Negative PCR Results Research Articles

Diagnostische Verfahren bei der Hepatitis C und ihre Zuverlässigkeit. Reliability of diagnostic procedures for hepatitis C

Abstract The diagnosis of HCV infection is often hampered by false positive reactivities in antibody screening assays. Therefore, confirmatory assays are necessary. Since most unspecific reactivities are only slightly above the cut-off value, the intensity of the reactivity should always be considered. Particularly in groups with low HCV risk such as blood donors, positive reactivities obtained by one test system can often be ruled out by re-testing with another format (e.g. an enzyme immunoassay instead of a microparticle enzyme immunoassay). Most confirmatory assays are based on a recombinant immunoblot assay (RIBA); these tests should always be done in patients diagnosed as HCV positive for the first time. Additionally, in many cases, tests for HCV RNA are necessary. Based on these results, an evaluation of the risk of transmission to household contacts is possible and in patients receiving antiviral treatment, the success can be monitored. A negative PCR result in patients showing high positive antibody reactivities does not necessarily represent loss of the virus since even in chronic carriers, viral replication can be intermittently very low. Immuno-compromised patients often show prolonged seroconversion so that antibody screening assays remain negative for a long time while the patients have high level viraemia. Therefore, we recommend regular RT-PCR in populations at risk such as patients on chronic dialysis. To evaluate patients for antiviral treatment, the determination of the genotype is necessary. The HCV genotype also has an influence on the course of liver disease in chronic carriers. Possible transmission of HCV, e.g. by blood products, can be elucidated by sequence analysis of the high variance region (HVR) of the virus.

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.

Accuracy of HCV-RNA PCR tests for diagnosis or exclusion of vertically acquired HCV infection

The aim of the study was to estimate the sensitivity, specificity, positive (PPV) and negative (NPV) predictive values, and likelihood ratios for HCV-RNA PCR tests for the early diagnosis or exclusion of HCV infection in vertically exposed children. Data were included for children with confirmed HCV infection status from a European multi-center study. Confirmation was dependent on antibody status at or beyond 18 months, the 'gold standard' measure of infection status against which the use of qualitative HCV-RNA PCR tests was assessed. Of the 547 children included in this analysis, 193 were HCV-infected and 354 were not. Sensitivity of the HCV-RNA PCR test was low at birth (22%), but increased to 85% by 6 months. Specificity of RNA PCR was constant over age at 98%. The PPV of the PCR test rose from 33% at birth to 78% at 9 months of age, while NPV ranged from 96% to 99%. The high positive likelihood ratios from 1 month of age indicate strong evidence to diagnose infection but the negative likelihood ratios were consistent with weak evidence to exclude infection. The results suggest that the first qualitative HCV-RNA PCR test should be delayed until after the first month of life given the low sensitivity in the first few weeks. Although a negative test result after this time indicates probable absence of infection, this should be confirmed with a negative anti-HCV antibody test between 9 and 15 months of age as negative PCR results can be observed in infected children with fluctuations in viremia.

Frequency of Molecular and PET/CT Complete Remissions in Patients with Multiple Myeloma after Autologous Followed by Reduced-Intensity Allogeneic Stem Cell Transplants.

Despite recent advances multiple myeloma (MM) remains an incurable hemat</INS>ologic malignancy with a median survival of about 4 years. Autologous stem cell transplant (SCT) followed by reduced-intensity allogeneic SCT (tandem auto-allo) is a promising new approach designed to combine maximal tumor reduction and graft-versus-myeloma effect. The definition of complete response (CR) after tandem auto-allo is based on conventional serologic, radiologic, and pathologic parameters (Blade criteria). Because more sensitive molecular and radionuclear techniques are available, we evaluated the predictive value of these techniques after tandem auto-allo. Our hypothesis is that minimal residual disease (MRD) after tandem auto-allo likely predicts relapse and that, if identified early, patients with MRD may be candidates for novel salvage therapies. We employed patient-specific clonal Ig gene polymerase chain reactions using marrow and blood cells, and whole-body positron-emission tomography/CT scans (PET/CT) to assess patients after tandem auto-allo. PCR detects and estimates molecular MRD. PET/CT may detect occult medullary and extramedullary (soft tissue) disease. In patients achieving CR by the Blade criteria, we determined the frequency with which PCR and PET/CT findings were concordant. We have treated 10 patients with MM, 8 men and 2 women, with tandem auto-allo. Median age was 50.5 years (range, 37–58). There was no peri-transplant mortality, but 3 patients have died of progressive disease at 278, 398, and 402 days post-allo. Seven patients are alive at a median of 337 days (101–1187) post-allo, one with persistent myeloma and one awaiting evaluation. The remaining 5 patients are in CR by standard criteria (immunofixation negative). Their current status is shown below.Age/GenderStatus, days post-alloPCR (marrow)PET/CT55MCR, d 1003Neg on d 201, 924Neg d 98337WCR, d 632Pos on d 452Neg d 19245MCR, d 337Neg on d 88, 200Equivocal d 22239MCR, d 319Pos on d 93, 177Pos d 29051MCR, d 172Neg on d 104Neg d 94In sum, 2/5 have negative PCR and PET/CT findings; 1/5 has negative PCR and equivocal PET/CT findings; and 2/5 have positive PCR, one with positive PET/CT findings. Of note, all had negative PCR results using peripheral blood cells. The equivocal PET/CT showed low-level uptake in areas of prior myeloma in the pelvis. The positive PET/CT showed increased uptake in cervical and portacaval lymph nodes, but whether these findings represent occult myeloma is unclear. Overall, based on intention-to-treat in this small series, the frequency of CR with concordant PCR and PET findings is in the 20% to 30% range; and the frequency with which patients may become candidates for novel early salvage regimens is similar. A systematic approach using these methods after tandem auto-allo clearly merits further investigation.

Long-Term Monitoring of Cancer-Free Subjects Carrying Non-Lymphoma Associated bcl2/IgH Rearrangements (NLABR): Prolonged Persistence of Clonal Populations Potentially Related to Follicular Lymphoma (FL).

Introduction: NLABRs are frequently observed in cancer free-subjects. We recently observed that NLABR-positive clones can persist up to 60 days (Ladetto et al, J Clin Oncol 2003). However the long-term kinetics and potential pre-neoplastic role of NLABR-carrying cells are unknown. To define the natural history of NLABR-positive clones, long term monitoring of cancer-free subjects carrying these lesions has been performed.Methods: 118 subjects undergoing periodical blood examinations for warfarin therapy were screened for the bcl-2/IgH translocation. PCR-positive subjects underwent subsequent monitoring at least once every three months. NLABR-positive clones were monitored using both nested and real time-PCR according to previously published approaches (Ladetto et al Exp Hematol 2001). Sequence homology of NLABRs has always been confirmed by direct sequencing of nested PCR products.Results: 15 NLABR-positive subjects were identified out of 118 (12.7%) subjects. NLABR-positive subjects were monitored for a median time of 13 months (mos) (range 3–30 mos) for a total number of 60 timepoints. In eight subjects (53%), NLABRs detected at study initiation were not detected again in follow-up samples. These eight subjects have been monitored for median period of 12 mos (range 3–28 mos). Follow-up samples in this group were usually PCR-negative, although transient PCR-positivity due to unrelated NLABRs were noticed in two samples. In seven subjects (47%), the same NLABR observed at study initiation was detected one or more times at follow-up. In four subjects, NLABRs detected at diagnosis were amplified in every available follow-up sample (three to seven samples were available for each subject). In three, NLABRs detected at diagnosis were amplified only in a fraction of follow-up samples while the remaining were PCR-negative. Overall, persistent NLABRs were followed on these subjects for a median time of 15 months (range 3–30). The median burden of persistent NLABRs assessed by real-time PCR was 33 rearrangements (rg)/106 diploid genomes (dg) (range <10–760), while the median burden of short-lived NLABRs was <10rg/106 dg (range <10–330). The number of NLABR-positive cells appeared to be rather stable in subjects with persistent NLABR-positive clones. In none of these subjects we could detect differences greater than 1 log among available follow-up samples. Subjects having mixed PCR-positive and PCR-negative results had a smaller tumor burden compared to those constantly PCR-positive. This is consistent with the presence of a small though persistent clonal population. Studies on selected populations showed that NLABR-positive cells were CD19-positive.Discussion: NLABR-positive clones are long-lived cell populations in approximately 50% of cases. Based on this finding it is reasonable to hypothesize the existence of a follicular lymphoma (FL)-related lymphoproliferation of undetermined significance. Since NLABRs occurs in more than than 10% of healthy subjects, this condition is expected to be highly prevalent in the general population (as observed in MGUS and CLUS) and of potential relevance for the pathogenesis of FL.

EVALUATION OF A PRE-EMPTIVE GANCICLOVIR THERAPY BASED ON THE USE OF CMV PP67 NASBA IN HIGH-RISK (R-/D+) PEDIATRIC KIDNEY TRANSPLANTED PATIENTS FOR THE PREVENTION OF CYTOMEGALOVIRUS DISEASE.

P814 Aims: Despite considerable progress in laboratory diagnosis of cytomegalovirus (CMV) infection and despite more active anti-viral agents, CMV infection remains an important source of morbidity, especially in high-risk CMV seronegative recipients (R-) of CMV seropositive organs (D+). Because of the high incidence of R- recipients in the pediatric population and the increasing rate of D+ donors, prophylactic ganciclovir administration is often advocated. We elected to use a different strategy: prophylactic administration of intravenous CMV immune globulin (CMV-IG) for high-risk (R-/D+) recipients over the first four months and pre-emptive use of intravenous ganciclovir therapy, guided by monitoring CMV pp67 mRNA in whole blood by the qualitative nucleic acid sequence-based amplification assay (NASBA). We would like to report our initial results. Methods: A retrospective study was conducted in 20 consecutive children (9 boys, 11 girls, aged 14.8 ± 2.6 years) who underwent successful renal transplantation (graft survival ≥ 6months) between April 2000 to June 2003. PCR NASBA monitoring was performed at the time of transplantatation, then every week until day 90, and bi-monthly during the second trimester. Intravenous ganciclovir (5 mg/kg twice a day) was initiated on first positive NASBA results and stopped after two consecutive weekly negative PCR results. CMV disease was defined according to standard criteria. Results: In children R-/D-, the NASBA assay was negative in all (0/9). It became positive post transplantation in 72% (8/11) R-/D+ children. The first positive PCR NASBA occurred between 25-115 days (mean: 56 days) post transplant. Two of the 11 (18%) high-risk children developed CMV disease (pneumonia in one child and thrombocytopenia in the other), simultaneously with the first positive PCR, as PCRs were negative one week before the onset of CMV disease. Of note, these two children were the only high-risk subjects who did not receive the complete CMV- IG treatment, because of infusion related side effects. In addition, one of them had been treated for acute rejection 15 days prior to CMV infection. Intravenous ganciclovir was administered for a mean duration of 20 days. Four patients experiencing CMV infection relapses, detected by the NASBA assay, received another course of antiviral therapy. Conclusion: Although CMV seroconversion in R-/D+ is common (72%) after renal transplantation, CMV disease is not (18%) when pre-emptive ganciclovir is used. PCR NASBA monitoring failed to predict the development of CMV disease in 2/11 D+/R- patients. Pp67 NASBA-based pre-emptive ganciclovir therapy, combined with prophylactic administration of CMV-IG in high-risk patients leads to a low rate of CMV disease, when CMV-IG prophylaxis treatment is completed. These results could be optimized by the addition of intravenous ganciclovir, as a targeted prophylaxis strategy, during the treatment of acute rejection in high-risk populations.

Rapid molecular typing of Tuber melanosporum, T. brumale and T. indicum from tree seedlings and canned truffles.

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115 degrees C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of "truffle or truffled" French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.

The use of real-time PCR in the diagnosis and monitoring of Mycoplasma haemofelis copy number in a naturally infected cat

A 5-year-old male neutered cat was diagnosed with severe anaemia due to acute Mycoplasma haemofelis infection. Inflammatory respiratory disease was present concurrently. The cat was treated successfully using a fresh transfusion of whole blood and a 6 week course of doxycycline. The patient made a rapid recovery although the allergic airway disease subsequently required specific therapy consisting of inhaled fluticasone and salbutamol. Real-time quantitative PCR assays confirmed the presence of M. haemofelis DNA copies in the blood at presentation. Repeat PCR assays showed a reduction in copy number during treatment and negative PCR results were obtained both 91 and 425 days after presentation. The report describes, for the first time, the use of real-time PCR in the diagnosis and monitoring of natural M. haemofelis copy number, as well as the induction of long-term negative PCR status.

Parameters affecting positive viral load after sperm wash on HIV seropositive patients

Introduction: Sperm wash followed by nested-PCR is the unique ART procedure that avoids the transmission of HIV in serodiscordant couples wishing to conceive children when the male partner is infected. Unfortunately, viral particles are not always completely eliminated from the semen after the wash, thus forcing to repeat the procedure until a negative PCR result is achieved. Our aim was to determine which male clinical and sperm parameters are related with the presence of HIV in washed sperm.

Diagnostic anténatal de la toxoplasmose

Polymerase chain reaction analysis applied in amniotic fluid has represented a major breakthrough for a more accurate, safe, simple and rapid result of prenatal diagnosis of congenital toxoplasmosis. However, given the limited but significant risk of fœtal loss associated with amniocentesis, such indication should be restricted to documented or highly suspected cases of maternal infections as assessed by appropriate serological methods and screening strategies during pregnancy. Knowledge of risks of transmission and infectious sequelae according to gestational age at maternal infection is also required for decisions of prenatal diagnosis as well as interpretation of results of PCR in amniotic fluid. In all cases of a negative PCR result, only a serological post-natal follow-up can rule out congenital infection.

DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes

This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose gel. DNA losses during glassmilk treatment can generate negative PCR results.

Skeletal Remains Presumed Submerged in Water for Three Years Identified Using PCR-STR Analysis

We describe the successful identification of the remains of a saponified body found in a dam by typing of nuclear DNA. Whereas DNA extracted from soft tissues yielded negative PCR results, DNA extracted from the bone by a slightly modified Qiagen procedure allowed the typing of sex (AMG locus) and of 10 additional STR loci. An identity document was found belonging to a man missing for 3 years and comparison of the results to the DNA profiles of his son and wife confirmed the identity. The longest delay reported until now for successful nuclear DNA genotyping after immersion in river water was 18 months. This case demonstrates a delay of up to 3 years.

Development of a sandwich ELISA and comparison with PCR for the detection of F11 and F165 fimbriated Escherichia coli isolates from septicaemic disease in farm animals

The P fimbriae F11 and F165 that have been demonstrated on Escherichia coli septicaemic strains in poultry and calves, respectively, possess a nearly identical major subunit that demonstrates a serological cross-reaction. A polyclonal antibody-based sandwich ELISA (sELISA) that was specific for both F11 and F165 fimbriated strains was compared with a PCR method to detect F11/F165 fimbriated strains, in a collection of E. coli strains isolated from diseased animals. Of 298 isolates tested, 36 were positive by PCR of which only 14 were sELISA positive. There were no sELISA positive but PCR negative results. The 36 PCR positive isolates comprised 11 avian strains of which 10 were sELISA positive, 20 bovine strains of which 4 were sELISA positive and 3 ovine strains, 1 porcine strain and 1 equine strain all of which were sELISA negative. The F11/F165 incidence of 10.7% in 103 poultry and 18.3% in 109 bovine isolates demonstrates a moderate level of these factors in E. coli septicaemic cases in Northern Ireland.

Screening for possible failure of herpes simplex virus PCR in cerebrospinal fluid for the diagnosis of herpes simplex encephalitis.

The objectives of this study were to evaluate the reliability of herpes simplex virus (HSV) PCR testing in cerebrospinal fluid (CSF) for the detection of herpes simplex encephalitis. This was done by examining retrospectively the clinical follow-up of a large group of patients tested routinely by HSV-PCR. In addition, an attempt was made to assess the incidence of herpes simplex encephalitis in a central European population. CSF samples from 1,427 patients from all Vienna hospitals were submitted for HSV-PCR testing during a period of 4 years and 8 months. Herpes simplex encephalitis was detected by PCR in 12 cases and by serological methods in one additional patient. Retrospective analysis of the course of disease, which was possible in 799 PCR-negative patients, led to the identification of three additional cases in which herpes simplex encephalitis appears to have occurred despite negative PCR results. Failure of the PCR in these patients is most likely due to the time of obtaining CSF during the course of disease. A high specificity of the assay was demonstrated by the lack of false positive results in any of the 708 cases in which other causes for the neurological symptoms had been identified in the follow-up. The incidence of herpes simplex encephalitis in the population of Vienna was between 1 case/469,000-577,000 individuals/year. The highest annual incidence was detected in the age group between 3 months and 3 years, which, however, could not be confirmed statistically.

Timing and interpretation of tests for diagnosing perinatally acquired hepatitis C virus infection.

The diagnosis of hepatitis C virus (HCV) infection in children born to HCV-infected women is based on serologic assays and HCV RNA measurement by PCR. Interpretation of the results of these tests is hampered by uncertainty about the age distribution of loss of maternal antibody and the sensitivity and specificity of PCR at different ages. On the basis of findings from a recent vertical transmission study, we estimated the posttest probability of a child's being infected or uninfected under several test result scenarios. These estimates may assist clinicians in assessing the likelihood of infection in an individual child and in using the currently available assays cost effectively.

A link between chronic asthma and chronic infection

Background: Asthma is a prevalent disease with marked effects on quality of life and economic societal burden. However, the cause of asthma and its pathophysiology are not completely defined. Recently, the possibility that chronic infection may play a role has been suggested. Objective: We sought to define the association between Mycoplasma and Chlamydia species and chronic asthma. Methods: We performed a comparison study of asthmatic patients and normal control subjects. Fifty-five patients with chronic stable asthma were compared with 11 normal control subjects by using PCR, culture, and serology for Mycoplasma species, Chlamydia species, and viruses from the nasopharynx, lung, and blood. Bronchoalveolar lavage cell count and differential, as well as tissue morphometry, were also evaluated. Computer-generated scoring for the degree of chronic sinusitis in asthmatic patients was additionally evaluated. Results: Thirty-one of 55 asthmatic patients had positive PCR results for Mycoplasma (n = 25) or Chlamydia species (n = 6), which were mainly found on lung biopsy specimens or in lavage fluid. Only 1 of 11 normal control subjects had positive PCR results for Mycoplasma species. The distinguishing phenotype between asthmatic patients with positive and negative PCR results was the significantly greater number of tissue mast cells in the group with positive results. Conclusion: A significant number of patients with chronic stable asthma demonstrate the presence of Mycoplasma species, Chlamydia species, or both in their airways, with the distinguishing feature of increased mast cell number. These findings need further delineation but may help us to understand the pathophysiology of asthma and new treatment options. (J Allergy Clin Immunol 2001;107:595-601.)

CD68+/CD83+/CD1a- dendritic cell subsets from patients with multiple myeloma are not infected with human herpesvirus 8.

Recently, a subset of dendritic cells with the phenotype CD68+/CD83+/CD1a-, present in patients with multiple myeloma (MM), was reported to be infected with human herpesvirus 8 (HHV-8). Therefore we wished to clarify whether HHV-8 infection might be related to the pathogenesis of MM. In an attempt to identify HHV-8 infected cells in patients with MM, long-term bone marrow cultures from 8 MM patients and dendritic cell cultures from 11 MM patients were established. In addition, fresh bone marrow aspirates from 10 MM and 10 patients with monoclonal gammopathy of undetermined significance (MGUS) were included in the study. All samples were analysed by a sensitive semi-nested PCR assay and were found to be consistently PCR negative. Phenotyping of day 7 dendritic cell cultures demonstrated the presence of a sufficient number of CD68+/CD1a- and CD83+/CD1a- cells. However, to exclude the presence of infrequent HHV-8 infected cells, the CD68+/CD1a- subset from 3 dendritic cell cultures was sorted in numbers of 105 for each PCR test, and again a negative PCR result was observed. This study documents that the CD68+/CD83+/CD1a- dendritic cells in patients with MM are not generally infected with HHV-8 and, as a consequence, it is unlikely that HHV-8 plays a role in the pathogenesis of MM.

Evaluation of minimal residual disease using reverse-transcription polymerase chain reaction in t(8;21) acute myeloid leukemia: a multicenter study of 51 patients.

Most studies using various reverse-transcription polymerase chain reaction (RT-PCR) techniques reported that the detection of the AML1-ETO fusion transcript was a common finding in long-term complete remission (CR) in acute myeloid leukemia (AML) with t(8;21) translocation. However, larger prospective studies with interlaboratory quality control may be important to investigate more precisely the clinical usefulness of studying minimal residual disease with RT-PCR in t(8;21) AML. We collected 223 marrow samples from 51 patients with t(8;21) AML diagnosed in five centers and tested all samples by two different RT-PCR techniques (a nested technique and a one-step technique, with a sensitivity of 10(-6) and 10(-5), respectively) in two different laboratories. Samples from 14 patients in long persistent CR (median follow-up duration, 112 months) were taken at least twice, and all were PCR-negative by both techniques. Samples were prospectively taken from 37 patients after achievement of first CR and/or second CR, before intensive consolidation treatment, and every 3 to 6 months after completion of therapy. Patients who converted to PCR negativity with the one-step technique (60%) or both techniques (48%) after CR achievement had a longer CR duration than those with persistently positive PCR results (two-sided log-rank test, P =.0001). Patients who became PCR-negative with the one-step technique before intensive consolidation (23%) had a lower relapse rate (11% v 72%) and a longer CR duration than those who remained persistently PCR-positive at that point (two-sided log-rank test, P =.0015). Patients with AML with t(8;21) in long-term remission were all PCR-negative. In prospectively studied patients, a good correlation was found between negative PCR results and absence of relapse. Early negative results with the one-step RT-PCR technique, before consolidation treatment, seemed to carry an especially good prognosis, suggesting that RT-PCR analysis could help in choosing the type of consolidation therapy in patients with t(8;21) AML.

Intérêt de la biologie moléculaire dans le diagnostic des infections vlrales du système nerveux central

The use of PCR and hybridization for the detection of viral nucleic acids in cerebrospinal fluid (CSF) currently represents the most reliable method for the meningoencephalitis diagnosis due to herpes viruses, enteroviruses, JC polyomavirus and HIV-1 primary infection. The molecular tools are less suitable for the postinfectious encephalitis associated with a systemic viral infection such as neurologic complications of measles, rubella, influenza and varicella, The speed and high sensitivity allow earlier diagnosis and specific treatment. The monitoring of the antiviral therapy is assessed by the development of quantitative amplification assays in CSF or by a negative PCR result. The pattern mutations conferring resistance and the genetic variability of viruses (HIV-1, Dengue virus) could be determined from different locations in the genome. Theses methods performed over the past several years as research tools have now a large scale application with the commercialization of some tests. But before becoming the first line diagnostic test, PCR of CSF could be conducted with control quality and evaluated protocols for avoiding false positive and negative results.

Panfungal PCR assay for detection of fungal infection in human blood specimens.

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient's blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.