Characterization of the Cytopathic Effects of Monkeypox Virus Isolated from Clinical Specimens and Differentiation from Common Viral Exanthems.

While the practice of viral culture has largely been replaced by nucleic acid amplification tests, circumstances still exist in which the availability of viral culture will allow for the diagnosis of infections not included in a provider's differential diagnosis. Here, we examine the cytopathic effects (CPE) and clinical data associated with 18 cases of monkeypox virus (MPXV) isolated from 19 clinical samples submitted for viral culture. During the study period, a total of 3,468 viral cultures were performed with herpes simplex virus (HSV) most commonly isolated (646/3,468; 18.6%), followed by MPXV (19/3,468; 0.6%) and varicella-zoster virus (VZV) (12/3,468; 0.4%). Most MPXV-positive samples were obtained from males (14/19) and taken from genital (7/19) or rectal lesions (5/19). Cycle threshold values of tested samples ranged from 15.3 to 29.0. Growth of MPXV in cell culture was rapid, yielding detectable CPE at a median of 2 days (range: 1 to 4) often with >50% of the monolayer affected in RMK, BGM, A549, and MRC-5 cell lines. As clinical features of MPXV, HSV, and VZV lesions may overlap, CPE patterns were compared between viruses. HSV CPE developed in a similar time frame (median: 2 days, range: 1 to 7) but was more often negative in RMK cells relative to MPXV. VZV grew more slowly (median: 9 days, range: 5 to 11) and demonstrated CPE affecting ≤25% of cell monolayers when positive. Viral culture remains an important tool for the detection of rare or emerging viral pathogens, particularly when high viral load specimens are easily obtained.

Contact Transmission of Vaccinia to an Infant Diagnosed by Viral Culture and Metagenomic Sequencing.

Targeted molecular diagnostic tests and accurate immunoassays have transformed the landscape of clinical virology, calling into question the usefulness of traditional viral culture. Here we present a case where viral culture, followed by metagenomic sequencing, was central to the diagnosis of an unexpected viral infection, with significant clinical and public health implications.

Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2.

BACKGROUND: Most herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing. OBJECTIVE: To enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet. STUDY DESIGN: The assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions. RESULTS: Results agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3-37% coefficient of variation based on IC50. CONCLUSIONS: Chemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing.

Limited utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for diagnosis of respiratory tract infections.

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.

Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus.

A high-throughput real-time RT-PCR assay was developed to amplify and detect a conserved region of the hemagglutinin gene of the 2009-H1N1 influenza A virus using a minor groove binder-conjugated hybridization probe. The assay was paired with a separate triplex real-time assay that detects influenza A via the matrix gene, influenza B and RSV in a multiplex format and compared with the Centers for Disease Control and Prevention (CDC) rRT-PCR assay using 143 samples. The 2009-H1N1 portion of the multiplex assay had 100% correlation with the CDC assay, while the triplex assay had a 99% agreement. An additional 105 samples collected from October to November 2009 were also tested using both the individual 2009-H1N1 and triplex assays. Of these 105 samples, eight were positive for the hemagglutinin target in the H1N1 assay and negative for the matrix target in the triplex assay. Discrepant analysis revealed single nucleotide polymorphisms within the matrix gene of 2009-H1N1 virus-positive samples. The limit of detection for the 2009-H1N1 assay was between 750 and 1,500 copies/reaction and no cross-reactivity with other respiratory pathogens was observed. Overall, this multiplexed format proved to be sensitive, robust and easy to use and serves as a useful tool for pandemic testing.

Utility of terminal hemadsorption for detection of hemadsorbing respiratory viruses from conventional shell vial cultures for laboratories using R-Mix cultures.

BACKGROUND: Many laboratories using R-Mix cell lines inoculate other shell vial cultures to improve the recovery of viruses, and in particular, perform terminal hemadsorption (THad) following 10-14 days of incubation to improve detection of respiratory viruses. We explored the cost-effectiveness and added benefits of THad on conventional shell vial cultures from respiratory samples for laboratories using R-Mix cell lines. OBJECTIVES: To determine if eliminating the practice of THad from conventional shell vial culture when R-Mix cultures are negative, would result in a significant reduction in the number of hemadsorbing respiratory viruses detected. STUDY DESIGN: THad results were retrospectively reviewed for 41,129 respiratory shell vial cultures that were set up concurrently with R-Mix cultures. RESULTS: Greater than 95% of hemadsorbing respiratory viruses were recovered by R-Mix standard protocol within 24h of inoculation, and only 5% were detected by THad at 10-14 days. CONCLUSION: The practice of hemadsorption at days 10-14 for conventional shell vial cultures from respiratory specimens should be discontinued for laboratories using R-Mix due to its low yield, questionable clinical impact of delayed results and additional costs.

Comparison of multiple shell vial cell lines for isolation of enteroviruses: a national perspective.

BACKGROUND: Isolation of enterovirus in cell culture is still utilized by clinical laboratories, for vaccine research, and for identifying serotypes for disease surveillance. Use of a combination of cell lines is recommended yet this practice has not been rigorously examined for shell vials. OBJECTIVES: To evaluate the growth predilection of enterovirus serotypes for certain shell vial cell lines in clinical samples received at a national reference laboratory. STUDY DESIGN: We retrospectively reviewed results of samples submitted for viral culture over a 3-year period. PMK, BGM, RD, A549, and MRC-5 cell lines grown in shell vials were inoculated and serotyped. RESULTS: Of 55,816 cultures, 1047 (1.9%) yielded enterovirus representing 18 serotypes. Echovirus 7, echovirus 9, and echovirus 30 were the most common serotypes recovered in 2002, 2003, and 2004, respectively. PMK and MRC-5 recovered the majority of enterovirus isolates; the addition of BGM and RD cells increased our recovery rate by 13%. For 52.6% of enteroviral isolates, cytopathic effect was found in only a single cell line. PMK and BGM cells were effective in isolating coxsackieviruses, and RD and MRC-5 were useful particularly in isolating echoviruses. CONCLUSIONS: A combination of shell vial cell lines is still recommended for recovery of enteroviruses.

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.

Sensitivity of respiratory virus culture when screening with R-mix fresh cells.

Use of R-Mix Fresh Cells has been shown to be a rapid and sensitive method for the detection and identification of respiratory viruses. We prospectively evaluated the impact of incorporation of R-Mix shell vials on the sensitivity and time to detection of seven respiratory viruses recovered in a comprehensive culture during the course of an entire respiratory season in a high-volume clinical laboratory. In this study, R-Mix shell vials were used as part of the culture of 3803 respiratory specimens. A total of 428 respiratory viruses were recovered. Staining of R-Mix vials after overnight incubation allowed initial detection of 274 of 279 influenza viruses, 33 of 38 parainfluenza viruses, 35 of 51 adenoviruses, and 52 of 60 respiratory syncytial viruses (RSVs). The time to reporting of all positive cultures after in-lab specimen receipt was 2.9 days on average and those initially detected in R-Mix cells were reported in 2.3 days on average. A combination of direct fluorescent-antibody (DFA) staining and virus culture was performed on a subset of 711 respiratory specimens. Of 152 viruses identified, 57 were observed only with DFA testing (55 RSV and 2 influenza A viruses) and 31 were recovered only in cell culture. After overnight incubation, R-Mix cells detected 87.1% of respiratory viruses not observed by DFA testing and 96.9% of viruses positive by both methods. The sensitivities of DFA testing and R-Mix cells for identification of influenza viruses were 70.5% and 96.7%, respectively. The R-Mix method detected influenza virus in 18 samples that were negative by DFA testing.

Emergence of echovirus type 13 as a prominent enterovirus.

In 2001, increased activity of the rarely detected enterovirus echovirus type 13 (E13) was observed in the United States. This article describes the epidemiologic, clinical, and genetic characteristics of E13 activity in the United States in 2001, compared with E13 activity abroad in 2000-2002. In the United States, E13 accounted for 376 (24%) of the 1584 enterovirus isolates reported in 2001 (29% of the reported isolates had a known serotype), compared with 74 isolates reported during 1970-2000. Five states reported aseptic meningitis outbreaks associated with E13, for a total of 521 cases. All characterized E13 isolates from the United States, Europe, Asia, and Oceania recovered in 2000-2002 were at least 95% identical to each other in VP1 capsid gene sequence, but they were genetically distinct from E13 isolates recovered before 2000. Continued surveillance of enteroviruses is important to alert physicians and public health officials to changes in disease trends and to improve efficiencies of clinical intervention.