Emergence and evolution of SARS-CoV-2 genetic variants during the Cuban epidemic.

The SARS CoV-2 D614G variant circulated in Cuba in 2020. New viral variants were detected after the opening of the border in November 2020. We show the results of the genomic surveillance in Cuba from December 28, 2020, to September 28, 2021 and their relationship to the epidemiological situation in the country. A total of 1,406 nasopharyngeal exudates from COVID-19 patients were processed for RNA extraction and the 1836 bp fragment of the spike gene was amplified and sequenced. The mutations present were determined using the GISAID database. Prevalence ratios were estimated by fitting Poisson univariate and multivariate regression models to investigate associations between SARS-CoV-2 variant group (VOC, non-VOC) and disease outcome. Seventeen genetic variants were detected including VOC Alpha, Beta, Gamma and Delta, one variant of interest (VOI) (Lambda) and two previous VOI (A.2.5.1 and Zeta/P.2). Beta (34.77%), Delta (24.89%) and D614G (19%) variants were the most frequently detected. By June, Delta increased in frequency, displacing Beta. Disease severity increased significantly with age and VOC (PR =1.98, IC 95%: 1.33-3.05, p <0.05). Genomic surveillance allowed us to identify the upsurge of novel variants. Coinciding with the higher epidemic period, multiple variants were co-circulating. Although we cannot rule out that failure in the transmission containment measures occurred, the increase in the number of cases associated with the circulation of several variants, particularly the Beta and Delta variants is highly suggestive. A greater association of Beta variant with clinical severity and Delta variant with a greater transmissibility was observed.

Evaluación de ensayos ultramicroanalíticos para la detección de anticuerpos contra el SARS-CoV-2 / Evaluation of ultramicroanalytic assays to detect antibodies against SARS-CoV-2

Introducción: En el presente trabajo se muestran los resultados de la validación de los ensayos serológicos in vitro para la detección de anticuerpos IgM, IgG y anticuerpos totales contra el SARS-CoV-2 UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 y UMELISA SARS-CoV-2 IgG desarrollados por el Centro de Inmunoensayo (CIE). Métodos: Se utilizaron paneles de muestras de suero de individuos negativos y de casos confirmados de COVID-19 para determinar el desempeño analítico de cada ensayo. Resultados: La especificidad clínica de los ensayos UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 y UMELISA SARS-CoV-2 IgG fue del 100 por ciento en todos los ensayos y la especificidad analítica fue de 100 por ciento para los dos primeros ensayos y del 93,1 por cientopara el último. La sensibilidad clínica fue de 64,3, 80,8 y 97,5 por ciento, respectivamente. El valor predictivo positivo fue de 100 por ciento en todos los ensayos, en tanto que el negativo osciló entre 83,3 y 95,2 por ciento. La concordancia fluctuó entre 92,4 y 96,9 por ciento y el índice kappa de todos los ensayos fue muy bueno. La sensibilidad de los ensayos se incrementó a 82,76, 96,5 y 100 por ciento, respectivamente, en las muestras de suero colectadas con más de 14 días de iniciado el cuadro clínico. Conclusiones: Los ensayos demostraron una elevada sensibilidad y especificidad, lo que permite contar con herramientas basadas en una tecnología desarrollada en Cuba que posibilita la realización de estudios serológicos, vigilancia epidemiológica y de otro tipo, incluyendo los relacionados con vacunas en una plataforma con amplia distribución nacional(AU)

Introduction: This paper shows the results obtained in the validation of in vitro serological assays to detect IgM, IgG antibodies, and total antibodies against SARS-CoV-2 UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 and UMELISA SARS-CoV-2 IgG developed by the Immunoassay Center. Methods: Panels of serum samples from negative and COVID-19 confirmed patients were used to determine the analytical performance of each assay. Results: UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 and UMELISA SARS-CoV-2 IgG assays demonstrated 100 percent clinical specificity for all assays; and 100 percent analytical specificity for the first two assays, and 93.1 percent for the last one. Clinical sensitivity was 64.3 percent, 80.8 percent and 97.5 percent, respectively. The positive predictive value was 100 percent in all assays, while the negative predictive value ranged from 83.3 percent to 95.2 percent. Concordance varied from 92.4 percent to 96.9 percent, and kappa index in every assay was very good. Assays sensitivity increased to 82.7 percent, 96.5 percent and 100 percent, respectively for serum samples collected more than 14 days after onset of the symptoms. Conclusions: The assays demonstrated high sensitivity and specificity, which allows us to have Cuban technology-based tools for serological, epidemiological surveillance, and other types of studies, including those related to vaccines on a platform with wide national distribution(AU)

Identification of human parvovirus B19 among measles and rubella suspected patients from Cuba.

Between September 2014 and December 2015, 298 sera from rash and fever patients from all over Cuba were investigated for specific IgM antibodies against measles, rubella, dengue, human parvovirus B19 (B19V) and human herpesvirus 6 (HHV6) using a commercial enzyme-linked immunosorbent assay kits. B19V IgM positive and equivocal samples were investigated by a polymerase chain reaction and genotyping. No measles, rubella or dengue cases were detected. HHV6-IgM antibodies were confirmed in 5.7% and B19V-IgM antibodies in 10.7% of the patients. A total of 31.3% of the B19V cases were between 5 and 9 years old and 34.4% were 20 years and older. The only B19V sequence obtained belonged to genotype 1a. Diagnosis was established for only 16% of the rash and fever patients, suggesting that other diseases such as Zika or Chikungunya may play a role.

Mumps epidemiology in Cuba between 2004 and 2015.

Vaccination with the measles, mumps and rubella vaccine decreased the mumps incidence in Cuba, but in 2006 and 2007 an outbreak with more than 1000 laboratory confirmed cases occurred, mainly among high school and university students. The objective of the study was to investigate mumps epidemiology in Cuba between 2004 and 2015 and provide an in-depth laboratory characterization of selected samples from mumps patients. Samples from 116 cases (throat swabs, urines, paired acute and convalescent serum samples) were tested for mumps-specific IgM antibodies by ELISA, in a hemagglutination inhibition assay (HIA) or by RT-PCR. IgM antibodies were found in 80.2% of cases. 48.3% of first sera were positive, 30 of which were collected within two days after symptom onset. Testing of all 116 paired sera by HIA showed seroconversion in 55.2% individuals and an at least fourfold increase in antibodies in 44.8% of cases. In 18 out of the 111 vaccinated people (16.2%) no IgM antibodies were detected, neither in the acute nor the convalescent sera, but 14 of them showed seroconversion by HIA and 4 had an at least fourfold increase of hemagglutinin antibody titers. In the RT-PCR, 23 acute phase sera, 4 throat swabs and 5 urines were positive. Detection of mumps-specific IgM antibodies by ELISA and additional diagnostic methods may be required in settings with high vaccination coverage rates.

Detection of rotavirus and other enteropathogens in children hospitalized with acute gastroenteritis in Havana, Cuba.

The aim of the study was to diagnose infections with rotavirus and other enteric pathogens in children under five years old with acute gastroenteritis and to identify the most common epidemiological and clinical characteristics of these pathogens. The study was conducted using 110 stool samples from the same number of children under five years old who were inpatients at three paediatric hospitals in Havana, Cuba, between October and December 2011. The samples were tested for rotavirus and other enteric pathogens using traditional and molecular microbiological methods. Pathogens were detected in 85 (77.3 %) of the children. Rotavirus was the most commonly found, appearing in 54.5 % of the children, followed by bacteria (29 %) and parasites (10.9 %). Other viral pathogens detected included adenovirus (6.4 %) and astrovirus (3.6 %). In rotavirus-positives cases, at least one other pathogen was detected, usually a bacterium (26.6 %). More than three episodes of watery diarrhea in 24 hours were observed in 78.3 % of the cases. Dehydration was found in 30 (50 %) rotavirus-positive children, of whom seven (11.6 %) were transferred to an intensive care unit due to complications of metabolic acidosis. Rotavirus was most commonly observed among children under 12 months old (65 %). The highest incidence of infection occurred in children who were under the care of a relative at home (78.3 %), had not been breastfed (65 %), or had been breastfed for less than six months (28.3 %). The genotype combinations most frequently found were G9P8 (28.3 %) and G1P8 (10 %). This study demonstrates the presence of rotavirus and other enteric pathogens as causes of gastroenteritis in hospitalized infants and young children in Cuba.

Identificación de infección primaria y secundaria al virus de la rubéola mediante la detección de la avidez de la IgG en muestras de un brote en el 2004 en Cuba / Identification of primary and secondary infection to rubella virus by the detection of IgG avidity in samples of an outbreak occurred in 2004 in Cuba

La rubéola es una enfermedad infectocontagiosa que afecta a niños y adultos jóvenes, y cuando esta se produce en el primer trimestre del embarazo ocasiona malformaciones congénitas en el feto. En el Laboratorio Nacional de Referencia de Sarampión, Rubéola y Parotiditis del Instituto de Medicina Tropical Pedro Kourí, se realizó la detección del índice de avidez relativo (IAR) de la IgG en 15 pares de sueros correspondientes a igual número de pacientes adultos que enfermaron durante un brote de rubéola ocurrido en dos provincias de Cuba en el 2004, los cuales fueron positivos a anticuerpos de tipo IgM específicos al virus, y en algunos se encontró seroconversión o un aumento de cuatro veces el título entre los sueros de la fase aguda y convaleciente con la técnica de inhibición de la hemaglutinación. En 13 (86,6 por ciento) de los pacientes estudiados se detectó infección primaria, mientras que en dos (13,3 por ciento) de ellos la infección fue secundaria. La detección de la avidez de la IgG constituye un método alternativo útil para distinguir una infección primaria de una secundaria al virus de la rubéola y fortalece el diagnóstico de esta enfermedad en Cuba(AU)

Rubella infectious diseases affect children and young adults and when they occur during the first trimester of pregnancy, cause congenital malformations in the newborn. In the National Reference Laboratory (NLR) of measles, rubella and mumps from the Tropical Medicine Institute Pedro Kourí, the IgG Avidity Index (AI) detection was carried out to 15 paired serum samples, which corresponded to same number of patients infected with this virus in two Cuban provinces in 2004. The samples were positive to virus- specific IgM antibodies, in addition seroconversion or an increase of > 4 fold of the titers in the sera of the acute and convalescence phase using the hemagglutination inhibition assay (HIA). The AI value showed a primary infection to rubella virus in 13 (86.6 per cent) of the patients studied, and a secondary infection in 2 (13.3 percent) of them. The IgG avidity antibody detection is a reliable alternative tool for distinguishing between primary and secondary rubella virus infection and for strengthing the diagnosis of this disease in Cuba(AU)

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation.

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Emergence of G9 as a predominant genotype of human rotaviruses in Cuba.

To study human rotaviruses in Cuban children up to 5 years old with acute diarrhea, a total of 64 faecal samples from two pediatric hospitals between 2006 and 2008 were analyzed. Thirty-nine samples (60.9%) were found positive for rotaviruses by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE), while four (6.2%) exhibited discordant results (ELISA positives/PAGE negatives). All the positive samples were genotyped by a reverse transcription-polymerase chain reaction (RT-PCR). The most prevalent G and P types were G1 (60.5%) and P[8] (90.6%), respectively. Among the rotaviruses detected, two long RNA patterns were identified by PAGE (L/A and L/B). The predominant genotype in 2006 and 2007 was G1/P[8] with detection rates 71.4% and 80%, respectively. In contrast, G9/P[8] was found at the highest rate (78.5%) in 2008. The phylogenetic analysis of VP7 genes of the ten representative strains indicated that Cuban G1/P[8] rotaviruses were close to those reported in the Americas, and G9/P[8] rotaviruses were located in the lineage of the emerging G9 strains spreading worldwide. This is the first molecular epidemiologic study of rotaviruses in Cuba, highlighting the current increase of the G9 rotavirus which has been recognized globally as an emerging genotype. The study reinforces the need for a systematic surveillance of the molecular epidemiology of rotaviruses.

Presencia de anticuerpos a sarampión, rubéola y parotiditis en una población cubana de 7 meses a 23 años / Presence of measles, rubella and parotiditis antibodies in a Cuban population aged 7-23

Se creó un programa a partir de 1988 por las autoridades de salud en Cuba, en el cual se trazó como meta eliminar el sarampión, la rubéola y la parotiditis. Sus bases fundamentales fueron alcanzar coberturas de vacunación superiores a 95 % con la vacuna triple viral (sarampión, rubéola y parotiditis), implantar un sólido sistema de vigilancia seroepidemiológica y lograr medidas de control de foco, con lo cual cesaría la transmisión del virus salvaje productor de estas entidades. Con el fin de conocer el estado inmune de la población a más de 10 años de iniciarse la vacunación con la triple viral, se realizó una encuesta seroepidemiológica a una muestra representativa de la población cubana entre 7 meses y 23 años. Se estudió un total de 1 593 muestras mediante la prueba de inhibición de la hemaglutinación. La prevalencia de anticuerpos detectada fue de 86,75 % para sarampión, 64,72 % para rubéola y 86 % para parotiditis. La inmunidad a los virus del sarampión y rubéola fue menor en el grupo de 6 a 10 años y en el caso de la parotiditis en los niños entre 2 y 5 años. Se sugirió incluir en el programa nacional de vacunación, una segunda dosis de la vacuna triple viral en los niños de 6 años o más, para eliminar la circulación de estos agentes infecciosos en la población.

The Cuban health authorites implemented a program in 1988 aimed at eradicating measles, rubella and parotiditis. Its fundamental bases were to attain vaccination coverages over 95 % with the viral triple vaccine (measles, rubella and parotiditis), to establish a solid system of seroepidemiological surveillance and to take measures to control the focus, with which the trasmission of the wild virus producing these entities would stop. In order to know the immune state of the population a seroepidemiological survey was done in a representative sample of the Cuban population from 7 months old to 23 years old. A total of 1 593 samples were studied by the hemagluttination inhibition test. The prevalence of antibodies detected was 86.75 % for measles, 64.72 % for rubella and 86 % for parotiditis. The immunity to the measles and rubella viruses was slower in the group aged 6-10 and in children aged 2-5 in the case of parotiditis. It was suggested to include in the national vaccination program a second dose of the viral triple vaccine in children 6 or over to eliminate the circulation of these infectious agents in the population.

Presencia de anticuerpos a sarampión, rubéola y parotiditis en una población cubana de 7 meses a 23 años / Presence of measles, rubella and parotiditis antibodies in a Cuban population aged 7-23

Se creó un programa a partir de 1988 por las autoridades de salud en Cuba, en el cual se trazó como meta eliminar el sarampión, la rubéola y la parotiditis. Sus bases fundamentales fueron alcanzar coberturas de vacunación superiores a 95 por ciento con la vacuna triple viral (sarampión, rubéola y parotiditis), implantar un sólido sistema de vigilancia seroepidemiológica y lograr medidas de control de foco, con lo cual cesaría la transmisión del virus salvaje productor de estas entidades. Con el fin de conocer el estado inmune de la población a más de 10 años de iniciarse la vacunación con la triple viral, se realizó una encuesta seroepidemiológica a una muestra representativa de la población cubana entre 7 meses y 23 años. Se estudió un total de 1 593 muestras mediante la prueba de inhibición de la hemaglutinación. La prevalencia de anticuerpos detectada fue de 86,75 por ciento para sarampión, 64,72 por ciento para rubéola y 86 por ciento para parotiditis. La inmunidad a los virus del sarampión y rubéola fue menor en el grupo de 6 a 10 años y en el caso de la parotiditis en los niños entre 2 y 5 años. Se sugirió incluir en el programa nacional de vacunación, una segunda dosis de la vacuna triple viral en los niños de 6 años o más, para eliminar la circulación de estos agentes infecciosos en la población(AU)

Role of the virology diagnosis laboratory in the surveillance of rubella virus Cuba 1988/2000.

In Cuba, on the basis of Measles Elimination Program, the incidence of this disease decline, and was necessary to test rubella virus as a possible etiology agent that produce fever and rash illness. To reach this goal, Cuba developed rubella elimination strategies with integrated epidemiologic and laboratory surveillance. In the country, the vaccination program against rubella started in 1982 by vaccinating 12-14 years old females, with a special surveillance program with laboratory study of all suspected cases. Through 1988-2000, the Serology Diagnosis Laboratory in the Virology Branch of Pedro Kouri Institute had the responsibility to do the measles and rubella surveillance and play a key roll in the elimination strategies of these diseases. For confirmation of all suspected cases, 8566 serum samples with the suspected diagnosis of measles or rubella from different provinces in Cuba were studied in the laboratory using different techniques as haemagglutination inhibition test (HIA), ultra micro analytic assay (UMA); and in 1995 by the newly introduced IgM ELISA, which was used taken only one sample in the acute phase of the disease. These techniques allowed knowing that the annual number of reported rubella cases in the country decreased substantially after the implementation, in 1986, of the second vaccine policy, that of vaccinating women of childbearing age. However, in 1989, was detected an outbreak of rubella virus infection that had occurred in young adults male 15-19 age groups in Matanzas' province. The last three indigenous cases of this disease were confirmed by our laboratory in 1995, after national vaccine coverage over 95%.

Detección de anticuerpos antirrábicos en personal de riesgo con el empleo de la técnica de neutralización por reducción del número de placas / Detection of antirabic antibodies in personnel at risk through the use of the neutralization technique by reducing the number of plaques

Se empleó la técnica de neutralización por reducción del número de placas (NRNP) para la detección de anticuerpos antirrábicos en personal de riesgo. Se estudiaron muestras de suero de individuos de alto riesgo y personas con antecedentes de vacunación y sin estos. La técnica de neutralización fue comparada con la prueba biológica en ratón y como resultado del estudio se obtuvo una concordancia de 100 por ciento. Se encontró que el sexo no influye en la respuesta de anticuerpos a este virus, aunque el contacto directo y la cantidad de veces que el individuo ha recibido la vacuna actúan positivamente en la respuesta inmune. La introducción de esta técnica en el laboratorio permite contar con una herramienta útil, en el seguimiento del personal que recibe vacunación preexposición y posexposición al virus rábico

Detección de anticuerpos antirrábicos en personal de riesgo con el empleo de la técnica de neutralización por reducción del número de placas / Detection of antirabic antibodies in personnel at risk through the use of the neutralization technique by reducing the number of plaques

Se empleó la técnica de neutralización por reducción del número de placas (NRNP) para la detección de anticuerpos antirrábicos en personal de riesgo. Se estudiaron muestras de suero de individuos de alto riesgo y personas con antecedentes de vacunación y sin estos. La técnica de neutralización fue comparada con la prueba biológica en ratón y como resultado del estudio se obtuvo una concordancia de 100 por ciento. Se encontró que el sexo no influye en la respuesta de anticuerpos a este virus, aunque el contacto directo y la cantidad de veces que el individuo ha recibido la vacuna actúan positivamente en la respuesta inmune. La introducción de esta técnica en el laboratorio permite contar con una herramienta útil, en el seguimiento del personal que recibe vacunación preexposición y posexposición al virus rábico(AU)

[Detection of antirabic antibodies in personnel at risk through the use of the neutralization technique by reducing the number of plaques]. / Detección de anticuerpos antirrábicos en personal de riesgo con el empleo de la técnica de neutralización por reducción del número de placas.

The neutralization technique by reducing the number of plaques (NRNP) for detecting antirabic antibodies in personnel at risk was used. Serum samples from individuals at high risk and from persons with vaccination antecedents and without them were studied. The neutralization technique was compared with the biological technique in mouses and as a result of the study it was obtained a concordance of 100%. It was found that sex does not influence on the response of the antibodies to this virus, although the direct contact and the number of times that the individual has received the vaccine act positively on the immune response. The introduction of this technique in the laboratory allows to have a useful tool for the follow-up of the personnel receiving the vaccine before and after being exposed to the rabies virus.

Presencia de anticuerpos al virus del sarampión en un grupo de pacientes con hemopatías malignas / Presence of antibodies to the virus of the measles in a group of patient with wicked hemopatías

Se estudiaron 225 monosueros de pacientes (121 adultos y 104 niños), con diferentes hemopatías malignas procedentes del Instituto de Hematología, a los cuáles se les realizaron las técnicas de inhibición de la hemaglutinación (IH) y neutralización para conocer la presencia de anticuerpos antisarampión. Todos estos pacientes tenían tratamiento inmunosupresor a la toma de la muestra y los niños habían recibido la vacuna triple viral (sarampión, rubéola y parotiditis) antes de ser diagnosticada la enfermedad. Se encontró que 51 (42,14 por ciento) de las muestras de los adultos fueron positivas por IH y de las 25 negativas, a las cuales se les realizó la técnica de neutralización, fueron positivas 18 (72 por ciento. De los 104 sueros correspondientes a los niños, 31 (29,8 por ciento) muestras resultaron positivas por IH y se estudiaron por neutralización 29 de las que habían resultado negativas. De todas las muestras 18,34 por ciento continuó negativo por ambos métodos. Con las 2 técnicas empleadas, la presencia de anticuerpos al virus del sarampión fue de 81,66 por ciento en los niños y de 90,78 por ciento en los adultos(AU)