Mark J. Espy

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.

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.

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.

Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens

ABSTRACT The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

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.

Duration of Influenza A Virus Shedding in Hospitalized Patients and Implications for Infection Control

OBJECTIVE To assess the duration of shedding of influenza A virus detected by polymerase chain reaction (PCR) and cell culture among patients hospitalized with influenza A virus infection. SETTING Mayo Clinic (Rochester, Minnesota) hospitals that cater to both the community and referral populations. METHODS Patients 18 years old and older who were hospitalized between December 1, 2004, and March 15, 2005, with a laboratory-confirmed (ie, PCR-based) diagnosis of influenza A virus infection were consecutively enrolled. Additional throat swab specimens were collected at 2, 3, 5, and 7 days after the initial specimen (if the patient was still hospitalized). All specimens were tested by PCR and culture (both conventional tube culture and shell vial assay). Information on demographic characteristics, date of symptom onset, comorbidities, immunosuppression, influenza vaccination status, and receipt of antiviral treatment was obtained by interview and medical record review. Patients were excluded if informed consent could not be obtained or if the date of symptom onset could not be ascertained. RESULTS Of 149 patients hospitalized with influenza A virus infection, 50 patients were enrolled in the study. Most patients were older (median age, 76 years), and almost all (96%) had underlying chronic medical conditions. Of 41 patients included in the final analysis, influenza A virus was detected in 22 (54%) by PCR and in 12 (29%) by culture methods at or beyond 7 days after symptom onset. All 12 patients identified by culture also had PCR results positive for influenza A virus. CONCLUSION Hospitalized patients with influenza A virus infection can shed detectable virus beyond the 5- to 7-day period traditionally considered the duration of infectivity. Additional research is needed to assess whether prolonging the duration of patient isolation is warranted to prevent nosocomial outbreaks during the influenza season.

Polyomavirus polymerase chain reaction as a surrogate marker of polyomavirus-associated nephropathy

Background. Polyomavirus-associated nephropathy (PVAN) is a significant cause of allograft loss after renal transplantation. A noninvasive assay that can guide the evaluation of PVAN would be of clinical value. We compared the utility of BK virus (BKV) polymerase chain reaction (PCR) and urine cytology in screening for concurrent PVAN. Methods. We used PCR to test urine and plasma samples from renal recipients simultaneously for BKV DNA. Additionally, we tested urine samples for decoy cells. Sample results were correlated with biopsy-proven PVAN. Receiver-operator characteristic curves were used to determine viral load thresholds associated with concurrent PVAN. Results. In this cross-sectional study, BKV viruria, viremia, and urinary decoy cells were detected in 24%, 9%, and 13% of renal recipients, respectively. Among 114 patients who had renal allograft biopsy, four (3.5%) were diagnosed with PVAN. Using pathology as gold standard for the diagnosis of PVAN, BKV viremia threshold of >1.6E+04 copies/mL had 100% sensitivity, 96% specificity, 50% positive predictive value, and 100% negative predictive value. A BKV viruria threshold of >2.5E+07 copies/mL had 100% sensitivity, 92% specificity, 31% positive predictive value, and 100% negative predictive value. In contrast, urine decoy cells had 25% sensitivity, 84% specificity, 5% positive predictive value, and 97% negative predictive value for the diagnosis of concurrent PVAN. Conclusion. BKV PCR may be a clinically useful noninvasive test to identify renal recipients with concurrent PVAN. BKV DNA >1.6E+04 copies/mL of plasma and >2.5E+07 copies/mL of urine were highly associated with concurrent PVAN whereas a negative PCR test makes the diagnosis of PVAN highly unlikely.

The pathogenesis of hepatitis C virus is influenced by cytomegalovirus

We investigated the effect of beta-herpesviruses on allograft failure and mortality, hepatitis C virus (HCV) replication, and liver histologic characteristics among 92 HCV-infected liver transplant recipients. Reactivation of cytomegalovirus (CMV) but not of human herpesvirus 6 (HHV-6) was independently associated with allograft failure and mortality (risk ratio, 3.71; 95% confidence interval, 1.64-8.39); allograft failure and mortality was observed in 48% of patients with CMV disease, 35% of patients with subclinical CMV infection, and 17% of patients without CMV infection (P=.0275). CMV reactivation was highly predictive of mortality (P<.001), regardless of whether it remained subclinical or evolved into CMV disease. Patients with CMV disease had a higher fibrosis stage (P=.05) and had a trend toward a higher hepatitis activity index (P=.10) and HCV load (P=.10) at 16 weeks after liver transplantation. The pathogenesis of HCV is influenced by its interaction with CMV but not with HHV-6.