Routine Viral Culture Research Articles

Diagnostic yield and clinical impact of routine cell culture for respiratory viruses among children with a negative multiplex RT-PCR result

BackgroundPolymerase chain reaction (PCR) is the reference standard for respiratory virus testing. However, cell culture may still have added value in identifying viruses not detected by PCR. ObjectivesWe aimed to estimate the yield and clinical impact of routine respiratory virus culture among children with a negative PCR result. Study designA retrospective cohort study was performed from Jan. 2013 to Sept. 2015. Respiratory samples from hospitalized or immunocompromised patients <18years old were routinely inoculated on traditional tube cell culture monolayers if they tested negative by a PCR assay for 12 respiratory viruses. We studied patients with a respiratory specimen negative by PCR and positive by culture. Duplicates and samples of sold services were excluded. Data on demographics, clinical history, laboratory findings, and patient management were collected from patients’ charts. Descriptive and multivariate statistics were performed. ResultsOverall, 4638 PCR-negative samples were inoculated in cell culture. Of those, 196 (4.2%) were cell culture positive, and 144 met study inclusion criteria. Most subjects (81.9%) were hospitalized. Mean age was 2.4±3.4years. The viruses most frequently isolated were cytomegalovirus (33.3%) and enteroviruses (19.4%). Cell culture results prompted a change in management in 5 patients (3.5%), all of whom had acyclovir initiated for localized HSV-1 infection. Four of these had skin or mucosal lesions that could be sampled to establish a diagnosis. ConclusionIn children, routine viral culture on respiratory specimens that were negative by PCR has low yield and minimal clinical impact.

Epidemiology of Enterovirus D68 in Ontario.

In August 2014, children’s hospitals in Kansas City, Missouri and Chicago, Illinois notified the Centers for Disease Control and Prevention (CDC) about increased numbers of pediatric patients hospitalized with severe respiratory illness (SRI). In response to CDC reports, Public Health Ontario Laboratories (PHOL) launched an investigation of patients being tested for enterovirus D-68 (EV-D68) in Ontario, Canada. The purpose of this investigation was to enhance our understanding of EV-D68 epidemiology and clinical features. Data for this study included specimens submitted for EV-D68 testing at PHOL from September 1, 2014 to October 31, 2014. Comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). EV-D68 was identified in 153/907 (16.8%) of patients tested. In the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with EV-D68 compared to those 20 and over, with peak positivity at ages 5–9 years. Cases were not more likely to be hospitalized than controls. Cases were more likely to be identified in September than October (OR 8.07; 95% CI 5.15 to 12.64). Routine viral culture and multiplex PCR were inadequate methods to identify EV-D68 due to poor sensitivity and inability to differentiate EV-D68 from other enterovirus serotypes or rhinovirus. Testing for EV-D68 in Ontario from July to December, 2014 detected the presence of EV-D68 virus among young children during September-October, 2014, with most cases detected in September. There was no difference in hospitalization status between cases and controls. In order to better understand the epidemiology of this virus, surveillance for EV-D68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data.

Immunohistochemical detection of virus through its nuclear cytopathic effect in idiopathic interstitial pneumonia other than acute exacerbation

Idiopathic interstitial pneumonias include complex diseases that have a strong interaction between genetic makeup and environmental factors. However, in many cases, no infectious agent can be demonstrated, and these clinical diseases rapidly progress to death. Theoretically, idiopathic interstitial pneumonias could be caused by the Epstein-Barr virus, cytomegalovirus, adenovirus, hepatitis C virus, respiratory syncytial virus, and herpesvirus, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect them. To test the hypothesis that immunohistochemistry provides more accurate results than the mere histological demonstration of viral inclusions, this method was applied to 37 open lung biopsies obtained from patients with idiopathic interstitial pneumonias. As a result, immunohistochemistry detected measles virus and cytomegalovirus in diffuse alveolar damage-related histological patterns of acute exacerbation of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia in 38 and 10% of the cases, respectively. Alveolar epithelium infection by cytomegalovirus was observed in 25% of organizing pneumonia patterns. These findings were coincident with nuclear cytopathic effects but without demonstration of cytomegalovirus inclusions. These data indicate that diffuse alveolar damage-related cytomegalovirus or measles virus infections enhance lung injury, and a direct involvement of these viruses in diffuse alveolar damage-related histological patterns is likely. Immunohistochemistry was more sensitive than the histological demonstration of cytomegalovirus or measles virus inclusions. We concluded that all patients with diffuse alveolar damage-related histological patterns should be investigated for cytomegalovirus and measles virus using sensitive immunohistochemistry in conjunction with routine procedures.

597: Clinical risk factors associated with mother-to-child transmission (MTCT) of hepatitis C virus (HCV)

597: Clinical risk factors associated with mother-to-child transmission (MTCT) of hepatitis C virus (HCV)

Laboratory Surge Response to Pandemic (H1N1) 2009 Outbreak, New York City Metropolitan Area, USA

The North Shore-Long Island Jewish Health System Laboratories serve 15 hospitals and affiliated regional physician practices in the New York City metropolitan area, with virus testing performed at a central reference laboratory. The influenza A pandemic (H1N1) 2009 outbreak began in this area on April 24, 2009, and within weeks respiratory virus testing increased 7.5 times. In response, laboratory and client service workforces were increased, physical plant build-out was completed, testing paradigms were converted from routine screening tests and viral culture to a high-capacity molecular assay for respiratory viruses, laboratory information system interfaces were built, and same-day epidemiologic reports were produced. Daily review by leadership of data from emergency rooms, hospital facilities, and the Health System Laboratories enabled real-time management of unfolding events. The ability of System laboratories to rapidly increase to high-volume comprehensive diagnostics, including influenza A subtyping, provided key epidemiologic information for local and state public health departments.

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

Background The rapid molecular diagnosis of enteroviral meningitis has been shown important for an adequate management of the patients. Objectives A new CE-marked real-time RT-PCR assay (ENTEROVIRUS R-gene™, Argene) was evaluated in two university hospital virology laboratories. Study design Reactivity, analytical sensitivity and specificity were evaluated using 54 prototype and 173 clinical human enterovirus (HEV) strains, a 12-sample HEV proficiency panel, and 30 non-HEV microorganisms. The clinical performance of the ENTEROVIRUS R-gene™ assay was evaluated by testing 197 cerebrospinal fluid (CSF) and 103 respiratory specimens, comparatively to the routinely used diagnostic techniques. Results Sixty-four out of the 65 HEV serotypes tested were detected. The analytical sensitivity ranged between 10 −2.64 and 10 2.39 TCID 50/50 μl. Cross-reactivity was observed with four human rhinoviruses. On 59 CSF specimens analyzed prospectively, the results of the ENTEROVIRUS R-gene™ assay showed a 94.8% concordance with those of the Smart enterovirus (EV) assay (Cepheid). On 138 CSF specimens tested retrospectively, the results of the ENTEROVIRUS R-gene™ assay showed a 97.1% concordance with those of either the GeneXpert EV assay (Cepheid) or the in-house RT-PCR HEV assays used at the time of specimen collection. On 103 respiratory specimens, the concordance between the results of the ENTEROVIRUS R-gene™ assay and those of the routine RT-PCRs or viral culture was 90.2% and 96.1% before and after retest, respectively. Conclusions The new test was found able to detect a large panel of enterovirus serotypes; it was sensitive when used on clinical specimens; and, easy and rapid to perform on a routine basis.

P.039 Is replacement of tAN cells in routine viral culture possible?

P.039 Is replacement of tAN cells in routine viral culture possible?

Neonatal Respiratory Tract Involvement by Trichomonas vaginalis: A Case Report and Review of the Literature

Only occasional cases of Trichomonas spp. infection have been reported in neonates, and these usually represent vaginal infections with Trichomonas vaginalis acquired during passage down the birth canal. We report the case of a 2-week-old girl who was brought by her mother to the Children's Emergency Clinic at our institution for symptoms of lethargy and poor appetite. The neonate was subsequently diagnosed with herpetic encephalitis and developed increasing respiratory difficulty, requiring intubation. Routine viral culture of a nasopharyngeal wash showed no viral organisms, but trichomonads were abundant microscopically on the viral culture medium. Molecular studies identified the organism as T. vaginalis. The significance of this organism as a neonatal respiratory pathogen and its contribution to neonatal respiratory distress are discussed.

Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered "true positives," the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

First Report of Dengue Virus Infection in a Bone Marrow Transplant Patient.

Dengue virus infections are a frequent cause of morbidity and mortality in certain endemic areas of the world but it has never been reported to occur in a bone marrow transplant patient. We describe a pediatric patient with ALL on her first bone marrow relapse undergoing allogeneic bone marrow transplant who developed sepsis and irreversible shock that on postmortem autopsy proved to be secondary to dengue virus infection Type 4. A 6 years old female with ALL underwent an allogeneic bone marrow transplant from her sibling (6/6 match) on Nov 10, 1994. She received TBI (1200 rads) and ARA-C (3gm/m2 per dose per 12 doses) as preparative regimen. She developed generalized skin erythema on day +5 followed by fever on day +6. The following days were characterized by continued fever spikes, severe mucositis, persistence of the erythematous skin rash (first degree burn like) and gross hematuria in spite of broad spectrum antibiotic coverage. Blood cultures were reported negative. She developed irreversible shock and finally died on day +11 post-transplant. At autopsy (Nov 22, 1994), Dengue virus type 4 was isolated from blood, ascitic fluid, liver and spleen by routine viral culture at San Juan C.D.C. Dengue Branch. Results were confirmed by polymerase chain reaction. Her donor also developed low grade fever the day after bone marrow donation. Further blood testing on donor (Dec 2, 1994) showed positive IgM antibodies against Dengue virus Type 4. The recipient viral cultures matches with donor acute titers for Dengue Type 4, suggesting infection through bone marrow transplant. Dengue virus infection may cause significant morbidity and mortality to blood and bone marrow recipients in endemic areas of the world.

Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay.

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.

Immunohistochemical and in situ detection of cytomegalovirus in lung autopsies of children immunocompromised by secondary interstitial pneumonia

Secondary interstitial pneumonia (SIP), a disease affecting patients immunocompromised by primary underlying diseases during their treatment in hospital, is frequently associated with cytomegalovirus (CMV) infection, a potentially treatable condition. However, in many cases, no infectious agent can be determined, and this clinical disease rapidly progresses to death. Theoretically, SIP could be caused by CMV, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect the virus. To test the hypothesis that immunohistochemistry (IH) and in situ detection by hybridization (ISH) provides more accurate results than the mere histological demonstration of CMV inclusions, these methods were applied to 37 autopsied lung sections obtained from children immunocompromised by primary underlying diseases and who died of SIP. As a result, the cases were subdivided into three groups: (1) children with SIP CMV inclusions (Diffuse alveolar damage—DAD-related) ( n=7); (2) children with SIP without classical viral inclusions (CMV-DAD-related) ( n=3); (3) children with SIP exhibiting nuclear cytopathic effect (not CMV-NSIP-related) ( n=27). In the first group, all three techniques yielded clearly positive results, whereas IH and ISH indicated that three of the children of the second group had CMV-related DAD without histological demonstration of CMV inclusions. In the third group, there were no positive CMV signals. These data indicate that DAD-related CMV infection is an important cause of SIP and of death in children immunosuppressed by primary underlying diseases, and that IH and in situ detection were more sensitive than the histological demonstration of CMV inclusions. A direct involvement of CMV in SIP exhibiting DAD is likely, but not in the non-specific interstitial pneumonia (NSIP) pattern. We conclude that all children with primary underlying diseases should be investigated for CMV SIP using sensitive IH and in situ tests in conjunction with histological routine procedures.

Three Sensitive Methods for the Detection of Cytomegalovirus in Lung Tissue of Patients with Interstitial Pneumonitis

Interstitial pneumonia after allogeneic bone marrow transplantation is frequently associated with human cytomegalovirus (HCMV) infection. However, in a considerable proportion of the cases, no infectious agent can be determined and the interstitial pneumonia is then classified as idiopathic. Hypothetically, idiopathic interstitial pneumonia could be caused by HCMV present in such small amounts or such conformation that the virus cannot be detected by routine histopathologic analysis or viral culture techniques. To check this hypothesis, three sensitive methods for HCMV detection (in situ hybridization, the polymerase chain reaction for HCMV-DNA detection, and immunohistochemistry for the detection of HCMV immediate early antigens) have been applied on lung tissue sections of bone marrow transplant patients who died with interstitial pneumonia. Three categories were distinguished: (1) patients with HCMV-related interstitial pneumonia (n = 5); (2) patients with idiopathic interstitial pneumonia (n = 10); and (3) patients with HCMV interstitial pneumonia who had been treated with antiviral therapy (n = 2). In the first category, all three techniques yielded clearly positive results, whereas these techniques indicated that one of the patients of the second category had HCMV-related pneumonia. In the third category no positive signals could be obtained. The presented data indicate that a direct involvement of HCMV in idiopathic interstitial pneumonia is unlikely. However, a PCR performed for Epstein-Barr virus (EBV) was positive in two patients with idiopathic interstitial pneumonia. These data indicate that the introduction of new sensitive techniques such as in situ hybridization, immunohistochemistry, and the polymerase chain reaction revives the interest for HCMV and other causative infectious agents in idiopathic interstitial pneumonia.

Polymerase chain reaction and viral culture techniques to detect HSV in small volumes of cerebrospinal fluid; an experimental mouse encephalitis study

A technique is described for the detection of HSV-DNA in very small volumes (5–10 μl) of cerebrospinal fluid (CSF). The method was evaluated in CSF samples of 4–6-week-old mice inoculated with HSV-1 via the corneal route. The sensitivity of the PCR assay was compared with results of spin-amplified viral culture with immunofluorescent visualization (SAC/IF), routine viral culture (RVC) and radioactive dot-blot hybridization (DBA) in CSF samples obtained from other mice. The results show the PCR to be superior over the other techniques: infectious virus or viral DNA in CSF was demonstrated by PCR, SAC/IF, RVC and DBA in 68, 55, 20 and 2.5%, respectively. These results show the feasibility of the PCR for a rapid, non-invasive diagnosis of human HSV-encephalitis.

Routine viral culture for pediatric respiratory specimens submitted for direct immunofluorescence testing.

From 1986 to 1987, 69 (25%) of 274 specimens from children with lower respiratory tract syndromes were positive for respiratory syncytial virus antigen by direct immunofluorescence assay (DFA). Comprehensive viral culture was performed on all 205 DFA-negative specimens, and 72 specimens yielded viruses; 5 specimens yielded multiple agents. Thus, 52% of specimens yielded a specific virus, supporting the routine use of viral culture. Isolates from the DFA-negative specimens included respiratory syncytial virus (n = 7), rhinovirus (n = 34), hemadsorbing viruses (n = 13), cytomegalovirus (n = 11), adenovirus (n = 8), enteroviruses (n = 3), and herpes simplex virus (n = 2). Although serologic confirmation is needed, cytomegalovirus may be an underappreciated cause of acute lower respiratory tract infection in normal children. Further studies must be conducted to document this possibility.

Immunofluorescence detection of herpes simplex virus type 1 antigen in cerebrospinal fluid cells of experimentally infected mice

Experimentally infected mice were used to assess the value of immunofluorescence (IF) procedures in cerebrospinal fluid (CSF) cells in comparison with routine viral culture of CSF for diagnosis of herpes simplex type-1 (HSV-1) encephalitis. Virus specific antigen was detected by immunofluorescence in the majority of CSF cells in 75% of infected animals. In contrast, only 20% of infected mice had positive viral cultures, which sometimes took as long as a week to show a cytopathological effect (CPE). It is concluded that antigen detection by immunofluorescence is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in CSF cells of experimentally infected mice. Our results put forward a plea for further studies using these techniques on CSF samples from patients with suspected HSV-1 encephalitis. The prerequisites for reliable interpretation of results have been defined in this study.

5 Infection of the fetus and the newborn: prevention, treatment and related handicap

Congenital infection is uncommon and the cause of only a small proportion of handicap seen in children but some infections may be preventable or even treatable. As an example, the congenital rubella syndrome first described in the 1940s is preventable by use of the vaccine but cases still occur. It is hoped that with the introduction of the measles, mumps, rubella immunization for young children, rubella will become as rare in the UK as it is in the USA. Cytomegalovirus is now a more common cause of handicap than rubella but no vaccine has been developed. Although antiviral drugs are available for herpes simplex virus and vaccinia, infection mortality in the newborn is high, even following the use of these agents; many HSV infections in the newborn arise following primary and asymptomatic maternal infections so that treatment may start late in the course of the illness. The obstetrician needs to understand the natural history as well as possible investigations available for congenital infections. There may be warning signs which require action, such as herpetic lesions in the genital tract of the mother. Less specific abnormalities during pregnancy, such as intra-uterine growth retardation and spontaneous onset of preterm labour, may point to congenital infection. This chapter describes both antenatal and postnatal management of the major congenital infections. We have included recent research data that should influence clinical practice; studies on HSV which suggest that, for women with a history of recurrent infection, routine viral culture of the genital tract at the end of pregnancy is unnecessary; reports from both the USA and the UK that rubella immunization performed inadvertently during early pregnancy has not resulted in the congenital rubella syndrome. The chapter would not have been complete without a discussion of human immunodeficiency virus, of concern to the obstetrician and midwife. There is still much to be learned about the natural history of this infection in both the mother and infant.

A PROSPECTIVE STUDY OF UNEXPLAINED NAUSEA AND VOMITING AFTER MARROW TRANSPLANTATION

We prospectively studied patients with enigmatic nausea and vomiting after allogeneic marrow transplantation to define the causes of this syndrome. Fifty consecutive episodes of persistent vomiting were investigated using physical examination and laboratory tests, endoscopic biopsies and brushings, and clinical follow-up for four weeks. Potential causes of vomiting were identified in 39 of the 50 cases (78%). Fifteen cases had gastrointestinal infections (mainly herpesviruses), 13 had unsuspected acute intestinal graft-versus-host disease (GVHD), 8 had intestinal infection plus acute GVHD, and 3 had other causes (subdural hematomas, bacteremia, and encephalitis). In the remaining 11 cases, no cause of vomiting was found. Endoscopy was necessary for diagnosis in 36 cases and required a combination of methods: routine histology, cytology, viral culture, and immunohistology using monoclonal antibodies to cytomegalovirus (CMV) and herpes simplex virus type 1. Patients with unexplained vomiting or intestinal GVHD had significant improvement of nausea and vomiting over the four-week observation period, but those with CMV did not (P = .01). We conclude that most allogeneic marrow transplant patients with enigmatic nausea and vomiting have gastrointestinal herpesvirus infections, acute GVHD, or both. Untreated CMV infections and persistent GVHD are associated with protracted vomiting in these patients.