Correction: Temporal Patterns of Influenza A and B in Tropical and Temperate Countries: What Are the Lessons for Influenza Vaccination?

[This corrects the article DOI: 10.1371/journal.pone.0152310.].

Large increase of rotavirus diarrhoea in the hospital setting associated with emergence of G12 genotype in a highly vaccinated population in Nicaragua.

Rotaviruses (RVs) are a major cause of severe diarrhoea in young children. Nicaragua introduced routine immunization with the pentavalent RV vaccine (RV5) in 2006, which greatly reduced the incidence of diarrhoea. A remaining concern has been the possible emergence of new RV strains to which the vaccination has less effect. In this study, 837 children with diarrhoea in hospital settings were investigated for RV between May 2011 and July 2013. RVs were subsequently typed by multiplex PCR and/or sequencing. Fecal anti-RV IgA titres for a subset of RV-infected (n = 137) and noninfected children (n = 52) were determined with an in-house enzyme-linked immunosorbent assay. The RV detection rate was 8% in 2011, followed by a sharp increase to 29% in 2012 and 19% in 2013. This was associated with emergence and predominance of genotype G12 RV, from 0% in 2011 to 66% in 2012 and 82% in 2013, infecting children from 1 month to 10 years of age. Two sequenced G12 strains showed a Wa-like genome with genotype G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, similar to the globally emerging G12 strains. Fecal anti-RV IgA analysis showed that most G12-infected and noninfected children had been in contact with either vaccine or wild RV strains, but such antibodies did not prevent symptomatic G12 infection. A marked increase of RV was evident in the hospital setting associated with a nationwide emergence and predominance of RV G12 genotype in a population with high RV5 vaccine coverage.

Clinical, epidemiologic, and virologic features of dengue in the 1998 epidemic in Nicaragua.

From July to December 1998, a hospital- and health center-based surveillance system for dengue was established at selected sites in Nicaragua to better define the epidemiology of this disease. Demographic and clinical information as well as clinical laboratory results were obtained, and virus isolation, reverse transcriptase-polymerase chain reaction, and serologic assays were performed. World Health Organization criteria were used to classify disease severity; however, a number of patients presented with signs of shock in the absence of thrombocytopenia or hemoconcentration. Therefore, a new category was designated as "dengue with signs associated with shock" (DSAS). Of 1,027 patients enrolled in the study, 614 (60%) were laboratory-confirmed as positive cases; of these, 268 (44%) were classified as dengue fever (DF); 267 (43%) as DF with hemorrhagic manifestations (DFHem); 40 (7%) as dengue hemorrhagic fever (DHF); 20 (3%) as dengue shock syndrome (DSS); and 17 (3%) as DSAS. Interestingly, secondary infection was not significantly correlated with DHF/DSS, in contrast to previous studies in Southeast Asia. DEN-3 was responsible for the majority of cases, with a minority due to DEN-2; both serotypes contributed to severe disease. As evidenced by the analysis of this epidemic, the epidemiology of dengue can differ according to geographic region and viral serotype.

Vector analysis of visual evoked potentials elicited by pattern reversal and photic stimuli.

OBJECTIVE: The 2D VEPs to pattern reversal (PR) and LED goggle were studied in order to obtain a stable parameter for the functional assessing of posterior visual pathways regardless of the stimulus type used. DESIGN AND METHODS: Apex c latency, bc segment amplitude (V), and bc vector orientation angle (theta) in voltage space were computed from VEPs recorded in 50 normal human beings and two patients with left posterior brain lesions, in an orthogonal Fpz-Oz and T3-T4 montage and displayed as a two channel Lissajous' trajectory. The effects of stimulus type and stimulated eye were analyzed in the normal group by a two-way ANOVA. RESULTS: The stimulated eye had no effect on any parameter. Apex c latency was slightly longer, and V was greater and more variable in the responses to goggle stimuli, but there was no significant difference in theta, oriented to mid-occipital scalp with very low variability for both stimulus types. The patients showed significant deviations of theta towards the affected hemisphere. CONCLUSIONS: The bc vector orientation (theta) is a stable parameter for the evaluation of the posterior visual pathways using both pattern reversal and LED stimuli, specially the latter, useful in unconscious or uncooperative patients.

Application of molecular typing techniques in the 1998 dengue epidemic in Nicaragua.

This report presents the results of applying the reverse transcriptase-polymerase chain reaction (RT-PCR) to the analysis of clinical specimens during the 1998 dengue epidemic in Nicaragua. The RT-PCR was validated through comparison with viral isolation, resulting in a sensitivity of 100% and a specificity of 90%. In-country application of the RT-PCR permitted the rapid identification of dengue-3 virus as the cause of the epidemic at the beginning of 1998 and the detection of the reintroduction of dengue-2 virus in the middle of the year. Nineteen isolates of dengue-3 and one of dengue-2 were characterized using the restriction site-specific (RSS)-PCR technique. This showed that the dengue-3 strain belonged to the "Sri Lanka" subtype and that the dengue-2 strain belonged to the "Jamaica" subtype, both of which have been associated with hemorrhagic dengue in the Americas. The application of these simple PCR-based strain typing methods in a country endemic for dengue virus infections can help to characterize the transmission dynamics of this important emerging infectious disease problem and provide this information to local health authorities in a timely manner so that appropriate control measures can be implemented.

Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR.

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

Prevalence and factors associated to the epidemic neuropathy in a population assisted by a family doctor in Cuba.

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.

[Validation of a ultramicroELISA for detecting antibodies against hepatitis B surface antigen]. / Validación de un ultramicroELISA de detección de anticuerpos contra el antígeno de superficie de la hepatitis B.

The results of a validation study of the ultramicroanalitical assay for the detection of antibodies against the hepatitis B surface antigen (UMELISA anti-HBsAg), which was carried out by comparing the results obtained with the Hepanostika anti-HBsAg, commercial diagnosis kit are presented. For this purpose, sera from the clinical assays of the Cuban recombinant vaccine against hepatitis B were used. With the first sera group (n = 30) it was obtained, 93.1% of sensitivity, 98.5% of specificity and a concordance of 94.3%. The correlation coefficient showed a similar trend of the results (p < 0.01) and no significant differences were found in the average geometrical titre (TPG) between both assays (p > 0.05). With the second group (n = 100), whose assays were carried out at the "Pedro Kouri" Institute of Tropical Medicine (PKI) and at the Immunoassay Center (IAC) simultaneously, it was observed a sensitivity of 96.25% in both centers, a specificity of 75% at the PKI and of 90% at the IAC, and a coincidence of 92% and 95%, respectively. The correlation coefficient presented similar values and there were no significant differences between the TPG obtained by the two methods (p > 0.05). The results attained show in general the validity of the new assay and the feasibility to put it into practice either for following up the infection, or for carrying out clinical assays of vaccine evaluations.

[Cuban epidemic neuropathy. III. Neutralizing antibodies to the strains isolated and to other enteroviruses in patients and healthy subjects]. / Neuropatía epidémica cubana. Parte III. Anticuerpos neutralizantes a cepas aisladas y otros Enterovirus en pacientes y personas sanas.

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.

[The use of ELISA and ultramicro ELISA in identifying a Coxsackie A9 strain isolated during the epidemic of neuropathy in Cuba]. / Utilización del ELISA y el ultramicroELISA en la identificación de una cepa de Coxsackie A9 aislada durante la epidemia de neuropatía en Cuba.

During 1993 an outbreak of epidemic neuropathy characterized by 2 major clinical forms: the optic neuropathy and peripheral neuropathy, occurred in our country. Although the cause of the disease was considered to be multifactorial (mainly due to neurotoxic agents and to nutritional deficiency) an Enterovirus was isolated from the cerebrospinal fluid of a patient (strain 47/93, IPK) which was subsequently classified as a Coxsackie A9 virus (Cox A9) by the neutralization test, and using pools of Lim Benyesh Melnick (LBM) serum. Results obtained during the application of ELISA and ultramicro ELISA techniques for the identification of this agent from the supernatant of a culture of infected cells are reported, as well as the detection of antibodies to the strain found within populations with high and low incidence rates of the disease.

[A comparison of the results obtained in IgM serology and anti-Coxsackie A9 (strain 47) neutralization in cases of epidemic neuropathy and their contacts]. / Comparación de los resultados obtenidos en la serología de IgM y la neutralización anti-Coxsackie A9 (cepa 47) en casos de neuropatía epidémica y sus contactos.

A number of 213 monosera from patients presenting with epidemic neuropathy and their contacts was studied by indirect immunofluorescence and neutralization tests with the aim of demonstrating the presence of serum IgM and neutralizing antibodies against the strain 47/93 IPK identified as a Coxsackie A9 virus. The mean age of the patients ranged from 20 to 59 years and positivity to both techniques was not found to be predominant for one or another sex. No significant difference was observed with respect to results obtained between patients and contacts with the techniques used for the study.

[Detection of immunoglobulin A in serum and saliva of patients with hepatitis A]. / Detección de inmunoglobulina A en suero y saliva de pacientes con hepatitis A.

Specific secretory serum IgA antibodies to the hepatitis A virus from samples from patients with clinical symptoms compatible to hepatitis A, their contacts, and healthy subjects were analyzed using an ELISA technique; results were compared with those of specific serum IgM antibodies to the hepatitis A virus. The following results were attained in 175 blood samples: coincidence by 98.8%; sensitivity by 96.8%; and specificity by 100%. Two cases were negative to IGA and positive to IGM. On comparing the presence of IGA in saliva with the presence of IGM in blood, coincidence was of 88.1%; sensitivity, of 40.9% and specificity, of 100%. Of the 22 cases with positive IGM in blood, only 9 showed specific IGA antibodies in the saliva. The 111 cases who had negative IGM in blood were also negative to IGA. The obtained data suggest that specific serum IGA antibodies to the hepatitis A virus are an indicator of a recent or occurrent infection due to this virus and thus it may be considered and alternative for the diagnosis of this disease.

[Development and evaluation of an ELISA for the detection of IgM antibodies against the hepatitis A virus]. / Desarrollo y evaluación de un ELISA para la detección de anticuerpos IgM contra el virus de la hepatitis A.

We present the results of the normalization of an IgM capture ELISA method for the diagnosis of type A viral hepatitis with reagents produced in the laboratory and its comparison with the "Diag-A-Hep" commercial ELISA. The results attained were: sensibility by 91%; specificity by 100%; and coincidence of the two systems by 97%. Results are discussed and their relationship with clinical symptoms and epidemiological characteristics is established. The results attained in 13 serum samples taken from patients seen during 2 acute viral hepatitis outbreaks agreed with clinical findings.

[Serologic marker as indicator of no circulation of poliovirus in Cuba]. / Marcador serológico como indicador de no circulación de poliovirus en Cuba.

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.

Further differences between brain-stem auditory potentials evoked by rarefaction and condensation clicks as revealed by vector analysis.

Vectorial analysis of the brain-stem auditory evoked potential (BAEP) elicited by either condensation and rarefaction stimuli was performed in 22 healthy subjects. Besides differences in latency of components III and V ('R' longer than 'C'), a significant difference was found between the directions of the vector calculated over the descending limb of wave III, with an ascending direction for 'C' responses and a contralateral direction for 'R' responses. The possible origins of these differences are discussed. It is concluded that the use of alternating or isolated single polarity clicks is not recommended, mainly when assessing integrity of central auditory pathways.