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1.
J Allergy Clin Immunol ; 153(1): 297-308.e12, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37979702

RESUMEN

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.


Asunto(s)
Síndrome Linfoproliferativo Autoinmune , Receptor fas , Humanos , Síndrome Linfoproliferativo Autoinmune/diagnóstico , Síndrome Linfoproliferativo Autoinmune/genética , Biomarcadores , Variaciones en el Número de Copia de ADN , Secuenciación del Exoma , Receptor fas/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Mutación
2.
Endocr Connect ; 12(10)2023 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-37523248

RESUMEN

Glucocorticoids represent a key element in the treatment of pediatric acute lymphoblastic leukemia (ALL) and lead to adrenal suppression. We aimed to assess the differential response profile of adrenal steroids in children with ALL during BFM (Berlin-Frankfurt-Münster) induction treatment. Therefore, we performed liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based steroid profiling of up to seven consecutive leftover morning serum samples derived from 11 patients (pts) with ALL before (day 0) and during induction therapy at days 1-5, 6-12, 13-26, 27-29, 30-35 and 36-40. 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11S), cortisol, 11-deoxycorticosterone (DOC), corticosterone and aldosterone were determined in parallel. Subsequently, steroid concentrations were normalized by multiples of median (MOM) to adequately consider pediatric age- and sex-specific reference ranges. MOM-cortisol and its precursors MOM-11S and MOM-17OHP were significantly suppressed by glucocorticoid treatment until day 29 (P < 8.06 × 10-10, P < 5.102 × 10-5, P < 0.0076, respectively). Cortisol recovered in one of four pts at days 27-29 and in two of five pts at days 36-40. Among the mineralocorticoids, corticosterone was significantly suppressed (P < 3.115 × 10-6). Aldosterone and DOC showed no significant changes when comparing day 0 to the treatment time points. However, two ALL patients with ICU treatment due to the sepsis showed significantly lower MOM-DOC (P = 0.006436) during that time and almost always the lowest aldosterone compared to all other time points. Suppression of mineralocorticoid precursors under high-dose glucocorticoid therapy suggests a functional cross talk of central glucocorticoid regulation and adrenal mineralocorticoid synthesis. Our data should stimulate prospective investigation to assess potential clinical relevance.

3.
Lancet Reg Health Eur ; 28: 100599, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37180743

RESUMEN

Background: Due to the high risk of severe infection among pediatric hematology and oncology patients, antimicrobial use is particularly high. With our study, we quantitatively and qualitatively evaluated, based on institutional standards and national guidelines, antimicrobial usage by employing a point-prevalence survey with a multi-step, expert panel approach. We analyzed reasons for inappropriate antimicrobial usage. Methods: This cross-sectional study was conducted at 30 pediatric hematology and oncology centers in 2020 and 2021. Centers affiliated to the German Society for Pediatric Oncology and Hematology were invited to join, and an existing institutional standard was a prerequisite to participate. We included hematologic/oncologic inpatients under 19 years old, who had a systemic antimicrobial treatment on the day of the point prevalence survey. In addition to a one-day, point-prevalence survey, external experts individually assessed the appropriateness of each therapy. This step was followed by an expert panel adjudication based upon the participating centers' institutional standards, as well as upon national guidelines. We analyzed antimicrobial prevalence rate, along with the rate of appropriate, inappropriate, and indeterminate antimicrobial therapies with regard to institutional and national guidelines. We compared the results of academic and non-academic centers, and performed a multinomial logistic regression using center- and patient-related data to identify variables that predict inappropriate therapy. Findings: At the time of the study, a total of 342 patients were hospitalized at 30 hospitals, of whom 320 were included for the calculation of the antimicrobial prevalence rate. The overall antimicrobial prevalence rate was 44.4% (142/320; range 11.1-78.6%) with a median antimicrobial prevalence rate per center of 44.5% (95% confidence interval [CI] 35.9-49.9). Antimicrobial prevalence rate was significantly higher (p < 0.001) at academic centers (median 50.0%; 95% CI 41.2-55.2) compared to non-academic centers (median 20.0%; 95% CI 11.0-32.4). After expert panel adjudication, 33.8% (48/142) of all therapies were labelled inappropriate based upon institutional standards, with a higher rate (47.9% [68/142]) when national guidelines were taken into consideration. The most frequent reasons for inappropriate therapy were incorrect dosage (26.2% [37/141]) and (de-)escalation/spectrum-related errors (20.6% [29/141]). Multinomial, logistic regression yielded the number of antimicrobial drugs (odds ratio, OR, 3.13, 95% CI 1.76-5.54, p < 0.001), the diagnosis febrile neutropenia (OR 0.18, 95% CI 0.06-0.51, p = 0.0015), and an existing pediatric antimicrobial stewardship program (OR 0.35, 95% CI 0.15-0.84, p = 0.019) as predictors of inappropriate therapy. Our analysis revealed no evidence of a difference between academic and non-academic centers regarding appropriate usage. Interpretation: Our study revealed there to be high levels of antimicrobial usage at German and Austrian pediatric oncology and hematology centers with a significant higher number at academic centers. Incorrect dosing was shown to be the most frequent reason for inappropriate usage. Diagnosis of febrile neutropenia and antimicrobial stewardship programs were associated with a lower likelihood of inappropriate therapy. These findings suggest the importance of febrile neutropenia guidelines and guidelines compliance, as well as the need for regular antibiotic stewardship counselling at pediatric oncology and hematology centers. Funding: European Society of Clinical Microbiology and Infectious Diseases, Deutsche Gesellschaft für Pädiatrische Infektiologie, Deutsche Gesellschaft für Krankenhaushygiene, Stiftung Kreissparkasse Saarbrücken.

4.
Haematologica ; 108(12): 3278-3286, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-37021527

RESUMEN

Although initial central nervous system (CNS) involvement is rarely detected in childhood acute lymphoblastic leukemia (ALL), risk-adapted CNS-directed therapy is essential for all patients. Treatment intensity depends on the initial CNS status. In the AIEOP-BFM ALL 2009 trial, patients with cytomorphologic detection of leukemic blasts in initial cerebrospinal fluid were classified as CNS2 or CNS3 and received five intrathecal doses of methotrexate (MTX) in induction therapy compared to patients with CNS1 status (no blasts detected) who received three doses. The impact of additional intrathecal (IT) MTX on systemic toxicity in induction therapy is unknown. Between June 1st 2010 and February 28th 2017, a total of 6,136 ALL patients aged 1-17 years were enrolled onto the AIEOP-BFM ALL 2009 trial. The effect of three versus five doses of IT MTX during induction therapy on the incidence of severe infectious complications was analyzed. Among 4,706 patients treated with three IT MTX doses, 77 (1.6%) had a life-threatening infection during induction as compared to 59 of 1,350 (4.4%) patients treated with five doses (P<0.001; Odds Ratio 2.86 [95% Confidence Interval 1.99-4.13]). In a multivariate regression model, treatment with additional IT MTX proved to be the strongest risk factor for life-threatening infections (Odds Ratio 2.85 [1.96-4.14]). Fatal infections occurred in 16 (0.3%) and 38 (1.6%) patients treated with three or five IT MTX doses, respectively (P<0.001). As the relevance of additional intrathecal MTX in induction for relapse prevention in CNS2 patients is unclear, doses of intrathecal therapy have been reduced for these patients. (Clinicaltrials.gov identifiers: NCT01117441 and NCT00613457).


Asunto(s)
Metotrexato , Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Humanos , Metotrexato/uso terapéutico , Quimioterapia de Inducción/efectos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Terapia Combinada , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos
6.
JMIR Res Protoc ; 11(6): e35774, 2022 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-35723906

RESUMEN

BACKGROUND: Because infections are a major driver of morbidity and mortality in children with hematologic or oncologic diseases, antimicrobials are frequently prescribed in pediatric oncology practice. However, excess or inappropriate use of antimicrobials is directly linked to the emergence of antimicrobial resistance. Although point-prevalence studies have examined the extent of antimicrobial use, a comprehensive qualitative evaluation of individual antimicrobial prescriptions remains lacking. OBJECTIVE: The aim of this study is to identify appropriate versus inappropriate antimicrobial use among pediatric cancer patients in a point-prevalence study, followed by an expert panel adjudication process and a subsequent report of these findings to participating centers. This study also aims to improve the quality of patient care by informing centers about discrepancies between internal standards of care and national guidelines. METHODS: Our point-prevalence study is performed at pediatric cancer centers in Germany and Austria. All patients under 18 years old who are hospitalized at the time of the study are included. As a supplement to the point-prevalence study, an expert panel is qualitatively assessing each of the antimicrobial prescriptions at the participating centers to review local guidelines and compare them with national guidelines. RESULTS: As of December 2021, the point-prevalence survey has been conducted at 30 sites and expert panel adjudication for qualitative assessment of each antimicrobial use is ongoing. Results of the study are expected in 2022. CONCLUSIONS: This is the first point-prevalence study conducted among pediatric cancer centers with an integrated, multistep, qualitative approach that assesses each antimicrobial prescription. The results of this study will inform possible interventions for internal guidelines and antimicrobial stewardship programs implemented at pediatric cancer centers. In addition, local guidelines will be compared with national guidelines. Furthermore, this study will contribute to the overall integration of antimicrobial stewardship principles and initiatives in pediatric oncology and hematology, thereby improving safety and quality of care for children and adolescents with cancer and blood disorders. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/35774.

7.
Klin Padiatr ; 233(3): 101-106, 2021 May.
Artículo en Alemán | MEDLINE | ID: mdl-33971688

RESUMEN

Immunocompromised children and adolescents receiving treatment for cancer have an increased risk for potentially life-threatening infectious complications such as blood stream infections with Gram-positive and Gram-negative pathogens. Therefore, several centers for Pediatric Hematology and Oncology administer antibacterial prophylaxis to these patients to lower morbidity and mortality. Two pediatric specific guidelines on antibacterial prophylaxis were recently published. One of these guidelines was drawn up by an international group of pediatric experts of Europe, North and South America and Australia. The other guideline was prepared by an European group convened at the Eighth European Conference on Infections in Leukaemia (ECIL-8). In this review article, the working groups "Infections" of the Society of Pediatric Oncology and Hematology (GPOH) and "Fever in the neutropenic host" of the German Society for Pediatric Infectious Diseases" (DGPI) summarize the available data from randomized studies, systematic reviews and meta-analyses on antibacterial prophylaxis as well of current data on the emergence of resistance and discuss methodological aspects and the recommendations of the two guidelines.


Asunto(s)
Enfermedades Transmisibles , Hematología , Neoplasias , Adolescente , Antibacterianos/efectos adversos , Niño , Enfermedades Transmisibles/tratamiento farmacológico , Europa (Continente) , Humanos , Neoplasias/tratamiento farmacológico
8.
Klin Padiatr ; 230(3): 115-121, 2018 Apr.
Artículo en Alemán | MEDLINE | ID: mdl-29589346

RESUMEN

Immunocompromised children and adolescents receiving treatment for cancer have a considerably increased risk for infection. Neutropenia is the most important single risk factor for infectious complications, and fever in neutropenia is considered as an emergency. Whereas guidelines for the management of fever in neutropenic adults have been established for decades, specific pediatric guidelines have not been developed until recently. As children differ in many aspects from adults such as in the underlying malignancy or in the availability and dosing of antimicrobial compounds, guidelines for pediatric patients are important. This article reviews similarities and differences between the recently published German interdisciplinary guideline of the German Societies of Pediatric Infectious Diseases and Pediatric Oncology and Hematology and a guideline developed by a panel of international experts for the management of fever in neutropenia in children and adolescents.


Asunto(s)
Fiebre/diagnóstico , Fiebre/terapia , Hematología/normas , Oncología Médica/normas , Neoplasias/complicaciones , Neutropenia/diagnóstico , Neutropenia/terapia , Guías de Práctica Clínica como Asunto/normas , Adolescente , Adulto , Niño , Hematología/métodos , Humanos , Oncología Médica/métodos , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Sociedades Médicas/normas
10.
Br J Haematol ; 179(2): 272-283, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28771662

RESUMEN

The prognosis in children with refractory or relapsed (r/r) T-cell acute lymphoblastic leukaemia (T-ALL) or lymphoblastic lymphoma (T-LBL) is poor. Nelarabine (Ara-G) has successfully been used as salvage therapy in these children, but has been associated with significant, even fatal, neurotoxicities. We retrospectively analysed 52 patients with r/r T-ALL/T-LBL aged ≤19 years who were treated with Ara-G alone (n = 25) or in combination with cyclophosphamide and etoposide (n = 27). The majority of patients (45/52) received 1-2 cycles of Ara-G. Seventeen patients (32·7%) had refractory disease, 28 (53·8%) were in first relapse and 7 (13·5%) were in second relapse. A response to Ara-G was achieved in 20 patients and 15 (28·8%) were in remission at last follow-up. Twelve patients (23·1%) had neurotoxic adverse effects (neuro-AE) of any grade, of whom 7 (13·5%) developed neurotoxicity ≥ grade III. The most frequent neuro-AEs were peripheral motor neuropathy (19·2%), peripheral sensory neuropathy (11·5%) and seizures (9·6%). Three patients died of central neuro-AE after 1-2 cycles of combination therapy. Patients with neurotoxicity were significantly older (median 15·17 years) than those without (10·34 years, P = 0·017). No differences were observed between mono- and combination therapy concerning outcome and neuro-AE. The incidence of neuro-AE was not associated with concurrent intrathecal therapy or prior central nervous system irradiation.


Asunto(s)
Arabinonucleósidos/efectos adversos , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/epidemiología , Síndromes de Neurotoxicidad/epidemiología , Linfocitos T , Adolescente , Adulto , Factores de Edad , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Arabinonucleósidos/administración & dosificación , Niño , Preescolar , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Etopósido/administración & dosificación , Etopósido/efectos adversos , Femenino , Estudios de Seguimiento , Neoplasias Hematológicas/patología , Humanos , Masculino , Síndromes de Neurotoxicidad/patología , Estudios Retrospectivos
11.
Blood ; 130(13): 1543-1552, 2017 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-28698205

RESUMEN

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene (MLL)-rearranged ALL were established in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Antibody CD19-DE was efficient in prolonging the survival of NSG mice in a minimal residual disease (MRD) model. The majority of surviving mice remained polymerase chain reaction (PCR)-MRD negative after treatment. When antibody therapy was initiated in overt leukemia, antibody CD19-DE was still efficient in prolonging survival of xenografted mice in comparison with nontreated control animals, but the effects were less pronounced than in the MRD setting. Importantly, the combination of antibody CD19-DE and cytoreduction by chemotherapy (dexamethasone, vincristine, PEG-asparaginase) resulted in significantly improved survival rates in xenografted mice. Antibody CD19-DE treatment was also efficient in a randomized phase 2-like PDX trial using 13 MLL-rearranged BCP-ALL samples. Macrophage depletion by liposomal clodronate resulted in a reversal of the beneficial effects of CD19-DE, suggesting an important role for macrophages as effector cells. In support of this finding, CD19-DE was found to enhance phagocytosis of patient-derived ALL blasts by human macrophages in vitro. Thus, Fc-engineered CD19 antibodies may represent a promising treatment option for infants and children with MLL-rearranged BCP-ALL who have a poor outcome when treated with chemotherapy only.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Animales , Anticuerpos/genética , Anticuerpos/uso terapéutico , Antígenos CD19/genética , Antígenos CD19/inmunología , Femenino , Xenoinjertos , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Lactante , Macrófagos/citología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Neoplasia Residual/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Tumorales Cultivadas
12.
Br J Haematol ; 170(4): 539-49, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25940575

RESUMEN

Haemophagocytic lymphohistiocytosis (HLH) in the context of malignancy is mainly considered a challenge of adult haematology. While this association is also observed in children, little is known regarding inciting factors, appropriate treatment and prognosis. We retrospectively analysed 29 paediatric and adolescent patients for presenting features, type of neoplasm or preceding chemotherapy, treatment and outcome. Haemophagocytic lymphohistiocytosis was considered triggered by the malignancy (M-HLH) in 21 patients, most of whom had T- (n = 12) or B-cell neoplasms (n = 7), with Epstein-Barr virus as a co-trigger in five patients. In eight patients, HLH occurred during chemotherapy (Ch-HLH) for malignancy, mainly acute leukaemias (n = 7); an infectious trigger was found in seven. In M- and Ch-HLH, median overall survival was 1·2 and 0·9 years, and the 6 month survival rates were 67% and 63%, respectively. Seven of 11 deceased M-HLH patients exhibited active malignancy and HLH at the time of death, while only two out of five deceased Ch-HLH patients had evidence of active HLH. To overcome HLH, malignancy- and HLH-directed treatments were administered in the M-HLH cohort; however, it was not possible to determine superiority of one approach over the other. For Ch-HLH, treatment ranged from postponement of chemotherapy to the use of etoposide-containing regimens.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Leucemia , Linfohistiocitosis Hemofagocítica , Enfermedad Aguda , Adolescente , Adulto , Niño , Preescolar , Supervivencia sin Enfermedad , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/mortalidad , Femenino , Humanos , Lactante , Recién Nacido , Leucemia/tratamiento farmacológico , Leucemia/mortalidad , Linfohistiocitosis Hemofagocítica/etiología , Linfohistiocitosis Hemofagocítica/mortalidad , Linfohistiocitosis Hemofagocítica/terapia , Masculino , Estudios Retrospectivos , Tasa de Supervivencia
13.
Pediatr Blood Cancer ; 62(9): 1656-60, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25833113

RESUMEN

Histiocytic sarcoma (HS) is a rare disease with poor prognosis which may develop subsequent to acute lymphoblastic leukemia (ALL). Here we report two children treated within the AIEOP-BFM ALL 2009 trial: one patient succumbed to fulminant hemophagocytic lymphohistiocytosis triggered by HS during ALL maintenance therapy, the other patient had a smoldering course of HS for over 2 years, and subsequently died after allogeneic stem cell transplantation. In both cases, HS and ALL were clonally related and apparent return of minimal residual disease (MRD) was detected by qPCR in bone marrow. Thus, HS should be considered in ALL when MRD appears to persist or reappear.


Asunto(s)
Sarcoma Histiocítico/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Transdiferenciación Celular , Células Clonales/patología , Resistencia a Antineoplásicos , Resultado Fatal , Reordenamiento Génico de Linfocito T , Genes p16 , Trasplante de Células Madre Hematopoyéticas , Sarcoma Histiocítico/genética , Sarcoma Histiocítico/terapia , Humanos , Linfohistiocitosis Hemofagocítica/etiología , Células Madre Neoplásicas/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Recurrencia
14.
Leuk Res Rep ; 3(2): 36-7, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24918065

RESUMEN

We report on an 8 year old boy with primary cardiac anaplastic large cell lymphoma (ALCL), in whom the diagnosis was challenging and who was treated with modified chemotherapy without radiation therapy according to the ALCL 99 study protocol [1]. Two years and 4 months after completion of therapy the boy is in complete remission with normal cardiac function.

15.
Trans R Soc Trop Med Hyg ; 101(12): 1253-64, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17905372

RESUMEN

This study describes an RT-PCR assay targeting the L RNA segment of arenaviruses. Conserved regions were identified in the polymerase domain of the L gene on the basis of published sequences for Lassa virus, lymphocytic choriomeningitis virus (LCMV), Pichinde virus and Tacaribe virus, as well as 15 novel sequences for Lassa virus, LCMV, Ippy virus, Mobala virus and Mopeia virus determined in this study. Using these regions as target sites, a PCR assay for detection of all known Old World arenaviruses was developed and optimized. The concentration that yields 95% positive results in a set of replicate tests (95% detection limit) was determined to be 4290 copies of Lassa virus L RNA per ml of serum, corresponding to 30 copies per reaction. The ability of the assay to detect various Old World arenaviruses was demonstrated with in vitro transcribed RNA, material from infected cell cultures and samples from patients with Lassa fever and monkeys with LCMV-associated callitrichid hepatitis. The L gene PCR assay may be applicable: (i) as a complementary diagnostic test for Lassa virus and LCMV; (ii) to identify unknown Old World arenaviruses suspected as aetiological agents of disease; and (iii) for screening of potential reservoir hosts for unknown Old World arenaviruses.


Asunto(s)
Infecciones por Arenaviridae/diagnóstico , Fiebre de Lassa/diagnóstico , Virus Lassa/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Aminoácidos , Animales , Infecciones por Arenaviridae/genética , Arenavirus del Viejo Mundo/clasificación , Arenavirus del Viejo Mundo/genética , Secuencia de Bases , ADN Viral/análisis , Haplorrinos , Humanos , Fiebre de Lassa/genética , Virus Lassa/genética , Datos de Secuencia Molecular , Sensibilidad y Especificidad
16.
J Clin Virol ; 32(3): 229-35, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15722028

RESUMEN

BACKGROUND: Five of the known arenaviruses cause viral hemorrhagic fever in humans and are classified as biosafety level 4 pathogens. Four of the viruses, namely Junin, Guanarito, Machupo, and Sabia, belong to clade B of New World arenaviruses that also comprises the nonpathogenic viruses Tacaribe, Cupixi, and Amapari. OBJECTIVES: To establish real-time reverse transcription (RT)-PCR assays for Junin and Guanarito virus based on fluorescence resonance energy transfer (FRET) probes, and a universal RT-PCR assay for all known clade B viruses with conventional read-out. RESULTS: Conserved sequences in the nucleoprotein gene were chosen as target sites for primers and FRET probes. A common set of primers was designed for all three assays. The assays were based on one-step RT-PCR reagents and were optimised with respect to analytical sensitivity using synthetic RNA templates. The real-time PCR assays detected about 0.5 and 5TCID(50) of cell culture-derived Junin and Guanarito virus, respectively. The universal clade B PCR amplified cell culture-derived RNA of Junin, Guanarito, Machupo, and Sabia virus (5-500TCID(50) per reaction), as well as RNA of Tacaribe, Cupixi, and Amapari virus. CONCLUSIONS: The PCR assays may be used as complementary diagnostic tests for pathogenic New World arenaviruses. The universal PCR assay could also be suitable for the detection of novel clade B arenaviruses in patients as well as in animal reservoirs.


Asunto(s)
Arenavirus del Nuevo Mundo/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Infecciones por Arenaviridae/diagnóstico , Arenavirus del Nuevo Mundo/clasificación , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Humanos , Nucleoproteínas/genética , Sensibilidad y Especificidad , Células Vero , Proteínas Virales/genética
17.
Virology ; 318(1): 153-68, 2004 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-14972544

RESUMEN

The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses.


Asunto(s)
Virus Lassa/genética , ARN Polimerasa Dependiente del ARN/química , Análisis de Secuencia de ADN , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , ARN Viral/química , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Células Vero , Proteínas Virales/genética
18.
N Engl J Med ; 348(20): 1967-76, 2003 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-12690091

RESUMEN

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role in causing SARS.


Asunto(s)
Brotes de Enfermedades , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Coronavirus/genética , ADN Viral/análisis , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , ARN Viral/sangre , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Homología de Secuencia de Ácido Nucleico , Síndrome Respiratorio Agudo Grave/epidemiología , Esputo/virología
19.
Virus Genes ; 24(2): 153-6, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12018706

RESUMEN

Genotype G of hepatitis B virus (HBV) has only recently been discovered. This report describes the full-length sequence of genotype G HBV (designated 235/01) isolated in Germany from a chronic HBV carrier. The patient was hepatitis B e antigen-positive, had high HBV DNA levels of about 10(10) copies/ml serum, lacked a measurable anti-HBc response, and was coinfected with human immunodeficiency virus type 1. Genome 235/01 showed characteristic genotype G-specific features: stop codons at codon 2 and 28 of the pre-C region and insertion of 36 nucleotides at the 5' end of the C gene. It was nearly identical (< or = 99.7% identity) to both genotype G genomes (B1-89 and FR1 from France) described so far, suggesting either close epidemiological link among European genotype G isolates or high genetic stability of genotype G.


Asunto(s)
Genoma Viral , Virus de la Hepatitis B/clasificación , Hepatitis B Crónica/virología , Filogenia , Análisis de Secuencia de ADN , ADN Viral/análisis , Variación Genética , Genotipo , Alemania , Infecciones por VIH/complicaciones , VIH-1 , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/complicaciones , Humanos , Masculino , Datos de Secuencia Molecular
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