Improved Detection of Bacterial Central Nervous System Infections by Use of a Broad-Range PCR Assay

A universal PCR assay for bacteria and fungi detected meningitis pathogens in 65% of 20 cerebrospinal fluid (CSF) samples from patients with suspected central nervous system (CNS) infections compared to a 35% detection rate by culture and/or microscopy methods. Thus, the PCR assay can improve the diagnosis rate of infective meningitis when standard methods provide a negative result.     

Comparison of a Commercial Multiplex Real-Time PCR to the Cell Cytotoxicity Neutralization Assay for Diagnosis of Clostridium difficile Infections

A commercial multiplex real-time PCR assay (Cepheid Xpert C. difficile assay) for the diagnosis of Clostridium difficile infection was evaluated. The sensitivity and specificity of the Cepheid assay were 97.1% and 93.0% for fresh stools, using the cell cytotoxicity neutralization assay as the reference. Using PCR ribotyping as the reference for ribotype 027 strains, the corresponding figures were 100% and 98.1%, respectively.Clostridium difficile infection (CDI) has increased in frequency and severity in North America and Europe over the last 5 years, largely due to the emergence of the epidemic PCR ribotype 027 strain (10, 11). The diagnosis of CDI is usually based on a clinical history of recent antimicrobial usage and diarrhea in combination with laboratory tests (9). Therefore, rapid and accurate microbiological diagnosis is urgently needed. The Cepheid Xpert C. difficile assay (Sunnyvale, CA) is a real-time multiplex PCR assay performed on the Cepheid GeneXpert Dx system. Proprietary primers specific for the toxin B gene (tcdB), binary toxin genes (cdtA and cdtB), and tcdC gene single-base deletion at nucleotide 117 were designed to detect toxigenic C. difficile and the presumptive PCR ribotype 027 strain. The purpose of this study was to evaluate the Cepheid Xpert C. difficile multiplex real-time PCR assay for the detection of toxigenic C. difficile strains and the presumptive ribotype 027.There were four serial investigations in the present study. In investigation 1, 205 frozen C. difficile strains collected during 2007 and 2008 were analyzed. In investigation 2, 195 frozen stool specimens belonging to different categories were selected based on direct cell cytotoxicity neutralization assay (CCNA) and toxigenic anaerobic culture results. Because PCR ribotype 027 is uncommon in Sweden, 40 frozen stool specimens collected in the United States were also analyzed. In investigation 3, 30 pairs of fresh-frozen stool specimens were analyzed. The fresh stool was analyzed within 24 h of collection, and then the leftover was stored at −20°C for 3 days and retested. In investigation 4, 220 consecutive fresh, unformed stool specimens (Bristol Stool Chart grade 5 to 7) from patients older than 2 years were analyzed within 24 h of collection. Eligible participants were those symptomatic patients who had a stool sample submitted to the Karolinska University Hospital for routine C. difficile testing.Unrepeated strains and stools were determined for C. difficile test by CCNA with a commercial C. difficile toxin/antitoxin kit (TechLab, Blacksburg, VA). For the stool specimens, anaerobic cultures on selective taurocholate cycloserine-cefoxitin-fructose agar plates were also performed (13). All isolates were typed by PCR ribotyping (19).Concurrently, the Cepheid Xpert C. difficile assay was performed according to protocols provided by the manufacturer. Each kit contained single-use disposable cartridges with integrated reaction chambers and reagents. A sterile Copan swab was dipped into the stool specimen or used to pick one fresh C. difficile colony from the blood agar plate, and the stool sample or colony was resuspended in sample buffer and then transferred to the cartridge. The cartridge was placed in the GeneXpert Dx module and run. Every PCR run included a sample-processing control and a probe check control. Each day, a positive control (C. difficile ATCC 9689 or the ribotype 027 strain) and a negative control (diluted C. difficile-negative stool sample) provided by Cepheid were tested. Results were automatically interpreted by the software as follows: “C. difficile positive,” “C. difficile 027 NAP1 presumptive positive,” “C. difficile negative,” “invalid,” “error,” or “no result.” If any of the test results was “invalid,” “error,” or “no result,” the sample was retested.CCNA and strain typing of the isolates were used as the reference standards for all of the investigations. If discrepant results for stool specimens were obtained with the Cepheid assay and CCNA, the results of CCNA for recovered isolates were taken into consideration. Sensitivity, specificity, positive and negative predictive values, and 95% confidence interval (CI) were calculated using SAS.The agreement between the Cepheid assay and CCNA for 205 isolates, 235 frozen stool specimens, and 220 fresh stools was 99.5%, 89.4%, and 93.6%, respectively (Table ​(Table1).1). Mixed populations of isolates with different toxin profiles (toxin B negative and toxin B positive) and different PCR ribotypes were found in four fecal samples. The real-time PCR cycle threshold values for frozen-thawed samples matched those for fresh samples (data not shown). In investigation 4, the initial evaluation of the stool specimens yielded 95.7% sensitivity and 87.3% specificity, respectively. The discrepancies between the assays are listed in Table ​Table2.2. Upon reevaluation by toxigenic anaerobic culture, the sensitivity and specificity increased to 97.1% and 93.0%, respectively.

TABLE 1.

Comparison between the Cepheid Xpert C. difficile assay and CCNA

Virological Diagnosis of Central Nervous System Infections by Use of PCR Coupled with Mass Spectrometry Analysis of Cerebrospinal Fluid Samples

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections.     

Clinical Impact of Multiplex Syndromic Panels in the Diagnosis of Bloodstream,Gastrointestinal, Respiratory,and Central Nervous System Infections

Recent technological advances are revolutionizing clinical microbiology. In place of the traditional diagnostic approach in which a clinician suspecting an infectious disease is required to test for the most likely etiology, perform follow-up testing for rarer causes, and then perhaps be left with a diagnosis of exclusion, many laboratories are now offering multiplex syndromic panels to provide a comprehensive diagnostic approach to bloodstream, gastrointestinal, respiratory, and central nervous system infections. In addition to simplifying testing and laboratory workflow, multiplex panels can improve patient care, promote antimicrobial stewardship, and assist with infection control and prevention. This review discusses the growing body of evidence demonstrating the clinical impact and cost-effectiveness of multiplex panels.     

Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens

We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively.     

Chronic Infection of the Rabbit Central Nervous System by a Slowly Growing Equine Herpesvirus

The spinal cords of rabbits were chronically infected by a slowly growing horse herpesvirus (a "cytomegalovirus") inoculated directly therein. Virus was recovered from the central nervous systems of some of such animals after more than 1 year. The virus could be reisolated from all the animals killed during the first few weeks after its injection; acute focal meningomyelitis was present with involvement of gray and white matter of the cervical, thoracic, and lumbar levels of the spinal cords of these rabbits, though the nerve cells themselves remained undamaged. Thereafter, reisolation of the virus became sporadic, and no damage to the spinal cord could be histologically discerned even in animals from which the virus was recovered. No paralytic or other clinical effects could be attributed to the infection.     

Evaluation of a Commercial PCR Kit for Diagnosis of Cytomegalovirus Infection of the Central Nervous System

We evaluated the AMPLICOR cytomegalovirus (CMV) PCR kit for the diagnosis of neurologic CMV infections on 43 positive and 112 negative archived cerebrospinal fluid specimens originally tested by an in-house PCR method. The AMPLICOR kit showed sensitivity and specificity of 95 and 100%, respectively, versus the home-grown assay, indicating its utility in this clinical setting.     

Diagnostics and Discovery in Viral Central Nervous System Infections

The range of viruses implicated in central nervous system disease continues to grow with globalization of travel and trade, emergence and reemergence of zoonoses and investments in discovery science. Diagnosis of viral central nervous system infections is challenging in that brain tissue, where the pathogen concentration is likely to be highest, is not readily obtained and sensitive methods for molecular and serological detection of infection are not available in most clinical microbiology laboratories. Here we review these challenges and discuss how they may be addressed using advances in molecular, proteomic and immunological methods.     

Development of Whole-Virus Multiplex Luminex-Based Serological Assays for Diagnosis of Infections with Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Homologs in Macaques

Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi''s sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.     

Comparative Evaluation of the Diagenode Multiplex PCR Assay on the BD Max System versus a Routine In-House Assay for Detection of Bordetella pertussis

This study looked at 128 nasopharyngeal aspirates (NPA) and 162 throat swabs (TS) tested with the Diagenode multiplex assay on the BD Max system versus our in-house Bordetella pertussis PCR. Sensitivity and specificity were 97.3% and 100% for NPA and 88.3% and 98% for TS, respectively. Of positive NPA, 42.1% were coinfected with respiratory viruses.