Thomas F. Smith

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.

Incidence, distribution, and outcome of episodes of infection in 100 orthotopic liver transplantations

Of 83 patients who underwent 100 orthotopic liver transplantations, 53 had a single transplant procedure and at least 6 months of follow-up. In this main study group of 53 patients, major infections developed in 28 (53%) (a mean of 1.8 major episodes per infected patient). Of 51 major infections, 27 were bacterial, 19 were viral, 3 were protozoan, and 2 were fungal. Of the 27 bacterial infections, 22 (81%) occurred in the first 2 months after transplantation. Of the 40 bacterial isolates in the 27 bacterial infections, gram-positive aerobic bacteria were isolated in 26 (65%), anaerobic bacteria in 8 (20%), and aerobic gram-negative bacteria in 6 (15%). Only 1 of 16 bacteremic episodes was due to a gram-negative aerobic bacterium. Cytomegalovirus (CMV) infection occurred in 30 of the 53 patients (57%) and was symptomatic and invasive in 18. CMV infection was diagnosed a mean of 26 days after transplantation. Infections due to Pneumocystis carinii occurred later (2 to 3 months after transplantation). Death from infection occurred in 4 of the 53 patients (8%). In the group of 16 patients with two or more liver transplantations, fungal infection occurred in 2 and CMV infection in 13. In all 16 patients who underwent more than one liver transplantation, a major infection developed. The observations made in the main study group were consistent with findings in 13 patients with one liver transplantation but less than 6 months of follow-up. Infection is a major complication after liver transplantation, generally occurring in the first 2 months. Our observations suggest that the use of selective bowel decontamination may be associated with a relatively lower incidence of gram-negative aerobic bacterial infections.

Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation

Cytomegalovirus (CMV) DNA load was analyzed as a marker for relapse of CMV infection in 24 solid organ transplant patients with CMV infection or disease who received a fixed 14-day course of intravenous ganciclovir. Viral load was measured in blood samples obtained before and at the completion of treatment. Eight (33%) of 24 patients developed relapsing CMV infection. Median pretreatment viral loads were higher in the relapsing group (80,150 copies/106 leukocytes) than in the nonrelapsing group (5500 copies/106 leukocytes; P=.007). The relapsing group also had persistent detectable viral DNA (median, 5810 copies/106 leukocytes) after treatment, whereas it was undetectable in the nonrelapsing group (P<. 0001). Primary CMV infection (seronegative recipients of seropositive organs, D+R-) was an independent marker for CMV relapse (P=.03), and these patients had higher pre- and posttreatment viral loads than did non-D+/R- patients (P<.0001 and P=.0014, respectively). CMV DNA load is a useful marker for individualizing antiviral treatment of CMV infection in solid organ transplant recipients.

Human β-Herpesvirus Interactions in Solid Organ Transplant Recipients

The replication of beta-herpesviruses-cytomegalovirus (CMV), human herpesvirus (HHV)-6, and HHV-7-and their association with CMV disease and response to antiviral therapy were prospectively investigated in 33 liver transplant recipients not given antiviral prophylaxis. CMV, HHV-6, and HHV-7 DNA were detected within 8 weeks after transplantation in 70%, 33%, and 42% of the patients, respectively. The univariate association between CMV disease and the 3 beta-herpesviruses was more significant by virus load quantitation than by qualitative detection of DNA. This association with high levels of CMV, HHV-6, and HHV-7 (P<.001,.022, and.001, respectively) occurred mainly in CMV-seronegative recipients of transplants from CMV-seropositive donors. Antiviral therapy with ganciclovir (Gcv) reduced the load of CMV and HHV-6 and HHV-7. These results suggest that CMV disease in transplant recipients is related to the unique interaction of the 3 beta-herpesviruses and is ultimately reduced after intravenous Gcv treatment.

Detection of Bacillus anthracis DNA by LightCycler PCR

ABSTRACT Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

The clinical use of various blood compartments for cytomegalovirus (CMV) DNA quantitation in transplant recipients with CMV disease.

BACKGROUND The quantitation of cytomegalovirus (CMV) DNA is a cornerstone in the management of CMV disease in transplant recipients. However, a consensus as to what is the optimal blood compartment for the detection and quantitation of CMV DNA in peripheral blood is nonexistent. METHODS With an automated quantitative assay, we have simultaneously quantified the CMV DNA load in whole blood (WB), plasma (PL), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) in 319 samples from 17 transplant recipients with 19 episodes of CMV disease that were treated with 2 weeks of intravenous ganciclovir. RESULTS Higher levels of CMV DNA were observed in WB than PL (PL minus WB mean difference, 0.67 log; 95% confidence interval, -1.02 to -0.32; P=0.0009). This observation was most evident before treatment with intravenous ganciclovir (pretreatment geometric mean CMV DNA was 45,412 copies per ml of WB vs. 14,995 copies per ml of PL). In contrast, the CMV DNA levels between PBL and PBMC were highly comparable throughout the course of CMV disease and its treatment. Intravenous ganciclovir exerted a uniform effect on the four blood compartments with no statistically significant difference in the degree and rate of CMV DNA decline between WB and PL and between PBL and PBMC. CONCLUSIONS Although our study demonstrates the adequacy of all blood compartments for CMV DNA quantification, the higher sensitivity of WB and its yield of higher CMV DNA render it an optimal sample for monitoring CMV DNA load during CMV disease in immunocompromised patients.

Risk factors for cytomegalovirus and severe bacterial infections following liver transplantation: a prospective multivariate time-dependent analysis.

Risk factors for cytomegalovirus and severe bacterial infections were studied prospectively by univariate, multivariate and time-dependent Cox model analysis in 79 consecutive liver transplant patients treated with selective bowel decontamination. Cytomegalovirus infection occurred in 39 patients (49%) and was symptomatic in 22 patients. Twenty-six patients (33%) developed at least one of 43 documented severe bacterial infections. In a multivariate analysis of risk factors for all cytomegalovirus infections, the cytomegalovirus seronegative recipient-cytomegalovirus seropositive donor group was the highest risk group (P < 0.001). Using the same analysis for risk factors for symptomatic cytomegalovirus infections, a prolonged prothrombin time (P < 0.005), a diagnosis of acute fulminant hepatitis as the underlying liver disease (P < 0.01) and a cytomegalovirus seronegative patient receiving a liver from a seropositive donor (P < 0.001) were significant. The treatment with OKT3 therapy (P < 0.008) and hepatic artery thrombosis (P < 0.02) were found to be significant risk factors in a time-dependent univariate analysis but were not independent risk factors when multivariate analysis was utilized. Significant risk factors for major bacterial infections (P < 0.03) using univariate analysis included a prolonged anesthesia, anhepatic and surgical times, as well as the transfusion of large amounts of fresh frozen plasma or autologous blood. In a multivariate analysis, only the transfusion of large amounts of fresh frozen plasma (P < 0.04) was a significant independent risk factor. Cytomegalovirus infection was a risk factor for the development of severe bacterial infections (P < 0.03) in a multivariate time-dependent analysis.

Role of the Laboratory in Diagnosis and Management of Cytomegalovirus Infection in Hematopoietic Stem Cell and Solid-Organ Transplant Recipients

Solid-organ and hematopoietic stem cell transplantation have become vital in the treatment of many illnesses that were previously considered fatal. In 1999, a total of 21,516 solid-organ transplant procedures were performed in the United States (Scientific registry, United Network for Organ Sharing

Selective Reactivation of Human Herpesvirus 6 Variant A Occurs in Critically Ill Immunocompetent Hosts

Reactivation of human beta-herpesviruses (cytomegalovirus [CMV], human herpesvirus [HHV]-6, and HHV-7) in nonimmunocompromised hosts is rare. Because these viruses are susceptible to reactivation by cytokines and stress-related mechanisms, the incidence of their reactivation was investigated among 120 patients during stress related to critical illness and compared with findings among 50 healthy volunteers. Human beta-herpesvirus DNA was found in 65% of critically ill patients (60% men; mean age, 63 years) who required admission to an intensive care unit for medical (40%) or surgical (53%) indications or trauma (7%). HHV-6 reactivation was higher in critically ill patients than in healthy volunteers (54/101 vs. 0/50; P=.001). All patients except 1 were confirmed as HHV-6 variant A (mean virus load, 5066 copies/10(6) peripheral blood leukocytes). The reactivation of HHV-6A did not affect disease severity and outcome. No significant reactivation of HHV-7 or CMV was demonstrated among the critically ill patients. These findings contribute to the less-defined epidemiology of HHV-6A infection.

Preemptive Use of Oral Ganciclovir to Prevent Cytomegalovirus Infection in Liver Transplant Patients: A Randomized, Placebo-Controlled Trial

The use of postdetection antiviral treatment of cytomegalovirus (CMV) as a strategy to prevent infection and disease in solid-organ transplant patients has not been evaluated by placebo-controlled trials. We carried out such a study in 69 patients who had received liver transplants and had positive results of CMV polymerase chain reaction within 8 weeks after transplantation but did not have concomitant CMV infection or disease. These patients were randomly assigned to receive placebo or oral ganciclovir for 8 weeks. CMV infection developed in 21% and disease developed in 12% of placebo recipients (P =.022), compared with 3% and 0%, respectively, among ganciclovir recipients (P =.003). Similarly, in the placebo arm, 55% and 36% of CMV-negative patients who received organs from CMV-positive donors developed CMV infection or disease, respectively (P =.02), compared with 11% and 0% of such patients in the ganciclovir arm (P <.01). Oral ganciclovir administered on CMV detection by PCR prevents CMV infection or disease after liver transplantation.