Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Citomegalovirus: modalidades broncopulmonares de presentación / Cytomegalovirus: detailed respiratory presentation

Citomegalovirus (CMV), a herpesviridae, due to its high transmission rate, is endemic, with wide world distribution, especially in underdeveloped countries. CMV is an important pathogen agent during embryological period, being the commonest cause of TORCH syndrome. Due to its capability of infecting different organs, it can have a variety of clinical presentations. The more frequent and severe forms affect the respiratory, gastrointestinal, central nervous system and the retina. At the respiratory system CMV manifests in different ways, depending on age and immunological state of the patient. It produces interstitial pneumonias in immunocompromised host; but occasionally affects immunocompetent patients as a first trimester pneumonia, whooping cough like syndrome or chronic interstitial lung disease. The diagnostic method in use, the shell vial accelerated cultivation of samples obtain by bronchoalveolar lavage is both highly sensitive and specific. Treatment with ganciclovir has improved prognosis. The objective of this article is to show the different clinical presentations of CMV infection of the respiratory system in pediatric patients and the response to treatment.

Citomegalovirus (CMV) es un herpesviridae, endémico de amplia distribución mundial, debido a su alta tasa de transmisión, especialmente en países de bajo nivel socioeconómico. CMV es un importante agente patógeno en el período embrionario-fetal, siendo el principal agente etiológico del síndrome de TORCH. Por su capacidad de infectar diferentes órganos puede producir una variedad de cuadros clínicos, las formas más graves y frecuentes afectan al aparato respiratorio, gastrointestinal, sistema nervioso central y la retina. En el aparato respiratorio CMV se manifiesta de diversas maneras, dependiendo de la edad y del estado inmunológico del huésped. En inmunodeprimidos produce neumonías intersticiales graves; sin embargo, ocasionalmente afecta a individuos inmunocompetentes manifestándose como neumonía del primer trimestre, síndrome coqueluchoídeo o enfermedad pulmonar intersticial crónica. En la actualidad el método diagnóstico de elección es el cultivo acelerado de shell vial de muestra obtenida mediante lavado broncoalveolar, presenta alta sensibilidad y especificidad. El tratamiento con ganciclovir ha mejorado el pronóstico. El objetivo de este artículo es mostrar las diferentes formas de presentación clínica del CMV en el aparato respiratorio en pediatría y la respuesta al tratamiento utilizado.

Comparison of Collagen-coated Polyethylene Terephthalate Disc Plate and Shell Vial Culture Method for the Isolation of Chlamydophila pneumoniae / 대한임상미생물학회지

BACKGROUND: Chlamydophila pneumoniae is one of the major respiratory infectious pathogens and can be accurately diagnosed by cell culturing. The author performed this study to compare the usefulness of the collagen-coated polyethylene terephthalate (PET) disc culture method and that of the shell vial method. METHODS: Twenty-nine sputums and 17 blood specimens collected from 46 patients for C. pneumoniae culture were inoculated into HeLa-229 cell monolayers cultured in shell vials and polyester plates. After incubation, they were stained using the indirect immunofluorescent method with genus-specific FITC-conjugated anti-chlamydia antibody. When both results were inconsistent, microimmunofluorescence results were used. RESULTS: HeLa-229 cells successfully formed monolayers in shell vials and collagen-coated PET plates in all cases. Positive inclusion bodies in HeLa-229 cells of shell vials and PET plates for C. pneumoniae culture were similarly stained with the indirect immunofluorescent method. Both methods showed consistent results with 20 positive and 22 negative cases. The total agreement between the PET plate and shell vial was excellent (91.3%, k=0.826). CONCLUSION: The collagen-coated PET disc culture method showed highly consistent results with that of the shell vial method, and no technical differences were experienced between the two methods. Therefore, the author concluded that the shell vial method could be replaced by the PET plate method for detection of C. pneumoniae.

Detection of Respiratory Viruses in Children by Multiplex Reverse Transcriptase PCR, Direct Immunofluorescence Assay, and Shell Vial Culture / 대한임상미생물학회지

BACKGROUND: Direct immunofluorescence assay (DFA) and shell vial culture (SVC) have been used to diagnose respiratory viral infections. Recently a multiplex reverse transcriptase PCR (mRT-PCR) for 12 respiratory viruses has been introduced. We evaluated the diagnostic usefulness of these methods. METHODS: Among 275 nasopharyngeal aspirates (NPAs) received from pediatric patients during the 3-month period from May through July, 2007, 122 samples were selected so as to include diverse viruses and varying numbers of DFA-positive cells for mRT-PCR. Also, the results of the 85 NPAs that had been analyzed by both DFA and SVC were reviewed retrospectively. RESULTS: Detection rates for the seven major respiratory viruses, respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1, 2, and 3, and adenovirus by DFA vs mRT-PCR were 32.0% and 55.7%, and by DFA vs SVC were 32.9% and 40.0%. A number of adenovirus detected by DFA vs mRT-PCR were 12 and 22, and by DFA vs SVC were 6 and 18. A number of RSV detected were 3 and 6, and 13 and 8, respectively. CONCLUSIONS: mRT-PCR detected the respiratory viruses at the highest rate, followed by SVC and DFA in a decreasing order. However, DFA and multiplex PCR were more sensitive than SVC for RSV, while SVC was more sensitive than the other methods for adenovirus.

Rapid Detection and Identification of Human Respiratory Syncytial Virus, Human Parainfluenza Virus Type 1, 2 and 3 by Single-tube Multiplex Reverse Transcription Polymerase Chain Reaction

Laboratory diagnosis of respiratory viral infection has traditionally been based upon virus isolation and/or viral antigen identification. Recently, more sensitive and specific nucleic acid detection methods by reverse transcription- polymerase chain reaction (RT-PCR) have been developed, however, conventional RT-PCR can identify only a single suspected virus. To identify the causative agents which belong to Paramyxoviridae of respiratory virus infections, we have developed a single-tube multiplex RT-PCR using four primer sets which can amplify respiratory syncytial virus and parainfluenza virus type 1, 2 and 3 simultaneously. Assay sensitivity of single-tube multiplex RT-PCR allowed a detection in the range of 3~500 TCID50 and there were no cross amplification among other respiratory viral agents based on the test using reference virus stocks. The single-tube multiplex RT-PCR was able to directly detect viruses in respiratory specimens, with virus being detected 11 of 80 samples as compared to 9 of 80 samples detected by indirect immunofluorescence or antigen detection following shell vial culture. This result suggests that the single-tube multiplex RT-PCR can be established as a more sensitive and rapid diagnostic application than shell vial assay for the detection of respiratory infection of Paramyxoviridae.

Epidemiological Analysis of Viral Respiratory Infections and Comparison of Isolation Rate of Various Clinical Specimens / 감염

BACKGROUND: We studied the epidemics of respiratory viral infections in Korea and examined various respiratory tract specimens for the presence of respiratory viruses, since the accuracy of rapid detection method depends, in part, on the source of the specimens. METHODS: Over a 24-month period, from March 1997 through February 1999, a total of 1,574 clinical specimens were submitted for the detection of respiratory viruses. A shell vial technique with commercially available monoclonal antibodies directed against respiratory viruses was used to detect respiratory syncytial virus, adenovirus, influenza virus, and parainfluenza virus in clinical specimens, which included throat swab, nasopharyngeal aspirate, tracheal aspirate, and bronchoalveolar lavage (BAL) fluid. RESULTS: Overall positive rate of respiratory viruses was 73/1574 (4.6%). Respiratory viruses were predominantly found between December and February. High incidences were observed among those younger than 2 years and those older than 50 years. The numbers of viral isolates were 3/69 (4.3%) for throat swab, 26/459 (5.7%) for nasopharyngeal aspirate, 11/315 (3.2%) for tracheal aspirate, and 30/528 (5.7%) for BAL fluid. CONCLUSIONS: Nasopharyngeal aspirate and BAL fluid appear to permit increased detection of the respiratory viruses compared with throat swab or tracheal aspirate. However, throat swab may be good specimen for the detection of influenza virus and parainfluenza virus.

Comparison of Direct Immunofluorescent Staining, Shell Vial Culture and Tube Culture for the Detection of Varicella-Zoster Virus in Skin Lesions / 대한임상병리학회지

BACKGROUND: For the diagnosis of varicella-zoster virus (VZV) infection, virus culture has been considered the reference method, but it gives delayed results and needs cell culture facilities. Shell vial culture is more rapid, but it also takes 2 days or more and needs cell culture. Immunofluorescent (IF) method has known to be rapid and sensitive. We compared the tube culture, shell vial culture, and direct IF method to find the most efficient diagnostic method. In addition, the MRC-5 cells were compared with A549 cells for the recovery of VZV in culture. METHODS: A total of 48 specimens were obtained from skin vesicles of patients with clinical herpes zoster. The vesicle smears were stained with FITC-conjugated monoclonal antibody. The vesicle aspirates obtained in 2 mL of viral transport media were inoculated into shell vials and tubes containing MRC-5 and A549 cell monolayers. After 48 h of incubation at 36degrees C the shell vials were stained with VZV-specific monoclonal antibody. The tubes were stained with the same antibody after 3 weeks or when the monolayer showed cytopathic effect. RESULTS: The positive rates of direct IF, shell vial culture, and tube culture were 67.5%, 87.5%, and 72.5% respectively. The positive rate of direct IF was increased to 96.4% when inadequate specimens for the direct IF were excluded. The MRC-5 and A549 cells showed no significant difference in the isolation rates of VZV in both shell vial and tube culture. CONCLUSIONS: Direct IF is the most rapid and practical method for the laboratory confirmation of VZV infection when the swab specimen is adequately obtained. The MRC-5 cells are recommended for the tube culture and A549 cells for the shell vial culture.

Early Detection of Human Cytomegalovirus DNA by PCR-ELISA / 대한임상병리학회지

BACKGROUND: Human cytomegalovirus (CMV) infections are common and occasionally severe in newborns, immunocompromised hosts, cancer patients, and recipients of organ transplant. Consequently, sensitive and rapid methods for CMV detection are of great diagnostic value since antiviral drugs have become available, which might be more effective upon early administration. We evaluated a polymerase chain reaction and enzyme-linked immunosorbent assay (PCR- ELISA) to detect human CMV infection as an aid in making a prompt diagnosis and a determination of therapeutic efficacy. METHODS: CMV DNA was amplified by single PCR, using primers chosen from genomic regions (major immediate-early [MIE] protein coding region), and the microwell plate hybridization assay was performed for specific detection of 5'-biotinylated PCR products using CMV-specific probes labeled with digoxigenin. A total of 35 clinical specimens from 14 patients who were suspected CMV infectious state was analyzed by PCR-ELISA and its results were compared with those of serum anti-CMV IgM, shell vial culture assay and PCR. RESULTS: The sensitivity for detection of PCR-amplified CMV DNA by the ELISA was 102 copies, which was ten-fold greater than ethidium bromide staining of agarose gels. The positive rates of 35 clinical specimens by serology, shell vial culture assay, PCR and PCR-ELISA were 37.9%, 40.0%, 60.0% and 68.6%, respectively. The OD ranges of 24 positive specimens by PCR-ELISA were from 0.042 to above 2.5. In follow-up studies of two patients with bone marrow transplantation, positive CMV results by PCR-ELISA earlier than by other methods including serologic method, shell vial culture assay and PCR. CONCLUSIONS: These results reveal that PCR-ELISA may show higher sensitivity and positive rate than serologic method, shell vial culture assay and conventional PCR. PCR-ELISA can be useful to manage CMV infection rapidly in patients at risk.

Detection of Cytomegalovirus Infection and IE Gene Variants in Renal Transplant Recipients by Shell Vial Culture and DNA Methods / 대한신장학회잡지

Cytomegalovirus (CMV) is a ubiquitous virus and its infections occur commonly after renal transplantation and immunosuppressive therapy. Early and accurate laboratory diagnosis of CMV infection in renal transplant is necessary but often difficult. To find optimal diagnostic methods for CMV infection, we compared shell vial culture and polymerase chain reaction (PCR) and Southern blot of PCR products. A total of 301 specimens of urine, blood neutrophils, tissues, or body fluids were obtained from 75 renal transplant recipients and were submitted to shell vial culture for CMV as well as DNA PCR using primers for immediate early(IE) gene of CMV. The human fibroblast cell line (MRC-5) was used to culture CMV and were examined with immunofluorescence staining using monoclonal antibody to the early antigen of CMV. The PCR products (274 and 379 bp) were detected by gel electrophoresis and ethidium bromide staining. When PCR products were not clearly visible on electrophoresis, PCR products were analyzed by Southern blot using IE gene probe. Sixty four(85.3%) of 75 renal transplant recipients showed CMV infection as analyzed by PCR and Southern blot as well as shell vial culture. On shell vial culture, CMV were detected in 81 specimens from 30(40%) renal transplant recipients in viremic state. On PCR and Southern blot analysis CMV were detected in 55 and 26 specimens, respectively from 59 patients. The sensitivity of culture and PCR to detect CMV infection were 42.4% and 83.3%, respectively. The results of two studies were concordant in 48%. PCR and Southern blot did not detect CMV in 10 and 5 culture proven CMV positive samples, respectively. Mutant CMV were found in 3 patients which showed 5-10 bp deletion in IE gene. Moreover, DNA sequencing analysis showed 5 mutant strains among 11 strains which appeared same by PCR prodcut. These results suggest that PCR followed by Southern blot may be more sensitive, but less specific than shell vial culture in the diagnosis of CMV disease. PCR followed by Southern blot may not detect mutant CMV. Combined analysis using both shell vial culture and PCR followed by Southern blot may be necessary to diagnose CMV infection in renal transplant recipients.

Study of Cytomegalovirus Infection in Renal Transplant Recipients Who had IgG CMV Antibody in the Pretransplantation Period / 대한신장학회잡지

Cytomegalovirus(CMV) infection occurs more frequently in renal transplant recipients than in the normal population. But the incidence and severity of CMV infection and antibody positivity are different between countries. We studied the incidence of CMV infection with a long term follow up in renal transplant recipients who were IgG CMV positive and whose donors were also IgG CMV positive preoperatively. We studied the difference and usefulness of various detection methods including IgM CMV antibody by ELISA, shell vial culture, and quantitative dual polymerase chain reaction(PCR). We also studied possible factors that may affect CMV infection and the function of the grafted kidney in CMV infected patients. This study included 36 patients, 20 males and 16 females, who received renal transplantation at Hanyang University Hospital between July, 1994 and March, 1995. IgG and IgM CMV antibodies were detected and shell vial cultures were performed in recipient candidates and donor candidates preoperatively. Postoperatively, we checked IgM CMV and performed shell vial cultures in renal transplant recipients every month after the operation and when CMV infections were suspected. We also performed dual PCR with endpoint titration to quantify the amount of CMV DNA. The total incidence of CMV infection was 30.6% (11 patients among 36 patients). Three patients had CMV disease, and only one patient was severe enough to need gancyclovir therapy. The average onset of infection was 12.9 weeks after the operation(earliest 5weeks-latest 33weeks). In all patients with CMV disease, CMV positivity appeared by all three detection methods. But detection time and duration of positivity were different. The amount of CMV DNA in patients with active CMV disease was higher than those of asymptomatic patients and one patient following antiviral therapy. Age, sex, donor type, HLA matching and rejection did not affect CMV infection. Incidence of CMV infection was higher in patients who were transfused within 3 weeks before the operation(6/8 vs 5/28, p=0.048). Changes of creatinine level from initial outpatient department(OPD) visit to last OPD visit which were corrected by OPD follow up time showed no significant difference between CMV infected and non-infected patients In conclusion, the incidence of CMV infection and disease was relatively low, and the degree of disease severity was mild. In our renal transplant recipients, CMV infection may not be a serious problem. Quantitative dual PCR using end-point titration is a good method to detect CMV infections and monitor the clinical course. Because it is easy to use, detect disease earlier and can quantify the amount of CMV DNA. Among various factors, transfusion affected CMV infection significantly in our patients. In an average of 231 days of OPD follow up time, CMV infected patients showed no impairment of grafted kidney function, but a more long term follow up is needed.

Human Cytomegalovirus (HCMV) Infection in Organ Transplant Recipients / 감염

BACKGROUND: Human cytomegalovirus (HCMV) is not eradicated from a host after a primary infection and persists in a latent form. When the immunological condition of the host is compromised, the virus can be reactivated and cause serious disease. Given that the prevalence of anti-HCMV IgG antibody positivity is over 95% in Korean adult population, the transplant recipients in Korea are likely to be a high risk of developing HCMV infection and diseases. METHODS: Fourteen bone marrow recipients, 44 kidney transplant recipients and 4 liver transplant recipients were evaluated for excretion of HCMV. Urine and blood were cultured by conventional method at the time of transplantation and at regular intervals thereafter. To evaluate sensitivity and specificity of shell vial assay for detecting HCMV, the specimens from 41 transplant recipients were cultured by using both shell vial assay and conventional virus culture. RESULTS: The frequency of HCMV infection was 50%(7/14) in allogeneic bone marrow (BM) recipients, 36%(16/44) in kidney recipients and 25%(1/4) in liver transplant recipients. HCMV diseases were observed in only 3 cases; 3 BM recipients developed interstitial pneumonitis. Sensitivity and specificity of shell vial assay for the detection of HCMV was 50%(3/6) and 91%(32/35), respectively. CONCLUSION: HCMV infections in organ transplant recipients were relatively common in Korea. HCMV started to be excreted in urine about 1 month after transplantation and were found in 33-50% of all recipients later on. Sensitivity of shell vial assay was so low that it needs to be complemented with other diagnostic methods.