Vaccine-mismatched influenza B/Yamagata lineage viruses in Cuba, 2012-2013 season.

Annual trivalent influenza vaccines contain one of influenza B lineages; influenza B/Victoria-lineage or influenza B/Yamagata viruses. Theoretically, these vaccines should protect against viruses expected to circulate in the next influenza season. The National Influenza Centers, based on surveillance data from National Reference Laboratories, selects the strains composing each annual trivalent or tetravalent vaccine. Nevertheless, in some epidemics, vaccine strains do not match genetically with circulating strains. The aim of the present study is to compare the HA1-domain of 42 influenza B viruses circulating in Cuba during the 2012-2013 season with the vaccine strain B/Wisconsin/01/2010-like virus from the B/Yamagata lineage, included in the 2012-2013 Northern-Hemisphere Influenza vaccine. The efficacy of the influenza vaccine was also estimated. The analysis of the present study indicates that the B/Victoria and B/Yamagata lineages co-circulated in Cuba in the 2012-2013 season. In 2012-2013 season, according to the sequences analysis, trivalent vaccine did not match with the circulating strains. The present study also detected amino acid substitutions which could have altered the antigenic properties of HA gene. The results presented here suggest the need to consider a possible introduction of tetravalent influenza vaccine in Cuba, as has been recommended by the WHO to ensure higher levels of protection.

New genetic variants of influenza A(H1N1)pdm09 detected in Cuba during 2011-2013.

Influenza A(H1N1)pdm09 virus has evolved continually since its emergence in 2009. For influenza virus strains, genetic changes occurring in HA1 domain of the hemagglutinin cause the emergence of new variants. The aim of our study is to establish genetic associations between 35 A(H1N1)pdm09 viruses circulating in Cuba in 2011-2012 and 2012-2013 seasons, and A/California/07/2009 strain recommended by WHO as the H1N1 component of the influenza vaccine. The phylogenetic analysis revealed the circulation of clades 3, 6A, 6B, 6C and 7. Mutations were detected in the antigenic site or in the receptor-binding domains of HA1 segment, including S174P, S179N, K180Q, S202T, S220T and R222K. Substitutions S174P, S179N, K180Q and R222K were detected in Cuban strains for the first time.

Prospective, comprehensive, and effective viral monitoring in Cuban children undergoing solid organ transplantation.

PURPOSE: In Cuba, viral monitoring in the post-transplant period was not routinely performed. The aim of this research is to identify the most frequent viruses that affect transplanted Cuban children, by implementing a viral follow-up during the post-transplant period. METHODS: The study population included all Cuban pediatric patients who underwent solid organ transplantation (SOT) between November 2009 and December 2012. A total of 34 transplanted pediatric patients of kidney (n = 11) and liver (n = 23) were prospectively monitored during a 34-week period for viral DNAemia and DNAuria by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus, herpes simplex virus type 1 and 2, varicella zoster virus, human herpesvirus 6, human adenovirus, and polyomaviruses (BKV and JCV) using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Viral genome of at least one virus was detected in 21 of 34 recipients, 18 patients excreted virus in urine while 12 presented DNAemia. CMV (41.2%) and BKV (35.3%) were the most frequent viruses detected during the follow-up. CMV was the virus mainly associated with clinical symptoms and DNAemia. Its excretion in urine (with cut off value of 219 copies/mL) was associated with detection in plasma (p < 0.001); furthermore, CMV viruria was predictive of CMV viremia (OR:8.4, CI:2.4-29.1, p = 0.001). There was no association between high viral load and clinical complications, due to the prompt initiation of preemptive ganciclovir. CONCLUSION: This comprehensive viral monitoring program effectively prevents the development of critical viral disease, thus urge the implementation of qRT-PCR as routine for viral monitoring of transplanted Cuban organ recipients.

First report on fatal myocarditis associated with adenovirus infection in Cuba.

Myocarditis is caused frequently by viral infections of the myocardium. In the past, enteroviruses (EV) were considered the most common cause of myocarditis in all age groups. Other viruses that cause myocarditis are adenovirus and influenza viruses. Parvovirus B19 infection is associated sometimes with myocarditis. Members of the Herpesviridae family, cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) have been associated occasionally with myocarditis. During an atypical outbreak of acute febrile syndrome, eight children, with ages from 5 months to 15 years, died in cardiogenic shock due to myocarditis in July-August 2005, in the city of Havana, Cuba. Nested polymerase chain reaction (nPCR) and nested reverse transcription-PCR (nRT-PCR) were carried out on fresh heart muscle and lung tissue to analyze the genomic sequences of adenovirus, CMV, HHV-6, herpes simplex virus, Epstein-Barr virus (EBV), varizella zoster virus, influenza virus A, B, C, respiratory syncytial virus (RSV) A and B, parainfluenza viruses, rhinoviruses, coronavirus, flaviruses and enteroviruses. Evidence was for the presence of the adenovirus genome in 6 (75%) of the children. Phylogenetic analyses of a conserved hexon gene fragment in four cases showed serotype 5 as the causal agent. No others viruses were detected. Histological examination was undertaken to detect myocardial inflammation. After exclusion of other possible causes of death, the results indicated that viral myocarditis was the cause of death in patients with adenovirus infection.

A myocarditis outbreak with fatal cases associated with adenovirus subgenera C among children from Havana City in 2005.

BACKGROUND: Among multiple causes of acute myocarditis, viral infection, especially that due to enteroviruses and adenoviruses, is the leading cause. In the summer 2005 an outbreak of a febrile syndrome accompanied by acute cardiac decompensation occurred in infants and young children in Havana City. Eleven patients had a rapid evolution of disease and there were 8 fatalities from cardiac failure secondary to myocarditis. OBJECTIVE: The aim of the present study was to determine the etiological agent responsible for this outbreak. STUDY DESIGN: Children admitted to the pediatric hospitals of Havana City from July 3 to August 2 with this clinical presentation were studied. Forty samples of necropsy tissue, cerebrospinal fluid, stools and serum were tested by molecular methods for 14 respiratory viruses, 6 herpesviruses and generic enteroviruses and flavirus and alfaviruses. Viral isolation was performed in A-549 cells. Isolated viruses were typed by sequence analysis. RESULTS: Adenovirus genome was detected in 6 of the 8 fatal cases-the lungs in 5 (63%) and the myocardium in 3 (37%). In two fatal cases, viral genome was detected in both lung and myocardium. Adenovirus was isolated in five fatal cases. In all three non-fatal cases, adenovirus genome was detected and adenovirus was isolated into two. Sequence analysis showed that adenovirus type 5 was the only isolate from fatal cases and adenovirus 1 the only isolate in non-fatal cases. No other viruses were found by PCR or isolation techniques. CONCLUSION: Adenovirus was the etiologic agent implicated in this myocarditis outbreak and adenovirus type 5 was associated with fatal outcome.

Respiratory syncytial virus group A and B genotypes and disease severity among Cuban children.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of serious lower tract infections in infants. Comorbid conditions such as chronic diseases and prematurity have been associated with greater severity illness, but virus genotypes and disease severity is still unknown. METHODS: Forty selected strains of RSV group A and B from Cuban infants with acute respiratory disease (ARD) over five seasons were studied. Viral RNA was extracted and polymerase chain reaction (PCR) was carried out using direct primers directed to parts of the nucleoprotein (N) and fusion (F) genes, respectively. Amplicons were digested using restriction fragment length polymorphism (RFLP) to define the association between virus and disease severity. Disease severity was assessed as very mild, mild, moderate, and severe. RESULTS: Three of six known N genotypes were detected. NP4 and NP3 were found more frequently; moreover, it was difficult to establish a relationship between N genotypes and disease severity. Five genotypes in F gene were found: F1, F2, F5, F9 and F11; F9 and F11 were associated with very mild disease, but F1 genotype appears to be associated with moderate to severe disease. CONCLUSIONS: At least five combinations of N and F genotypes circulated in the studied infants in Cuba. Patients with F1NP4 genotype showed moderate to severe disease. Relationship between genotypes and disease severity was established.

Diferentes patrones de circulación dentro de los subgrupos A y B del virus sincitial respiratorio humano en algunas provincias de Cuba / Different patterns of circulation in subgroups A and B of human respiratory syncytial virus in some provinces of Cuba

Se realizó la secuenciación nucleotídica de la región del tercio C-terminal de la proteína G de 37 muestras de exudados nasofaríngeos, de niños menores de 1 año provenientes de algunas provincias de Cuba durante 5 períodos epidémicos (1995-2000), para conocer los patrones de circulación de cepas del virus sincitial respiratorio humano; el cual se clasifica en 2 subgrupos antigénicos A y B, y cada uno contiene múltiples variantes. El subgrupo A circuló durante todos los años, el subgrupo B se detectó solamente durante el año 2000. Dentro del subgrupo A se observó la presencia de cepas con 2 tamaños diferentes de la proteína G (297 aa y 298 aa), mientras que para el subgrupo B fue observado un único tamaño (295 aa). El análisis filogenético permitió identificar 5 y 2 genotipos dentro de los subgrupos A y B, respectivamente. Los virus de Cuba se relacionaron filogenéticamente con cepas de otras partes del mundo. Dentro del subgrupo A se encontraron 2 cepas, las cuales fueron muy similares a la cepa prototipo Long. Casi todas las cepas del año 2000 de ambos subgrupos, se agruparon filogenéticamente con cepas que circularon en Sudáfrica durante ese mismo período

Diferentes patrones de circulación dentro de los subgrupos A y B del virus sincitial respiratorio humano en algunas provincias de Cuba / Different patterns of circulation in subgroups A and B of human respiratory syncytial virus in some provinces of Cuba

Se realizó la secuenciación nucleotídica de la región del tercio C-terminal de la proteína G de 37 muestras de exudados nasofaríngeos, de niños menores de 1 año provenientes de algunas provincias de Cuba durante 5 períodos epidémicos (1995-2000), para conocer los patrones de circulación de cepas del virus sincitial respiratorio humano; el cual se clasifica en 2 subgrupos antigénicos A y B, y cada uno contiene múltiples variantes. El subgrupo A circuló durante todos los años, el subgrupo B se detectó solamente durante el año 2000. Dentro del subgrupo A se observó la presencia de cepas con 2 tamaños diferentes de la proteína G (297 aa y 298 aa), mientras que para el subgrupo B fue observado un único tamaño (295 aa). El análisis filogenético permitió identificar 5 y 2 genotipos dentro de los subgrupos A y B, respectivamente. Los virus de Cuba se relacionaron filogenéticamente con cepas de otras partes del mundo. Dentro del subgrupo A se encontraron 2 cepas, las cuales fueron muy similares a la cepa prototipo Long. Casi todas las cepas del año 2000 de ambos subgrupos, se agruparon filogenéticamente con cepas que circularon en Sudáfrica durante ese mismo período(AU)

[Different circulation patterns of subgroups A and B of human respiratory syncytial virus in some provinces of Cuba]. / Diferentes patrones de circulación dentro de los subgrupos A y B del virus sincitial respiratorio humano en algunas provincias de Cuba.

The nucleotide sequencing of the protein G C-terminal region of 37 samples taken from nasopharyngeal washings of under one-year old children from some Cuban provinces was made for 5 epidemic periods (1995-2000) to find out the circulation patterns of strains of human respiratory syncytial virus that is classified in two antigenic subgroups known as A and B; each of them contains multiple variants. Subgroup A has circulated during all these years but subgroup B was detected only in the year 2000. The presence of strains with two different sizes of protein G (297 aa and 298 aa) was observed whereas subgroup B showed only one size (295 aa). Phylogenetic analysis allowed identifying 5 and 2 genotypes within subgroups A and B respectively. Viruses present in Cuba were phylogenetically related to the strains of other parts of the world. Subgroup A comprised two strains very similar to Long prototype strain. Almost all the strains of both subgroups in the year 2000 phylogenetically related with strains that circulated in South Africa during the same period.

Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba

Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100 percent) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus

Variabilidad antigénica de cepas de virus sincitial respiratorio aisladas en ciudad de la Habana mediante ELISA utilizando anticuerpos monoclonales / Antigenic variance of strains of the espiratory sincitial virus isolated in Habana city by eans of ELISA using antibodies

Se realizó el estudio de caracterización y análisis de la variabilidad antigénica con un panel de 10 anticuerpos monoclonales que reconocen a epítopes lineales de la protéina G, a 15 cepas de Virus Sincitial Respiratorio aisladas en dos brotes ocurridos en Ciudad Habana entre los meses de octubre a enero (1994-1995) y octubre (1996), respectivamente. Para ello se utilizaron dos cepas de referencia: la Cepa Long y la Cepa A/Mon/3/88. El ELISA fue el método empleado para los ensayos. Los resultados mostraron la variabilidad de la proteína G entre los aislamientos y su relación antigénica con la cepa prototipo Long, además de confirmar que todas las cepas que habían circulado en estos períodos correspondían antigénicamente al Subgrupo A

Incidencia de los Adenovirus en las conjuntivitisvirales / Incidence of Adenovirus on viral conjunctivitis

Se llevó a cabo un estudio de incidencia de los Adenovirus en las conjuntivitis virales, para lo cual se realizó un diseño muestraly se tomaron muestras de exudado conjuntival a 150 pacientes con diagnóstico deconjuntivitis de presuntiva causa viral, en el Hospital Oftalmológico "Ramón Pando Ferrer" en el período comprendido entre julio y diciembre de 1994. Las muestras fueron inoculadas en cultivo celular y a las que presentaron un efecto citopatogénico sugestivo de infección por Adenovirus, se les realizó la técnica de inmunofluorescencia indirecta (IFI). Se usaron anticuerpos monoclonales contra Adenovirus, lo que permitió identificarlas como pertenecientes a la familia Adenoviridae. Se utilizó la técnica de hemaglutinación, con hematíes de monos y ratas como sistema indicador, para agrupar los aislamientos previamente identificados mediante la técnica de IFI, después se les realizó el análisis por enzimas de restricción del genoma viral, lo cual posibilitó la clasificación en tipos. Los resultados de este estudio mostraron una incidencia de los Adenovirus en las conjuntivitis virales de 20 por ciento con un intervalo de confianza del 14-26 por ciento y un índice de confiabilidad de 95 por ciento. Se demostró que el serotipo 37 fue el que produjo conjuntivitis con mayor frecuencia(AU)##o

Incidencia de los Adenovirus en las conjuntivitis virales / Incidence of Adenovirus on viral conjunctivitis

Se llevó a cabo un estudio de incidencia de los Adenovirus en las conjuntivitis virales, para lo cual se realizó un diseño muestral y se tomaron muestras de exudado conjuntival a 150 pacientes con diagnóstico de conjuntivitis de presuntiva causa viral, en el Hospital Oftalmológico "Ramón Pando Ferrer" en el período comprendido entre julio y diciembre de 1994. Las muestras fueron inoculadas en cultivo celular y a las que presentaron un efecto citopatogénico sugestivo de infección por Adenovirus, se les realizó la técnica de inmunofluorescencia indirecta (IFI). Se usaron anticuerpos monoclonales contra Adenovirus, lo que permitió identificarlas como pertenecientes a la familia Adenoviridae. Se utilizó la técnica de hemaglutinación, con hematíes de monos y ratas como sistema indicador, para agrupar los aislamientos previamente identificados mediante la técnica de IFI, después se les realizó el análisis por enzimas de restricción del genoma viral, lo cual posibilitó la clasificación en tipos. Los resultados de este estudio mostraron una incidencia de los Adenovirus en las conjuntivitis virales de 20 por ciento con un intervalo de confianza del 14-26 por ciento y un índice de confiabilidad de 95 por ciento. Se demostró que el serotipo 37 fue el que produjo conjuntivitis con mayor frecuencia(AU)

Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping

The aim of this study was to develop a polymerase chain reation (PCR) for detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respirary ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100 per cent sensitivity and 80 per cent specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

Evaluación de un conjugado anti-IgG de ratón-fluoresceína mediante técnicas de inmunofluorescencia indirecta y citometría de flujo / Evaluation of an anti-IgG conjugate of mouse-fluorescin by the indirect immunofluorescent techniques and flow citomerty

Se purificó una inmunoglobulina G de ratón a partir de suero por cromatografía de afinidad en proteína A. Con esta preparación se inmunizaron los conejos cuyos sueros fueron capaces de reconocer al antígeno inyectado mediante inmunodifusión doble. Los anticuerpos fueron precipitados del suero de conejo y purificados mediante cromatografía de intercambio iónico. Esta preparación fue conjugada a isotiocianato de fluorescencia según la tecnología convencional. El conjugado obtenido fue evaluado con las cepas de referencia de virus Parainfluenza 1, 2, 3; Adenovirus; virus sincitial respiratorio y virus influenza A y B por una técnica de inmunofluorescencia indirecta y muestras positivas de VIH mediante citometría de flujo. En ambos casos se utilizaron anticuerpos monoclonales específicos. Se evaluaron muestras clínicas de pacientes con infección respiratoria aguda

Aplicación de la reacción en cadena de la polimerasa para la identificación del virus sincitial respiratorio / Application of the polymerase chain reaction to identify the respiratory syncytial virus

Se desarrolló la reacción en cadena de la polimerasa (RCP) para la identificación del virus sincitial respiratorio (VSR) con el uso de la cepa de referencia. La alta sensibilidad y la especifidad obtenidas en nuestro trabajo demuestran la utilidad de la RCP para la detección del genoma del VSR y su aplicación en el diagnóstico