Integrating indicator-based and event-based surveillance data for risk mapping of West Nile virus, Europe, 2006 to 2021.

BackgroundWest Nile virus (WNV) has an enzootic cycle between birds and mosquitoes, humans being incidental dead-end hosts. Circulation of WNV is an increasing public health threat in Europe. While detection of WNV is notifiable in humans and animals in the European Union, surveillance based on human case numbers presents some limitations, including reporting delays.AimWe aimed to perform risk mapping of WNV circulation leading to human infections in Europe by integrating two types of surveillance systems: indicator-based and event-based surveillance.MethodsFor indicator-based surveillance, we used data on human case numbers reported to the European Centre for Disease Prevention and Control (ECDC), and for event-based data, we retrieved information from news articles collected through an automated biosurveillance platform. In addition to these data sources, we also used environmental data to train ecological niche models to map the risk of local WNV circulation leading to human infections.ResultsThe ecological niche models based on both types of surveillance data highlighted new areas potentially at risk of WNV infection in humans, particularly in Spain, Italy, France and Greece.ConclusionAlthough event-based surveillance data do not constitute confirmed occurrence records, integrating both indicator-based and event-based surveillance data proved useful. These results underscore the potential for a more proactive and comprehensive strategy in managing the threat of WNV in Europe by combining indicator- and event-based and environmental data for effective surveillance and public health response.

Seropositivity of West Nile virus among acute febrile patients in Ilorin, Nigeria.

INTRODUCTION: West Nile Virus (WNV), a member of Flaviviridae family, is one of the most widely distributed arboviruses in the world. In developing countries like Nigeria, fever resulting from the WNV infection is often presumptively ascribed to malaria or typhoid due to misdiagnosis and low-level awareness of the viral infection. This study determined the prevalence of WNV IgM and IgG antibodies among febrile patients in the Ilorin metropolis. MATERIALS AND METHODS: A total of two hundred (200) blood samples were collected from consenting patients and each serum was screened for anti-WNV IgM and IgG antibodies using indirect enzyme-linked immunosorbent assay (ELISA). Statistical correlation and logistic regression analysis were conducted. RESULTS: Overall, 6% (12/200) anti-WNV IgM seropositivity rate was recorded amongst the acute febrile patients with higher prevalence (6.30%) in females than in males (5.45%). Anti-WNV IgG positivity rate of 52% (104/200) was recorded, with 50.67% positivity rate in males and 38.95% in female participants. The convalescence phase posited by the 5.4% (11/200) co-detection of anti-WNV IgG and IgM antibodies among the participants was recorded. A statistical correlation was noticed with the age and religion of respondents to WNV serological positivity while gender, occupation, use of mosquito nets and formal education had no positive correlation at p < 0.05. However, based on odd ratio at 95% CI and logistic regression coefficients, the evaluated risk factors such as blood transfusion, residency, malaria parasite, and proximity to stagnant water and bush were significant to anti-WNV IgG and IgM positivity. CONCLUSION: The findings of this study show the circulation of WNV in the study area. There is an urgent need for clinicians/physicians to include screening for the West Nile virus in cases of febrile patients before the commencement of treatment.

[Opsoclonus-myoclonus syndrome associated with West Nile virus]. / Opsoklonus-mioklonus sindrom, obuslovlennyi likhoradkoi Zapadnogo Nila.

Opsoclonus is oculomotor dyskinesia characterized by rapid, repetitive conjugate eye movements that are involuntary, arrhythmic, chaotic, and multidirectional (horizontal, vertical, and torsional components). Most common cause of the symptom is paraneoplastic process. It is combined with myoclonus usually with the development of opsoclonus-myoclonus syndrome. Viral etiology is one of the possible causes of the of this syndrome, which is presented in the following case. A 26-year-old man was admitted to an infectious hospital suspected by encephalitis. After a 2-day febrile fever the patient developed balance problem, nausea, vomiting, tremors in the limbs and head, sensations of jerking of eyeballs. The neurological examination revealed opsoclonus, myoclonic jerking in the limbs, neck and trunk muscles, severe static and dynamic ataxia, there was no consciousness changes or altered mental stature. Cerebrospinal fluid examination revealed a pleocytosis (24 cells), increased protein levels (1.1 g/l). MRI of the brain was normal. After excluding of typical neuroinfections the patient was tested for West Nile fever. Elevated titers of IgG and IgM for West Nile fever virus were detected, as well as positive PCR for virus RNA in the cerebrospinal fluid. Patient was treated by acyclovir, an antibiotic and dexamethasone but severe neurological symptoms were persisted for 2 weeks with inability of sitting and walking. Then the symptoms gradually began to improve, rehabilitation was included with total recovery during the next 2 months. The doctors should be aware for possibility of neuroinvasive form of West Nile fever as the etiology of opsoclonus-myoclonus syndrome.

Neuroinflammation-Induced SIADH in West Nile Virus Infection: An Uncommon Neurological and Endocrine Manifestation.

BACKGROUND West Nile virus (WNV) is a vector-borne flavivirus that is typically transmitted by Aedes and Anopheles mosquitos. WNV infection typically presents with symptoms consistent with viral meningitis, which include fever, headache, and meningeal signs. WNV infections are typically self-resolving, with symptoms lasting 3-10 days. Although uncommon, patients with WNV infection can be afflicted with hyponatremia, although the etiology is unclear. Because of encephalitis, neuroinflammation may be involved in the deterioration of adrenal signaling, leading to salt wasting. CASE REPORT We present the case of a 75-year-old man who presented with headache, neck pain, photophobia, and viral illness symptoms. He was found to be profoundly hyponatremic, concerning for SIADH. He had a sodium drop to 117 mmol/L, leading to further lethargy and confusion. He received 3% saline, fluid restriction, and salt tablets, and more common causes of SIADH were ruled out. MRI revealed the absence of the posterior pituitary bright spot. He was subsequently found to have positive WNV titers and improved with continued supportive treatment. CONCLUSIONS WNV presents a unique challenge to providers due to its difficult diagnosis and association with hyponatremia. There is no clear role for specific medical treatment such as corticosteroids vs IV immunoglobulins. Supportive care is recommended for those testing positive for WNV. Clinicians should consider the possibility of viral infections, such as WNV, in patients living in endemic areas who present with unexplained hyponatremia, cognitive symptoms, and relevant history.

Vector-Borne Diseases Potpourri.

The Intergovernmental Panel on Climate Change has reported that the prevalence of vector-borne diseases has increased in recent decades and that the prevalence of malaria, Lyme disease, dengue, and, in particular, West Nile virus infection are expected to increase further if control measures are not strengthened. (1)(2) This review article summarizes the epidemiology, various clinical manifestations, and management strategies of these vector-borne diseases with increasing prevalence both in the United States and worldwide.

Ocular manifestations of West Nile virus.

PURPOSE OF REVIEW: Over the past two decades, the ophthalmic manifestations of West Nile virus have been more clearly established in the literature. This review aims to summarize its diagnosis and pathogenesis, with a focus on its clinical appearance, characteristic imaging features, and management. RECENT FINDINGS: Ocular manifestations of West Nile virus present early in the disease course and are more common in cases with severe neurological involvement. The use of optical coherence tomography (OCT), optical coherence tomography angiography (OCT-A), fundus autofluorescence (FAF), fluorescein angiogram (FA), and indocyanine green angiography (ICGA) can aid in its diagnosis and management. SUMMARY: West Nile virus infection may present with ocular findings that include anterior uveitis, vitritis, retinitis, chorioretinitis, and optic neuropathy; visual prognosis can range from excellent to poor depending on severity of involvement and the presence of secondary complications, such as occlusive vasculitis and macular ischemia. Diagnosis may be aided by multimodal imaging assessment. The ophthalmologist should have a high clinical suspicion for ocular involvement in cases of severe systemic disease.

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.

Catastrophic Case of West Nile Virus Rhombencephalitis in AIDS.

West Nile Virus (WNV) belongs to the Flaviviridae family of viruses. It was first isolated and identified in 1937. Patients typically present with flu-like symptoms or are asymptomatic; however, neuroinvasive West Nile can lead to significant neurological impairment. Herein presented is a catastrophic case of WNV rhombencephalitis in a male patient newly diagnosed with AIDS. This report sheds light on the potential for severe neurological complications in co-infected patients and emphasizes the importance of early recognition.

West Nile virus meningitis presenting with migrainous headache.

West Nile Virus (WNV) infection is a clinical picture that is transmitted from wild birds, its natural host, to humans through mosquitoes and generally shows an asymptomatic course. Influenza-like WNV fever is frequently seen in symptomatic individuals, and a neuroinvasive course is more rarely observed. Neuroinvasive WNV has a broad-spectrum profile of neurological signs and symptoms. WNV meningitis is one of the most common neuroinvasive forms of WNV, and it does not differ clinically and radiologically from other viral meningitis. Secondary headaches, which can mimic primary headaches, are an infectious factor that should be kept in mind in the etiology, especially in cases presenting in the summer months. In this study, a case of WNV meningitis presenting with a headache of migrainous character is presented.

Early rise of West Nile fever in Israel, June 2024.

This report describes an unusual surge of West Nile fever in Israel in June 2024, during which 125 cases were diagnosed, compared with 4 cases on average during June in previous years (2014-23). Of the cases, 64 (62.1%) had neuroinvasive disease and 12 (9.6%) died; the 2024 case fatality rate was not significantly elevated vs the average rate in 2014-23. The early rise could be related to a temperature increase in spring and early summer of 2024.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Bilateral multifocal chorioretinitis as the only presentation of acute West Nile virus infection: a case report.

BACKGROUND: To describe a case of bilateral multifocal chorioretinitis as the only presentation of acute West Nile virus (WNV) infection in the absence of neurological involvement. CASE PRESENTATION: A 78-year-old Italian woman was admitted to our emergency department because she noticed blurry vision in both eyes. She did not report fever, fatigue, or neurological symptoms in the last few days. Multimodal imaging showed the presence of bilateral hyperfluorescent lesions with a linear distribution, that corresponded to hypocyanescent spots on indocyanine green angiography. Antibody serology showed the presence of IgM antibodies, IgG antibodies, and ribonucleic acid (RNA) for WNV. Magnetic resonance imaging (MRI) of the brain ruled out central nervous system involvement. Three months later, the patient reported spontaneous resolution of her symptoms and remission of the chorioretinal infiltrates. CONCLUSIONS: In endemic areas, it is important to think of acute WNV infection as an explanatory etiology in cases of multifocal chorioretinitis, even without neurological involvement.

[Arbovirus - a threat to transfusion safety in Spain: a narrative review]. / Arbovirus como amenaza para la seguridad transfusional en España: revisión narrativa.

Arboviruses represent a threat to transfusion safety for several reasons: the presence of vectors and the notification of autochthonous cases in our region, the recent increase in the number of cases transmitted through blood and/or blood component transfusion, the high prevalence rates of RNA of the main arboviruses in asymptomatic blood donors, and their ability to survive processing and storage in the different blood components. In an epidemic outbreak caused by an arbovirus in our region, transfusion centres can apply different measures: reactive measures, related to donor selection or arbovirus screening, and proactive measures, such as pathogen inactivation methods. The study of the epidemiology of the main arboviruses and understanding the effectiveness of the different measures that we can adopt are essential to ensure that our blood components remain safe.

A Specific Pattern of Routine Cerebrospinal Fluid Parameters Might Help to Identify Cases of West Nile Virus Neuroinvasive Disease.

BACKGROUND: Viral meningitis/encephalitis (ME) is a rare but potentially harmful disease. The prompt identification of the respective virus is important to guide not only treatment but also potential public health countermeasures. However, in about 40% of cases, no virus is identified despite an extensive diagnostic workup. The aim of the present study was to analyze demographic, seasonal, and routine cerebrospinal fluid (CSF) parameters in cases of viral ME and assess their utility for the prediction of the causative virus. METHODS: Demographic data, season, and routine CSF parameters (total leucocytes, CSF cell differentiation, age-adjusted CSF/serum albumin ratio, and total immunoglobulin ratios) were retrospectively assessed in cases of viral ME. RESULTS: In total, 156 cases of acute viral ME (74 female, median age 40.0 years) were treated at a tertiary-care hospital in Germany. Specific viral infections were detected in 93 (59.6%) cases. Of these, 14 (9.0%) cases were caused by herpes simplex virus (HSV), 36 (23.1%) by varicella-zoster virus (VZV), 27 (17.3%) by enteroviruses, 9 (5.8%) by West Nile virus (WNV), and 7 (4.5%) by other specific viruses. Additionally, 64 (41.0%) cases of ME of unknown viral etiology were diagnosed. Cases of WNV ME were older, predominantly male, showed a severe disruption of the blood-CSF-barrier, a high proportion of neutrophils in CSF, and an intrathecal total immunoglobulin M synthesis in the first CSF sample. In a multinominal logistic regression analysis, the accuracy of these CSF parameters together with age and seasonality was best for the prediction of WNV (87.5%), followed by unknown viral etiology (66.7%), VZV (61.8%), and enteroviruses (51.9%). CONCLUSIONS: Cases with WNV ME showed a specific pattern of routine CSF parameters and demographic data that allowed for their identification with good accuracy. These findings might help to guide the diagnostic workup in cases with viral ME, in particular allowing the timely identification of cases with ME due to WNV.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

BACKGROUND: Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. METHODS: In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4 h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. RESULTS: A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. CONCLUSIONS: The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

[West Nile Infection and Kidney Disease: Description of Two Clinical Cases in Peritoneal Dialysis Patients].

The West Nile Virus (WNV), an RNA arbovirus, has been transmitted by wild birds and conveyed by ticks and mosquitoes, with wide diffusion all over the world; it is not transmitted from human to human. It can give clinical symptoms only in a minority of infected subjects such as fever, headache, muscle tiredness, visual disturbances, drowsiness, convulsions and muscle paralysis; in the most serious cases even potentially fatal encephalitis. In the literature there are few reports on WNV infection in patients with kidney diseases: here we report our experience on two patients on peritoneal dialysis infected by WNV with a revision of the literature.

Automated molecular detection of West Nile Virus in mosquito pools using the Panther Fusion system.

West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation. This study demonstrates the successful optimization of a WNV multiplex LDT (assay targets: ENV and NS1 genes) for high-throughput environmental surveillance testing of mosquito pool homogenates on the Panther Fusion System. Analytical sensitivity of the assay was 186 and 744 copies/PCR reaction for the ENV and NS1 targets, respectively. To assess the performance of this assay, a total of 80 mosquito pools were tested, including 60 previously tested pools and 20 spiked negative mosquito pools. Among the 60 previously tested specimens, the Panther Fusion WNV LDT demonstrated 100% positive and negative agreement with the CDC West Nile RT-qPCR assay. The Panther Fusion WNV LDT also detected all 20 spiked specimens. The Panther Fusion WNV LDT assay was successfully developed and optimized for high throughput testing with similar performance to the previously used CDC West Nile RT-qPCR assay.

West Nile virus seropositivity in Alanya, a coastal city in the Mediterranean region of Turkey.

BACKGROUND: West Nile virus (WNV)-related illness is a global health problem. Understanding the seropositivity rates and identifying the risk factors related to WNV in various animal species including humans is crucial for the implementation of effective prevention strategies. OBJECTIVES: Assess the rate of seropositivity and the risk factors associated with WNV seropositivity. DESIGN: Descriptive, cross-sectional. SETTING: Microbiology and virology departments in a veterinary college. PATIENTS AND METHODS: In a sample of healthy human participants in Alanya, located close to regions where WNV activity has been detected, anti-WNV IgG antibody detection was performed using enzyme-linked immunosorbent assays. The positive results were confirmed by virus neutralization tests (VNTs). The sample was compared with a second group of age- and gender-matched healthy subjects selected from a previous cross-sectional study. MAIN OUTCOME MEASURES: Determination of the seropositivity and risk factors that were associated with WNV in healthy humans. SAMPLE SIZE: 87 in current study; 356 in previous study. RESULTS: The first group of 87, which had a high risk of encountering vector mosquitoes, had a positivity rate of 8% (7/87), whereas positivity in the second group was 4.5% (16/356; P=.181). In the entire sample, the anti-WNV IgG antibody was positive in 23 out of 443 (5.2%) samples by the ELISA test. Among these 23 samples, ten were confirmed as positive using VNTs. Therefore, the WNV IgG seropositivity was 2.3% (10/442). Confirmed IgG seropositivity rates were higher among male (3.8%) than female participants (0.9%; P=.054) and among adults aged ≥45 years (4%) than those aged 18-44 years (0.8%; P=.048). CONCLUSION: This study highlights the presence of WNV infection in the research region. More comprehensive and multidisciplinary studies are required to increase our knowledge about this zoonotic infection including risk factors in line with the One Health approach. LIMITATIONS: Small sample size.

West Nile Virus Infection in a Pekin Duck ( Anas platyrhynchos domesticus ) Developer Flock.

Developer ducks are ducks being reared for breeding. Like breeder candidate chickens, they are raised with appropriate light and feed programs. A commercial Pekin duck (Anas platyrhynchos domesticus) developer flock experienced an extraordinary, elevated mortality event at 6 wk of age. Weekly mortality rate that week was 162 ducklings out of a flock of 6420 (2.5%). Mortality jumped to 988 (15.4%) ducklings the next week. On first elevated mortality, six dead ducks from that flock were submitted for diagnostic investigation at Michigan State University Veterinary Diagnostic Laboratory. Myocarditis, pale striping or diffuse pallor of the epicardium, was grossly evident in five of the six submitted ducklings. All of the ducklings had hydropericardium, three had ascites, and three had congested meninges. Histology confirmed myocarditis with myocardial necrosis. Cerebrum and brainstem had lymphocytic vasculitis with rare neuronal necrosis in affected areas, as well as Purkinje cells in the cerebellum. West Nile virus was confirmed by PCR the day after submittal and by immunohistochemistry soon thereafter.

Reporte de caso- Infección por el virus del Nilo occidental en una parvada en desarrollo de patos Pekin (Anas platyrhynchos domesticus) reproductores. Los patos reproductores en desarrollo son patos que se crían para la reproducción. Al igual que los pollos candidatos para reproducción, se crían con programas de iluminación y alimentación adecuados. Una parvada comercial en desarrollo de pato Pekín (Anas platyrhynchos domesticus) experimentó un evento de mortalidad elevada y extraordinaria a las seis semanas de edad. La tasa de mortalidad semanal de esa semana fue de 162 patitos de una parvada de 6420 (2.5%). La mortalidad se elevó a 988 (15.4%) patitos la semana siguiente. En el primer aumento de mortalidad, seis patos muertos de esa bandada fueron enviados para una investigación de diagnóstico en el Laboratorio de Diagnóstico Veterinario de la Universidad Estatal de Michigan. La miocarditis, caracterizada por rayas pálidas o palidez difusa del epicardio, fue evidente en cinco de los seis patitos presentados. Todos los patitos mostraron hidropericardio, tres tenían ascitis y tres tenían meninges congestionadas. La histología confirmó miocarditis con necrosis miocárdica. El cerebro y el tronco del encéfalo tenían vasculitis linfocítica con rara necrosis neuronal en las áreas afectadas, así como de las células de Purkinje en el cerebelo. El virus del Nilo Occidental se confirmó mediante PCR el día después de la llegada al laboratorio y mediante inmunohistoquímica poco tiempo después.