Evaluation of the National Case Report Form for dengue and timely case notification before the co-circulation of multiple arboviruses in Barranquilla, Colombia / Evaluación del formulario nacional de informe de caso para dengue y de la puntualidad en la notificación del caso antes de la co-circulación de múltiples arbovirus en Barranquilla-Colombia

ABSTRACT Objective To evaluate the case report forms and times elapsed between the surveillance steps for dengue virus (DENV) infection in a large Colombian city before the emergence of other arbovirus epidemics. Materials and Methods The descriptive epidemiology of DENV infection cases was analyzed from 2009 to 2013. The completeness of the case report forms filed at the Primary Units of Data Generation (PUDG) were evaluated, as well as the accuracy and suitability of the tests (PPV: positive predictive value). The average time-lags between each step were then calculated. Results There were 7.3, 12.38, 4.66, 6.25 and 29.9 annual cases of dengue infection per 10 000 inhabitants in 2009 to 2013, respectively. In this study, only 57.76% of the cases were classified correctly by the physicians and 26.32% of them were questioned about their home conditions and whether their family/friends had similar symptoms. Patients visited a clinic/hospital on average 4.76 days after developing symptoms and the health system was notified on average 1.75 days later, while 70.6% of them were reported within the one-day target period. There were only minor changes in case reporting times even during a DENV epidemic. Some (12.85%) of the case forms were later modified (average 16.7 days). In the period 2009-2013, the IgM confirmed PPV was 58.60%, while 20 mandatory criteria were absent on more than 25% of the forms. Conclusions The system was accurate, simple, flexible, stable and acceptable, but a number of ways are suggested to improve this case detection and reporting system.(AU)

RESUMEN Objetivo Evaluar los formularios de informe de casos y los tiempos entre los pasos de vigilancia para el dengue en una ciudad colombiana antes de la aparición de otras epidemias de arbovirus. Materiales y Métodos Se analizó la epidemiología descriptiva entre 2009 y 2013. Se evaluó la integridad de los formularios de informes de casos, registrados en las Unidades Primarias de Generación de Datos, así como el valor predictivo (VPP) de las pruebas diagnósticas. Se calcularon los intervalos de tiempo promedio entre cada paso de la vigilancia. Resultados Hubo 7.3, 12.38, 4.66, 6.25 y 29.9 casos anuales por cada 10 000 habitantes en 2009-2013, respectivamente. Solo el 57.76% de los casos fueron clasificados correctamente por los médicos, el 26.32% de ellos fueron interrogados sobre las condiciones de su hogar y si sus familiares/amigos tenían síntomas similares. Los pacientes se presentaron a una clínica/hospital en promedio 4.76 días después de desarrollar síntomas y el sistema de salud fue notificado en promedio 1.75 días más tarde, mientras que el 70.6% de ellos se informaron dentro del período objetivo de un día. Algunos (12.85%) de los formularios de casos se modificaron posteriormente (promedio de 16.7 días). Desde 2009-2013, el VPP confirmado por IgM fue de 58.60%, mientras que veinte criterios obligatorios estuvieron ausentes en más del 25% de los formularios. Conclusiones El sistema fue preciso, simple, flexible, estable y aceptable, pero sugerimos varias formas de mejorar este sistema de detección e informe de casos.(AU)

Evaluation of the National Case Report Form for dengue and timely case notification before the co-circulation of multiple arboviruses in Barranquilla, Colombia.

OBJECTIVE: To evaluate the case report forms and times elapsed between the surveillance steps for dengue virus (DENV) infection in a large Colombian city before the emergence of other arbovirus epidemics. MATERIALS AND METHODS: The descriptive epidemiology of DENV infection cases was analyzed from 2009 to 2013. The completeness of the case report forms filed at the Primary Units of Data Generation (PUDG) were evaluated, as well as the accuracy and suitability of the tests (PPV: positive predictive value). The average time-lags between each step were then calculated. RESULTS: There were 7.3, 12.38, 4.66, 6.25 and 29.9 annual cases of dengue infection per 10 000 inhabitants in 2009 to 2013, respectively. In this study, only 57.76% of the cases were classified correctly by the physicians and 26.32% of them were questioned about their home conditions and whether their family/friends had similar symptoms. Patients visited a clinic/hospital on average 4.76 days after developing symptoms and the health system was notified on average 1.75 days later, while 70.6% of them were reported within the one-day target period. There were only minor changes in case reporting times even during a DENV epidemic. Some (12.85%) of the case forms were later modified (average 16.7 days). In the period 2009-2013, the IgM confirmed PPV was 58.60%, while 20 mandatory criteria were absent on more than 25% of the forms. CONCLUSIONS: The system was accurate, simple, flexible, stable and acceptable, but a number of ways are suggested to improve this case detection and reporting system.

Estimate of the reproduction number of the 2015 Zika virus outbreak in Barranquilla, Colombia, and estimation of the relative role of sexual transmission.

BACKGROUND: In 2015, the Zika arbovirus (ZIKV) began circulating in the Americas, rapidly expanding its global geographic range in explosive outbreaks. Unusual among mosquito-borne diseases, ZIKV has been shown to also be sexually transmitted, although sustained autochthonous transmission due to sexual transmission alone has not been observed, indicating the reproduction number (R0) for sexual transmission alone is less than 1. Critical to the assessment of outbreak risk, estimation of the potential attack rates, and assessment of control measures, are estimates of the basic reproduction number, R0. METHODS: We estimated the R0 of the 2015 ZIKV outbreak in Barranquilla, Colombia, through an analysis of the exponential rise in clinically identified ZIKV cases (n=359 to the end of November, 2015). FINDINGS: The rate of exponential rise in cases was ρ=0.076days-1, with 95% CI [0.066,0.087] days-1. We used a vector-borne disease model with additional direct transmission to estimate the R0; assuming the R0 of sexual transmission alone is less than 1, we estimated the total R0=3.8 [2.4,5.6], and that the fraction of cases due to sexual transmission was 0.23 [0.01,0.47] with 95% confidence. INTERPRETATION: This is among the first estimates of R0 for a ZIKV outbreak in the Americas, and also among the first quantifications of the relative impact of sexual transmission.

A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes.

BACKGROUND: Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. METHODS: Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. RESULTS: Optimal quenching of endogenous human serum peroxidases was attained using 3% H(2)O(2) in H(2)0 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays.DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. CONCLUSIONS: This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed by staff with a basic level of training and storage at low temperatures (e.g., -80°C) is not necessary. These simple, inexpensive (US$ 0.05/sample), robust, sensitive and relatively rapid assays, using improved MAbs such as MAb 2C4.6, should be ideal for the diagnosis of all DENV serotypes in DENV endemic regions.

Cross-sectional study of Leptospira seroprevalence in humans, rats, mice, and dogs in a main tropical sea-port city.

Samples were collected from 128 symptomatic humans, 83 dogs, 49 mice, and 20 rats (Rattus rattus: 16; Rattus norvegicus: 4) in neighborhoods where human leptospirosis have been reported within the principal sea-port city of Colombia. Seroprevalences were assessed against 19 pathogenic, 1 intermediate pathogenic, and 1 saprophytic Leptospira serogroups. Pathogenic Leptospira were confirmed using conventional Leptospira-specific polymerase chain-reaction and pulsed-field gel electrophoresis analysis was used for serovar identification. Seroprevalences of 20.4%, 12.5%, 25.0%, 22.9%, and 12.4% were obtained against one to seven different serogroups in mice, R. rattus, R. norvegicus, dogs, and humans, respectively. The DNA was confirmed to be from pathogenic Leptospira by detecting the lipL32 gene in 12.5%, 3.7%, and 0.03% of the R. rattus, dog, and human samples, respectively. The first genetically typed Colombian isolate was obtained from a rat and identified as Leptospira interrogans serovar Icterohaemorrhagiae/Copenhageni.

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies.

BACKGROUND: Epitope-mapping of infectious agents is essential for pathogenesis studies. Since polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) are always polyspecific and can react with multiple epitopes, it is important to distinguish between specific and non-specific reactions. Relative antibody discriminating specificity (RADS) values, obtained from their relative ELISA reactions with L-amino acid peptides prepared in the natural versus reverse orientations (x-fold absorbance natural/absorbance reverse = RADS value) may be valuable for this purpose.PAbs generated against the dengue type-2 virus (DENV-2) nonstructural-1 (NS1) glycoprotein candidate vaccine also reacted with both DENV envelope (E) glycoproteins and blood-clotting proteins. New xKGSx/xSGKx amino acid motifs were identified on DENV-2 glycoproteins, HIV-1 gp41 and factor IXa. Their potential roles in DENV and HIV-1 antibody-enhanced replication (AER) and auto-immunity were assessed.In this study, a) RADS values were determined for MAbs and PAbs, generated in congeneic (H2: class II) mice against DENV NS1 glycoprotein epitopes, to account for their cross-reaction patterns, and b) MAb 1G5.3 reactions with xKGSx/xSGKx motifs present in the DENV-4 NS1, E and HIV-1 glycoproteins and factor IXa were assessed after the introduction of amino acid substitutions, deletions, or intra-/inter-cysteine (C-C) bridges. RESULTS: MAbs 1H7.4, 5H4.3, 3D1.4 and 1G5.3 had high (4.23- to 16.83-fold) RADS values against single epitopes on the DENV-2 NS1 glycoprotein, and MAb 3D1.4 defined the DENV complex-conserved LX1 epitope. In contrast, MAbs 1G5.4-A1-C3 and 1C6.3 had low (0.47- to 1.67-fold) RADS values against multiple epitopes. PAb DENV complex-reactions occurred through moderately-high (2.77- and 3.11-fold) RADS values against the LX1 epitope. MAb 1G5.3 reacted with xSGKx motifs present in DENV-4 NS1 and E glycoproteins, HIV-1 gp41 and factor IXa, while natural C-C bridge formations or certain amino acid substitutions increased its binding activity. CONCLUSIONS: These results: i) were readily obtained using a standard 96-well ELISA format, ii) showed the LX1 epitope to be the immuno-dominant DENV complex determinant in the NS1 glycoprotein, iii) supported an antigenic co-evolution of the DENV NS1 and E glycoproteins, and iv) identified methods that made it possible to determine the role of anti-DENV PAb reactions in viral pathogenesis.

Simple Prognostic Criteria can Definitively Identify Patients who Develop Severe Versus Non-Severe Dengue Disease, or Have Other Febrile Illnesses.

BACKGROUND: SEVERE DENGUE DISEASE (SDD) (DHF/DSS: dengue hemorrhagic fever/dengue shock syndrome) results from either primary or secondary dengue virus (DENV) infections, which occur 4 - 6 days after the onset of fever. As yet, there are no definitive clinical or hematological criteria that can specifically identify SDD patients during the early acute febrile-phase of disease (day 0 - 3: < 72 hours). This study was performed during a SDD (DHF/DSS) epidemic to: 1) identify the DENV serotypes that caused SDD during primary or secondary DENV infections; 2) identify simple clinical and hematological criteria that could significantly discriminate between patients who subsequently developed SDD versus non-SDD (N-SDD), or had a non-DENV fever of unknown origin (FUO) during day 0 - 3 of fever; 3) assess whether DENV serotype co-infections resulted in SDD. METHODS: First serum samples, with clinical and hematological criteria, were collected from 100 patients during the early acute febrile-phase (day 0 - 3: < 72 hours), assessed for DENV or FUO infections by IgM- and IgG-capture ELISAs on paired serum samples and by DENV isolations, and subsequently graded as SDD, N-SDD or FUO patients. RESULTS: IN THIS STUDY: 1) Thirty-three patients had DENV infections, predominantly secondary DENV-2 infections, including each SDD (DHF/DSS) case; 2) Secondary DENV-2/-3 and DENV-2/-4 serotype co-infections however resulted in N-SDD; 3) Each patient who subsequently developed SDD, but none of the others, displayed three clinical criteria: abdominal pain, conjunctival injection and veni-puncture bleeding, therefore each of these criteria provided definitively significant prognostic (P < 0.001) values; 4) Petechia, positive tourniquet tests and hepatomegaly, and neutrophilia or leukopenia also significantly identified those who: a) subsequently developed SDD versus N-SDD, or had a FUO; b) subsequently developed SDD versus N-SDD; c) subsequently developed N-SDD versus FUOs, respectively. CONCLUSIONS: This is the first report of simple definitively prognostic criteria for SDD patients, including the first assessment and confirmation of conjunctival injection. The three definitive clinical criteria used alone, or supported by the other four criteria, could be essential for specifically identifying those patients needing prompt hospital-based therapies to lessen or avert SDD, without unnecessary hospitalization of the other patients. KEYWORDS: Dengue virus; Severe dengue; Dengue fever; Diagnostic; Criteria; Hemorrhage; Shock.

The NS1 glycoprotein can generate dramatic antibody-enhanced dengue viral replication in normal out-bred mice resulting in lethal multi-organ disease.

Antibody-enhanced replication (AER) of dengue type-2 virus (DENV-2) strains and production of antibody-enhanced disease (AED) was tested in out-bred mice. Polyclonal antibodies (PAbs) generated against the nonstructural-1 (NS1) glycoprotein candidate vaccine of the New Guinea-C (NG-C) or NSx strains reacted strongly and weakly with these antigens, respectively. These PAbs contained the IgG2a subclass, which cross-reacted with the virion-associated envelope (E) glycoprotein of the DENV-2 NSx strain, suggesting that they could generate its AER via all mouse Fcγ-receptor classes. Indeed, when these mice were challenged with a low dose (<0.5 LD50) of the DENV-2 NSx strain, but not the NG-C strain, they all generated dramatic and lethal DENV-2 AER/AED. These AER/AED mice developed life-threatening acute respiratory distress syndrome (ARDS), displayed by diffuse alveolar damage (DAD) resulting from i) dramatic interstitial alveolar septa-thickening with mononuclear cells, ii) some hyperplasia of alveolar type-II pneumocytes, iii) copious intra-alveolar protein secretion, iv) some hyaline membrane-covered alveolar walls, and v) DENV-2 antigen-positive alveolar macrophages. These mice also developed meningo-encephalitis, with greater than 90,000-fold DENV-2 AER titers in microglial cells located throughout their brain parenchyma, some of which formed nodules around dead neurons. Their spleens contained infiltrated megakaryocytes with DENV-2 antigen-positive red-pulp macrophages, while their livers displayed extensive necrosis, apoptosis and macro- and micro-steatosis, with DENV-2 antigen-positive Kuppfer cells and hepatocytes. Their infections were confirmed by DENV-2 isolations from their lungs, spleens and livers. These findings accord with those reported in fatal human "severe dengue" cases. This DENV-2 AER/AED was blocked by high concentrations of only the NG-C NS1 glycoprotein. These results imply a potential hazard of DENV NS1 glycoprotein-based vaccines, particularly against DENV strains that contain multiple mutations or genetic recombination within or between their DENV E and NS1 glycoprotein-encoding genes. The model provides potential for assessing DENV strain pathogenicity and anti-DENV therapies in normal mice.

Evaluation of a rapid water-surface sweeping method to accurately estimate numbers of Aedes aegypti (Diptera: Culicidae) late larval stages in large water-storage containers: comparison with pupal estimates.

INTRODUCTION: Since the methodologies used to calculate Stegomyia indices have been shown to be inadequate for assessing the risk of dengue virus transmission and targeting Aedes aegypti control strategies, new surveillance methods are needed. OBJECTIVE: To evaluate the water-surface sweeping method in combination with calibration factors to estimate the total number of Ae. aegypti late larval stages (L3/L4) in large water-storage containers at different temperatures at which transmission of dengue virus occurs. MATERIALS AND METHODS: Calibration factors were derived based on the proportion of L3/L4 recovered from a predetermined number of larvae using a net of specific dimensions and water-storage containers of different capacities and water levels in semi-field conditions and at four different altitudes (14, 358, 998 and 1,630 meters above sea level). The calibration factors obtained at 14 masl were then fully validated in a field study site at this altitude. RESULTS: Four calibration factors were derived at 14 masl (28-30°C) that were used to estimate the total L3/L4 numbers in large water storage containers greater than 20 L (n=478) at 1/3, 2/3 and full water-levels. This methodology was accurate and robust within and between the 10 pairs of field workers who applied it. Different calibration factors were, however, derived to accurately estimate the total L3/L4 numbers at each of the study sites located at 358, 998 and 1,630 masl, where average temperatures were 19°C, 24°C, and 26°C respectively. CONCLUSIONS: The accurate estimates of L3/L4 numbers calculated using the water surface sweeping method can be useful for evaluating intervention strategies directed against the larval stages.

Evaluation of a rapid water-surface sweeping method to accurately estimate numbers of Aedes aegypti (Diptera: Culicidae) late larval stages in large water-storage containers: comparison with pupal estimates / Evaluación de un método de barrido rápido sobre la superficie para estimar el número total de estados larvarios tardíos de Aedes aegypti (Diptera: Culicidae) en depósitos de agua de grandes capacidades: comparación con estimativos de pupas

Introduction. Since the methodologies used to calculate Stegomyia indices have been shown to be inadequate for assessing the risk of dengue virus transmission and targeting Aedes aegypti control strategies, new surveillance methods are needed. Objective. To evaluate the water-surface sweeping method in combination with calibration factors to estimate the total number of Ae. aegypti late larval stages (L3/L4) in large water-storage containers at different temperatures at which transmission of dengue virus occurs. Materials and methods. Calibration factors were derived based on the proportion of L3/L4 recovered from a predetermined number of larvae using a net of specific dimensions and water-storage containers of different capacities and water levels in semi-field conditions and at four different altitudes (14, 358, 998 and 1,630 meters above sea level). The calibration factors obtained at 14 masl were then fully validated in a field study site at this altitude. Results. Four calibration factors were derived at 14 masl (28-30°C) that were used to estimate the total L3/L4 numbers in large water storage containers greater than 20 L (n=478) at 1/3, 2/3 and full water-levels. This methodology was accurate and robust within and between the 10 pairs of field workers who applied it. Different calibration factors were, however, derived to accurately estimate the total L3/L4 numbers at each of the study sites located at 358, 998 and 1,630 masl, where average temperatures were 19°C, 24°C, and 26°C respectively. Conclusions. The accurate estimates of L3/L4 numbers calculated using the water surface sweeping method can be useful for evaluating intervention strategies directed against the larval stages.

Introducción. Las metodologías usadas para calcular los índices de Stegomyia son inadecuadas para evaluar el riesgo de transmisión del virus del dengue y, tampoco, permiten enfocar estrategias de control de Aedes aegypti, por lo cual se requiere desarrollar nuevos métodos para la vigilancia. Objetivo. Evaluar el método de barrido del agua superficial combinado con factores de calibración para estimar el número total estadios larvarios tardíos (L3/L4) de Ae. aegypti en depósitos de grandes capacidades a diferentes temperaturas de transmisión del virus del dengue. Materiales y métodos. Los factores de calibración se derivaron de la proporción de L3/L4 recolectadas con una malla de dimensiones específicas y a partir de un número conocido de larvas, en depósitos de diferentes capacidades y niveles de agua, en condiciones de campo simuladas y a cuatro altitudes diferentes (14, 358, 998 y 1.630 metros sobre el nivel del mar). Los factores de calibración obtenidos a 14 msnm fueron plenamente validados en el campo a esa altitud. Resultados. Se derivaron cuatro factores de calibración a 14 msnm (28°C-30°C) los cuales se emplearon para estimar el número total de L3/L4 en depósitos con capacidades mayores a 20 L (n=478) y a niveles de agua de un tercio, dos tercios y lleno. Esta metodología fue precisa y sólida en los 10 pares de trabajadores que aplicaron el método y entre ellos. Sin embargo, diferentes factores de calibración fueron derivados para estimar con precisión los números totales de L3/L4 en cada uno de los sitios de estudio localizados a 358, 998 y 1.630 msnm, donde las temperaturas promedio fueron de 19°C, 24°C y 26°C, respectivamente. Conclusión. La estimación precisa del número total de L3/L4 usando el barrido descrito permite proponer el uso de este método para evaluar estrategias de control dirigido a contra estados larvarios.

Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

Monoclonal antibodies that bind to common epitopes on the dengue virus type 2 nonstructural-1 and envelope glycoproteins display weak neutralizing activity and differentiated responses to virulent strains: implications for pathogenesis and vaccines.

The abilities of monoclonal antibodies (MAbs) that bind to defined sequential epitopes on the dengue virus (DENV) nonstructural-1 (NS1) glycoproteins to cross-react with epitopes on the DENV envelope (E) glycoproteins were investigated. In this study, some of these MAbs cross-reacted with the DENV type 2 (DENV-2) E glycoprotein and with synthetic peptides representing X-ray crystallographically confirmed surface-exposed regions on this glycoprotein. MAb 1G5.3 cross-reacted with the flavivirus-conserved 101-WGNGCGLFG-109 fusion sequence, the 273-SSGNL-277 DENV-2 hinge region sequence, and the 156-GKHGKEIKIT-165 sequence of virulent DENV-2 strains. MAb 1G5.4-A1-C3 cross-reacted with the 67-NTTTESRCPT-76 and 156-GKHGKEIKIT-165 sequences of virulent DENV-2 strains, the 338-EIMDLDNRHV-347 sequence from a highly virulent DENV-2 (M2) strain, and two epitopes on a virulent DENV-3 strain (288-KMDKLELKG-296 and 323-RVEYRGEDAP-332), which all contained target ELK/KLE-type motifs (underlined). These MAbs showed reduced cross-reactions with the corresponding sequences from weakly pathogenic strains of all four DENV serotypes and had either no (MAb 1G5.4-A1-C3) or weak (MAb 1G5.3) neutralizing activity against them. MAb 1G5.3 more strongly neutralized DENV-2 strains with higher pathogenic capacities, while MAb 1G5.4-A1-C3 showed increasing neutralizing titers against the virulent DENV-3 strain and the moderately virulent and highly virulent (M2) DENV-2 strains. These cross-reactions with the E glycoprotein accord with the observation that MAb 1G5.3 caused dramatic and lethal antibody-enhanced replication (AER) of a DENV-2 strain in vivo. Together with in vivo AER studies of these DENV strains using MAb 1G5.4-A1-C3, these results may account for the increased pathogenic capacities of such strains, which is likely to have important implications for pathogenesis and vaccines.

Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans: implications for diagnosis, pathogenesis, and vaccine design.

Antibodies generated to the purified dengue type 2 virus (D-2V) nonstructural-1 (NS1) protein in mice and rabbits were compared with those generated to this protein in congeneic (H-2 class II) mouse strains and humans after D-2V infections. Unlike the profiles observed with the rabbits, similar antibody reaction profiles were generated by mice and humans with severe D-2V disease (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Many of these epitopes contained the core acidic-hydrophobic-basic (tri-amino-acid; ELK-type) motifs present in the positive or negative orientations. Antibody responses generated to these ELK/KLE-type motifs and the epitope LX1 on this protein were influenced by class II molecules in mice during D-2V infections; but these antibodies cross-reacted with human fibrinogen and platelets, as implicated in DHF/DSS pathogenesis. The core LX1 epitope (113YSWKTWG119), identified by the dengue virus complex-specific monoclonal antibody (MAb) 3D1.4, was prepared so that it contained natural I-Ad-binding and ELK-type motifs. This AFLX1 peptide, which appropriately displayed the ELK-type and LX1 epitopes in solid-phase immunoassays, generated a similar, but lower, immunodominant anti-ELK-motif antibody reaction in I-Ad-positive mice, as generated in mice and humans during D-2V infections. These antibody responses were much stronger in the high-responding mouse strains and each of the DHF/DSS patients tested and may therefore account for the association of DHF/DSS resistance or susceptibility with particular class II molecules and autoantibodies, antibody-stimulating cytokines (e.g., interleukin-6), and complement product C3a being implicated in DHF/DSS pathogenesis. These results are likely to be important for the design of a safe vaccine against this viral disease and showed the AFLX1 peptide and MAb 3D1.4 to be valuable diagnostic reagents.

Altered enzyme-linked immunosorbent assay immunoglobulin M (IgM)/IgG optical density ratios can correctly classify all primary or secondary dengue virus infections 1 day after the onset of symptoms, when all of the viruses can be isolated.

We compared dengue virus (DV) isolation rates and tested whether acute primary (P) and acute/probable acute secondary (S/PS) DV infections could be correctly classified serologically when the patients' first serum (S1) samples were obtained 1 to 3 days after the onset of symptoms (AOS). DV envelope/membrane protein-specific immunoglobulin M (IgM) capture and IgG capture enzyme-linked immunosorbent assay (ELISA) titrations (1/log(10) 1.7 to 1 log(10) 6.6 dilutions) were performed on 100 paired S1 and S2 samples from suspected DV infections. The serologically confirmed S/PS infections were divided into six subgroups based on their different IgM and IgG responses. Because of their much greater dynamic ranges, IgG/IgM ELISA titer ratios were more accurate and reliable than IgM/IgG optical density (OD) ratios recorded at a single cutoff dilution for discriminating between P and S/PS infections. However, 62% of these patients' S1 samples were DV IgM and IgG titer negative (or=2.60 and <2.60) discriminatory IgM/IgG OD (DOD) ratios on these S1 samples than those published previously to correctly classify the highest percentage of these P and S/PS infections. The DV isolation rate was highest (12/12; 100%) using IgG and IgM titer-negative S1 samples collected 1 day AOS, when 100% of them were correctly classified as P or S/PS infections using these higher DOD ratios.

Comportamiento de la leptospirosis en el departamento del Atlántico (Colombia) Enero de 1999 a marzo del 2004 / Behavior of leptospirosis in Atlantic Department (Colombia)January 1999 –March 2004

Objetivos: Describir el comportamiento epidemiológico de la leptospirosis en el departamento delAtlántico (Colombia), de enero de 1999 a marzo del 2004.Metodología: Estudio descriptivo. Se analizaron 970 muestras únicas de pacientes sospechosos deinfecciones con Leptospira en el Laboratorio Departamental del Atlántico mediante AglutinaciónMicroscópica (MAT), usando como antígenos los serovares Icterohemorragiae, Pomona, Canícola,Hardjo, Grippotyphosa y Hardjo-bovis de Leptospira interrogans.

Objective: This study was performed to describe the epidemiological situation of Leptospira in theDepartament of Atlantico (Colombia), from January 1999 to March 2004.Methods: A descriptive study was performed. A total of 970 single serum samples from patients withsuspected Leptospira infections, were analyzed using the microscopic agglutination test (MAT). Theserovars of Icterohaemorrhagiae, Pomona, Canicola, Hardjo, Grippotyphosa and Hardjo-bovis belongingto L. interrogans, were used as antigens. Information about clinical presentation based on epidemiologicalsheets, visits to patients and climatological data were obtained.Results: The 9,7percent samples were IgM positive for Leptospira and the most prevalent was the serovarIcterohaemorrhagiae (62percent), followed by Hardjo (12.8percent). Most of the patients were male (61percent) between 15 and 45 y.o. The most common presenting features in these patients were (91.7percent), myalgia (72 percent), vomit/nausea (70.8 percent), headache (68.1 percent) and icterichia (63.9 percent). 8.6percent of the cases were severe, associated to infections with the serovar Icterohemorragiae and their symptomathology was similar to the Weil ìs syndrome; no fatalities were registered. The highest incidences were recorded during the years 2003 (23),2001(21) and 2002 (18) especially during the rainy season (August-November). Barranquilla reportedthe highest number of cases (46) followed by Soledad (25), Puerto Colombia (6) and Galapa...

Investigation of relationships between Aedes aegypti egg, larvae, pupae, and adult density indices where their main breeding sites were located indoors.

Aedes aegypti (L.) density indices obtained in a dengue fever (DF) endemic area were compared. One hundred and twenty premises, in an urban area of Colombia where dengue type-1 and type-2 virus cocirculated, were randomly selected and sampled for 7 months. The geometric mean monthly numbers (density index, DI) of Ae. aegypti eggs (ODI), 4th instar larvae (LDI), pupae (PDI), and adults (ADI) were calculated based on the use of ovitraps, nets, and manual aspirators, respectively. A negative temporal correlation was observed between the LDI and the ODI (r = -0.83, df = 5, and P < 0.01). Positive temporal correlations were only observed between the LDI and the PDI (r = 0.90, df = 5, and P < 00.5) and the Breteau and House indices (r = 0.86, df = 5, and P < 0.01). No other correlations were found between these indices and any of the other density indices or the incidence of suspected DF cases in residents, the temperature, the rainfall, or seasonal fluctuations. Our results were, therefore, probably due to the most productive Ae. aegypti breeding sites (large water containers) being located indoors within this study area. The number of adult female Ae. aegypti/person (n = 0.5) and pupae/person (n = 11) in our study area were lower and dramatically higher than the transmission thresholds previously reported for adult and pupae, respectively. Because there were confirmed DF cases during the study period, the transmission threshold based on the Ae. aegypti pupae was clearly more reliable. We found that the mean ovitrap premise index (OPI) was 98.2% during this study and that the mean larval (L-4th instars) premise index (LPI) was 59.2%, and therefore we suggest that the OPI and LPI would be more sensitive methods to gauge the effectiveness of A. aegypti control programs.

An inexpensive intervention for the control of larval Aedes aegypti assessed by an improved method of surveillance and analysis.

A sampling method coupled with statistical calibration factors was developed to accurately assess the numbers of larvae and pupae of Aedes aegypti in large water-storage containers of variable capacities and water levels. Aedes aegypti productivity in different types of breeding sites found in an urban study area in central Colombia was assessed and compared. In this study, water-storage tanks and drums were found to comprise 79% of the containers positive for larval Ae. aegypti, which contributed to 93 and 92% of the total production of populations of 4th-stage larvae and pupae, respectively. These main breeding sites of Ae. aegypti were found at an indoor to outdoor ratio of 2.4:1 and no correlation was found between temporal fluctuation of populations of larval Ae. aegypti and monthly rainfall. Netted lids that used inexpensive local materials were designed to prevent oviposition by Ae. aegypti. During a 6-month trial period, 56% of inspected containers had netted lids correctly in place. Of these, 78% had no mosquito larvae. Because only 37% of uncovered containers were free of mosquito larvae, a significant difference was demonstrated when these inexpensive mechanical barriers were used (chi2 = 138.7; P < 0.001). These netted lids and the improved methods described to assess the productivity of larval and pupal Ae. aegypti in this study are now being used in combination with other strategies to assess and control these populations of dengue virus vectors in the main port city on the Atlantic Coast of Colombia.

Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography.

Phylogenetic analysis of the Flavivirus genus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. These phylogenies identified mosquito-borne, tick-borne and no-known-vector (NKV) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. The mosquito-borne flaviviruses revealed two distinct epidemiological groups: (i) the neurotropic viruses, often associated with encephalitic disease in humans or livestock, correlated with the Culex species vector and bird reservoirs and (ii) the non-neurotropic viruses, associated with haemorrhagic disease in humans, correlated with the Aedes species vector and primate hosts. Thus, the tree topology describing the virus-host association may reflect differences in the feeding behaviour between Aedes and Culex mosquitoes. The tick-borne viruses also formed two distinct groups: one group associated with seabirds and the other, the tick-borne encephalitis complex viruses, associated primarily with rodents. The NKV flaviviruses formed three distinct groups: one group, which was closely related to the mosquito-borne viruses, associated with bats; a second group, which was more genetically distant, also associated with bats; and a third group associated with rodents. Each epidemiological group within the phylogenies revealed distinct geographical clusters in either the Old World or the New World, which for mosquito-borne viruses may reflect an Old World origin. The correlation between epidemiology, disease correlation and biogeography begins to define the complex evolutionary relationships between the virus, vector, vertebrate host and ecological niche.