Pathophysiology of chikungunya virus infection associated with fatal outcomes.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.

Norovirus in children under 2 years of age: an epidemiological study in Panama during the COVID-19 pandemic.

Introduction: Norovirus infection is a common cause of acute gastroenteritis (AGE). Surveillance activities are important to aid investigation into effective norovirus control strategies, including vaccination. Here, we report ancillary findings related to the incidence, prevalence, and etiology of AGE caused by norovirus in Panama after adjustment of study methodology to comply with national coronavirus disease 2019 (COVID-19) mandates. Methods: In January 2020, children aged <2 years began enrolling into an epidemiological study in Panama to estimate the burden of norovirus in preparation for evaluating upcoming prevention strategies. This included an observational, longitudinal, community-based AGE surveillance study and a hospital-based AGE surveillance study. For the longitudinal study, healthy children aged 5-18 months were enrolled from January 6 through March 23, 2020, with a follow-up of approximately 6 months. The last participant was contacted on September 23, 2020. For the hospital-based study, starting on January 21, 2020, children aged <2 years who were admitted to the Hospital del Niño Dr. José Renán Esquivel in Panama City due to AGE were evaluated. The last sample was collected on September 29, 2020. Collected stool samples were tested for norovirus as well as astrovirus, sapovirus, and various enteropathogens. Unfortunately, this study was disrupted by the subsequent implementation of disease transmission control procedures for the COVID-19 pandemic, and the study methodology was revised to comply with COVID-19 mandates. Results: In the longitudinal surveillance cohort [N = 400 (Chiriquí, n = 239; Panama, n = 161)], a total of 185 AGE episodes were documented (Chiriquí, n = 85; Panama, n = 100) resulting in an overall AGE incidence of 11.6 (95% CI: 9.99-13.4) episodes per 100 child-months. The norovirus-related AGE incidence was 0.3 (95% CI: 0.10-0.73) episodes per 100 child-months (5/185 AGE episodes) and the prevalence of norovirus was 4.6% (13/282 stool samples collected). In the hospital-based surveillance cohort, at least one pathogen was detected in 50% of samples (44/88 stool samples collected) and norovirus prevalence was 6.8% (6/88 stool samples collected). Discussion: This report demonstrates how the occurrence of the COVID-19 pandemic hindered the conduct of clinical trials. However, this also created unique research opportunities to investigate the potential impact of pandemic control measures on the etiology of infectious diarrheal disease.

Detection of parechovirus A in respiratory, gastrointestinal, and neurological clinical samples of pediatric patients from Panama (2014-2015).

Parechovirus A (PeV-A, Parechovirus, Picornaviridae) are human pathogens associated with mild to severe gastrointestinal and respiratory diseases in young children. While several studies have investigated the association of PeV-A with human disease, little is known about its epidemiology or detection in Latin America. Between the years 2014 and 2015, a total of 200 samples were collected from Panamanian pediatric patients aged < 16 years old exhibiting symptoms associated with respiratory (n = 64), gastrointestinal (n = 68), or neurological (n = 68) diseases. These samples were gathered from patients who had previously received negative diagnoses for the main respiratory viruses, rotavirus, and neurological viruses like herpes virus, enterovirus, and cytomegalovirus. The presence of PeV-A was analyzed by real time RT-PCR.Eight positive PeV-A infections (4.0%, 95% CI: 1.7 to 7.7) were detected: two in respiratory samples (3.0%, 95% CI: 0.3 to 10.8), five in gastrointestinal samples (7.3%, 95% CI: 2.4 to 16.3), and one in cerebrospinal fluid (1.5%, 95% CI: 1.4 to 7.9). The study provides evidence of PeV-A circulation in Panama and the data collectively, remarked on the importance of considering PeV-A in the Panamanian pediatric diagnostic landscape, especially when conventional testing for more common viruses yields negative results.

Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Detection of Leishmania RNA Virus 1 in Leishmania (Viannia) panamensis Isolates, Panama.

We detected Leishmania RNA virus 1 (LRV1) in 11 isolates of Leishmania (Viannia) panamensis collected during 2014-2019 from patients from different geographic areas in Panama. The distribution suggested a spread of LRV1 in L. (V.) panamensis parasites. We found no association between LRV1 and an increase in clinical pathology.

SARS-CoV-2 reinfection with a virus harboring mutation in the Spike and the Nucleocapsid proteins in Panama.

We report a case of reinfection by SARS-CoV-2 with the second virus harboring amino acid changes in the Spike protein (141-143del, D215A, ins215AGY, L452R, D614G), orf1a, helicase, orf3a, and Nucleocapside. The virus associated with the reinfection, from an endemic lineage containing the S:L452R immune escape mutation, was circulating in Panama at the time.

Expansion and Refinement of Deep Sequence-Coupled Biopanning Technology for Epitope-Specific Antibody Responses in Human Serum.

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases.

Molecular Epidemiology of Dengue in Panama: 25 Years of Circulation.

Dengue virus (DENV) is the most prevalent arbovirus in terms of human public health importance globally. In addition to DENV epidemiological surveillance, genomic surveillance may help investigators understand the epidemiological dynamics, geographic distribution, and temporal patterns of DENV circulation. Herein, we aimed to reconstruct the molecular epidemiology and phylogeny of DENV in Panama to connect the epidemiological history of DENV dispersal and circulation in Latin America. We retrospectively analyzed the epidemiological data obtained during 25 years of DENV surveillance in Panama. DENV was reintroduced in Panama in 1993 after a 35 year absence of autochthonous transmission. The increase in the number of total dengue cases has been accompanied by an increase in severe and fatal cases, with the highest case fatality rate recorded in 2011. All four serotypes were detected in Panama, which is characterized by serotype replacement and/or co-circulation of multiple serotypes. Phylogenetic analysis of datasets collected from envelope (E) gene sequences obtained from viruses isolated from human sera demonstrated that circulating viruses were highly diverse and clustered in distinct clades, with co-circulation of clades from the same genotype. Our analyses also suggest that Panamanian strains were related to viruses from different regions of the Americas, suggesting a continuous exchange of viruses within the Americas.

Zika Virus-Associated Cerebellitis with Complete Clinical Recovery.

Zika virus (ZIKV) was first detected in the Americas in Brazil in 2015, with a rapid spread to surrounding countries. In Panama, the outbreak began in November 2015 in an indigenous community located on the Caribbean side of the country. Zika virus is typically associated with a diffuse rash, fever, and conjunctivitis. It can rarely cause neurologic manifestations, most commonly microcephaly and Guillain-Barré syndrome. Encephalitis and acute encephalomyelitis are known complications, but ZIKV-associated cerebellitis has yet to be reported in the literature. Herein, we report a case of cerebellitis in a patient infected with ZIKV. This patient developed severe frontal headache and vertigo on the third day of illness, and dysarthria and ataxia on the fifth day. After 1 week of hospitalization, the patient completely recovered. The laboratory serological diagnosis was complicated because of the detection of antibodies against dengue, suggesting a secondary flavivirus infection.

Distribution of influenza virus types by age using case-based global surveillance data from twenty-nine countries, 1999-2014.

BACKGROUND: Influenza disease burden varies by age and this has important public health implications. We compared the proportional distribution of different influenza virus types within age strata using surveillance data from twenty-nine countries during 1999-2014 (N=358,796 influenza cases). METHODS: For each virus, we calculated a Relative Illness Ratio (defined as the ratio of the percentage of cases in an age group to the percentage of the country population in the same age group) for young children (0-4 years), older children (5-17 years), young adults (18-39 years), older adults (40-64 years), and the elderly (65+ years). We used random-effects meta-analysis models to obtain summary relative illness ratios (sRIRs), and conducted meta-regression and sub-group analyses to explore causes of between-estimates heterogeneity. RESULTS: The influenza virus with highest sRIR was A(H1N1) for young children, B for older children, A(H1N1)pdm2009 for adults, and (A(H3N2) for the elderly. As expected, considering the diverse nature of the national surveillance datasets included in our analysis, between-estimates heterogeneity was high (I2>90%) for most sRIRs. The variations of countries' geographic, demographic and economic characteristics and the proportion of outpatients among reported influenza cases explained only part of the heterogeneity, suggesting that multiple factors were at play. CONCLUSIONS: These results highlight the importance of presenting burden of disease estimates by age group and virus (sub)type.

Genotypes of human metapneumovirus circulating during 2010-2012 in children from Panama.

Human metapneumovirus (HMPV) is a common causative agent of severe respiratory tract infections in children under 5 years old, the elderly and immunocompromised patients, being responsible for 5-15% of all viral respiratory infections requiring hospitalization. Though HMPV was included in the surveillance program for respiratory viruses in 2010, its genotype distribution remains unknown. Herein, 45 positive samples to HMPV from children ≤5 years old were characterized by phylogenetic analysis based on N gene sequence. Results showed the co-circulation of four sub-lineages: A2a (8.8%), A2b (55.5%), B1 (15.6%), and B2 (20%), demonstrating the genetic heterogeneity of HMPV circulating in Panamá.

The reintroduction of DENV-2 in 2011 in Panama and subsequent outbreak characteristic.

The circulation of the South-east Asian/American (AS/AM) dengue 2 virus (DENV-2) genotype in the Americas has been associated with a high rate of severe disease. From 1993, the year DENV was reintroduced in Panama, until 2011 there were 29 dengue-associated deaths, 17 of which occurred in 2011, the most severe outbreak with a case fatality rate (CFR) of 44% (17 deaths out of 38 severe dengue cases). During this outbreak DENV-2 was reintroduced into the country, whereas over the prior five years DENV-1 and -3 were predominant. Herein, we describe the 2011 Panama outbreak and genetically characterize the Panamanian DENV-2 strains, which were associated with severe dengue disease in Panama. Our results suggest that the DENV-2 isolates from this outbreak belonged to the AS/AM genotype sub-clade 2BI and were genetically close to viruses described in the outbreaks in Nicaragua, Honduras, Guatemala and Mexico from 2006-2011. Sub-clade 2BI has previously been associated with severe disease in Nicaragua during outbreaks from 2005-2007.

Genetic variability of human respiratory syncytial virus group B in Panama reveals a novel genotype BA14.

In Panama, human respiratory syncytial virus (HRSV) is responsible of 20-40% of acute respiratory infections in children under 5 years old. Currently, little is known about the genetic variability of HRSV in Central America and the Caribbean. Recently, we reported the genetic variability of HRSV-A, however; no studies on HRSV-B in Panama have been described yet. In this study, 24 sequences of Panamanian HRSV-B, from children (<5 years) with acute respiratory infections (ARI), collected from July 2008 to November 2012 were analyzed. All sequences share the characteristic 60-nt duplication of the BA strains. Six Panamanian strains grouped with the BA10 genotype and 12 samples clustered together in a separate monophyletic clade with an aLRT support value of 0.92 and an intra-group p-distance less than 0.07. This fulfills the criteria to consider a new genotype in HRSV, which we named BA14 genotype. Another six strains remain unclassified, but closely related to BA9, BA11, or the new BA14 genotypes, according to their genetic p-distance. Different amino acid substitutions in the Panamanian HRSV-B strains were observed, some previously described and others found only on Panamanian strains. This study contributes to the knowledge of the genetic variability and evolution of HRSV in Central America.

Clinical Manifestations of Punta Toro Virus Species Complex Infections, Panama, 2009.

An investigation in Panama found that Punta Toro virus species complex (PTVs) may contribute to febrile illnesses with symptoms mirroring those of dengue fever. However, further studies are needed to determine if PTV infection causes only a mild disease or if it can have more serious manifestations in some patients.

Characteristics of seasonal influenza A and B in Latin America: Influenza surveillance data from ten countries.

INTRODUCTION: The increased availability of influenza surveillance data in recent years justifies an actual and more complete overview of influenza epidemiology in Latin America. We compared the influenza surveillance systems and assessed the epidemiology of influenza A and B, including the spatio-temporal patterns of influenza epidemics, in ten countries and sub-national regions in Latin America. METHODS: We aggregated the data by year and country and characteristics of eighty-two years were analysed. We calculated the median proportion of laboratory-confirmed influenza cases caused by each virus strain, and compared the timing and amplitude of the primary and secondary peaks between countries. RESULTS: 37,087 influenza cases were reported during 2004-2012. Influenza A and B accounted for a median of 79% and, respectively, 21% of cases in a year. The percentage of influenza A cases that were subtyped was 82.5%; for influenza B, 15.6% of cases were characterized. Influenza A and B were dominant in seventy-five (91%) and seven (9%) years, respectively. In half (51%) of the influenza A years, influenza A(H3N2) was dominant, followed by influenza A(H1N1)pdm2009 (41%) and pre-pandemic A(H1N1) (8%). The primary peak of influenza activity was in June-September in temperate climate countries, with little or no secondary peak. Tropical climate countries had smaller primary peaks taking place in different months and frequently detectable secondary peaks. CONCLUSIONS: We found that good influenza surveillance data exists in Latin America, although improvements can still be made (e.g. a better characterization of influenza B specimens); that influenza B plays a considerable role in the seasonal influenza burden; and that there is substantial heterogeneity of spatio-temporal patterns of influenza epidemics. To improve the effectiveness of influenza control measures in Latin America, tropical climate countries may need to develop innovative prevention strategies specifically tailored to the spatio-temporal patterns of influenza in this region.

Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses.

Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets.

Unusual pattern of chikungunya virus epidemic in the Americas, the Panamanian experience.

BACKGROUND: Chikungunya virus (CHIKV) typically causes explosive epidemics of fever, rash and polyarthralgia after its introduction into naïve populations. Since its introduction in Panama in May of 2014, few autochthonous cases have been reported; most of them were found within limited outbreaks in Panama City in 2014 and Puerto Obaldia town, near the Caribbean border with Colombia in 2015. In order to confirm that Panama had few CHIKV cases compared with neighboring countries, we perform an epidemiological analysis of chikungunya cases reported from May 2014 to July 2015. Moreover, to understand this paucity of confirmed CHIKV cases, a vectorial analysis in the counties where these cases were reported was performed. METHODS: Chikungunya cases were identified at medical centers and notified to health authorities. Sera samples were analyzed at Gorgas Memorial Institute for viral RNA and CHIKV-specific antibody detection. RESULTS: A total of 413 suspected cases of CHIKV infections were reported, with incidence rates of 0.5 and 0.7 per 100,000 inhabitants in 2014 and 2015, respectively. During this period, 38.6% of CHIKV cases were autochthonous with rash and polyarthralgia as predominant symptoms. CHIKV and DENV incidence ratios were 1:306 and 1:34, respectively. A phylogenetic analysis of E1/E2 genomic segment indicates that the outbreak strains belong to the Asian genotype and cluster together with CHIKV isolates from other American countries during the same period. Statistical analysis of the National Vector Control program at the district level shows low and medium vector infestation level for most of the counties with CHIKV cases. This index was lower than for neighboring countries. CONCLUSIONS: Previous training of clinical, laboratory and vector workers allowed a good caption and detection of the chikungunya cases and fast intervention. It is possible that low/medium vector infestation level could explain in part the paucity of chikungunya infections in Panama.

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

INTRODUCTION: Determining the optimal time to vaccinate is important for influenza vaccination programmes. Here, we assessed the temporal characteristics of influenza epidemics in the Northern and Southern hemispheres and in the tropics, and discuss their implications for vaccination programmes. METHODS: This was a retrospective analysis of surveillance data between 2000 and 2014 from the Global Influenza B Study database. The seasonal peak of influenza was defined as the week with the most reported cases (overall, A, and B) in the season. The duration of seasonal activity was assessed using the maximum proportion of influenza cases during three consecutive months and the minimum number of months with ≥80% of cases in the season. We also assessed whether co-circulation of A and B virus types affected the duration of influenza epidemics. RESULTS: 212 influenza seasons and 571,907 cases were included from 30 countries. In tropical countries, the seasonal influenza activity lasted longer and the peaks of influenza A and B coincided less frequently than in temperate countries. Temporal characteristics of influenza epidemics were heterogeneous in the tropics, with distinct seasonal epidemics observed only in some countries. Seasons with co-circulation of influenza A and B were longer than influenza A seasons, especially in the tropics. DISCUSSION: Our findings show that influenza seasonality is less well defined in the tropics than in temperate regions. This has important implications for vaccination programmes in these countries. High-quality influenza surveillance systems are needed in the tropics to enable decisions about when to vaccinate.

Epidemiology of Emergent Madariaga Encephalitis in a Region with Endemic Venezuelan Equine Encephalitis: Initial Host Studies and Human Cross-Sectional Study in Darien, Panama.

BACKGROUND: Neurotropic arboviral infections are an important cause of encephalitis. A zoonotic, vector-borne alphavirus, Madariaga virus (MADV; formerly known as South American eastern equine encephalitis virus), caused its first documented human outbreak in 2010 in Darien, Panama, where the genetically similar Venezuelan equine encephalitis virus (VEEV) is endemic. We report the results of a seroprevalence survey of animals and humans, illustrating contrasting features of MADV and VEEV ecology and epidemiology. METHODS: Small mammals were trapped in 42 sites in Darien, Panama, using Sherman traps, Tomahawk traps, and mist nets for bats. Blood was tested for the presence of neutralizing antibodies to MADV and VEEV. In addition, bird sera collected in 2007 in Chagres, Panama, were tested for MADV and VEEV neutralizing antibodies. Viremia was ascertained by RT-PCR. Human exposure to these two viruses was determined by IgG ELISA, followed by plaque reduction neutralization tests. To identify relevant risk factors for MADV or VEEV exposure, logistic regression analysis was performed, and the most parsimonious model was selected based on the Akaike information criterion. RESULTS: The animal survey yielded 32 bats (16 species), 556 rodents (12 species), and 20 opossums (4 species). The short-tailed cane mouse (Zygodontomys brevicauda) found abundantly in pasture and farms, had the highest MADV seroprevalence (8.3%). For VEEV, the shrub and forest-dwelling long-whiskered rice rat (Transandinomys bolivaris) had the highest seroprevalence (19.0%). Viremia was detected in one animal (Z. brevicauda). Of the 159 bird sera (50 species) tested, none were positive for either virus. In humans (n = 770), neutralizing antibodies to MADV and VEEV were present in 4.8% and 31.5%, respectively. MADV seropositivity was positively associated with cattle ranching, farming, and fishing. Having VEEV antibodies and shrubs near the house diminished risk. Age, forest work, farming and fishing were risk factors for VEEV, while having MADV antibodies, glazed windows, waste pick-up and piped water were protective. CONCLUSION: Our findings suggest that the short-tailed cane mouse and the long-whiskered rice rat serve as hosts for MADV and VEEV, respectively. The preferred habitat of these rodent species coincides with areas associated with human infection risk. Our findings also indicate that MADV emerged recently in humans, and that the transmission cycles of these two sympatric alphaviruses differ spatially and in host utilization.