Shell Vial Assay Research Articles

Expression of Concern: Shell-vial Assay in Diagnosis of Disseminated BCG Infection in an Immunodeficient Child.

The Pediatric Infectious Disease Journal ():10.1097/INF.0000000000003757, November 10, 2022. | DOI: 10.1097/INF.0000000000003757

Advances in laboratory assays for detecting human metapneumovirus.

Human metapneumovirus (HMPV) is one of the major causes of acute respiratory tract infection (ARI) and shows high morbidity and mortality, particularly in children and immunocompromised patients. Various methods for detecting HMPV have been developed and applied in clinical laboratories. When reviewing the literature, we found that polymerase chain reaction (PCR)-based assays have been most frequently and consistently used to detect HMPV. The most commonly used method was multiplex reverse transcriptase-PCR (RT-PCR; 57.4%), followed by real-time RT-PCR (38.3%). Multiplex RT-PCR became the more popular method in 2011–2019 (69.7%), in contrast to 2001–2009 (28.6%). The advent of multiplex PCR in detecting broader viral pathogens in one run and coinfected viruses influenced the change in user preference. Further, newly developed microarray technologies and ionization mass spectrometry were introduced in 2011–2019. Viral culture (including shell vial assays) and fluorescent immunoassays (with or without culture) were once the mainstays. However, the percentage of studies employing culture and fluorescent immunoassays decreased from 21.4% in 2001–2010 to 15.2% in 2011–2019. Meanwhile, the use of PCR-based methods of HMPV detection increased from 78.6% in 2001–2010 to 84.8% in 2011–2019. The increase in PCR-based methods might have occurred because PCR methods demonstrated better diagnostic performance, shorter hands-on and run times, less hazards to laboratory personnel, and more reliable results than traditional methods. When using these assays, it is important to acquire a comprehensive understanding of the principles, advantages, disadvantages, and precautions for data interpretation. In the future, the combination of nanotechnology and advanced genetic platforms such as next-generation sequencing will benefit patients with HMPV infection by facilitating efficient therapeutic intervention. Analytical and clinical validation are required before using new techniques in clinical laboratories.

Development of a real-time quantitative polymerase chain reaction assay for the detection of congenital human cytomegalovirus infection in urine samples

A TaqMan real-time quantitative PCR (qPCR), based on amplification of HCMV UL54 gene specific sequence, was developed and compared with shell vial viral culture assay, the gold standard technique for the diagnosis of congenital HCMV infection, using urine samples collected from 110 newborns. The results indicate that this qPCR is slightly more sensitive than shell vial assay suggesting that qPCR may be considered a useful alternative for diagnosing congenital HCMV infection.

Spotted fever group rickettsia closely related to Rickettsia monacensis isolated from ticks in South Jeolla province, Korea

Rickettsia monacensis, a spotted fever group rickettsia, was isolated from Ixodes nipponensis ticks collected from live-captured small mammals in South Jeolla province, Korea in 2006. Homogenates of tick tissues were inoculated into L929 and Vero cell monolayers using shell vial assays. After several passages, Giemsa staining revealed rickettsia-like organisms in the inoculated Vero cells, but not the L929 cells. Sequencing analysis revealed that the ompA-small part (25-614 bp region), ompA-large part (2849-4455 bp region), nearly full-length ompB (58-4889 bp region) and gltA (196-1236 bp region) of the isolates had similarities of 100%, 99.8%, 99.3% and 99.5%, respectively, to those of R. monacensis. Furthermore, phylogenetic analysis showed that the isolate was grouped into the cluster in the same way as R. monacensis in the trees of all genes examined. These results strongly suggest that the isolate is closely related to R. monacensis. As far as is known, this is the first report of isolation of R. monacensis from ticks in Korea.

Enhanced detection of infectious salmon anaemia virus using a low‐speed centrifugation technique in three fish cell lines

Infectious salmon anaemia (ISA), caused by ISA virus (ISAV), is a serious disease of farmed Atlantic salmon, Salmo salar L. Recently, molecular- and immunofluorescent-based techniques have become powerful diagnostic tools for ISAV detection, but culture-based techniques remain the gold standard. A disadvantage of ISAV culture is that the incubation time required before cytopathic effect (CPE) is observed in cell monolayers. To decrease time until CPE is observed, a low-speed centrifugation technique was applied to existing standard operating procedures for ISAV culture in three fish cell lines. Time until CPE observation was compared in CHSE, SHK and ASK cells, treated or not treated with low-speed centrifugation after inoculation with ISAV. Low-speed centrifugation treatment significantly enhanced observable cell infection. Compared to control cells, the length of time until ISAV CPE observation decreased in centrifuged ASK and CHSE cells. Low-speed centrifugation was also incorporated into a modified clinical shell vial assay. At 48h post-inoculation with approximately 20 viral particles, ISAV was detected by an immunofluorescence antibody test in treated ASK and SHK1 cells but not in control cells. Finally, this enhanced viral adsorption assay performed in ASK cells demonstrated higher sensitivity than a real-time RT-PCR assay performed on RNA isolated from ISAV-spiked salmon kidney homogenates.

Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus.

Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4(+) T-cell activation and proliferation, as well as a sustained increase in CD4(+) T cells coexpressing CCR5 and α4β7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.

Universal Screening for Congenital Cytomegalovirus (C-CMV) Using Real-Time Polymerase Chain Reaction (RT-PCR) from Umbilical Cord Blood

Objectives: C-CMV infection affects 0.4-2% of newborn infants in Israel, most of whom are asymptomatic. Of these, 10-20% will subsequently develop hearing impairment and might have benefitted from early detection by universal neonatal screening.Methods: We retrospectively analyzed the results of a universal screening C-CMV program using RT-PCR from umbilical cord blood, carried out at the Sheba Medical Center, Israel over a 1-year period. C-CMV was confirmed by urine CMV culture (Shell-vial assay). All confirmed cases were further investigated for C-CMV manifestations by means of head ultrasound, complete blood count, liver enzyme measurement, ophthalmology examination and thorough hearing investigation and follow up.Results: From June 1, 2009 to May 31, 2010, 11,022 infants were born at the Sheba Medical Center, 8,105 of whom (74%) were screened. Twenty three (0.28%) samples were positive for CMV and all except one were confirmed by urine culture. Two more infants, who had not been screened, were found positive after clinical suspicion. All 24 infants were further investigated, and three (12.5%) were found positive for central nervous system involvement (including hearing impairment) and offered intravenous ganciclovir for six weeks. Of these 24 infants, 18 (82%) infants would not otherwise have been diagnosed.Conclusion: The relatively low incidence of C-CMV detected in our screening program probably reflects the low sensitivity of cord blood screening. Other screening methods using urine or saliva should be further investigated. Nevertheless, this screening program detected a non-negligible number of infants that could benefit from early detection.

English

Ethyl acetate, chloroform, butanol and methanol extracts of the aerial parts ofConyza Canadensis L. Cronquist were investigated for their antiviral activity against human cytomegalovirus (HCMV) AD-169 and Cox-B3 viruses by modification of the widely used shell-vial assay. The results showed that butanol and methanol extracts had the most potent antiviral activity against HCMV and Cox-B3 viruses.   Key words: Conyza canadensis (L.) Cronquist, antiviral activity, human cytomegalovirus (HCMV), Coxsackie B virus type 3 (CoxB-3).

Cytomegalovirus Infections in Non-Immunocompromised and Immunocompromised Patients in the Intensive Care Unit

Infection, inflammatory response, activation of coagulation cascade and sepsis are tightly interconnected. In the initial phase, sepsis is characterized by a pro-inflammatory state, while in the late phase, by an anti-inflammatory state which favors cytomegalovirus reactivation. Cytomegalovirus infection would accentuate the sepsis-induced immunologic effects increasing the risk for other infections. The rate of CMV infection is 17% in critically ill nonimmunocompromised patients, up to 30% in hematopoietic stem cell transplant and up to 60% in solid organ transplant recipients. Cytomegalovirus infection in critically ill patients is associated with prolonged ventilator support, nosocomial infections, prolonged hospital and/or ICU stay and increased mortality. In immunocompromised patients, cytomegalovirus causes direct effects (viral syndrome, pneumonia, meningo-encephalitis, and gastro-intestinal tract involvement) and indirect (immunomodulatory) effects. These indirect effects would predispose the patients to secondary infections, delay immune recovery after hematopoietic stem cell transplant, and increase the risk of EBV-related B-cell lymphoproliferative disease and allograft rejection. Cytomegalovirus serology is not useful for the diagnosis of active infections. Cytomegalovirus culture is impractical for clinical purposes. The shell vial assay has low sensitivity. pp65 antigen is a sensitive and specific diagnostic method. Real-time PCR is more sensitive and specific (earlier detection) than pp65 antigen test and it is a more reliable marker to monitor the clearance of viremia. Ganciclovir and valganciclovir are the first-line antiviral therapies for the treatment of immunocompromised patients, while foscarnet and cidofovir are reserved mainly for treatment of ganciclovir-resistant cytomegalovirus infections.

Association of Human bocavirus with Respiratory Infections in Outpatients and in Patients Attended at a Reference Hospital

The role of Human bocavirus (HBoV) in human infectious disease is unclear due to the frequent detection of this virus in association with other respiratory viruses with a recognized pathogenic role in acute respiratory infection. We have analyzed the impact of HBoV in outpatients and in patients requiring hospitalisation or emergency attention for acute respiratory infections. Respiratory viruses were investigated by real-time PCR, direct antigen detection and/or viral culture by shell-vial assay. Nasopharyngeal aspirates, BAL and/or sputum samples from patients attended at a reference hospital, and nasal/throat swabs from outpatients were used. Respiratory viruses were detected in 660 samples (47%). HBoV detection rate was 12.6%, only preceded by respiratory syncytial virus (25%). Co-detections were observed in 12.9% of samples, and HBoV was present in 81% of them. Similar detection rates of HBoV were obtained in individuals with positive and negative results for other respiratory viruses (12.5% and 12.7%, respectively). The crossing point value was taken as a measure of HBoV viral load. Higher HBoV loads were observed in children, and in patients from the hospital. HBoV viral load was not associated with symptoms of upper respiratory tract infection or lower respiratory tract disease. Although HBoV is frequently detected in respiratory specimens, there is a poor association between HBoV-positive specimens and clinical parameters. A clinical impact of HBoV in respiratory infection probably occurs in few cases.

Trivalent Inactivated Influenza Vaccine in African Adults Infected With Human Immunodeficient Virus: Double Blind, Randomized Clinical Trial of Efficacy, Immunogenicity, and Safety

Data on the efficacy of trivalent, inactivated influenza vaccine (TIV) in HIV-infected adults, particularly in Africa, are limited. This study evaluated the safety, immunogenicity, and efficacy of TIV in HIV-infected adults. In Johannesburg, South Africa, we undertook a randomized, double-blind, placebo-controlled trial involving 506 HIV-infected adults. Subjects included 157 individuals who were antiretroviral treatment (ART) naive and 349 on stable-ART. Participants were randomly assigned to receive TIV or normal saline intramuscularly. Oropharyngeal swabs were obtained at illness visits during the influenza season and tested by shell vial culture and RT PCR assay for influenza virus. Immune response was evaluated by hemagglutinin antibody inhibition assay (HAI) in a nested cohort. The primary study outcome involved vaccine efficacy against influenza confirmed illness. This trial is registered with ClinicalTrials.gov, number NCT00757900. The efficacy of TIV against confirmed influenza illness was 75.5% (95% CI: 9.2%-95.6%); with a risk difference of 0.18 per 100 person-weeks in TIV recipients. Among TIV recipients, seroconversion, measured by HAI titers, was evident in 52.6% for H1N1, 60.8% for H3N2, and 53.6% for influenza B virus. This compared with 2.2%, 2.2%, and 4.4% of placebo recipients (P < .0001). The frequency of local and systemic adverse events post-immunization was similar between study groups. TIV immunization is safe and efficacious in African HIV-infected adults without underlying co-morbidities. Further evaluation of effectiveness is warranted in severely immunocompromized HIV-infected adults and those with co-morbidities such as tuberculosis.

Detection of respiratory viruses from patients with influenza like illness in Guangzhou using centrifugation-enhanced shell vials method between January and June, 2009

Objective To evaluate the application of high-throughput shell vial assay in a clinical laboratory for detection of respiratory viruses from patients with ILI in Guangzhou between January and June, 2009. Methods Six hundred and fifty-two pharyngeal swab specimens were taken from ILI patents. Centrifugation-enhanced shell vials including 4 cell lines (MDCK, Hep-2, LLC-MK2 and MRC-5) were used for culture of respiratory viruses for 2-3 days. The cultures were identified by observation of cytopathic effect (CPE) , hemmaglution or hemmadsorption test as well as immunofluorescence staining. Results A total of 161 swab samples (24.69% ,161/652) were shown to have any one of the 5 common respiratory viruses including influenza A viruses ( 38. 51% , 62/161 ), influenza B virus ( 54. 65% , 88/161 ), parainfluenza viruses (4. 96% , 8/161 ) , adenovirus ( 1. 24% , 2/161 ), and respiratory syncytial virus (0. 62% ,1/161). The turnaround time was 2d for influenza viruses, 3d for adenovirus and parainfluenza viruses respectively. Conclusions (1) The shell vial method was effective, rapid and high throughout for the detection of respiratory viruses in clinical laboratories.(2)Influenza viruses were dominant in the swab samples from patients with ILI in Guangzhou between January and June with the highest appearance in the summer influenza B vires was the most common pathogen in patients with ILI in this study. Key words: Orthomyxoviridae; Vires cultivation; Respiratory tract infections

Comparison of Rickettsia conorii growth within different cell lines by real-time quantitative PCR

Rickettsia conorii is a gram-negative bacterium responsible for Mediterranean spotted fever, a rickettsiosis transmitted to humans by tick bite inoculation of rickettsiae into the skin [1]. The isolation of rickettsial isolates from various samples including blood, skin or infected ticks can be achieved on shell vial assays [2]. This approach, initially developed for R. conorii, was later successfully applied for several strains, including R. prowazekii and R. felis, the major drawback being related to the selection of convenient animal cell lines and to the choice of appropriate growth condition parameters, among which are temperature, culture medium, and fetal calf serum proportion. The development of standardised culture conditions allowing efficient growth of various strains would be advantageous to investigate the differences among various species of rickettsiae [3]. Moreover, a feature of the post-genomic era is the requirement of important amounts of bacteria for large-scale investigations such as proteomic and transcriptomic approaches [4]. The aim of this work was thus to quantify the growth of R. conorii within three different cell lines and using real-time quantitative PCR (qPCR). Eukaryotic cells used in this study were the African green monkey kidney cells (Vero cell, American Type Culture Collection ATCC CRL 1587) and the murine fibrobastic L929 cell line (ATCC CCL1), both maintained in Eagle’s minimum essential medium (MEM; Invitrogen, Paisley, UK) supplemented with 4% fetal bovine serum (FBS; Invitrogen) and 1% L-glutamine (Invitrogen). We also used 3T3 cell lines (ATCC CCL163) maintained in Dulbecco minimum essential medium (DMEM; Invitrogen) supplemented with 10% FBS and 1% L-glutamine. Confluent cells grown on coverslips were infected with 10 rickettsiae resuspended in 300 lL of the appropriate medium. After centrifugation (3452 · g; 15 min), the supernatants were removed, and following a gentle washing, 500 lL of fresh buffer was added in each shell vial and further incubated at 37 C and under 5% CO2. This point was considered as the starting point (t0) of infection. For each kinetic point, samples were processed four times, two coverslips being used for Gimenez or immunofluorescence staining while quantification of bacteria was carried out on remaining shell vials. DNA was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) and amplification was performed with the 16sRNA primers forward 5¢-TGATGAAGGCCTTAGGGTTG-3¢ and reverse 5¢-TAAACCGCCTACGCACTCTT3¢ using the LightCycler system together with the SYBR Green master mix (Roche Diagnostics, Mannheim, Germany). In some experiments, the determination of the gene copy numbers was also performed using the probes targeting rickA, a gene exclusive for bacteria from the Rickettsia genus (F 5¢-AGTTACAAAAGATAGTAAGTAA-3¢; R 5¢-CCRGYTTTTTAA CCGTAGTAG-3¢). The yield of qPCR was assessed by plotting the cycle threshold values vs. the log10 numbers of input DNA copies, the standard curve being made with genomic DNA of R. conorii (from 1–10 copies) as template and using the same primers. The qPCR data obtained indicated that entrance of R. conorii into the three eukaryotic cell lines tested in this study was not significantly different. Thus, under our experimental conditions, almost all bacteria were taken up into the host cell, the percentages being 94 ± 8%, 80 ± 6.5% and 84 ± 3.2% for Vero, L929 and 3T3 cell lines, respectively. Despite a similar invasion efficacy, we thereafter observed variable replication rates (Fig. 1). Thus, 96 h after the cell infection, the number of R. conorii appeared around six times higher in L929 than in Vero cells or 3T3. These results were confirmed with qPCR assays targeting rickA, as well as through the optical Corresponding author and reprint requests: P. Renesto, Unite des Rickettsies, URMITE IRD-CNRS 6236, Faculte de Medecine, 27 Bd Jean Moulin, 13385 Marseille, France E-mail: prenesto@embl.fr

Adenovirus in Solid Organ Transplant Recipients

Adenoviruses are nonenveloped, double-stranded DNAviruses associated with a wide range of clinical syndromesin humans (1). There are 52 immunologically distinct typesof adenoviruses that are further classified into one of7 (A–G) subgroups based on hemagglutinin properties,DNA homology, oncogenic potential in rodents and clin-ical disease (Table 1) (2,3). Adenoviruses typically causeself-limited respiratory, gastrointestinal or conjunctival dis-ease throughout the year without significant seasonal vari-ation. Adenovirus infections are most common amongchildren, people living in close quarters (such as collegestudents and military recruits), and immunocompromisedpatients (1).Adenovirus is the third most important viral infection fol-lowing liver transplantation in children, occurring in 10%of 484 pediatric liver transplant recipients in one series(4). A single series has reported an incidence of 5.8%of 191 adult liver transplant recipients (5). Symptomaticdisease (ranging from self-limited fever, gastroenteritis orcystitis to devastating illness with necrotizing hepatitis orpneumonia) occurred in over 60% of the infected patients.Symptomatic infections typically occurred within the first3 months after transplantation. The frequency of invasiveadenovirus infections after pediatric liver transplantationappeared to decrease with use of tacrolimus-based im-munosuppression(6);similardataarenotavailableforadultliver transplant recipients.Adenovirus infection in other organ recipients is less wellcharacterized. A recent prospective study in adult SOT re-cipients demonstrated measurable adenovirus DNAemiain 7.2% of 263 recipients in the first posttransplant year;nearly 60% of these patients were completely asymp-tomatic with the remaining patients manifesting gastroin-testinal (primarily diarrhea), respiratory or vague, nonspe-cific symptoms (7). In contrast to outcomes found in thisprospective study, case reports and small series identifythe fact that adenoviral infection can be clinically signif-icant in these patients. Severe adenoviral infection hasbeen reported after lung transplantation often leading tofatal disease (8–10). The presence of adenovirus DNA incardiac biopsies after pediatric heart transplantation wassignificantly associated with poor graft survival in one se-ries (11). Adenovirus also has been associated with hemor-rhagiccystitisandgraftdysfunctioninadultrenaltransplantrecipients (12). It has also been found in high rates after pe-diatric intestinal transplantation, though frequently in theabsence of obvious clinical disease or associated pathol-ogy (13). As adenovirus, like cytomegalovirus (CMV), canbe latent and can reactivate asymptomatically, ascribing acausative role in the pathologic process may sometimesbe difficult.Sites and clinical manifestations of adenoviral disease intransplant recipients appear to vary according to the typeof organ transplanted, with frequent involvement of theallograft. In liver transplant recipients, infection with aden-ovirusserotype5typicallyresultsinhepatitiswithamedianonset of 55 days after transplant (4,5), while serotypes 1and 2 are more commonly associated with pneumonia.Adenoviral pneumonia is associated with graft loss, deathor progression to obliterative bronchiolitis for lung trans-plant recipients (8). In one study of pediatric heart trans-plant recipients, detection of adenoviral genome copies inmyocardial biopsy specimens was predictive of adverseclinical events including coronary vasculopathy and graftloss (OR 4.7 compared to adenovirus negative patients;95% confidence interval 1.3–17.1) (11). The strength ofthis association and its pathogenesis remain to be clari-fied. Enterocolitis occurs more commonly in small bowelrecipients and may mimic rejection (7,14). Hemorrhagiccystitis and nephritis are described in renal transplant re-cipients (15). Disease in other organ recipients has beendescribed rarely.

Bacteriostatic and Bactericidal Activities of Tigecycline against Coxiella burnetii and Comparison with Those of Six Other Antibiotics

The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 microg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; linezolid and clarithromycin ranged from 2 to 4 microg/ml; and ciprofloxacin ranged from 4 to 8 microg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 microg/ml.

P.046 Serological micro-array for simultaneous detection of antibodies to measles, mumps and rubella viruses

Background: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. Objectives: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. Study design: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). Results: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24–48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. Conclusions: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.

Características clínicas y epidemiológicas de las infecciones respiratorias causadas por el metapneumovirus humano en pacientes pediátricos

Human Metapneumovirus (hMPV) was first identified in 2001 in respiratory samples from children and adults with acute respiratory tract infection. The aim of this prospective study was to determine the clinical and epidemiological characteristics of pediatric patients with an acute respiratory tract infection and exclusive isolation of hMPV in respiratory samples (December 2005-January 2007). All respiratory tract samples were submitted to rapid antigen detection against respiratory syncytial virus (RSV) and influenza A and B viruses. To isolate respiratory viruses, samples were inoculated in various cell lines using the shell vial culture assay. To isolate hMPV, the LLC-MK2 cell line was used. Only antigen-negative samples were studied for the presence of hMPV. Over the study period, 32 hMPV were isolated from different patients, accounting for 1.7% of all samples studied for this virus (only RSV-negative samples, 1,791) and 1.5% of all samples studied. Peak incidence was found between December and March of the two years studied. Of the 32 patients in whom hMPV was detected, 17 (53.2%) were female and 15 (46.8%) male. Mean age was 12.5 months (range, 1 month-4 years). The most frequent clinical symptoms were fever (90.6%), cough (87.5%), rhinorrhea and bronchiolitis (46.8%). Three patients (9.4%) were hospitalized. Respiratory infection caused by hMPV is considered an emergent disease in pediatric patients. The clinical manifestations are very similar to those of RSV infection; hence, only virological study can establish the definite etiological diagnosis.

Diagnosis & management of cytomegalovirus infections in the GI tract

Cytomegalovirus (CMV) infection of the GI tract is a common problem in patients immunosuppressed by HIV or organ transplantation. The esophagus and colon are the most common sites of involvement. Recent developments in diagnosis, such as immunohistochemical staining, shell vial assay and PCR, aid in early detection and predicting the prognosis of CMV disease in susceptible individuals. Although current drugs have limitations in terms of toxicity, drug interactions and resistance, newer drugs appear promising.

Duration of Influenza A Virus Shedding in Hospitalized Patients and Implications for Infection Control

To assess the duration of shedding of influenza A virus detected by polymerase chain reaction (PCR) and cell culture among patients hospitalized with influenza A virus infection. Mayo Clinic (Rochester, Minnesota) hospitals that cater to both the community and referral populations. Patients 18 years old and older who were hospitalized between December 1, 2004, and March 15, 2005, with a laboratory-confirmed (ie, PCR-based) diagnosis of influenza A virus infection were consecutively enrolled. Additional throat swab specimens were collected at 2, 3, 5, and 7 days after the initial specimen (if the patient was still hospitalized). All specimens were tested by PCR and culture (both conventional tube culture and shell vial assay). Information on demographic characteristics, date of symptom onset, comorbidities, immunosuppression, influenza vaccination status, and receipt of antiviral treatment was obtained by interview and medical record review. Patients were excluded if informed consent could not be obtained or if the date of symptom onset could not be ascertained. Of 149 patients hospitalized with influenza A virus infection, 50 patients were enrolled in the study. Most patients were older (median age, 76 years), and almost all (96%) had underlying chronic medical conditions. Of 41 patients included in the final analysis, influenza A virus was detected in 22 (54%) by PCR and in 12 (29%) by culture methods at or beyond 7 days after symptom onset. All 12 patients identified by culture also had PCR results positive for influenza A virus. Hospitalized patients with influenza A virus infection can shed detectable virus beyond the 5- to 7-day period traditionally considered the duration of infectivity. Additional research is needed to assess whether prolonging the duration of patient isolation is warranted to prevent nosocomial outbreaks during the influenza season.

Testing of Diagnostic Methods for Detection of Influenza Virus for Optimal Performance in the Context of an Influenza Surveillance Network

Influenza surveillance networks must detect early the viruses that will cause the forthcoming annual epidemics and isolate the strains for further characterization. We obtained the highest sensitivity (95.4%) with a diagnostic tool that combined a shell-vial assay and reverse transcription-PCR on cell culture supernatants at 48 h, and indeed, recovered the strain.