Shell Vial Culture Research Articles

Detection of Human Metapneumovirus in Clinical Samples by Immunofluorescence Staining of Shell Vial Centrifugation Cultures Prepared from Three Different Cell Lines

Monoclonal antibody MAb-8 was evaluated for detection of human metapneumovirus (HMPV) in shell vial centrifugation cultures (SVCC). Detection of HMPV was similar in A549, HEp-2, and LLC-MK2 SVCC, and MAb-8 staining was optimal on day 2 postinoculation. Availability of SVCC for HMPV will be of significant benefit to clinical laboratories.

Clinical Utility of Cytomegalovirus Viral Load in Bronchoalveolar Lavage in Lung Transplant Recipients

The utility of cytomegalovirus (CMV) viral load (VL) by quantitative hybrid capture assay (Q-HCA) was investigated in bronchoalveolar lavage (BAL) from lung transplant recipients and compared with BAL cultures and blood VL. Forty-three consecutive BAL samples from 27 lung transplant recipients were analyzed. All samples had shell vial (SV) cultures in addition to Q-HCA. Histopathology was done on all lung tissues, and immunohistochemistry (IHC) in those with positive CMV cultures. Fifteen (56%) lung transplant recipients had both positive BAL SV cultures and BAL VL. Five of 15 had CMV pneumonitis with a VL in BAL >500 000 copies/mL (mean: 1638 450). Ten patients without CMV pneumonitis had VL in BAL <500 000 copies/mL (mean 81 820, p = 0.002). High VL in BAL and blood invariably meant CMV pneumonitis, but 2 patients with CMV pneumonitis had high BAL VL but relatively low blood VL. Initial CMV seronegativity was associated with pneumonitis (4/5 vs. 1/10; p = 0.004) and higher BAL CMV VL (p = 0.03). High CMV BAL or blood VL did not correlate with acute rejection or development of bronchiolitis obliterans syndrome (BOS). High CMV VL in BAL in lung transplant recipients is strongly associated with CMV pneumonitis, and may be more predictive than peripheral blood viral load.

Evaluation of a commercially available reverse transcription-PCR enzyme immunoassay (Enterovirus Consensus kit) for the diagnosis of enterovirus central nervous system infections

A commercial reverse transcription (RT)-PCR amplification method was compared with culture for the diagnosis of enterovirus meningitis. In total, 99 cerebrospinal fluid (CSF) specimens were examined with the Enterovirus Consensus kit and shell vial culture. RT-PCR allowed the amplification of enterovirus cDNA and its detection in a microtitre plate by hybridisation. Clinical information and CSF analysis were used to resolve the discrepancy in results. The detection limit of the RT-PCR assay was determined with the Third European Union Concerted Action Enterovirus Proficiency Panel. There were 34 true-positive CSF specimens. Of these, RT-PCR detected 33 (sensitivity 97%), while culture detected 19 (sensitivity 54.5%). RT-PCR failed to detect one culture-positive specimen that contained inhibitors. When samples from the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, the RT-PCR method gave identical results to those expected. The Enterovirus Consensus kit was rapid and statistically more sensitive than culture (p < 0.01) for the detection of enteroviruses in CSF, and may offer considerable benefits in the clinical management of patients with enterovirus meningitis.

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.

Cytomegalovirus Infections Following Reduced-Intensity Allogeneic Stem Cell Transplantation.

Reduced intensity allogeneic stem cell transplantation is increasingly used for patients who are at high risk for mortality following conventional full-intensity regimens. Defining the risk of CMV-related events in this growing modality has a significant implication in future immunologic interventions to improve their transplant outcomes. Therefore, we analyzed 160 patients at risk for CMV (R+ and/or D+) who received a reduced intensity transplant (RIT) between May 2000 and February 2004 from either a matched-related (n=91) or unrelaed donor (n=69) under the City of Hope IRB protocol #02047. The median age of the cohort was 51 (range: 15–71) and recipient/donor CMV serostatus was; R+D+=87, R+D-=44, R-D+=29. Conditioning regimens were reduced but myeloablative (fludarabine + melphalan or busulfan) in 124 or truly non-myeloablative (TBI 200cGy +/− fludarabine) in 36 patients. Fourteen received a bone marrow graft and the remaining 146 received PBSC graft. GVHD prophylaxis consisted of cyclosporine and mycophenolate +/− short course of methotrexate. CMV reactivation was monitored by shell vial blood culture (BC) twice a week from d21 until d100 or longer if clinically indicated. Additional monitoring with quantitative PCR was available in 57 patients. Preemptive therapy with ganciclovir was started at the first positive BC at 5mg/kg twice a day for a week, followed by maintenance therapy for 5 weeks. Of 160 patients, at the time of analysis 94 patients are alive with a median follow up of 12.2 months. The actuarial probabilities of overall survival, disease-free survival, and relapse for the entire cohort at 1 year were 55.9 %, (95%CI: 51.1–60.5), 40.4% (95% CI: 37.1–43.7), and 40.1% (95%CI: 34.8–46.2), respectively. Grade 2–4 acute GVHD occurred in 91 (56%) patients. Eighty-one patients developed CMV reactivation (actuarial probability of 52+/−4%) at the median of 44 days post-transplant (range: d20-d216, 8 after d100). Of these, 42 (52%) had either persistent or recurrent reactivation. Nineteen patients (12%) developed CMV disease (8 with pneumonitis, 11 with GI tract) at the median onset of 56 days (range d28-d591: 5 after d100). Seven of these 19 patients had both BC and PCR data, which were consistent each other in all but one. CMV viremia itself did not predict poorer survival (p=NS), but CMV disease was associated with lower overall survival (37% vs. 65%, p=0.02). The univariate analysis revealed two major findings: 1) CMV seropositive patients were at seven times the ‘hazard' (HR: 7.2, 95%CI: 2.3–22.9) for CMV reactivation compared to those who were CMV seronegative (p<0.001); and 2) patients who received a true non-myeloablative regimen were at decreased ‘risk' for CMV reactivation compared to those who received fludarabine + melphalan or busulfan (HR: 0.42, 95%CI: 0.2–0.8) (p<0.01). In multivariate analysis these factors remained significant.In summary, our data demonstrate the significant incidence of CMV reactivation and disease in RIT, but true non-myeloablative transplants may have lower risk. Future immunologic or pharmacologic interventions should include RIT patients.

A Randomized Trial of Short-Course Preemptive Ganciclovir Compared with Conventional Ganciclovir Therapy To Prevent Cytomegalovirus Infection after Allogeneic Stem Cell Transplantation.

We studied the efficacy and safety of a short course of ganciclovir to prevent cytomegalovirus (CMV) infection in allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In general, preemptive ganciclovir is administered twice daily for 1–2 weeks (induction phase) and then once daily until 6 days after cessation of viremia and antigenemia (maintenance phase). We randomly assigned allogeneic HSCT recipients who had positive shell vial culture for CMV or CMV antigenemia to receive preemptive ganciclovir therapy with or without induction phase (5 mg/kg body weight, twice a day for one week). 32 and 34 patients were randomized to the standard treatment arm and the short course arm, respectively. There were no statistically significant differences in patient characteristics such as age, sex, type of disease, type of transplantation, seropositivity of CMV antibody in donor, seropositivity of CMV antibody in recipient, type of conditioning regimen or GVHD prophylaxis regimen between two groups. The median time to clearance of CMV viremia and antigenemia was 7 d and 9 day, respectively (P = 0.333). Seven patients in the short course arm failed for viral clearance after preemptive therapy and were cross-overed to the standard preemptive therapy, which resulted in clearance of antigenemia and viremia in all patients. The toxicity profile was higher in the standard treatment arm with 5 neutropenia and 1 drug-induced hepatitis whereas there was only 1 neutropenia in the short-course arm. Of 5 patients who experienced neutropenia, 2 died of sepsis. This study shows that the preemptive ganciclovir therapy with maintenance phase alone may be equally effective to the standard treatment arm with less toxicity profile in prevention of CMV disease in allogeneic HSCT recipients. The omission of induction phase in CMV preemptive therapy may allow patients to avoid unnecessary hospital admissions.

Prospective evaluation of a dot-blot enzyme immunoassay (Directigen RSV) for the antigenic detection of respiratory syncytial virus from nasopharyngeal aspirates of paediatric patients

This study investigated the efficacy of a commercial enzyme immunoassay (Directigen RSV, ColorPAC) in comparison with the shell vial culture method (using Hep-2 cells) for the detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates from children with bronchiolitis. During the period 1995-2002, 4950 samples were examined. RSV was detected in 1660 (33.5%) samples, with a sensitivity of 80.9%, a specificity of 97.5%, a positive predictive value of 93.8%, a negative predictive value of 91.6%, and a testing efficiency value of 92.2% compared with shell vial culture. In 83 (5%) samples, the ColorPAC was positive and the shell vial assay was negative. Of these, 71 (85.6%) were false-negative by cell culture. The true false-positive results obtained by ColorPAC represented only 0.7% of all RSV-positive samples. In general, no statistically significant differences were detected between the different months and epidemic periods studied. Compared with ColorPAC, the shell vial culture method displayed a sensitivity of 95.8% and a specificity of 100%. Overall, the ColorPAC assay was an acceptable, simple and rapid method for the antigenic detection of RSV in paediatric respiratory samples.

Significance of nested PCR and quantitative real time PCR for cytomegalovirus detection in renal transplant recipients

Immunocompromised renal transplant recipients are susceptible to severe cytomegalovirus (CMV) infection that makes its detection important in clinical practice. A total of 536 blood and 536 serum samples from 67 renal transplant recipients who had previously been diagnosed with terminal renal insufficiency were studied for CMV infection. In all samples, serology, shell vial culture, antigenaemia and nested polymerase chain reaction (PCR) in blood and serum were tested, and a real-time quantitative PCR was run on 90 specimens. Sixty-seven blood donors were used as controls. The results show that the quantitative real-time PCR assay could be of great interest for predicting CMV disease, and to monitor the onset of pre-emptive therapy.

Community-acquired respiratory viruses

Community-acquired respiratory viruses

Evaluation of the ELVIS plate method for the detection and typing of herpes simplex virus in clinical specimens

This study was conducted to assess the reliability of a commercial enzyme-linked viral inducible system (ELVIS) (Diagnostic Hybrids, Inc., Athens, OH) for rapid detection and typing of herpes simplex virus (HSV). Results using ELVIS were compared to those of shell vial culture (SVC) and HSV detection with monoclonal antibodies and an immunoperoxidase stain plus typing with MicroTrak direct fluorescent antibodies (Trinity Biotech PLC, Wicklow, Ireland). Specimens yielding discrepant HSV results were tested by polymerase chain reaction (PCR); those with discrepant typing results were stained with Simulfluor (Chemicon, Temecula, CA). Of the 206 samples tested, 144 were negative and 54 were HSV-positive by both methods (agreement, 96.1%). Five specimens were positive by ELVIS but negative by SVC; 3 of these were positive and 2 were negative by HSV PCR. Both of the latter were the result of mechanical problems early in the study. Three specimens were positive by SVC but negative by ELVIS; all 3 were positive by HSV PCR. After resolution of discrepancies, the sensitivity and specificity for detection of HSV were 95.0% and 100% for SVC, respectively, and 95.0% and 98.6% for ELVIS. Of the 46 HSV-positive samples that were typed, 26 were called type 2 and 18 were type 1 by both methods (agreement, 95.7%). The 2 specimens with discrepant results were called HSV-2 by SVC, staining with MicroTrak, and HSV-1 with ELVIS; both of these were type 2 when stained with the Simulfluor reagent. ELVIS is a reliable alternative to SVC for rapid detection and typing of HSV.

ESCV Meeting calendar

Background: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. Objectives: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. Study design: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. Results: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. Conclusions: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.

Diagnosis of herpes simplex virus-1 keratitis: Comparison of Giemsa stain, immunofluorescence assay and polymerase chain reaction

Purpose. To evaluate three different diagnostic tests against the gold standard of viral isolation, in the diagnosis of HSV-1 keratitis.Methods. Corneal scrapings from 170 patients with clinically suspected HSV keratitis were tested by; 1) Giemsa staining procedure for the presence of multinucleated giant cells and lymphocytes, 2) immunofluorescence assay for HSV-1 antigen, 3) polymerase chain reaction (PCR) for HSV-1 DNA and 4) virus isolation by shell vial culture in SIRC (Rabbit corneal epithelial cell line). The results of the former three tests were compared among 14 cases that were culture positive and 156 cases that were culture negative for HSV-1.Results. The sensitivity of PCR was 100%, while IFA and Giemsa had sensitivities of 85.7% and 57.1% respectively. The specificity of PCR, IFA and Giemsa were found to be 67.9%, 85.3% and 85.9% respectively.Conclusions. In the present study, a combination of PCR and immunofluorescence assay appears to be the most suitable choice of tests for diagnosis of HSV-1 keratitis, while detection of MNGC by Giemsa staining procedure may give us a presumptive diagnosis of suspected viral infection.

Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults.

Respiratory syncytial virus (RSV) accounts for the majority of respiratory virus infections, producing high mortality rates in immunocompromised patients with hematologic malignancies. The available methods for the rapid detection of RSV by antigen detection or PCR either lack sensitivity, require complex laboratory manipulation, or have not been evaluated in this patient population. To assess the applicability of a TaqMan-based real-time PCR technique for the detection of RSV A and B in immunocompromised adults, we developed a rapid, sensitive detection method that simultaneously detects RSV A and B and can be applied in routine diagnostics. The specificity of the assay was assessed using a panel of reference strains of other respiratory viruses and RSV. Electron microscopy-counted stocks of RSV A and B were used to develop a quantitative PCR format. Eleven copies of viral RNA could be detected for RSV A strain Long, and 14 copies could be detected for RSV B strain 9320, corresponding to 50% tissue culture infective doses of 0.86 and 0.34, respectively. The assay was evaluated on 411 combined nose and throat swabs derived from immunocompromised adults with or without signs of respiratory tract infection. The diagnostic efficacy of the TaqMan PCR determined on the clinical samples showed that this real-time PCR technique was substantially more sensitive than the combination of conventional viral culture and shell vial culture. None of the clinical specimens derived from patients without signs of respiratory illness were found to be positive for RSV by real-time TaqMan PCR.

Seven years' experience of isolation of Rickettsia spp. from clinical specimens using the shell vial cell culture assay.

Annals of the New York Academy of SciencesVolume 990, Issue 1 p. 371-374 Seven Years' Experience of Isolation of Rickettsia spp. from Clinical Specimens Using the Shell Vial Cell Culture Assay G. VESTRIS, G. VESTRIS Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorJ. M. ROLAIN, J. M. ROLAIN Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorP. E. FOURNIER, P. E. FOURNIER Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorM. L. BIRG, M. L. BIRG Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorM. ENEA, M. ENEA Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorJ. Y. PATRICE, J. Y. PATRICE Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorD. RAOULT, Corresponding Author D. RAOULT Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceAddress for correspondence: Didier Raoult, Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Voice: 33-04-91-32-43-75; fax: 33-04-91-38-77-72; Didier.Raoult@medecine.univ-mrs.fr.Search for more papers by this author G. VESTRIS, G. VESTRIS Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorJ. M. ROLAIN, J. M. ROLAIN Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorP. E. FOURNIER, P. E. FOURNIER Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorM. L. BIRG, M. L. BIRG Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorM. ENEA, M. ENEA Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorJ. Y. PATRICE, J. Y. PATRICE Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceSearch for more papers by this authorD. RAOULT, Corresponding Author D. RAOULT Unité des Rickettsies CNRS UMR-A 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, FranceAddress for correspondence: Didier Raoult, Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Voice: 33-04-91-32-43-75; fax: 33-04-91-38-77-72; Didier.Raoult@medecine.univ-mrs.fr.Search for more papers by this author First published: 18 July 2008 https://doi.org/10.1111/j.1749-6632.2003.tb07394.xCitations: 24Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume990, Issue1RICKETTSIOLOGY: Present and Future DirectionsJune 2003Pages 371-374 RelatedInformation

Risk factors for admission and the role of respiratory syncytial virus-specific cytotoxic T-lymphocyte responses in children with acute bronchiolitis.

Risk factors for admission of children with acute bronchiolitis have remained controversial. Technological advances in the measurements of cytotoxic T-lymphocyte (CTL) activity, enable respiratory syncytial virus (RSV)-specific CTL activity to be studied in infants with bronchiolitis for the first time. We evaluated risk factors for admission of children with acute bronchiolitis and determined the role of CTL responses in those infected with RSV. Children between 3 and 24 months of age presenting with bronchiolitis to the paediatric outpatient department at King Edward VIII Hospital, Durban, over a 1-year period were enrolled. Management included clinical evaluation, nasopharyngeal aspiration, standard treatment and hospitalisation if indicated. Secretions were tested with monoclonal antibodies for RSV and pooled respiratory viruses; shell vial cultures were also established. Permission was requested from parents of RSV-infected subjects for blood draws for specific cytotoxic T-cell assays and CD4/CD8 cells on admission and repeat CTL on day 7. Viruses were identified in 55 of the 114 subjects studied (48.2%). RSV was seen in 41 cases (74.5%). Twenty-three infants (20.2%) required admission. Risk factors associated with inpatient admissions on univariate analysis included younger mean age (7.6 months v. 10.1 months), overcrowding (p < 0.01) and indoor exposure to products of combustion of cooking fuels (p = 0.05). Only the former two were significant on multivariate analysis. RSV-specific CTL responses were obtained in 21 children (51.2%). Responses were either very weak (N = 7) or negative (N = 14) on day 0 and did not alter significantly on day 7. The mean CD4/CD8 ratios in this group were 2.27:1. The highest frequency of CTL was directed against the proteins M4/5/6', with counts ranging from 100 to 400 spot forming cells (sfc)/million. Measures to address risk factors identified in this study may decrease the need for hospitalisation from bronchiolitis. The lack of RSV-specific CTL responses in peripheral blood of immunocompetent RSV-infected children suggest an alternative method of induction of immunity or compartmentalisation of immune cells.

Síndrome de mononucleosis infecciosa en pacientes adolescentes y adultos

Infectious mononucleosis syndrome, characterized in typical cases by fever, sore throat, tonsillar exudates, cervical adenopathies and atypical lymphocytosis is associated in most cases to Epstein-Barr virus (EBV) infection. Other potential causes for this syndrome are acute cytomegalovirus (CMV), Human Immunodeficiency Virus, Toxoplasma gondii or Human Herpes virus 6 infection. These alternative etiologies evolve with a modified clinical picture that includes sometimes leukopenia or rash. Diagnosis of EBV is easily accomplished by atypical lymphocytosis (> 10%), positive heterophil antibodies and IgM antibodies directed against the EB viral capsid antigen (VCA). The latter is needed for cases without positive heterophil antibodies. Acute CMV infection is the second most important cause and can be diagnosed by CMV antigen detection, PCR or shell vial culture of blood samples, although experience with these tests among immunocompetent patients in primary care settings is sparse. Acute primary HIV infection is an important cause for this syndrome and should not be neglected when other causes are discarded. Third or fourth generation HIV ELISA tests, p24 antigen or HIV-PCR detection in blood samples allow recognition of this agent from the second or third week of inoculation. T. gondii and human herpes virus 6 infection can be diagnosed by serological methods. Evolution of EBV or CMV infection is favorable with infrequent complications

Lower respiratory tract infections associated with influenza A and B viruses in an area with a high prevalence of pediatric human immunodeficiency type 1 infection.

Despite the high burden of pediatric HIV-1 infection in developing countries, there are few data on the clinical course of influenza virus-associated lower respiratory tract infection (LRTI) in these children. To define and compare the clinical course of HIV-1-infected and -uninfected African children hospitalized with influenza virus associated severe LRTI. Children with severe LRTI were prospectively recruited between March, 1997, and March, 1999, as part of a broader study evaluating the etiology and outcome of this condition in hospitalized HIV-1-infected and -uninfected children. The results of children in whom influenza A or B virus was identified by immunofluorescent antibody staining after shell vial culture are reported. Viruses isolated were typed by hemagglutination inhibition assays. Twenty-five (21.6%) of the 116 children hospitalized with severe LRTI in whom influenza A or B virus was identified were HIV-1-infected. HIV-1-infected children were older than uninfected children (mean age +/- SD 17.4 +/- 10.8 months vs. 10.2 +/- 8.9 months; P = 0.002). HIV-1-infected children were more likely to have an underlying medical illness (in addition to HIV-1 infection) predisposing them to more severe LRTI (32.0% vs. 13.2%; P = 0.03). HIV-infected children were also more likely to have indirect evidence of bacterial coinfection, including chest radiographic evidence of confluent alveolar consolidation (78.9% vs. 35.1%, P = 0.006), and were less likely be wheezing (8.0% vs. 31.9%, P = 0.01). However, there was no difference in the clinical outcome of HIV-1-infected and -uninfected children. The duration of hospitalization [median (range) 5 (2 to 33) days vs. 4 (0 to 21) days, P = 0.08] and the mortality rates (8.0% vs. 2.2%, P = 0.20) were similar between HWV-1-infected and -uninfected children. HIV-1-infected children hospitalized with severe LRTI associated with influenza virus have an outcome similar to that of HIV-1-uninfected children even in the absence of antiretroviral or anti-influenza virus treatment.

Comparison of the sensitivity of a 24 h-shell vial assay, and conventional tube culture, in the isolation of Herpes simplex virus - 1 from corneal scrapings.

BackgroundHerpes simplex keratitis is a sight threatening ocular infection. A rapid and specific diagnosis is essential for the institution of specific antiviral therapy and to avoid complications that can arise from misdiagnosis and inappropriate treatment. Though a variety of techniques are available, isolation of Herpes simplex virus 1 (HSV-1) in culture provides the most reliable and specific method, and is considered as the gold standard in laboratory diagnosis of herpes simplex keratitis. We report a comparative study of the sensitivity of a 24 h-shell vial assay and conventional tube culture in the isolation of HSV-1 from corneal scrapings.MethodsA total of 74 corneal scrapings obtained from 74 patients with a clinical suspicion of herpes simplex keratitis submitted for the isolation of HSV-1, were simultaneously inoculated into shell vial and tube cultures employing the vero cell line. Shell vial and tube cultures were terminated at 24 h and fifth day respectively. Isolation of HSV-1 was confirmed employing an indirect immunofluorescence assay.ResultsHSV-1 was isolated from 24/74 (32.4%) specimens employing both the methods. Sensitivity of both the techniques were found to be similar (20/24, 83.3%) (P = 1.0).ConclusionA 24 h-shell vial assay is a rapid alternative technique in comparison to the time consuming conventional tube cultures for the isolation of HSV-1, especially from corneal scrapings for the laboratory diagnosis of herpes simplex keratitis.

Prevention of Cytomegalovirus Infection and Disease After Lung Transplantation: Results Using a Unique Regimen Employing Delayed Ganciclovir

Many lung transplant programs employ lengthy regimens of IV ganciclovir therapy to prevent disease due to cytomegalovirus (CMV). In 1994, we introduced a regimen of delayed ganciclovir prophylaxis for CMV infection. This consisted of 2 weeks of IV ganciclovir therapy, initiated 3 to 4 weeks after transplantation, with subsequent viral monitoring and preemptive therapy as needed. When not receiving ganciclovir, patients received oral acyclovir, 800 mg tid, for 6 months. CMV-seronegative patients with seropositive donors also received four doses of CMV hyperimmune globulin. This study analyzes the CMV outcomes of 54 patients who received the delayed regimen compared to 33 historical control subjects who received only acyclovir prophylaxis (n = 28) or oral acyclovir and 2 to 4 weeks of ganciclovir early after transplantation (n = 5). CMV detection was by shell vial culture or IgG seroconversion; after 1996, CMV detection was by blood antigenemia. The diagnosis of CMV disease also required a typical clinical syndrome or pathologic evidence of CMV. The main outcome was the actuarial incidence of CMV infection and disease. In order to account for the effect of other important risk factors for CMV infection, the time to CMV infection and disease was also studied as dependeant variables in a Cox proportional-hazard analysis, with the delayed regimen and other important risk factors as independent variables. The delayed regimen reduced the actuarial incidence of CMV infection from 80 to 48% (p < 0.001) and CMV disease from 31 to 10% (p < 0.01). No seropositive patient receiving the delayed regimen developed CMV disease. Twelve of the 54 patients in the study group required additional IV antiviral treatment, but the total use of ganciclovir averaged only 18 days per patient. In a Cox proportional-hazards model, the use of delayed ganciclovir was the only factor that showed a significant association with freedom from CMV infection (hazard ratio [HR], 0.43; 95% confidence interval [CI], 0.24 to 0.75; p = 0.003) and CMV disease (HR, 0.29; 95% CI, 0.10 to 0.86; p = 0.03). A regimen of CMV prophylaxis employing 2 weeks of IV ganciclovir initiated 3 to 4 weeks after lung transplantation followed by virologic monitoring and preemptive therapy as needed provides good protection against CMV disease.

Enteroviral meningitis in Northern Jordan: prevalence and association with clinical findings.

During the summer-autumn of 1999, 390 specimens of cerebrospinal fluid were taken from infants and children younger than 15 years of age. They were suspected of having meningitis and were admitted to Princess Rahma Hospital, Northern Jordan. They were investigated for the presence of enteroviruses using shell vial culture and indirect immunofluorescence assays. Most cases (46.9%) occurred in children younger than 1 year of age in which males represented 71.9%. The common symptoms were fever, vomiting, and headache. Enteroviruses were isolated from 32 (8.2%) cases, coxsackievirus B types 2, 4, and 5 from 15 (46.9%) cases, and echovirus 9 (31.3%) was the most common identified serotype. The virus isolation rate was directly proportional to the number of leukocytes in the cerebrospinal fluid. However, enteroviral isolation was demonstrated in 4 (12.5%) of 32 cerebrospinal fluid specimens without pleocytosis. Leukocyte differential count revealed a predominance of polymorphonuclear cells in 71.4% of the cases. Hospitalization ranged from 1 day to 25 days with a mean of 7 days. The majority of enterovirus-infected patients (88.9%) were treated with at least one type of antibiotic. These results emphasize the importance of shell vial culture assay for diagnosing enteroviruses, especially in laboratories that do not have access to advanced techniques such as polymerase chain reaction.