Mary F. Jones

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

SUMMARY Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Seroconversion to Human Herpesvirus 6 following Liver Transplantation Is a Marker of Cytomegalovirus Disease

Human herpesvirus 6 (HHV-6) infection is common after transplantation; HHV-6 is known to interact with other viruses and induce immunosuppression. Whether HHV-6 plays a role in the occurrence of cytomegalovirus (CMV) infection after transplantation was investigated. In a cohort of 247 liver transplant recipients, HHV-6 seroconversion was identified as a significant risk factor for development of symptomatic CMV infection (P < .001), including CMV organ involvement (P < .001), even in the presence of the other significant risk factors: D+/R- CMV serologic status (P < .001) or use of OKT3 after transplantation (P = .002). Subgroup analysis indicated that HHV-6 seroconversion was significantly associated with symptomatic CMV infection in the D+/R+ but not in the D+/R- CMV serologic group (P < .001 and P = .11, respectively). These results indicate that HHV-6 seroconversion is a marker for CMV disease after transplantation and suggest that additional studies using more sensitive diagnostic techniques are warranted to determine the relationship between HHV-6 and CMV infection after transplantation.

Human herpesvirus 6 seronegativity before transplantation predicts the occurrence of fungal infection in liver transplant recipients.

BACKGROUND Invasive fungal infection has a major impact on the morbidity and mortality of liver transplant recipients. Human herpesvirus (HHV)-6 infection after transplantation is associated with an immunosuppressive state and the development of cytomegalovirus disease. Because cytomegalovirus infection is a risk factor for invasive fungal infection after transplantation, we have examined whether HHV-6 and fungal infection are associated after transplantation. METHODS Pretransplantation sera from 247 consecutive liver transplant recipients were analyzed for IgG to HHV-6. Thirty-three (13%) HHV-6-seronegative recipients were identified. Six of 33 (18%) seronegative recipients experienced fungal infection as compared with 15 of 214 (7%) seropositive recipients (P=0.034). RESULTS In a univariate analysis of risk factors for fungal infection, pretransplantation seronegativity to HHV-6 (P=0.034), intraoperative cryoprecipitate requirements greater than the 75th percentile (P=0.035), reoperation (P=0.005), biliary stricturing postoperatively (P=0.046), and gastrointestinal or vascular complications postoperatively (P=0.030) were identified as significant risk factors. Moreover, in pairwise multivariate analysis, pretransplantation HHV-6 seronegativity remained a significant variable even in the presence of each of the other variables. CONCLUSIONS These results suggest that HHV-6 seronegativity before transplantation is a valuable clinical marker that identifies patients at risk for developing fungal infection after transplantation.

Quantitative Real-Time Polymerase Chain Reaction for Evaluating DNAemia due to Cytomegalovirus, Epstein-Barr Virus, and BK Virus in Solid-Organ Transplant Recipients

Testing for cytomegalovirus-, Epstein-Barr virus-, and BK virus-specific gene targets in specimens from solid-organ transplant recipients for DNA by quantitative real-time polymerase chain reaction has been implemented in many diagnostic facilities. This technology provides rapid, accurate, and reproducible results for early detection, monitoring, and medical management of patients with these infections. Because these assays are becoming commonly used in clinical practice, the technical variables associated with specimen processing (e.g., nucleic acid extraction, gene target, and result reporting), amplification, and unique patient characteristics (e.g., age, sex, underlying diseases, immune status, and immunosuppressive regimens received) are factors that may influence the understanding and interpretation of test results. We emphasize the need for standardization of existing variables through parallel comparative and proficiency testing, uniform units for expressing results, to provide for clinical correlation with the results of these molecular assays.

Evaluation of three commercial latex agglutination kits and a commercial enzyme immunoassay for the detection of cryptococcal antigen.

We compared the performance of the Meridian CALAS, Wampole Crypto-LA, Murex Cryptococcus latex agglutination assay, and the Meridian Premier EIA for the detection of cryptococcal antigen in serum and CSF. The assays demonstrated similar performance characteristics based on concordance values > or = 93% but important differences were noted in endpoint titers.

Prospective Comparison of Cell Cultures and Nucleic Acid Amplification Tests for Laboratory Diagnosis of Chlamydia trachomatis Infections

ABSTRACT Specimens submitted in M5 medium for cell culture detection of Chlamydia trachomatis were tested by nucleic acid amplification testing (NAAT) and in cell cultures. Of 35 (genital) and 26 (nongenital) specimens positive for C. trachomatis, 21 and 14 specimens, respectively, were detected exclusively by NAAT. NAAT is significantly (P < 0.0001) more sensitive than cell culture and should be considered the new “gold standard” for the laboratory diagnosis of C. trachomatis infections.

Inhibition Controls for Qualitative Real-Time PCR Assays: Are They Necessary for All Specimen Matrices?

ABSTRACT A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Laboratory diagnosis of viral hepatitis.

Both serologic and molecular assays are useful in the diagnosis of viral hepatitis. They may detect early infections before other signs of disease appear, differentiate acute from chronic infections, and detect persistence of viremia or verify development of immunity. Molecular assays may also be used to monitor responses to antiviral therapy, and in the future, be a primary method to screen blood and organ donors (NAT). EIA serologies are used to diagnose acute HAV infections or establish immune status. Similar immunoassays are used to detect HBV infections, verify persistence of antigenemia and degree of infectivity, and indicate immunity (including the response to vaccination). HBV molecular assays can shorten the diagnostic window period, verify persistence of viremia, including monitoring response to antiviral therapy, and be useful in NAT screening of donors. Molecular assays play a major role in HCV diagnosis where serologic tests can document past or present infection but cannot differentiate one from the other. A variety of molecular tests can be used as sensitive (and early) detectors of viremia (and serve as confirmatory tests for positive serologies and as donor NAT methods), document its persistence as an indicator of chronic infection, and monitor responses to antiviral therapy. Both qualitative and quantitative molecular assays are available, and their efficient use requires familiarity with the sensitivity and dynamic ranges of each method.

Variability in parvovirus B19 IgG levels in intravenous immunoglobulin samples

Parvovirus B19 infection is an important cause of chronic anemia in patients infected with human immunodeficiency virus (HIV). Chronic infection is related to impaired humoral immunity (Kurtzman et al. 1987) and can be treated successfully with intravenous gamma globulin infusion (Kurtzman et al. 1989a). Treatment failures have been described clinically and, assuming that parvovirus viremia is the cause of the anemia, then failure may be attributable to failure to clear the virus or to subsequent relapse. Early treatment failures could be the result of immunoglobulin preparations that contain insufficient antiparvovirus activity. Gamma globulin preparations are manufactured using pooled serum. Marked variability in the levels of antibody to various infectious agents, such as cytomegalovirus (Roy and Grundy 1992) and hepatitis B virus (Hoofnagle et al. 1975), in these preparations have been reported. We analyzed the antibody concentration to human parvovirus B19 in a number of gamma globulin preparations in an attempt to define the range of B19 IgG antibody levels.