[Accuracy and diagnostic utility of IgM in Bartonella henselae infections]. / Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae.

INTRODUCTION: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. OBJECTIVE: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. MATERIAL AND METHODS: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). RESULTS: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. CONCLUSIONS: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae / Accuracy and diagnostic utility of IgM in Bartonella henselae infections

Introduction: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. Objective: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. Material and Methods: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). Results: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. Conclusions: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

Introducción: El diagnóstico de laboratorio de la enfermedad por arañazo de gato (EAG) se basa en la determinación de anticuerpos específicos anti-Bartonella henselae por distintas técnicas. El CDC de E.U.A. recomienda como estándar de oro la IgG mediante inmunofluorescencia (IF). Objetivo: Determinar la exactitud y utilidad diagnóstica de la IgM anti-B. henselae por IF para EAG. Material y Método: En suero de 108 pacientes a quienes se realizó IgG anti-B. henselae por sospecha de EAG, se determinó la presencia de IgM específica, en forma separada y ciega por dos profesionales de laboratorio ampliamente entrenados. Se calculó sensibilidad (S), especificidad (E), valores predictores positivo (VPP) y negativo (VPN) y likelihood ratio (LR) para una IgM positiva (LR+) y negativa (LR-). Resultados: En 37 pacientes con IgG anti-B. henselae positiva, la IgM fue positiva en 16 y negativa en 11; en 71 pacientes con IgG negativa, la IgM fue negativa en 69 y positiva en 2. Por consiguiente, la IgM presentó S 43%, E 97%, VPP 88%, VPN 77%, LR(+) 15 y LR(-) 0,58. Conclusiones: Los resultados sugieren que una IgM positiva apoya el diagnóstico de EAG, pero una negativa no permite descartarlo. Por tanto, la IgG debe seguir considerándose como el estándar de oro para el diagnóstico de infecciones por B. henselae.

Infection of human monocyte-derived dendritic cells by ANDES Hantavirus enhances pro-inflammatory state, the secretion of active MMP-9 and indirectly enhances endothelial permeability.

BACKGROUND: Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking. METHODS: A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC). RESULTS: Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-ß. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC. CONCLUSIONS: Primary human DCs, that are primarily targeted by hantaviruses can productively be infected by ANDV and subsequently induce direct effects favoring a proinflammatory phenotype of infected DCs. Finally, based on our observations, we hypothesize that soluble factors secreted in ANDV-infected DC supernatants, importantly contribute to the endothelial permeability enhancement that characterize the HCPS.

Andes virus antigens are shed in urine of patients with acute hantavirus cardiopulmonary syndrome.

Hantavirus cardiopulmonary syndrome (HCPS) is a highly pathogenic emerging disease (40% case fatality rate) caused by New World hantaviruses. Hantavirus infections are transmitted to humans mainly by inhalation of virus-contaminated aerosol particles of rodent excreta and secretions. At present, there are no antiviral drugs or immunotherapeutic agents available for the treatment of hantaviral infection, and the survival rates for infected patients hinge largely on early virus recognition and hospital admission and aggressive pulmonary and hemodynamic support. In this study, we show that Andes virus (ANDV) interacts with human apolipoprotein H (ApoH) and that ApoH-coated magnetic beads or ApoH-coated enzyme-linked immunosorbent assay plates can be used to capture and concentrate the virus from complex biological mixtures, such as serum and urine, allowing it to be detected by both immunological and molecular approaches. In addition, we report that ANDV-antigens and infectious virus are shed in urine of HCPS patients.

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation.

The HCV internal ribosome entry site (IRES) spans a region of approximately 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg(2+) ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.

Influence of extrahepatic viral infection on the natural history of hepatitis C.

HCV is primarily hepatotropic, but there is mounting evidence pointing to infection and replication of extrahepatic sites. Here we evaluated the occurrence of HCV infection of peripheral blood mononuclear cells (PBMC) and explored the possible association between viral extrahepatic infection and the natural history of the disease. Forty seven Chilean, HCV infected, treatment naïve patients were included in the study. HCV RNA was isolated from plasma and PBMC and subsequently reverse transcribed, amplified and sequenced. Most patients harbored HCV 1b genotype and the most common route of infection showed to be blood transfusion. HCV RNA was readily detected in PBMCs of 34 out of the 47 patients (72%). We report that HCV sequences found in PBMC differ from those in plasma of the same subjects strongly suggesting HCV compartmentalization. In addition, we found that patients with detectable HCV RNA in PBMC had a tendency for being more likely cirrhotic [OR 3.8 (95% CI: 0.98 to 14)]. In conclusion, this study provides further arguments for the existence of HCV infection of extrahepatic sites and suggests that extrahepatic infection could be a factor influencing the natural history of the disease.

In vivo induction of cellular and humoral immune responses by hybrid DNA vectors encoding simian/human immunodeficiency virus/hepatitis B surface antigen virus particles in BALB/c and HLA-A2-transgenic mice.

To improve the immunogenicity of epitopes derived from Gag proteins of simian immunodeficiency virus (SIV) and from the envelope (Env) protein of human immunodeficiency virus type 1 (HIV-1), we have designed hybrid DNA vaccines by inserting sequences encoding antigenic domains of SIV and HIV-1 into the hepatitis B virus envelope gene. This gene encodes the hepatitis B surface antigen (HBsAg) capable of spontaneous assembly into virus-like particles that were used here as carrier. Injections of hybrid vectors encoding B-cell epitopes from the gp41 and the gp120 envelope proteins of HIV-1 induced specific humoral responses in BALB/c mice. Furthermore, high frequencies of IFN-gamma-secreting CD8+ T cells specific for various antigenic determinants of SIV-Gag were observed after intramuscular injections of hybrid DNA vectors in BALB/c mice. Genetic immunization of HLA-A2.1-transgenic mice with HIV-Env/HBsAg-encoding DNA generated a strong CTL response and IFN-gamma-secreting CD8+ T lymphocytes specific for HIV-1 envelope-derived peptide. H-2d-restricted HBs-specific T-cell responses dominated over SIV-Gag responses in BALB/c mice whereas HLA-A2-restricted HIV-Env response was enhanced after fusion with HBsAg. These data demonstrate that different B and T-cell epitopes of vaccine-relevant viral antigens can be expressed in vivo as fusion proteins with HBsAg but that the optimal immunogenicity may differ strikingly between individual epitopes.

Efficient priming of simian/human immunodeficiency virus (SHIV)-specific T-cell responses with DNA encoding hybrid SHIV/hepatitis B surface antigen particles.

Recent efforts to design an human immunodeficiency virus type 1 (HIV-1) vaccine candidate have focused on means of eliciting anti-viral T-cell responses. We tried to improve the immunogenicity of DNA vaccines by designing hybrid DNA constructs encoding hepatitis B surface antigen (HBsAg) fused to antigenic domains of simian/human immunodeficiency virus (SHIV 89.6P). Immunisation with hybrid DNA induced both effector and long-lasting precursor T-cells. Following boosting with a recombinant modified vaccinia Ankara (rMVA) producing full-length SIV and HIV antigens, it appeared that priming with hybrid DNA had increased virus-specific T-cell responses in terms of both the number of virus-specific IFN-gamma-secreting T-cells and virus-specific lymphoproliferation. After intrarectal challenge with SHIV 89.6P, immunised animals demonstrated early control of SHIV 89.6P replication and stable CD4+ T-cell counts.