and October 2008. The seroprevalence against HIV, HBV, HCV, rubeola, T. gondii, T. pallidum and T. cruzi was determined in every pregnant immigrant, as well as in a representative group of Spanish pregnant women. RESULTS: A total of 2526 immigrant and 157 Spanish pregnant women were studied. None of the Spanish and 0.5% of the foreigners showed antibodies against HIV; 18.9% of them were Sub-Saharan women. Antigen HBs was detected in 2% of the immigrant women and in 1.1% of the Spanish women. Asian women had the highest rate of type B Hepatitis (10.9%). There was 0.9% of type C Hepatitis among the immigrants and 1% among the Spanish. Within the cases with RPR ≥ 1/8, 1.6% were immigrants, most of whom were Latin American. Thirty-one per cent of the immigrants showed antibodies against T. gondii (37.5% from Central America, 2.5% from the Far East). More than 95% of the Spanish women had antibodies against Rubella, this being lower in the rest of the areas (75.5% in Sub-Saharan Africa). T. cruzi infection was detected in 12.1% of the Bolivian women studied. CONCLUSION: The prevalence of mother-to-child transmitted infections depends on the origin of pregnant women. Knowledge of these differences may lead to improved control these diseases.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts.

OBJECTIVES: CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. METHODS: Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5'CXCL12 -1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. RESULTS: CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the -1.4 kb promoter. CONCLUSIONS: Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

QuantiFERON-TB Gold Plus Assay Specificity in Children and Adolescents With Suspected Tuberculosis-A Multicenter Cross-sectional Study in Spain.

In this cross-sectional study of 284 children and adolescents with clinically or radiologically suspected tuberculosis in a low-endemic country, the QuantiFERON-TB Gold Plus assay specificity, sensitivity, positive predictive value and negative predictive value were 91.5%, 87.3%, 86.4%, and 91.2%, respectively. The specificity was higher than that observed in tuberculin skin tests performed simultaneously, but similar to previous-generation interferon-gamma release assays.

Hepcidin treatment in Hfe-/- mice diminishes plasma iron without affecting erythropoiesis.

BACKGROUND: Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. MATERIALS AND METHODS: We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe(-/-)) and wild-type mice. RESULTS: Hepcidin reverted the high plasma iron concentrations in Hfe(-/-) mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe(-/-) mice. Hepcidin administration did not disturb erythropoiesis in either Hfe(-/-) or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe(-/-) and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. CONCLUSION: Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe(-/-) mice.

Immunohistochemical detection of intravascular platelet microthrombi in patients with lupus nephritis and anti-phospholipid antibodies.

OBJECTIVES: To evaluate whether the use of platelet immunohistochemistry (IHC) markers improves the sensitivity of histological methods to detect microthrombosis in SLE nephritis and aPLs and to analyse the clinicopathological correlations of microthrombosis in this setting. METHODS: Kidney biopsy specimens from 65 patients with SLE, including 36 with positive aPLs, were studied by IHC using antibodies against platelet glycoproteins CD41 and CD61. Clinical data at the time of kidney biopsy and during a mean follow-up of 7.5 years after biopsy were recorded and analysed with regard to histological or IHC data. RESULTS: Histological lesions previously defined as APS nephropathy were found in 33% of the SLE kidney biopsies and were not associated with positive aPLs. Microthrombi detected as intravascular CD61(+) platelet deposits were present in 43% of the tissues and were significantly associated with positive aPLs, but not with histological APS nephropathy, nephritis manifestations nor with renal outcome. Histological APS lesions but not CD61(+) microthrombi correlated with an older age at nephritis presentation, previous cardiovascular risk factors and worse renal outcome. CONCLUSIONS: Immunodetection of intravascular CD61(+) platelet aggregates is more sensitive than histological evaluation to detect acute microthrombosis and provides a better correlation with aPLs in SLE patients. In contrast, histological lesions consistent with APS nephropathy were not associated with aPLs but with cardiovascular risk factors and worse renal outcome.

Linezolid-containing Treatment Regimens for Tuberculosis in Children.

BACKGROUND: In recent years, there is an increasing interest in the use of linezolid for the treatment of tuberculosis (TB). METHODS: Patients less than 18 years of age who received linezolid within the Spanish Pediatric TB Network from 2001 to 2016 were retrospectively included. Treatment characteristics, adverse events (AEs) and outcomes were analyzed. RESULTS: Fifteen children were included (53% male) with a median age of 3.6 years [interquartile range (IQR): 1.6-6.2]. Median follow-up was 54 months (IQR: 38-76). The reasons for linezolid use were drug-resistant TB in 8 (53%) patients, drug-induced liver injury in 5 (33%) patients and chronic liver disease in 2 (13%) patients. Four children (26%) were on immunosuppressive therapy when TB was diagnosed. Five children (33%) were diagnosed with extrapulmonary TB. The median duration of linezolid treatment was 13 months (IQR: 7.5-17). Nine patients had 13 linezolid-related AEs. Hematologic toxicity was observed in 8 patients (53%) and gastrointestinal intolerance in 3 patients (20%). In 2 patients, linezolid dose was reduced, and in 2 patients, linezolid was discontinued because of AEs. A 2-year-old girl went back to her country of birth and was lost to follow-up. No relapses were observed among the other 14 patients (93%). CONCLUSIONS: Linezolid may be considered when treating children with drug-resistant TB but also in the cases of patients with chronic liver disease or drug-induced liver injury. However, AEs should be closely monitored. Further studies are needed to determine the optimum dosage and the optimal duration of linezolid treatment in children.

Immunoregulatory properties of vasoactive intestinal peptide in human T cell subsets: implications for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease whose pathogenesis is not completely understood. Unbalanced Th1/Th2 T-cell polarization has been suggested to play a pathogenetic role and therefore, modulation of T-cell polarization is a potential therapeutic target. Vasoactive intestinal peptide (VIP) is a broadly distributed peptide that exerts anti-inflammatory and immunomodulatory effects, in the collagen-induced arthritis (CIA) murine model of RA, and ex vivo, in synovial cells from RA patients. In the present study, we have found that polyclonal stimulation of peripheral blood lymphocytes (PBL) from RA patients produces higher levels of inflammatory mediators and lower levels of Th1 cytokines than PBL from healthy controls; moreover, VIP has negligible effects on inflammatory mediators and Th1 cytokines produced by PBL from healthy controls but favours Th2 profile and enhanced IL-10 production after stimulation. VIP increases the levels of IL-10 and IL-4 in the supernatant of human CD4(+)CD45RA(+) cells cultured in a non-conditioned or a Th2-conditioned situation. In contrast, VIP does not modify the production of these cytokines in a Th1-conditioned medium. In summary, VIP can differentially modify the functional capacity of human lymphocytes by inducing Th2/Treg differentiation depending on their previous phenotype.

[Paediatric research in Spain: Challenges and priorities. INVEST-AEP Platform]. / La investigación en Pediatría en España: retos y prioridades. Plataforma INVEST-AEP.

Research is the cornerstone of medical progress. Paediatric research has its own nuances and represents an additional challenge due to the intrinsic characteristics of the paediatric population compared with adults. Despite the tremendous importance of childhood health and its impact during adulthood, society is still not convinced about the importance of conducting research in paediatrics. This also applies to paediatricians themselves, who think about research as a discipline that does not directly involve them. The Spanish Academy of Paediatrics has developed a specific research platform- INVEST-AEP- to try to help and answer the challenges associated with paediatric research in the society This article reflects the current status of paediatric research in Spain, and the goals achieved over the last few years due to the effort of paediatric researchers. In addition, a deeper analysis is provided as regards: a) the barriers that represent a hurdle for the development of broad and competitive paediatric research in our day to day work; b) the limited incentives and specific pre- and post-doctoral training; c) the high clinical burden for paediatricians or; d) the lack of specific infrastructure and dedicated funding for paediatrics. The mission, vision and values of INVEST-AEP are to develop an accessible roadmap for the development and implementation of paediatric research in Spain for the next few years.

Optimising stable retroviral transduction of primary human synovial fibroblasts.

Fibroblast like synoviocytes are the main resident cells in normal joints and are known to play a major role in the pathogenesis of rheumatoid arthritis. Efficient gene targeting of fibroblast like synoviocytes (FLS) is a major goal of current ex vivo gene therapy approaches for the treatment of rheumatoid arthritis. However, there is a need to improve viral systems capable of delivering genes to human rheumatoid fibroblasts and attempts have been made to develop a protocol for high efficiency, reproducible gene transfer using a replication-defective retrovirus vector. The effects of different experimental conditions were examined as well as those related to cellular and viral features on the efficiency of transducing the retroviral-driven expression of enhanced green fluorescent protein (EGFP) to FLS harvested from patients with rheumatoid arthritis. The optimal method established involved a double round of infection by centrifugation with a resting period of 4h between rounds. This approach led to the transduction of 30-70% of FLS obtained from nine patients with rheumatoid arthritis. Consistent transduction efficiencies were achieved in repeat assays such that it could be inferred that the variations observed were attributable to the specific characteristics of each cell line. This simple protocol renders a consistent and reproducible efficiency of rheumatoid fibroblast transduction and makes stable gene targeting using non-replicating retrovirus derived vectors an affordable option for the treatment of rheumatoid arthritis.

The impact of Bacille Calmette-Guérin shortage on immunisation practice and policies in Europe - A Paediatric Tuberculosis Network European Trials Group (ptbnet) survey.

BACKGROUND: Recent reports indicate an ongoing BCG shortage that may influence immunisation practice. This study aimed to determine current availability of BCG vaccine across Europe, and implications on immunisation practices and policies in Europe. METHODS: Web-based survey among Paediatric Tuberculosis Network European Trials Group (ptbnet) members, between May and October 2015. RESULTS: Twenty individuals from 13 European countries participated. Ongoing shortages were reported in eight countries routinely using BCG (8/11, 73%). As a consequence of the shortage, BCG was not given as completely unavailable in some countries (2/8, 25%), was given only whenever available (1/8, 13%), or only in certain regions of the country (1/8, 13%). Strategies reported to reduce loss of immunisation were administration to selected high-risk individuals (2/8, 25%), or cohorting vaccinees on specific days to maximise the use of multi-dose vials (3/8, 38%). Authorities in two countries each were considering a change of manufacturer/supplier (2/8, 25%). CONCLUSIONS: The BCG shortage in Europe leads to significant changes in immunisation policies including changes of BCG vaccine strain and manufacturer. In addition, infants and children eligible for immunisation are at risk of not receiving BCG. To ensure necessary BCG immunisations, collaboration between national health agencies and vaccine manufacturers is crucial.