Clinical and diagnostic virology最新文献

Background: Most of the Coxsackie virus A strains are difficult to identify using traditional diagnostic methods such as virus isolation followed by neutralization with type-specific antisera. For the laboratory diagnoses of infections with Coxsackie viruses A, inoculation into newborn mice has traditionally been the method of choice. However, such investigations are complicated and time-consuming.

Objectives: To develop a reverse transcriptase (RT) and polymerase chain reaction (PCR) assay for specific detection of Coxsackie viruses A.

Study design: A total of 43 clinical specimens containing Coxsackie viruses A, B or echoviruses were investigated retrospectively. Nineteen samples were Coxsackie virus A positive, whereas 24 samples were positive for Coxsackie viruses B or echoviruses. Thirteen non-typable specimens from eight patients were also included, since they were characterized as enterovirus-like by electron microscopy.

Results: All the specimens containing Coxsackie virus A were positive with the Coxsackie virus A PCR assay. In addition, five out of eight samples characterized as enterovirus-like by electron microscopy were PCR positive. The PCR assay did not amplify Coxsackie viruses B or echoviruses identified in our laboratory.

Conclusion: The RT-PCR protocol established here should provide a useful alternative to the complicated and time-consuming diagnostic method based on live animals.

Background: The recent development of biosensor technologies for biospecific interaction analysis enables the monitoring of a variety of molecular reactions in real time by surface plasmon resonance (SPR). If the ligand is a biotinylated single stranded DNA, this technology could monitor DNA-DNA hybridization. This approach could be of great interest in virology, since the hybridization step is oftenly required to confirm specificity of molecular diagnosis.

Objectives: To determine whether real-time molecular diagnosis of human immunodeficiency virus type I (HIV-1) could be performed using biosensors and SPR technology.

Study design: Specific hybridization of a biotinylated HIV-1 oligonucleotide probe immobilized on a sensor chip to single stranded DNA obtained by asymmetric polymerase-chain reaction (PCR) was determined using the BIAcore biosensor.

Results: Direct injection of asymmetric PCR to a sensor chip carrying an internal HIV-1 oligonucleotide probe allows detection of hybridization by SPR using biosensor technology. This enabled us to apply a real-time, one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR.

Conclusion: The procedure described in this study for HIV-1 detection is simple, fast (PCR and SPR analyses take 30 min), reproducible and could be proposed as an integral part of automated diagnostic systems based on the use of laboratory workstations and biosensors for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Background: RSV-shedding during an RSV-infection declines dramatically after the first week of infection. It could be of interest to be able to diagnose RSV-infection for a longer period of time by detection of specific RSV-IgM and RSV-IgA in nasopharyngeal aspirates (NPA) in order to minimize unnecessary antibiotics.

Objectives: To evaluate an ELISA to detect specific RSV-IgM and RSV-IgA in NPA as a supplement to RSV-antigen detection.

Study design: A total of 104 NPA from 101 children (median age 9 months) with acute respiratory disease (group 1) admitted to hospital and consecutive NPA (collected on day 0, 7, 14, 30 and 60) from 11 children (median age 3 months) with a proven RSV infection (group 2) were collected. All NPA from group 1 were analysed for RSV-antigen, RSV-IgM and RSV-IgA. NPA from group 2 were analysed for RSV-IgM and RSV-IgA.

Results: Thirty-five NPA in group 1 were positive for RSV-antigen and 64 were positive for RSV-IgM. When ‘true’ RSV infection was defined by the detection of RSV-antigen and/or RSV-IgM the RSV-antigen test alone found 44% and the RSV-IgM test alone found 80%. In group 2 $\text{8}\text{11}$ (73%) had an excellent RSV-IgM response day 7, the rest responded later. Only $\text{5}\text{11}$ (46%) had a less pronounced RSV-IgA response on day 7, three cases responded later and three did not respond at all. RSV-IgM disappeared in $\text{8}\text{11}$ (73%) and RSV-IgA in $\text{7}\text{8}$ (88%) between day 30–60.

Conclusions: Specific RSV-IgM is a valuable supplement to RSV-antigen detection for the diagnosis of acute and recent RSV infection.

Background: Human cytomegalovirus (CMV) is the most common cause of congenital and perinatal infections throughout the world. High prevalence of CMV infection is associated mainly with universal breast feeding practices rather than with crowding or poverty. Perinatal CMV infection usually follows a benign course in immunocompetent individuals, but the virus remains latent or persistent in the host cell thereafter.

Objective: As a clinical diagnosis of CMV infection is generally not easy, rapid and accurate laboratory diagnosis of CMV infection is required for appropriate patient management. Numerous anti-CMV compounds including ganciclovir, foscarnet and cidofovir have been reported, most of them are unlikely in a clinical trial for perinatal CMV infection in immunocompetent individuals. Understanding the epidemiology of CMV is a key element in the development of strategies for prevention and treatment of infection.

Study design: This review focuses on recent advances in the diagnosis and treatment of perinatal CMV infections in infants.

Conclusions: Entirely new approaches to prevention and treatment of CMV infections in infants are necessary, including antiviral interventions and the development of a vaccine strategy.

Background: Texas is in the midst of two independent epizootics of rabies, involving coyotes (Canis latrans) and domestic dogs (Canis familiaris) in southern Texas and grey foxes (Urocyon cinereoargenteus) in west central Texas. The domestic dog/coyote (DDC) and grey fox (TF) rabies virus variants cannot be differentiated by antigenic typing with currently available monoclonal antibodies. These two variants also cannot be distinguished from a third variant, Sonora dog (SD) rabies, that is not enzootic in Texas, but occasionally occurs in animals along the western border with Mexico.

Objectives: To determine a method for the differentiation of the DDC, TF and SD variants, which is essential for epidemiologic monitoring of the Oral Rabies Vaccination Program (ORVP), a program instituted to control rabies in coyotes and grey foxes in Texas.

Study Design: Primers complementary to nucleoprotein sequence of either the DDC or TF rabies virus permit specific reverse transcription and amplification by polymerase chain reaction. In addition, general primers, which recognize a broad range of rabies variants, used in conjunction with a restriction digest for the differentiation of DDC, TF or SD rabies virus were investigated.

Results and Conclusions: Of 122 specimens tested with specific primers, 111 (91%) were specifically identified as either DDC (33 samples) or TF (78 samples). Overly stringent conditions, enzyme inhibitors, or limiting RNA may account for the 11 non-amplifications. Amplification of RNA under less stringent conditions, with primers recognizing a broad range of rabies variants followed by digestion with either restriction enzyme Desulfovibrio desulfuricans I (DdeI) or Haemophilus influenzae Rf. (HinfI), was used to identify the 11 isolates that did not amplify with specific primers (6 DDC, 4 TF and 1 SD). In addition to these 11 isolates, the less stringent method of amplification, followed by enzyme digestion has identified a total of 125 additional specimens (26 DDC, 94 TF and 5 SD) that were not tested by variant-specific amplification. These data provide a means to track the spread of the different rabies virus variants and allow the ORVP to plan its vaccine disbursement by defining the two epizootic boundaries.

Background: The polymerase chain reaction (PCR) applied in diagnostic and epidemiologic investigations is very useful for sensitivity, specificity and time saving.

Objectives: We have developed a method for the detection of genomic RNA of two different species of virus, the influenza A virus (IA) and the respiratory syncytial virus (RS), which are responsible for clinical similarities. We applied this multiplex RT-PCR protocol on clinical specimens.

Study design: We describe a method which allows rapid diagnosis by performing a single retro-transcriptase (RT) reaction associated with the PCR (multiplex RT-PCR) on different genomes in a single sample. We have evaluated the sensitivity and the specificity of the multiplex test on positive controls, then, on RNA extracted from clinical specimens harvested from 15 children with respiratory symptoms during the spring-winter season 1997.

Results and conclusions: The multiplex RT-PCR protocol, applied to respiratory specimens, allows the investigation of RNA IA virus and RS virus in a single sample at the same time. The detection of the etiologic viral agent is rapid and it is possible to evaluate incidental simultaneous infections.

Background: As the incidence of rubella has diminished, the proportion of unspecific rubella IgM reactivity among all samples with rubella IgM reactivity has increased. It is important to distinguish IgM reactivity caused by primary infection from that caused by reinfection or persistence, especially in pregnant women, as termination of pregnancy should be considered when primary rubella is diagnosed during the first trimester.

Objectives: To elucidate the changes over time of the avidity of rubella IgG antibodies after acute rubella infection.

Study design: Serial samples, 84, were collected from 15 patients up to 4–5 months after acute rubella infection. Rubella specific IgG avidity was tested by the eluting principle adding 35 mM diethylamine to the washing buffer of a commercially available rubella IgG ELISA. As controls, 137 samples from women with remote rubella and 94 samples from patients with a rubelliform rash, were tested.

Results: The avidity index increased steadily in all patients during the observation time. A low avidity index (<40%) was seen up to 6 weeks after onset of rash. A high avidity index (>60%) was not observed until 13 weeks after infection and only in four of the 15 patients during the observation time.

Conclusions: An increase of rubella IgG antibody avidity was seen during the whole observation time but was most pronounced during the first 3 months after onset of rash. Measurement of rubella IgG avidity is a good supplemental test for cases with rubella IgM reactivity to confirm or exclude a recent rubella infection.

Background: Monitoring of human cytomegalovirus (HCMV) load by quantification of antigenemia, viremia and DNAemia is helpful in the management of HCMV infections in immunocompromised patients. In fact, threshold values of these viral parameters are associated with the emergence of clinical symptoms. In addition, the response to antiviral treatment is revealed by a decrease in viral load or virus disappearance from blood.

Objectives: Aim of this study was to compare HCMV DNA quantification in blood of immunocompromised patients by an `in house' developed quantitative PCR (Q-PCR) assay and the commercially available Murex Hybrid Capture™ System (HCS).

Study design: HCMV DNA was quantified in 95 blood samples from 12 heart and heart-lung transplant recipients and 27 AIDS patients using both techniques. For HCS analysis 3.5 ml whole blood were utilized, whereas Q-PCR was performed using 1×10⁵ peripheral blood leukocytes (PBL). HCMV DNA levels obtained by HCS and Q-PCR were expressed as number of genome equivalents (GE)/ml whole blood or 1×10⁵ PBL, respectively. Results from HCS and Q-PCR were compared and submitted to statistical analysis. In addition, HCMV DNA values were compared to levels of antigenemia and viremia.

Results and Conclusions: Sensitivity of HCS, antigenemia and viremia with respect to Q-PCR were 37.2, 79.5 and 33.3%, respectively. Specificity was 100% for all techniques. On average, samples positive by Q-PCR only, contained low amounts of HCMV DNA. In particular, 45 (91.8%) out of 49 samples negative by HCS and positive by Q-PCR showed <500 GE/1×10⁵ PBL. A significant correlation was found between quantitative DNA levels in samples positive by both HCS and Q-PCR (n=29, R=0.693, P<0.01). HCS positivity was associated to significantly higher DNA values as determined by Q-PCR as well as to significantly higher antigenemia and viremia levels. A decrease in DNAemia levels was observed using both HCS and Q-PCR after antiviral treatment. Given that the great majority of blood samples missed by HCS contain low levels of HCMV DNA which are not clinically significant, HCS seems very promising as an alternative to HCMV DNA quantification by PCR in solid organ transplant recipients and AIDS patients.

Background: Previous studies have reported the role of enteroviruses in chronic diseases, using in-house RT-PCR protocols. A well-standardized PCR assay (Amplicor enterovirus, Produits Roche) designed for the diagnosis of enterovirus meningitis in cerebrospinal fluids (CSF) was recently described.

Objectives: To evaluate this commercially-available PCR assay for the detection of enterovirus in intestinal biopsies.

Study design: In order to obtain large quantities of infected material, eight mice were inoculated orally with 2×10⁵ 50% tissue culture infective doses (TCID₅₀) of coxsackievirus B3 (CBV3); two mice were sacrificed every day from day 1 to day 4 post-infection. Stool specimens and small bowel fragments were taken from infected animals and controls. Four protocols of RNA extraction from intestinal tissue were compared. Extracted RNA was then tested by the Amplicor assay and by a seminested in-house PCR.

Results: The best results were obtained with a commercial reagent using a combination of guanidium thiocyanate and phenol (TRI Reagent, Sigma). This procedure allowed the detection of enteroviral RNA in intestinal samples of 7/8 and 8/8 infected mice by Amplicor assay and seminested PCR, respectively, whereas only five samples were tested positive by conventional cell culture. When tested on serial dilutions of CBV3 mixed with intestinal tissue, a sensitivity of 0.2 TCID₅₀/mg was achieved with both PCR assays.

Conclusions: The data demonstrate that the Amplicor enterovirus assay, which is designed to avoid false-positive amplifications, can be used, with a slight modification of the RNA extraction step, for the detection of enterovirus in specimens different from CSF such as intestinal tissue.