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Background: The penetrance of type 1 diabetes mellitus (T1DM) in a genetically susceptible population is largely determined by environmental influences amongst which the human enteroviruses are prominent putative factors., Aim/hypothesis: The aim of this study was to determine the occurrence of enterovirus RNA in serum of children with type 1 diabetes at onset and ICA-positive subjects in a population with low incidence of type 1 diabetes and high circulation of enteroviruses., Subjects and Methods: Serum samples were collected from children with newly diagnosed type 1 diabetes (n = 34); islet autoantibody-positive (n = 32) and -negative (n = 31) first-degree relatives of type 1 diabetic patients; and control subjects (n = 194). Enteroviral RNA was assessed using a highly sensitive reverse transcriptase-polymerase chain reaction method., Results: The frequency of positive signals corresponding to enteroviral sequence amplifications were higher in newly diagnosed T1DM children (9/34, 26.5%) and islet autoantibody-positive first-degree relatives (5/32, 15.6%) than in their corresponding matched controls (2/68, 2.9%, p = 0.0007 and 0/64, 0.0%, p = 0.0033, respectively). The presence of enteroviral RNA appeared to be associated with severe diabetic ketoacidosis at onset (pH < 7.1, p = 0.0328) and high ICA titres ( > or = 20 JDF units, p < 0.05)., Conclusions: Despite there is a high circulation of enteroviruses and a low type 1 diabetes incidence in the Cuban population, the presence of enteroviral RNA is associated with type 1 diabetes and beta-cell autoimmunity and is similar to European countries in which this scenario is reversed.

Background: Recent outbreaks of poliomyelitis caused by vaccine-derived virus have raised concerns that vaccine-derived poliovirus may continue to circulate after eradication. In these outbreaks, the virus appears to have replicated for > or =2 years before detection. Early detection is critical for an effective response to these outbreaks. Although acute flaccid paralysis (AFP) surveillance will remain the standard for poliovirus detection, wastewater sampling could be a useful supplement. In this study, we evaluated the sensitivity of wastewater sampling by concurrently collecting stools from children aged < 3 years attending two neighbourhood clinics in Havana, Cuba, and wastewater from the same neighbourhoods., Methods: Sample collection was begun during the third week after the national immunization campaign, continued weekly through the seventh week, and was repeated during weeks 15 and 19. Virus detection and titration were performed using both cell culture and polymerase chain reaction techniques., Results: Wastewater sampling was found to be at least as sensitive as stool sampling under these conditions. Poliovirus was isolated from children through week 7, suggesting that viral shedding reached undetectable levels between weeks 8 and 14. The last virus-positive wastewater sample was collected during week 15., Conclusions: Wastewater sampling under the conditions studied can be a sensitive supplement to AFP surveillance. Similar studies under different conditions are needed to determine the role of wastewater sampling in post-eradication surveillance.

Background: With substantial progress made toward polio eradication, developing the appropriate strategy for discontinuing global oral poliovirus vaccine (OPV) after global eradication becomes increasingly important. At issue is the theoretical risk of independent circulation of potentially virulent OPV-derived strains. Because Cuba uses OPV only in mass campaigns, it represents an ideal site to assess vaccine-derived poliovirus persistence., Methods: Infants born after the 1997 biannual mass campaigns were evaluated for past (neutralizing antibody) or current (virus excretion) evidence of vaccine-derived poliovirus exposure. We obtained sera and/or stool specimens from 861 infants; a second serum from 218 infants., Results: All stool specimens were poliovirus negative. Of 762 infants, 113 (14.8%) had initially detectable poliovirus type 1 antibody, 193 (25.3%) type 2, and 94 (12.3%) type 3. A precipitous antibody decline occurred in initially positive sera., Conclusions: Our results suggest that in a country with high population immunity, vaccine-derived virus is unlikely to establish ongoing circulation.

The eradication of poliomyelitis in Cuba, for which effective vaccines had to be acquired, is reviewed in this article. The strategy for eradication was based on mass immunization campaigns for the annual delivery of two doses of trivalent Sabin oral poliovirus vaccine (OPV). Except during the first campaign in 1962, the ages of the children for immunization were determined through national serological surveys of the entire country, including rural and urban areas. The interruption of wild virus transmission had been suspected since 1967 in Cuba, and since 1970 no studies have detected any wild virus. The important role of political and social organizations in the success of the programme and in the execution of the mass immunization campaigns is underscored. Countries that have successfully interrupted poliovirus circulation should maintain high immunization coverage for as long as there are other countries in the world where poliovirus still exists.

The vaccine against measles came into use in Cuba in 1971. During the seventies, a new early strategy for measles control was established, and it was followed by further efforts in the early eighties. Despite improvements to the control program, disease outbreaks continued to occur. In 1986, after examining the experience acquired through the control initiatives that were already in place, a new measles vaccination strategy was adopted. In time, the new vaccination strategy against measles came to have three main components: first, a single vaccination "catching-up" campaign targeting children 1 to 14 years of age. Second, efforts were made to achieve and maintain high vaccine coverage through mandatory vaccination services for 12-month-old children ("maintenance vaccination"). Finally, periodic "follow-up" campaigns were carried out for children 2 to 6 years of age. Steps were taken, for the purpose of monitoring the progress made so far toward eliminating measles, to strengthen disease surveillance systems, including the screening of suspected cases. The "catching-up" and "follow-up" campaigns both achieved greater than 98% coverage within targeted age groups. The routine vaccination program has also maintained high coverage. The high population immunity against measles that has been attained through these vaccination strategies has resulted in a rapid decrease in the incidence of the disease. From 1989 to 1992, less than 20 laboratory-confirmed cases were reported annually. In Cuba, the last case confirmed through serologic screening was reported in July 1993. Cuba's strategy for measles elimination has interrupted disease transmission and kept the causal virus from circulating on the island. Cuba's experience with measles elimination suggests that if an appropriate vaccination strategy is applied, measles can be globally eradicated.

The genomic characterization of the strain of light cytopathic effect by nucleotide sequence is presented. It was possible to sequence 90% of the strain of light cytopathic effect. A close relationship with Coxsackie A9 was observed. The greatest mutations occurred in the region of the genome that codifies for the structural proteins. Therefore, it may be considered as a variant of Coxsackie A9 never reported before.

Of the 115 patients who suffered from epidemic neuropathy and received attention from March 16 de April 30, 1993, at the Center for Medical and Surgical Research (CMSR), in Havana City, 114 controls were selected at random in the community. In the univariate analysis it was found a significant association mainly with the smoking habit (odds ratio = 3.45 [95% confidence interval 1.85 to 6.35)] and with the consumption of edible fat of uncertified procedence (odds ratio = 2.77 [95% confidence interval 1.34 to 5.8]). All were ratified in the multivariate logistical regression analysis. Association (odds ratio = 8.8 [95% confidence interval 2.58 to 30.55] with the presence of antibodies against the Coxsackie virus A-9 strain 47 was also found among the 182 individuals who had serological test. It seems that the toxic factor derived from the smoking habit, together with the deficiency of some nutrients due to the changes occurred in the diet, as well as the contact with the isolated virus in one of the patients facilitated the clinical expression of this disease and therefore in the origin of the epidemics.

An increase of the cases of viral meningoencephalitis was reported in our country from October to December, 1995. 23 faeces specimens were received from 2 provinces aimed at obtaining the viral isolation by their inoculation in cell culture and in newborn mice at the same time in order to compare the sensitivity of both systems in isolation. A higher number of isolations was obtained in cell culture. The cell culture from kidney of a normal adult green African monkey (Vero) proved to be more sensitive. 18 isolations were obtained and identified as Coxsackievirus B5. An anatomical and pathological study was conducted in inoculated mice and injuries compatible with the enteroviral infection were observed.

An etiopathogenic hypothesis is explained taking into consideration the most significant results of the research performed on neuropathy as well as the latest knowledge about the infections produced by Enterovirus. The hypothesis allows to make a logical interpretation of these results; however, the new aspects included make it controversial in the light of the present knowledge to pathogeny by Enterovirus. Further research is needed to confirm this hypothesis.

During the epidemic of neuropathy occurred in Cuba from 1992 to 1994 viral agents antigenically related to the Coxsackie viruses were isolated from the cerebrospinal fluid of patients. To establish a function of these viruses in the etiopathogenesis of the disease, strains 47/93 IPK identified as Coxsackie A9 and the strain 44/93 of light cytopathic effect were selected to conduct a study of their antigenic characteristics by Western Blot. It was proved that there is an antigenic relationship between both strains and it was also demonstrated the absence of structural proteins in their native form among the agents of light cytopathic effect. According to these results, it is stated the possibility that persistence may be a mechanism through which these viruses take part in the etiopathogenesis of epidemic neuropathy in Cuba.

A series of experiments was made at the Virology Department of the "Pedro Kourí" Institute of Tropical Medicine aimed at obtaining new evidences on the possible antigenic relations existing between the viruses isolated from patients with epidemic neuropathy and the structures of the human nervous system. According to the results it may be finally inferred that the persistence and/or autoimmunity may be considered as mechanisms through which the studied viruses participate in the etiopathogenesis of the epidemic neuropathy in Cuba. For future experiments it is very important to identify the possible viral epitopes involved in the molecular mimicry that are responsible for the probable autoimmune mechanisms or for the viral persistence.

During the epidemic neuropathy occurred in Cuba from 1992 to 1993, viral isolations antigenically connected with Coxsackie viruses were obtained from the cerebrospinal fluid of patients. Virological, epidemiological, toxicologic, nutritional, immunological and histopathological investigations were made. Though the disease was related to toxic and nutritional factors, it has been impossible to identify the cause of the epidemic. Taking into consideration the results of the different investigations, we have formulated a comprehensive and multifactorial hypothesis to explain the physiopathological mechanism of the participation of the isolated viruses as mediators in a process of autoimmunity of the pathogeny of the disease.

An increase in the reported cases of viral meningoencephalitis (VME) was detected in October and November, 1995, compared with the same period of 1994. 43 stock specimens from children with this diagnosis were received at the Laboratory of Enterovirus from the "Pedro Kourí" Institute of Tropical Medicine. 23 isolations (53.4%) were obtained and identified as Coxsackievirus B5. Besides, in 43 matched sera investigated by the neutralization test against some Enteroviruses, 21 proved to be positive (48.8%) to the isolated agent. This allowed us to affirm, supported by the clinical picture and by epidemiology, that we are in the presence of an VME outbreak produced by Coxsackievirus B5.

With the outbreak of an epidemic neuropathy (EN) of unknown ethiology, a study of the prevalence and factors associated to the disease was carried out in the Zamora community, municipality of Marianao, Havana City. There were 11 patients identified with EN for a prevalence rate of 1.7/100. The major risk group was the one between 45 and 64 years of age, female sex, black skin. In the univaried analysis, a high prevalence ratio (PR) was found linked to beverage intake (PR = 5.32 95%; confidence intervals (CI) = 1.2-24.4), frequent drugs intake (PR = 6.59; CI = 1.8-24.6), consumption of edible of non certified fats (PR = 4.48; CI = 1.2-16.7) and the smoking habit (PR = 3.6; CI = 1.1-12.2). More than 73/100 (CI = 68.7-78.3) of the people under serologic study were infected with Coxsakie virus A-9 (strain 47) isolated from a patient still under research. It seems there are many factors like linder intake, antecedents of hemorrhagic conjunctivitis, nutritional aspects and others that, with the virus isolated were associated with this epidemiologic situation.

Results which allow to state the existence of antigenic relationships between viruses isolated from the cerebrospinal fluid of patients presenting with epidemic neuropathy and structures of the human central nervous system are reported. These evidences were obtained by 2 different and independent ways: 1) by the double diffusion method in agarose, immunoblot analysis, and immunohistochemical analysis it was confirmed that antibodies induced by isolated viruses react with antigens of the central and peripheral nervous system, 2) serum obtained by the immunization of a rabbit with human brain extract neutralizes the same viruses as those neutralized by hyperimmune sera obtained by isolations. The possible role of viruses as mediators of an autoimmune process in the pathogenesis of the disease is discussed.

From the end of 1991 to June, 1993, an epidemic neuropathy affecting 50,963 persons occurred in Cuba. Two clinical forms of the disease were observed: the optic form (with or without peripheral manifestations, 52% of the cases) and the peripheral form (48%). The epidemiological studies revealed nutritional disorders, mainly a vitamin B complex deficiency due to economic difficulties faced by the country in the "special period". Smoking habit and alcohol consumption were identified as risk factors, particularly for the most severe forms of the disease. In the virological studies of the cerebrospinal fluid (CSF) a Coxsackie 9 virus was isolated in Vero cells in 4% of the cases, as well as another agent with mild cytopathogenic effect in 80% of the samples. Sixty seven percent of the CSF samples inoculated to lactating mice caused the disease or the death of the animals. The viral persistence was confirmed in the cerebrospinal fluid during al least 21 days in a group of patients, and for a year in another group.

With the aim of characterizing antigenically isolations producing mild cytopathogenic effect obtained from the cerebrospinal fluid of patients presenting with epidemic neuropathy, neutralization tests and western blot analysis were performed using hyperimmune sera of patients and hyperimmune sera of rabbits. It was confirmed that isolations with mild cutopathogenic effect studied have the same antigenic characteristics and that they are related to Coxsackie A9 and B4 viruses. Structural proteins were not detected in the strains with mild cytopathogenic effects, only antigens having a high molecular weight which were considered as precursor proteins for viral replication were confirmed.

Determinations of neutralizing antibodies to the strain 47/93 IPK (CA9) and to the strain 590 were performed in serum samples from patients presenting with epidemic neuropathy and from a group of seemingly healthy subjects. Determinations were also done in the reference strains CA9 and CB1-6 by the microneutralization technique. Patients and their contacts showed significantly higher percentages of neutralizing antibodies to the strain 47/93 than the control group and residents of municipalities with a low rate of the disease. This difference was also confirmed regarding the geometric mean titres with the use of the reference strains CA9 and CB2-4. An increased circulation of the strain 47/93 within the infantile population from 1981 to 1993 was evidenced. Patients exhibited significantly lower percentages and geometric mean titres of neutralizing antibodies to the strain 590 than the control group, despite the fact that in 25/28 certain agents having a mild cytopathogenic effect had been isolated. The possibility of two mechanism of neutralization is stated and an hypothesis on the mechanism by which these viruses may be involved in the pathophysiology of the disease is formulated.

Fifty-six outbreaks of hepatitis A infection were studied in City of Havana between January and September, 1991. In 34 of these, the presence of the hepatitis A virus (HAV) was confirmed, either by the detection of specific serum IGM antibodies to the HAV or by the detection of the antigen in the feces of the subjects under study. Diagnosis was not made in some of the outbreaks due to the insufficient number of samples sent to the laboratory. Of the 453 blood serum samples under study, 126 were positive for IGM antibodies to HAV (27.8%): a there was a good correlation between the presence of this immunoglobulin and the levels attained by the alanyl aminotransferase enzyme (ALT). Only in 19 feces samples, of the 263 under study, was the HAV antigen detected, which accounted to 7.2%; it was demonstrated that this marker is not useful for the diagnosis of an outbreak of hepatitis A infection.

The immunogenicity of trivalent oral poliovirus vaccine (TOPV), which is less effective in tropical than in temperate areas, may potentially be improved in several ways, including increasing the number of doses. Little information is available on TOPV when more than 6 doses are given. The situation in Cuba provides a unique opportunity to relate the seroprevalence of neutralizing antibodies to the dose of TOPV because Cuba has not reported culture-confirmed poliomyelitis since 1973 and TOPV is only administered in twice yearly 1-week mass immunization campaigns. Sera from 2000 children nationwide were studied for neutralizing antibody among children who received 0, 2, 4, 6 and 8 doses of TOPV. These doses were administered in the period 1989-91, when TOPV (from the USSR) was being used with 500,000, 200,000, and 300,000 median tissue-culture-infecting doses (TCID50) for types 1, 2 and 3, respectively--the 5:2:3 formulation. Seroprevalence of neutralizing antibody after two TOPV doses was 91.5% for type 1, 90.8% for type 2, and 45.9% for type 3. Seroprevalence of type-3 neutralizing antibody after 6 doses remained low (73.4%), but increased to 83.5% after 8 doses (P < 0.05). Although 16.5% of the children remained unprotected for type-3 infection even after 8 doses, mass campaign immunization strategies were sufficient to eradicate the transmission of wild poliovirus in Cuba. Because the seroprevalence of type-1 neutralizing antibody was high (91.5%) after two campaign doses, additional studies using different formulations are needed to determine whether simultaneous improvement in the type-3 response to two campaign doses can be achieved.

We present the results of the in vitro action of alpha and gamma interferons and of Intacglobin and Igegam against the 47/93/IPK (Coxsackie A9) strain isolated from the cerebrospinal fluid of a patient with epidemic neuropathy. The in vitro studies showed that the two interferons inhibited the replication of this agent; they also showed the presence of antibodies to it in the Intacglobin and Igegam. The results attained demonstrated that the use of these compounds could be effective for the treatment of this entity.

The surveillance of the circulation of the wild poliovirus is an essential element for the eradication of poliomyelitis. The Pan-American Health Organization emphasises on the etiological study of acute flaccid paralysis in order to achieve this aim. The characteristics of our program against this entity allowed to use the presence of antibodies in a sample obtained in February from the infant population born on July during the previous year as criteria to know the circulation of wild poliovirus. In 727 infants studies in the country (5% of the total number of births during July), 99.1% showed no antibodies with a titre equal or greater than 1:10. Low titres found in 7 infants were considered as a remnant from maternal transmission. Results obtained represent a strong evidence of the fact that there is no environmental circulation of poliovirus during the period which had the greatest incidence of the disease.

The causative agent of an hemorrhagic conjunctivitis outbreak was assessed through the study of 18 conjunctival exudates using the indirect immunofluorescence technique and isolation in cell culture of human embryo fibroblasts. In addition, 395 paired serum samples were studied and Enterovirus 70 was found to be responsible for the outbreak.

Two methods for the diagnosis of rotavirus are compared with 120 stool samples from children under two years of age who had been hospitalized in Centro Habana Children's Hospital with a clinical diagnosis of acute diarrheal disease and 30 samples of healthy children of the same age group who served as controls. The methods used were latex agglutination (Rotalex, commercially-available kit from ORION DIAGNOSTIC ESPOO, Finland) and polyacrylamide gel electrophoresis. Electronic microscopy was used to define those cases whose results did not coincide with the previous techniques. Comparison was made based on the sensitivity and specificity, cost and easiness of performance. Rotalex was less specific but more sensitive than electrophoresis in polyacrylamide gel. The latter is less expensive, since reactants are available at a lower price in dollars and have no expiration date, is very useful in epidemiologic and outbreak studies, since it detects subgroups of circulating rotavirus and it is a painstaking technique (1-20 samples in 10 hours). Rotalex is simple, rapid (1-20 samples in 30 minutes) and allows individual determinations; therefore, we consider its application in hospitals is feasible.

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10 -3 and 5.80 × 10 -3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.