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PubMed2005

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR.

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.

Adenoviruses, Human↗
PubMed2003

Quantification of CCR5 mRNA in human lymphocytes and macrophages by real-time reverse transcriptase PCR assay.

CCR5, a beta-chemokine receptor, plays an important role in human immunodeficiency virus (HIV) infection of human immune cells, as it is a primary coreceptor for HIV entry into macrophages. We have applied a newly developed real-time reverse transcriptase PCR (RT-PCR) assay for the quantification of CCR5 mRNA in human blood immune cells. The CCR5 real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 10(2) and 10(6) copies of the CCR5 mRNA transcripts per reaction. The assay is highly reproducible, with an intra-assay coefficient of variation of the threshold cycle of less than 2.07%. When used for quantification of CCR5 mRNA in human monocyte-derived macrophages (MDM) and peripheral blood lymphocytes (PBL), the assay has precision and reproducibility. MDM expressed higher levels of CCR5 mRNA than did PBL. Thus, this assay has the potential and a wide application for the investigation of the role of CCR5 in inflammatory diseases and viral infections, including HIV disease.

Blood Cells↗
PubMed1998

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 +/- 1 degree C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.

Agar↗
PubMed2002

Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

Adult↗
PubMed2006

Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G.

The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

DNA, Viral↗
PubMed2006

A genotype-independent real-time PCR assay for quantification of hepatitis B virus DNA.

Accurate quantification of hepatitis B virus (HBV) DNA levels is important for monitoring patients with chronic HBV infection and for assessing their responses to antiviral therapy. This study aimed to develop a real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the known HBV genotypes. An "in-house" real-time PCR assay using primers and a TaqMan probe in a highly conserved region of the HBV surface gene was designed. The assay was standardized against a WHO standard and validated against plasmids of HBV genotypes A through H. The linear quantification range was approximately 5 x 10(0) to 2.0 x 10(9) IU/ml. Results of samples from patients infected with HBV genotypes A through H tested using our real-time "in-house" PCR assay showed an excellent correlation with those of the Cobas Amplicor HBV Monitor (R2=0.9435) and the Cobas TaqMan HBV (R2=0.9873) tests. We have established a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA levels. Further studies of additional samples are ongoing to validate the genotype independence of our assay.

DNA Primers↗
PubMed1999

Reproducibility and performance of the second-generation branched-DNA assay in routine quantification of human immunodeficiency virus type 1 RNA in plasma.

We examined the reproducibility of a second-generation branched-DNA (bDNA) assay (Quantiplex HIV RNA 2.0) for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma by retesting 325 specimens on separate runs and on different lots. The performance of the bDNA test was also assessed by data analysis obtained during routine testing of 15,365 specimens. Upon retesting, 96 and 86% of specimens displaying RNA levels above 5,000 and between 500 and 5, 000 copies/ml, respectively, showed less than a 0.3 log10 (twofold) difference with their initial values. Assay variability was found to increase as viral load decreased. Overall, the bDNA version 2.0 assay was found to be a reproducible and efficient test for routine quantification of HIV-1 RNA in plasma.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Effect of multiple freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology.

Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (P < 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable.

DNA, Viral↗
PubMed2000

Development of a TT virus DNA quantification system using real-time detection PCR.

Although TT virus (TTV) was isolated from a cryptogenic posttransfusion hepatitis patient, its pathogenic role remains unclear. It has been reported that the majority of the healthy population is infected with TTV. To elucidate the differences between TTV infection in patients with liver diseases and TTV infection in the healthy population, a quantification system was developed. TTV DNA was quantified by a real-time detection PCR (RTD-PCR) assay on an ABI Prism 7700 sequence detector. With this system, TTV DNA was quantified in 78 hepatitis C virus (HCV)-infected patients (63 with elevated serum alanine aminotransferase [ALT] levels and 15 with normal ALT levels) and in 70 voluntary blood donors (BDs). The quantification range was 2.08 to 7.35 log copies/ml. The intra-assay and interassay coefficients of variation were 0.37 to 6.33% and 0.60 to 7.07%, respectively. The mean serum TTV DNA levels in the HCV-infected patients with both elevated and normal ALT levels and BDs were 3.69 +/- 0.89, 3.45 +/- 0.76, and 3.45 +/- 0.67 log copies/ml, respectively. Comparison of the serum TTV DNA levels among the HCV-infected patients revealed that they were not related to the serum ALT and HCV core protein levels or to the histopathological score on liver biopsy. This study showed that (i) the RTD-PCR assay for the detection of TTV was accurate and had a high degree of sensitivity, (ii) the mean serum TTV DNA level was similar among HCV-infected patients, irrespective of their ALT level, and also among BDs, and (iii) a high serum TTV DNA level does not affect the serum ALT and HCV levels or liver damage in HCV-infected patients.

Blood Donors↗
PubMed2000

Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system.

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans and A. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 10(1) and 10(4) Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR-enzyme-linked immunosorbent assay analysis.

Aspergillosis↗
PubMed2000

Quantitative competitive reverse transcription-PCR for quantification of dengue virus RNA.

A quantitative competitive reverse transcription-PCR assay was developed to quantify dengue virus RNA in this study. The main features include a primer pair targeting a highly conserved region in the capsid and the addition of competing RNA that contains an internal deletion to provide a stringent internal control for quantification. It can be utilized to quantify RNA isolated from the four dengue virus serotypes but not RNA isolated from other flaviviruses, including Japanese encephalitis virus and hepatitis C virus, both prevalent in Asia. It can also be used to quantify dengue virus RNA isolated from the plasma of infected individuals. The sensitivity of the assay was estimated to be 10 to 50 copies of RNA per reaction, and twofold differences in virus titer are distinguishable. This assay is a convenient, sensitive, and accurate method for quantification and can be used to further understanding of the pathogenesis of dengue virus infection.

Culture Media↗
PubMed2001

Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.

Adolescent↗
PubMed2001

New LightCycler PCR for rapid and sensitive quantification of parvovirus B19 DNA guides therapeutic decision-making in relapsing infections.

Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically >or=5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.

Child↗
PubMed2001

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry.

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

Animals↗
PubMed2002

Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR.

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R(2) = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

Animals↗
PubMed2003

Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients.

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

Aspergillosis↗
PubMed2003

Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer.

We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10(2) to 10(7) HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.

Cervix Uteri↗
PubMed2004

Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity.

A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 10(6) nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.

Animals↗