Clinical and diagnostic virology最新文献

Background: Poliovirus (PV) is the etiologic agent of paralytic poliomyelitis, which is sometimes followed, after decades of clinical stability, by new symptoms, including progressive muscular atrophy, collectively known as the post-polio syndrome. This raises the question of possible PV persistence in post-polio patients.

Objective: To test the capacity of PV to establish persistent infections in human cells, three models were developed.

Study design: This review focuses on the viral and cellular parameters involved in persistent PV infection.

Results: Many PV strains, which are generally lytic in primate cell lines, are able to establish persistent infections in human neuroblastoma cells. During persistent infection, PV mutants (PVpi) are consistently selected, and several of their capsid substitutions occur at positions known to be involved in PV–PV receptor interactions. PVpi have a particular property: they can establish persistent infections in non-neural HEp-2 cells. PV can also persistently infect primary cultures of human fetal brain cells and the majority of cells which survive infection belong to the neuronal lineage.

Conclusions: The results obtained with the three models of persistent PV infection in human cells suggest that several mechanisms are used by PV to establish and maintain persistent infections in neural and non-neural cells. The interactions of the virus with its receptor seem to be a key-step in all cases. In the future, the elucidation of the etiology of the post-polio syndrome will require the characterization of PV sequences having persisted for decades in post-polio patients.

Background: Environmental agents such as viruses have been identified as potentially important determinants of insulin-dependent diabetes mellitus (IDDM). Enterovirus infections, Coxsackievirus B especially, could be linked to the β cell damaging process and to the onset of clinical IDDM.

Objectives: Enteroviral (EV) infection and β cell autoimmunity were studied in adult patients at the onset of IDDM.

Study design: A total of 14 newly diagnosed-IDDM patients with ketosis or ketoacidosis were compared to, anteriorly diagnosed IDDM patients with metabolic decompensation, non-IDDM patients with metabolic decompensation and healthy adults. EV infection was studied by genomic RNA detection in whole blood using a RT-PCR assay. In order to assess the level of β cell autoantibodies at the time of the initial metabolic decompensation, serum specimens from IDDM patients were tested for GAD65 antibodies and islet cell antibodies (ICAs).

Results: Coxsackie B3 or B4 virus genome was detected and genotyped in five of 14 (35.7%) newly diagnosed IDDM patients and in one of 12 (8%) patients in the course of IDDM. By contrast, none of the 12 non-IDDM patients and none of the 15 healthy adults was positive for enterovirus RNA detection in whole blood. Positive GAD65 antibodies and ICAs assays were not significantly correlated to a positive EV-RNA detection.

Conclusion: The present study demonstrates that Coxsackie B virus RNA sequences can be detected in the peripheral blood from adult patients at the onset or in the course of IDDM and suggests that a Coxsackie B virus infection could initiate or accelerate β cell autoimmune damaging process.

Serologic case-control studies have suggested an association between coxsasckie group B viruses and insulin-dependent diabetes mellitus (IDDM). New investigations have identified enteroviral nucleic acid in the peripheral blood mononuclear cells of newly-diagnosed patients with IDDM. The disease pathogenesis is dependent on several factors including the genetics of the host, strain of virus, activation status of autoreactive T-cells, upregulation of pancreatic MHC-1 antigens, molecular mimicry between viral and beta cell epitopes and direct islet cell destruction by viral cytolysis. Epitopes (IDDM-E1 and E2) on glutamate decarboxylase 65 (GAD65) are the most common targets for antibody and cellular-mediated autoimmune beta cell destruction.

Background: Although Enterovirus (EV) do not persist in the tissue, which is essential to maintain autoimmunity, they have been associated as the cause of chronic autoimmunity in some cases of insulin dependent diabetes mellitus (IDDM). Convincing reports, demonstrating persistent EV infections in the pancreases, are rare.

Objectives: To determine the role of EV in IDDM, a mouse model was tested and in situ polymerase chain reaction (ISPCR) developed. The major problem of ISPCR are the high amounts of non-specific staining. In the current study we developed an ISPCR protocol which minimised non-specific staining and allowed the accurate localisation of the viral RNA in the tissue.

Study design: Five mice were infected with coxsackievirus group B4, sacrificed 7 weeks later and the pancreases were harvested. The EV nucleic acid were localised and detected in the pancreases by ISPCR.

Results: In the current study non-specific staining of ISPCR, due to DNA repair and diffuse artefacts, were minimised and the EV nucleic acids were localised in the β cells of the endocrine pancreases in all five diabetogenic mice.

Conclusion: This study demonstrates an association of viral RNA with the development of diabetes in mice and the usefulness of ISPCR to determine the role of EV in IDDM.

Background: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H^2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed.

Objectives: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype.

Study design: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5′-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant.

Results: There are five candidates for attenuation: (1) A–G at base 580 in the 5′-NTR; (2) A–T at base 690 in the 5′-NTR; (3) CG–GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA–GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A–G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5′-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation.

Conclusions: The A–G mutation at 2916 (Asp to Gly at amino acid 155 in VP1) is a strong candidate for attenuation. It is located at the top of the receptor binding cleft and mutation of the Asp to a Gly may destabilise the receptor binding site.

Background: Insulin-dependent diabetes mellitus (IDDM) has a long subclinical period characterised by gradually progressing autoimmune damage of insulin producing beta-cells. Clinical IDDM is manifested when 90% of beta-cells have been destroyed. Several studies have indicated that enterovirus infections, coxsackievirus B (CVB) infections especially, are frequent at the manifestation of clinical IDDM suggesting that they can precipitate the symptoms of IDDM in individuals who already have an advanced beta-cell damage. Recently, the first prospective studies have been published suggesting that enterovirus infections can also initiate the process several years before clinical IDDM. This implies that enterovirus infections may have a crucial role in the pathogenesis of human IDDM.

Objective: The recent findings have brought up the question whether the time has come when a causal association between enterovirus infections and IDDM could finally be confirmed. This review focuses on this question summarising the current knowledge and the prospects of future research.

Study design: Review of the recent progress in studies evaluating the role of enterovirus infections in human IDDM.

Conclusions: The currently available information supports the assumption that the role of enterovirus infections may be more important than previously estimated. Enterovirus infections are obviously associated with increased risk of IDDM, but whether this association reflects causal relationship remains to be confirmed in future studies. Prospective birth-cohort studies will be among the most important ones giving important data on the etiologic fraction of enterovirus infections, the properties of diabetogenic virus variants and the mechanisms of beta-cell damage.

Background: Infection with HTLV-I is etiologically linked with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However some patients with chronic progressive paraparesis resembling HAM/TSP have been shown to be infected with HTLV-II.

Objective: To clarify the role of each of these human retroviruses in the etiology of HAM/TSP in São Paulo, Brazil.

Study design: A detailed serological and molecular analysis of HTLV-I/II infection was performed in a cohort of 19 patients with HAM/TSP attending a neurological clinic.

Results: Plasma samples analyzed for anti-HTLV-I/II antibodies using a Western blot assay, comprising HTLV-I (rgp46^I)- and HTLV-II (rgp46^II)-specific recombinant env epitopes, demonstrated reactivity to rgp46^I and hence were typed as seropositive for HTLV-I. Presence of HTLV genomic sequences in peripheral blood mononuclear cells (PBMC) was sought after by PCR using consensus primers SK 110 and SK 111 for the pol region of HTLV proviral DNA followed by hybridization with type-specific probes—SK 112 (HTLV-I) and SK 188 (HTLV-II). Southern blots from all individuals hybridized with SK 112 but not with SK 188, further confirming HTLV-I infection. Cocultivation of PBMC from eight of these patients with activated lymphocytes from normal individuals resulted in active viral production, detected as presence of soluble p24^gag antigen in culture supernatants. Investigation of risk factors for HTLV-I infection in these individuals revealed that five out of 19 patients studied (26.3%) had received blood transfusions previous to disease onset.

Conclusions: We demonstrate HTLV-I as the only viral type involved in the etiology of HAM/TSP in a cohort from São Paulo, Brazil, and emphasize that prevention measures, including widespread routine screening of blood donations for HTLV should be conducted in Brazil.

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.

Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.

Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.

Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.

Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.

Background: Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Fetal damage is mostly linked to maternal primary infection. It is therefore important to differentiate primary from non-primary infection in pregnant females. IgM tests often used for this purpose are not reliable enough.

Objective: To evaluate an HCMV–IgG urea-elution assay for its ability to distinguish primary from non-primary infection. In this assay, soaking the antigen–antibody complex with an urea containing solution frees antibodies with low avidity but has no influence on those with high avidity. An avidity index (AI) was calculated: AI=(OD with urea/OD without urea)×100.

Study design: HCMV–IgG avidity was measured on a single serum of 79 patients with past infection (pregnant women, graft recipients and blood donors) and of 63 patients (78 sera) with documented seroconversion (pregnant women and graft recipients). Sixty-one pregnant women positive or equivocal for HCMV–IgM but without a documented seroconversion were included in this study.

Results: Most (72/79) of the patients with past infection had an AI>65% and all but one had an AI>50%. In pregnant women, in the case of a primary infection within the past 3 months, AI are usually (51/53)<50% and never>65%. Among the IgM positive pregnant women who lack a seroconversion history, 38 had AI>65% suggestive of an infection that had occured at least 3 months earlier, 11 had an AI in a grey area between 50 and 65% and 12 had an AI<50%, suggestive of a recent primary infection.

Conclusions: In pregnant women, measurement of the IgG avidity may help to date a HCMV infection, an AI >65% highly suggests a past infection while an AI <50% corresponds to a recent primary infection.