Detección y cuantificación de virus dengue 2 en lisado celular y plasma de niños por qPCR en tiempo real usando un estuche comercial y el equipo EcoTM System-Illumina

Methods  for  Dengue  virus  (DENV)  diagnosis  in  endemic areas  are  greatly  needed.  One of  them  is  the  real-time polymerase  chain  reaction  (qPCR)  that  also  enables  to quantitate  the  viral  genome.  Kits of  qPCR  for  DENV  are expensive and restrict their use to a small number of qPCR devices, which limits the application of the technique. Here, we evaluated the performance of a commercial kit of qPCR to DENV-2 detection  on  a  locally  available  qPCR  device (EcoTM System, Illumina), not cited by the kit manufacturer.VERO-76 cells lysate and plasma from children, both with confirmed ongoing DENV-2 infection, were evaluated. As specificity control, cell lysates and plasma  from  children infected  with  DENV-1,  and  uninfected  lysate,  were  also included. The reactions were simultaneously evaluated in an Applied Bio systems  7300  device.  The standard  curve generated  by  EcoTM  was  robust  (R2=  0.99)  with  low variability in the replicates (<10%). The reaction efficiency was high (88.8%) and signal was only obtained in lysates and plasma infected  with  DENV-2.  There was  a  strong positive  correlation  (R2=  1.0,  P=  0.0028)  between  the number of copies of viral RNA in the samples detected by both  qPCR  devices.  Thus, the use  of  the  evaluate  kit  for detection of DENV-2 here tested can be extended to EcoTM. With  this  work,  technological  capacity  for  DENV  study  in an endemic zone is greatly strengthened.Métodos para diagnóstico de dengue virus (DENV) en zonas endémicas son altamente necesarios. Uno de ellos es la reacción en cadena de polimerasa en tiempo real (qPCR), método que permite además la cuantificación del genoma viral. Los estuches comerciales de qPCR para DENV son costosos y restringen su uso a un número pequeño de dispositivos de qPCR, limitando la aplicación de la técnica. Aquí se evaluó el desempeño de un estuche comercial de qPCR para la detección de DENV-2 en un dispositivo de qPCR (EcoTM System, Illumina) localmente disponible, no listado por el fabricante del estuche. Lisado de células VERO-76 y plasma de niños, ambos con infección confirmada por DENV-2, fueron evaluados. Como controles de especificidad, lisado celular y plasma de niños infectados con DENV-1, además de lisado no infectado, fueron también incluidos. Las reacciones fueron además evaluadas simultáneamente en un equipo Applied Biosystem 7300, uno de los recomendados por el fabricante del estuche. La curva estándar generada por el EcoTM fue robusta (R2= 0.99), con baja variabilidad en las réplicas (<10%). La eficiencia de la reacción fue buena (88.8%) y sólo hubo amplificación en los lisados y plasma de niños infectados con DENV-2. Hubo una fuerte correlación positiva (R2= 1.0, P=0.0028) entre el número de copias de ARN viral en las muestras detectadas por los dos dispositivos de qPCR usados. Así, el uso del estuche para detección de DENV-2 aquí probado puede extenderse al EcoTM de forma segura. Este trabajo fortalece la capacidad tecnológica para el estudio de DENV en un área endémica

Patterns of Local and Systemic Cytokines in Bacterial Meningitis and its Relation with Severity and Long-Term Sequelae

Bacterial meningitis (BM) is a pyogenic infection present in the subarachnoid space, potentially fatal and frequently associated with neurological sequelae. During BM, cytokines (CTs) are locally produced. We sought to determine the CTs’ clinical role as disease severity predictors in adults, which is not completely clear. Using a bead-based flow cytometric assay, levels of six CTs were determined in cerebrospinal fluid (CSF) and plasma from 18 adult BM patients and 19 uninfected controls. Long-term neurological sequelae were evaluated using the Glasgow Outcome Scale (GOS). All evaluated CTs were higher in CSF than in plasma, and the levels of CSF interleukin (IL)-6, IL-8, IL-10, IL-1β, and tumor necrosis factor-α and plasma IL-10 and IL-12p70 were significantly higher in patients with severe sepsis than with sepsis, suggesting an association with clinical severity. There was a strong negative correlation between CSF IL-6 and plasma IL-12p70 with GOS score, supporting the possible role of these CTs in the development of neurological long-term sequelae. These findings could be helpful to identify candidates to receive neuroprotective treatments and early physiotherapy schemes

CXCR3 and CXCR5 are highly expressed in HIV‐1‐specific CD8 central memory T cells from infected patients

International audienceNew ways of characterizing CD8+ memory T cell responses in chronic infections are based on the measurement of chemokine receptor expression (CXCR3, CXCR5, and CX3CR1). We applied these novel phenotyping strategies to chronic HIV infection by comparing healthy donors (HDs), HIV-infected patients receiving antiretroviral therapy (ART), and spontaneous HIV controllers (HICs). In all groups, the memory cells exhibited high proportion of CXCR3+ cells. Proportions of CXCR5+ and CX3CR1+ cells were preferentially observed among central memory cells (Tcm) and effector memory cells (Tem) respectively. Chronic controlled HIV infection impacted the chemokine receptor profile of both HIV-specific and nonspecific CD8+ T cells. In total CD8+ T cells, the proportions of CXCR3- CXCR5- CX3CR1- Tcm and Tem were lower in HIV-infected patients than in HDs with subtle differences between ART and HICs. Such phenotyping strategy also revealed differences in exhaustion and senescence phenotypes, the CXCR3+ CXCR5+ CX3CR1- being more exhausted and senescent than the CXCR3+ CXCR5- CX3CR1- Tcm fraction. Among HIV-specific CD8+ T cells, the vast majority of Tcm cells were CXCR3+ and CXCR5+ cells in contrast with their nonspecific counterparts. In conclusion, the addition of migration markers contributes to better characterize Tcm/Tem compartment

Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort

© 2020 Bosch et al. Background Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1–4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiat-ing DENV serotypes and none are currently commercially available. Methodology/Principle findings We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitiv-ity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 sero-typing were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07–100%. Significance Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions