Conventional Tube Cell Culture Research Articles

Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan

BackgroundEnteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder.MethodsStool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases.ResultsNPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers.ConclusionThe present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.

Comparison of conventional viral cultures with direct fluorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children.

Because of the widespread availability of rapid viral antigen testing, many institutions never adopted a routine practice of ordering viral cultures to detect community-acquired respiratory viruses (CRVs). The ease of performing complete viral studies in our on site laboratory allowed us to assess the clinical implications of the absence of conventional culture results in previously healthy hospitalized children with CRV infections. From June 1997 through May 2000, the results of direct immunofluorescence assay (DFA) of 1069 nasopharyngeal swab (NP) specimens were compared with simultaneously inoculated conventional tube cell cultures for detection of CRVs. In addition the medical records of 140 previously healthy infants and children hospitalized for management of lower respiratory tract infections caused by culture-proved CRVs were reviewed. Viruses were isolated or detected by DFA or viral culture or both in 468 (30%) of the 1557 NP samples evaluated. The most common CRV isolated was respiratory syncytial virus (49%), followed by parainfluenza viruses (15%), influenza A viruses (14%), rhinoviruses (8%), adenoviruses (4%), enteroviruses (4%) and influenza B viruses (1%). Of the 1069 NP specimens for which both viral culture and rapid antigen testing were performed, 190 specimens were DFA-positive and culture-positive, 7 specimens were DFA-positive and culture-negative, 35 specimens were DFA-negative and culture-positive and 837 specimens were DFA-negative and culture-negative. The overall sensitivity, specificity, positive predictive value and negative predictive value of DFA were 84, 99, 96 and 96%, respectively. Of the 140 hospitalized patients with culture-proved viral cultures (89 respiratory syncytial virus, 22 influenza A, 20 parainfluenza virus and 9 adenovirus), the mean duration of hospital stay was 3.6 days, and the mean time for viral cultures to become positive was 7.7 days (P < 0.001, signed rank test). One hundred twenty (86%) viral cultures did not become positive until after the patient had been discharged from the hospital. In no case was the clinical decision regarding the patient's treatment or discharge from the hospital based on the results of viral culture. We conclude that positive viral cultures have no impact on clinical decision making and management of healthy children during hospitalization for illness attributable to community-acquired respiratory viruses.

Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections.

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (n = 197; 23.1%) than cell cultures (n = 150) for the routine laboratory detection of herpes simplex virus infections.

Use of monoclonal antibodies for rapid diagnosis of respiratory viruses in a community hospital.

An indirect fluorescence antibody (IFA) procedure was used for the rapid detection of respiratory viruses in direct clinical specimens and for determining the epidemiology of viruses in a community hospital setting. Viral respiratory diseases were monitored for 10 consecutive respiratory seasons. The Bartels Viral Respiratory Screening and Identification Kit is an IFA method that contains pooled and individual monoclonal antibodies for seven common respiratory viruses. Compared with 8,670 conventional tube cell cultures, IFA staining of direct patient specimens had an overall sensitivity of 84.2% and a specificity of 87.7%. Yearly epidemics of respiratory syncytial virus were seen with alternating short and long intervals between successive periods when virus was isolated. Epidemics following short intervals were more severe. Influenza A virus epidemics occurred yearly, and influenza B virus activity was seen generally every other year. When influenza A and influenza B viruses were cocirculating in a given season, the months of peak activity of one virus were always within 1 month of the peak activity of the other virus. Parainfluenza virus type 1 was detected in the autumn of odd-numbered years, and parainfluenza type 2 virus was seen usually in the autumn of even-numbered years. Parainfluenza type 3 virus and adenovirus were the most ubiquitous agents, with peak incidence occurring in the late winter to spring.

Detection of Respiratory Syncytial Virus in Nasopharyngeal Secretions by 24-Well Plate Precentrifugation Assay Using a Monoclonal Antibody Against F Protein

Background Respiratory syncytial virus (RSV) is responsible for 50% of all bronchiolitis and 25% of pneumonia cases during the first month of life. Detection of the RSV antigen by immunofluorescence in exfoliated nasal epithelium or by other methods in nasopharyngeal swabs is useful in the potentially infected patient because results are available within a few hours. In contrast, RSV antigen detection in cell culture may require as much as 3 weeks. Methods Three methods for detection of respiratory syncytial virus in 131 clinical respiratory specimens from patients with acute respiratory disease and bronchiolitis were compared utilizing the following: a precentrifugation immunofluorescence assay using Hep-2 cells, indirect immunofluorescence assay, and conventional tube cell culture using Hep-2 cells. Results Respiratory syncytial virus was identified in 36 specimens by the three methods previously described. The virus was recovered in 41 (31.3%) samples by precentrifugation immunofluorescence assay, 40 (30.5%) were identified by the immunofluorescence technique, and 38 (29.0%) cases were positive by conventional cell culture. The sensitivity of the precentrifugation assay in relation to the immunofluorescence technique was 90%, the specificity 94.5%, and the agreement, 96.2%. A positive predictive value of 90.2% was obtained. Sensitivity, specificity, agreement, and positive predictive values obtained by the precentrifugation assay variant compared to the conventional cell were 90.8%, 94.5%, 93.1%, and 87.8%, respectively. Conclusions The precentrifugation immunofluorescence assay method was as sensitive as the remainder of the methods used in our study and represents a valid alternative for rapid detection of respiratory syncytial virus in clinical samples.

Detection of mumps virus in clinical specimens by rapid centrifugation culture and conventional tube cell culture

Conventional tube cell culture was compared with a 2 day and 5 day spin-amplified shell vial indirect immunofluorescence assay for the detection of mumps virus in swabs from the area of Stensen's duct. The sensitivity and specificity of the shell vial assay were 95.9 and 100%, respectively. The shell vial detected 66.3% of the positive cultures within 2 days of inoculation while the first positive results were available by conventional tube cell culture after 3 days (1.6%) reaching 72.4% of all culture positive specimens after 7 days. These data suggest that a centrifugation shell vial indirect immunofluorescence assay may be useful for rapid detection of mumps virus in clinical specimens.

Diagnosis of cytomegalovirus infection in HIV-infected patients with respiratory disease

Background: The role of the cytomegalovirus (CMV) in the respiratory morbidity and mortality of HIV-infected patients remains unclear. This is due in part to difficulties in making an accurate and rapid diagnosis. There has been a limited number of studies, often with few or no AIDS patients, on the use of DNA–DNA in situ hybridization (ISH) and polymerase chain reaction to diagnose CMV respiratory infection directly on bronchoalveolar fluid samples. Objectives: To compare the centrifugation culture (CC), ISH, and nested-primer polymerase chain reaction (npPCR) techniques on bronchoalveolar fluid for the diagnosis of respiratory CMV infection. Study design: Samples were obtained prospectively from a group of 35 HIV-infected homosexual men evaluated for pneumonia at a university hospital. Sensitivity, specificity, and predictive values of the three techniques were measured and compared, using the conventional roller tube cell culture (CRTC) as the gold standard. Results: Sensitivity, specificity, positive and negative predictive values were as follows: 86%, 86%, 90%, and 80% for the CC; 5%, 100%, 100%, and 41% for ISH; and 86%, 57%, 75%, and 73% for npPCR. Of the six false positive samples by npPCR, two were positive by CC (none by ISH). If the latter were considered true positives, the specificity and positive predictive values of npPCR would increase to 67% and 83%, respectively. Conclusions: CC appeared to be the best of the three techniques compared in this study for diagnosis of respiratory CMV infection in HIV-infected patients. The sensitivity and predictive values of DNA–DNA ISH were very poor. Results with npPCR were acceptable, and this technique may be considered in situations when rapid diagnosis of CMV infection is necessary.

Evaluation of rapid culture centrifugation method for adenovirus detection in stools

Routine laboratory testing for adenovirus (Adv) requires a procedure that is rapid and reliable, especially for samples from children and immunosuppressed patients, when diarrhea may signal the onset of severe gastrointestinal disorders. An improved culture technique for Adv isolation, using centrifugation step of 24-well plates and needing only 48 h incubation, was evaluated for 382 stool samples. This technique was compared with conventional tube cell culture and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Adv was isolated in 36 samples (9.4%) by rapid culture, in 32 (8.4%) by conventional culture, and in 42 samples (11%) using genus-specific ELISA. A total of 30 isolates were found to be Adv positive in both rapid and conventional cultures, and half of the Adv-positive rapid culture isolates were identified as serotypes 40 41 using a type-specific ELISA. The improved culture method considerably reduces incubation time and also offers a slightly enhanced sensitivity to Adv serotypes. Combined with appropriate cell lines adapted to the isolation of enteric adenoviruses, it therefore constitutes a valuable laboratory test particularly useful in the diagnosis of gastroenteritis.

Comparison of MRC-5 and A-549 cells in conventional culture tubes and shell vial assays for the detection of varicella-zoster virus

A-549 and MRC-5 cells were compared for the detection of varicella-zoster virus (VZV) by conventional tube cell culture and by shell vial assay using fresh specimens. VZV was detected in 33 (32.4%) of 102 specimens by one or all of these methods. In conventional tube culture, seven (21.2%) of 33 were positive in MRC-5 cells and 24 (72.7%) of 33 were positive in A-549 cells, a threefold increase in sensitivity. In shell vial assay, 30 (90.9%) of 33 were positive in MRC-5 cells and 31 (93.9%) of 33 were positive in A-549 cells. The samples tested by A-549 shell vial assay had more positives showing plague formation. The two shell vial procedures gave the highest levels of sensitivity. A-549 cells provided a more sensitive method of detecting VZV in clinical specimens in both conventional tubes and shell vial assays than did MRC-5 cells.

NEW DEVELOPMENTS IN THE DIAGNOSIS OF VIRAL DISEASES

Major technical advances have occurred, especially in the last 5 years, in the laboratory diagnosis of viral infections. Immunologic detection of immediate early antigens in specimens such as bronchoalveolar lavage fluid and blood inoculated into shell vial cell cultures, particularly for herpesvirus (cytomegalovirus, herpes simplex virus, varicella-zoster virus), has provided results 16 to 48 hours after inoculation rather than the several days required for recognition of cytopathic effects in conventional tube cell cultures. Similarly, cytomegalovirus viremia can be detected directly by immunostaining of peripheral blood leukocytes with commercially available reagents the same day the specimen is submitted to the laboratory. Single-test membrane immunoassays have provided rapid (15 minutes) detection of viral antigens (respiratory syncytial virus, rotavirus, influenza virus type A). In the near future, diagnostic virology laboratories will be expected to monitor viral strains for susceptibility to the growing list of antiviral drugs. Amplification of nucleic acid sequences of viruses from cerebrospinal fluid or tissue, which generally does not yield isolates by conventional diagnostic techniques, has added a new dimension to the laboratory diagnosis of viral infection.

Diagnostic virology--then and now.

In the past five years, technologic advances in the shell vial assay and expanding availability of rapid membrane EIA tests have allowed over 90% of the viruses detected in our laboratory to be reported within 24 h postinoculation. PCR technology promises to add a new practical dimension to diagnostic virology especially for the detection of viruses in CSF, tissues, and blood. Extension of these diagnostic capabilities from investigative protocols to general laboratories for routine use will be our biggest challenge and be based on considerations of cost, licensing, and availability of this technology in "kit" formats.

Rapid detection of polyomavirus BK by a shell vial cell culture assay

Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.

Multisite evaluation of a monoclonal antibody reagent (Syva) for rapid diagnosis of cytomegalovirus in the shell vial assay

A pre-cytopathic effect (CPE) monoclonal antibody reagent (Syva Co., Palo Alto, Calif.) was evaluated in four laboratories for the rapid detection of cytomegalovirus (CMV) in shell vial cell cultures at 16 to 24 h and 40 to 48 h postinoculation. Results were compared with those obtained by inoculation of the specimen into conventional tube cell cultures that were examined for the presence of typical CMV CPE and subsequently tested by reaction with the monoclonal antibody reagent in an indirect immunofluorescence test. Of 937 specimens, CMV was positive in 184 (20%). CMV was detected twice as frequently in shell vials only (n = 29) as in conventional tube cell cultures (n = 14). Pre-CPE shell vial assay was 91% sensitive (range, 84 to 98%) and 96% specific (range, 93 to 98%) compared with the detection of CPE in conventional tube cell cultures. Overall, 137 of 166 (83%) and 143 of 166 (86%) of the CMV strains were detected at 16 to 24 h and 40 to 48 h postinoculation, respectively. The Syva reagent produced sensitive and specific results for the rapid detection of CMV infection in shell vial cell cultures and reliably confirmed the presence of the virus as detected by CPE in conventional tube cell cultures.

Rapid Detection of Cytomegalovirus by 24-Well Plate Centrifugation with the Use of a Monoclonal Antibody to an Early Nuclear Antigen

Two methods for the detection of cytomegalovirus (CMV) in 457 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells seeded on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for both 16-18 hours (EA-1) and four days (EA-4); and (2) conventional tube cell culture. CMV was identified in 50 (11%) specimens from 34 different patients. EA-1 and EA-4 had positive results for CMV in 32 (64%) and 36 (73%) of the specimens, respectively. Positive inclusions were present on only one coverslip in 31% of the cases by EA-1 and in 10% by EA-4. The number of inclusions was not necessarily predictive of tissue culture results. CMV was recovered by conventional tissue culture from 27 specimens (54%) after an average of 17 days (range, 6-26 days). One specimen, positive for CMV by EA-4, yielded herpes simplex virus (HSV), and from 9 of the 407 CMV-negative specimens, another virus was recovered: HSV from 6 specimens and varicella zoster virus, adenovirus, and enterovirus from one specimen each. CMV was detected in significantly more specimens by EA-4 than by tissue culture (P = 0.037). However, there was no significant difference in the detection of CMV between EA-1 and EA-4 or between EA-1 and conventional culture. The authors' data suggest that for maximum recovery of CMV from clinical specimens, both an early antigen assay and conventional tissue culture should be performed. For urine specimens it appears that inoculation of two coverslips followed by staining after overnight incubation is adequate. To optimize the yield of the early antigen assay when testing specimens other than urine, the authors recommend inoculating three coverslips, two of which should be stained after overnight incubation, and, if necessary, the third coverslip could be stained after a more prolonged incubation period.

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.

Detection of herpes simplex virus in clinical specimens using a DNA probe after centrifugal inoculation of A549 cells

Two methods for detection of herpes simplex virus (HSV) in 216 clinical specimens were compared: (a) 24-well plate centrifugation using A-549 cells followed by nucleic acid hybridization (Ortho Diagnostic Systems, Inc., Raritan, NJ) after incubation for 16 to 18 h, and (b) conventional tube cell culture using A-549 cells. HSV was identified by conventional tube cell culture in 44 of 216 specimens (20%) and in 36 specimens (17%) by the centrifugation-hybridization method ( P < 0.01). HSV was recovered by tissue culture from all specimens positive by centrifugation-hybridization. The sensitivity, specificity, and positive and negative predictive values of the centrifugation-hybridization technique for detection of HSV in clinical specimens were 82, 100, 100, and 96%, respectively. Centrifugal inoculation of A549 cells in 24-well plates followed by nucleic acid hybridization after overnight incubation should not replace conventional tube cell culture for detection of HSV in clinical specimens.

Early diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA‐DNA hybridisation assay

One hundred forty-eight urine specimens were collected from 47 renal transplant and dialysis patients and screened for the detection of cytomegalovirus (CMV). Diagnosis of CMV infection was suggested in 17 out of 47 patients (36.2%) by more than one of the five methods used. DNA hybridisation assay (DNA HA) using 32P-labelled probe detected CMV DNA in 15 (31.9%) of 47 patients, whereas virus isolation on conventional tube cell cultures (CTC), immunofluorescence incorporating monoclonal antibodies on centrifugation vial cultures (IF), complement fixation test (CFT), and electron microscopy (EM) yielded positive results in only nine (19.2%), 12 (25.2%), 11 (23.4%), and one (2.1%) of 47 patients, respectively. The significance of these results obtained by DNA HA lies not only in the apparent increase in number of patients diagnosed, but also in both early and rapid detection of CMV DNA. More importantly, the DNA HA is highly specific in that it correlates accurately with clinical and laboratory data characteristic of CMV disease. In respect of clinically manifest CMV disease, the specificity of DNA HA, CTC, IF, CFT, and EM was 87.5, 43.7, 56.3, 43.7, and 6.3%, respectively. These advantages of DNA HA make it the test of choice for early diagnosis of CMV infections in immunosuppressed patients.

Combined use of sonication and monoclonal antibodies for the detection of early and late cytomegalovirus antigens in centrifugation cultures

A total of 157 clinical specimens was inoculated into shell vials and conventional tube cell cultures containing confluent monolayers of human embryonic lung fibroblasts (HELF). Of 31 clinical cytomegalovirus (CMV) isolates, 30 specimens (96.8%) were positive by the immunofluorescence method on centrifugation vial cultures (CVC-IF), whereas the cytopathic effects (CPE) of CMV were detected in only 14 specimens (45.2%) in conventional tube cell cultures (CCC), P < 0.001 and in 22 specimens (70.9%) in centrifugation vial cultures (CVC-P), P < 0.1. Significantly more fluorescent foci were detected in centrifugation cultures inoculated with sonicated urine samples ( P < 0.001). CVC-P is more sensitive than CCC for the diagnosis of CMV ( P < 0.05), and a highly significant difference was observed when we compared the mean day to initial detection of CPE ( P < 0.001). For optimal detection of CMV, both CVC-IF and CVC-P should be used for the laboratory diagnosis of this virus infection.

Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and Ficoll-Paque/Macrodex methods.

Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.

Indirect Immunofluorescence Assay for Rapid Diagnosis of Influenza Virus

Monoclonal antibodies were used to detect influenza A and B in clinical specimens by an indirect immunofluorescence assay using Madden-Darby canine kidney monolayers in shell vials. Twenty-nine influenza virus isolates, including 23 type A and six type B, were detected in conventional tube cell culture at an average time of 4.2 days. Of these isolates, 18 (62%) were detected by indirect immunofluorescence 24 hours after inoculation, and 24 isolates (83%) were detected at 48 hours. Of 30 culture-negative specimens tested, influenza A was detected in one specimen by immunofluorescence. The shell vial assay is useful for rapid detection of influenza types A and B in clinical specimens, and the sensitivity is increased by staining at 48 hours in addition to 24 hours.