Development of a multiple internal control for clinical diagnostic real-time amplification assays.

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.

Early detection and identification of commonly encountered Candida species from simulated blood cultures by using a real-time PCR-based assay.

In a recent study, Candida species in clinical blood samples were detected using a real-time PCR-based method (Maaroufi et al, J Clin Microbiol 2003, 41:3293-3298). For the present study, we evaluated the efficiency of this method as an adjunct to the BACTEC blood culture system to early detection of positivity and negativity of simulated low candidemias. We first established an in vitro correlation between the inoculum of the most frequently encountered Candida species and the time to positivity of these microorganisms. Then, aliquots from blood culture bottles infected with a final average candidal inoculum of 3.18 colony-forming units (CFU)/culture bottle (range, 1 to 6 CFU) were collected at increasing incubation times, and DNA was extracted and submitted to the TaqMan-based PCR assay. To optimize this assay, we evaluated the effect of adding 0.5% bovine serum albumin (BSA) to DNA extracts and found that it decreased the effects of inhibitors. Using specific probes for the tested Candida species, the PCR assay was positive on blood culture aliquots collected from the BACTEC system after a minimum culture turnaround time (TAT) of 3.11 +/- 1.24 hours. Addition of BSA to PCR reaction mixtures improves the TAT (1.84 +/- 0.41 hours). Hence, the combination of DNA "amplification" in the culture bottles by normal growth with an additional DNA amplification by PCR might be a reliable tool facilitating the early diagnosis of low candidemias.

Practical use of GenoType® HelicoDR, a molecular test for Helicobacter pylori detection and susceptibility testing.

Compared to culture-based method, the sensitivity, specificity, positive, and negative predictive values of the GenoType(®) HelicoDR for detecting Helicobacter pylori resistance were, respectively, 100, 86.2, 89.7%, and 100% to clarithromycin as well as 82.6, 95.1, 90.5%, and 90.7% to fluoroquinolones. This molecular assay detected a mixture of genotypes and could successfully analyze biopsies without transport/storage limitations.

Preemptive management of Epstein-Barr virus reactivation after hematopoietic stem-cell transplantation.

BACKGROUND: Epstein-Barr virus (EBV) reactivation after hematopoietic stem-cell transplantation can lead to posttransplant lymphoproliferative disease (PTLD), which carries a high mortality rate. Among therapeutic and prophylactic options being developed, B-cell depletion with monoclonal antibodies is encouraging. Because viral load after transplantation is correlated with PTLD occurrence, we developed a preemptive attitude based on polymerase chain reaction (PCR)-guided rituximab administration. METHODS: We monitored 115 transplant patients with a quantitative PCR for EBV DNA performed on whole-blood samples. Criteria for treatment initiation were a single PCR above 40,000 DNA genome copies per milliliter (gCop/mL) or two rising values above 10,000 gCop/mL. Weekly rituximab infusion at the dose of 375 mg/m was administered until negative PCR results were available. We evaluated the incidence of EBV reactivation and PTLD development. RESULTS: Nineteen patients (16.5%) met the criteria for treatment. Incidence of reactivation was the same in high-risk and standard-risk patients (12 vs. 7, P=0.38). One patient developed PTLD after discontinuation of therapy due to a serious adverse event. No other serious adverse events were noticed. Viral load disappeared after a median of three cycles of therapy, and weekly monitoring allowed prompt intervention. No PTLD-related death was observed, all-cause mortality in the treated population was 68%. CONCLUSIONS: Our PCR-guided and rituximab-based preemptive approach to avoid PTLD after allogeneic hematopoietic stem-cell transplantation is feasible but probably overtreated patients. Prospective trials are strongly needed, they should use uniform PCR techniques and consider higher threshold values for treatment initiation.

Real-time PCR for determining capsular serotypes of Haemophilus influenzae.

A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (10(1) CFU/PCR) was higher than that for type e (10(3) CFU/PCR) and types d and f (10(4) CFU/PCR), and a broader dynamic range was obtained (5 to 8 log(10) units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections.

Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay.

Molecular diagnosis based on genomic amplification methods such as real-time PCR assay has been reported as an alternative to conventional culture for early detection of invasive candidiasis. However, a major limitation of the molecular method is the difficulty associated with breaking fungal cell walls since the DNA extraction step still requires more than half of a working day. It has been suggested that PCR detection of free template DNA in serum is preferred over the use of whole blood for the diagnosis of systemic candidiasis. In this study, two conventional procedures (the first [the HLGT method] consists of boiling sera in an alkaline guanidine-phenol-Tris reagent, and the second [the PKPC method] uses proteinase K digestion, followed by organic extraction) and three commercially available kits for DNA isolation were evaluated for sensitivity, purity, cost, and use of template for most clinically important Candida species in a TaqMan-based PCR assay. To optimize these procedures, we evaluated the effect of adding 0.5% bovine serum albumin to DNA extracts and found that it decreased the effects of inhibitors. The QIAamp DNA blood kit did significantly shorten the duration of the DNA isolation but was among the most expensive procedures. Furthermore, the QIAamp DNA blood kit proved to be as sensitive as the HLGT DNA isolation method for PCR amplification from 52 serum samples from hematology or oncology patients with clinically proven or suspected systemic Candida infections. All PCR-positive samples showed approximately the same Candida species load by both procedures (100% correspondence), whereas one discordant result was obtained between PCR and blood culture.

Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay.

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.