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Background: We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer., Methods and Results: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease., Conclusion: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

"High-risk" human papillomaviruses (HPVs) are associated with intraepithelial neoplasia and cancer of the uterine cervix. HPV has also been found in nonmelanoma skin cancer (NMSC), especially in squamous cell carcinomas (SCCs) of immunosuppressed patients. Recently, lesions of psoriasis have been shown to harbor HPV, and patients with psoriasis often have a history of extensive therapy with ultraviolet radiation (UVR). UVR is the major known risk factor in the occurrence of NMSC, in which HPV may be a cofactor for SCC. We report an otherwise healthy, nonimmunosuppressed patient with psoriasis who had a history of extensive exposure to UVR and experienced multiple SCCs on UV-exposed body sites. By the polymerase chain reaction method, we detected HPV in 5 of 9 SCCs. Automated sequencing showed HPV types 12 and 17. Only 1 of 3 normal skin specimens was HPV positive (HPV type 17). This positive specimen was from UV-exposed skin; one of the two HPV-negative, normal skin specimens was located on a body site not exposed to sun. In addition, HPV type 62 was found in a brush specimen of the uterine cervix. This case report suggests an association between psoriasis, HPV infection, and UVR exposure, in onset of SCC.

Bartonella henselae has been implicated as a significant cause of HIV-associated dementia. We attempted to confirm this association by utilizing the database of the San Diego HIV Neurobehavioral Research Center, which collects longitudinal neurocognitive and laboratory data on over 500 HIV-infected participants. Utilizing an immunofluorescent assay we found that 11% of 177 subjects, half of whom had documented neurocognitive decline, were seropositive for B. henselae. There was no correlation between B. henselae seropositivity and neurocognitive decline. The role of B. henselae in HIV-associated dementia remains ambiguous.

At Mayo Medical Center (Rochester, MN), surveillance rectal (and other-site) cultures have been routinely collected from liver transplant recipients as part of a selective bowel decontamination program. Beginning in 1995, vancomycin-resistant enterococcus (VRE) colonization and infection were identified in Mayo Clinic liver and kidney transplant patients through our surveillance cultures. The purpose of this study is to describe the natural history of VRE colonization in this patient population. Fifty-two patients with VRE colonization (predominantly with a single vanB clone) were identified from September 1995 through December 1997. Five hundred ninety cultures were reviewed for this study (mean, 11.3 cultures/patient). The median time from initial VRE colonization to the last surveillance culture obtained was 306 days (range, 1 to 1,393 days). VRE infection was documented in 6 patients (11.3%). Eighteen patients (35%) met the criteria for clearance of VRE colonization, defined as VRE-negative rectal culture results on at least 3 consecutive occasions greater than 1 week apart. However, VRE was detected on subsequent surveillance cultures from 2 of these patients (11% relapse rate). Of the remaining 34 patients, 16 remained colonized with VRE and 18 did not meet the definition for clearance of VRE colonization because of incomplete follow-up. This study documents that VRE colonization usually persists for months to years in liver and kidney transplant patients.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Objective: The E6 regions of the oncogenic human papillomaviruses (HPVs) are important in carcinogenesis and immune recognition. We examined the E6 DNA sequence from HPV-16-associated cervical cancers to determine the frequency and degree of variation from the consensus sequence in selected populations., Methods: Samples positive for HPV-16 were analyzed using polymerase chain reaction followed by automated DNA sequencing: 62 from U.S. women, 20 each from Italian and Indian women, and 21 from Thai women., Results: Of 151 codons, 18 contained 24 base substitutions, reflecting the overall conserved nature of this region. The HPV-16 E6 region from U. S. women showed considerably more sequence variation than that from European and Asian women. Five patterns common to U.S. and European and Asian samples accounted for 78% of all tumor-associated viruses. The E6 regions known to be involved in p53 binding and degradation are involved with a surprising degree of sequence variation, whereas the carboxy end of the molecule is highly conserved., Conclusions: The area of greatest sequence variation includes a proposed human leukocyte antigen interaction site. A novel large deletion in one sample results in loss of all functional regions of E6. These findings were analyzed for possible significance with regard to immune selection and functional importance of the carboxy end of the E6 protein., (Copyright 2000 Academic Press.)

Objective: Because many patients with chronic viral hepatitis do not progress to end-stage liver disease, it is possible that host factors such as human leukocyte antigen (HLA) differences are important. Our aims were to determine HLA marker-specific rates of progression to liver transplantation among patients with chronic hepatitis C; and to determine if polymerase chain reaction (PCR)-based HLA DRB1 typing can be performed on stored serum samples., Methods: Forty-two hepatitis C virus RNA-positive liver transplant patients and 87 untransplanted patients were included in a Cox proportional hazards model to test whether the occurrence of certain HLA DRB1 markers were associated with progression to liver transplantation. HLA DRB1 typing was performed on stored serum samples using a PCR method., Results: There were no differences among the HLA DRB1 markers with regard to the HLA marker-specific rate of progression to transplantation among patients with chronic hepatitis C., Conclusions: HLA DRB1 markers do not appear to be associated with progression of disease in chronic viral hepatitis C. It is possible to perform PCR-based HLA DRB1 typing on stored frozen serum samples.

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy (n = 18) or were treatment naive (n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

Warts can be difficult to diagnose and to treat in the setting of human immunodeficiency virus (HIV) infection. A 37-year-old woman with a background of HIV presented with a large verrucous plaque involving her right foot. Human papillomavirus (HPV)-66 was identified in the lesional skin biopsy sample and in scrapings obtained from her cervix. The wart rapidly responded to topical cidofovir therapy. HPV-66 is a novel HPV type to be associated with verruca vulgaris. Topical cidofovir should be further investigated as an alternative treatment modality for verruca vulgaris.

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.

Objective: Recently a novel DNA virus (TT virus) has been identified in Japan and shown to be associated with elevated aminotransferase levels after blood transfusion. The exact role of TTV in the pathogenesis of liver disease is yet to be established. Our aim was to determine the prevalence and role of TTV in the pathogenesis of elevated transaminases in healthy blood donors in the absence of markers for viral hepatitis A-C., Methods: Stored sera were collected from 99 healthy blood donors with elevated alanine amino transferase (ALT) values that were discovered at the time of blood donation. A total of 146 samples were obtained from healthy donors with normal ALT values who were used as controls. None of the patients or controls had a history of blood transfusion or had clinical signs of acute or chronic hepatitis. Serological markers for hepatitis A, hepatitis B, and hepatitis C viruses were negative. TTV DNA was amplified and detected using polymerase chain reaction followed by gel electrophoresis., Results: Five of 99 (5%) samples obtained from donors with elevated ALT had TTV DNA detected by PCR, as compared to one of 146 (0.7%) of those with normal ALT (p = 0.006). Among those with elevated ALT, mean ALT values in patients with TTV (296 +/- 305 U/L) were higher than in patients without TTV (95 +/- 37 U/L), but the difference was not statistically significant (p = 0.08). The two samples with highest ALT values (both >450 U/L) were among the five samples with detectable TTV DNA in serum., Conclusions: Although TTV is not likely to explain the majority of elevated ALT cases in otherwise healthy blood donors, TTV infection may potentially be associated with some cases. Based on these findings, we propose that the role of TTV in the pathogenesis of acute and chronic liver diseases merits further investigation.

After orthotopic liver transplantation (OLT), patients with chronic hepatitis C virus (HCV) infection show nearly universal persistence of viremia and reinfection of the liver, but identifying the point at which the liver is reinfected morphologically can be difficult. One tool that may potentially be useful to detect reinfection is reverse transcriptase-polymerase chain reaction (RT-PCR), which has proven to be highly sensitive for detecting HCV RNA in formalin-fixed paraffin-embedded liver tissue. Our purpose was to gain insight into the time frame of HCV reinfection by assaying for HCV RNA in serial posttransplant liver biopsy specimens. Our study population consisted of 14 patients who underwent liver transplantation for hepatitis C and had confirmed HCV RNA in pretransplant serum, absence of HCV RNA in donor livers, and available consecutive posttransplant liver allograft specimens. We performed RT-PCR for HCV RNA in serial posttransplant liver biopsy specimens, beginning at 1 week until at least one biopsy from each tested positive. HCV RNA was detected in liver tissue by RT-PCR in 1-week post-OLT liver samples in 6 of 14 (42.8%) patients, the earliest being 5 days post-OLT. Eventually, each of the remaining eight samples became RT-PCR positive as well; the first detections occurred in these at 3 weeks (three cases), 4 weeks (three cases), 48 weeks (one case), and 144 weeks (one case). Histologic identification of hepatitis C recurrence was relatively insensitive in relation to these molecular data. These data suggest that (1) HCV RNA reinfection is nearly universal after liver transplantation in patients with chronic hepatitis C infection, (2) molecular reinfection by HCV occurs at a variable interval post-OLT, with the majority of allograft livers reinfected as early as 1 week, and (3) morphologic features of hepatitis C are usually appreciable at the time of "molecular" recurrence.

A significant percentage of human cancers worldwide are associated with infections due to known viruses, including human papillomaviruses (cervical cancer and other skin cancers), human T-lymphotropic viruses (adult T-cell leukemias and lymphomas in endemic areas), hepatitis B virus (liver cancer), and Epstein-Barr virus (Burkitt lymphoma and nasopharyngeal carcinoma). The fraction of human cancers attributable to infection may now need to be revised in light of the fact that new viral associations have been discovered and other nonviral associations have been identified. This article addresses the increasingly recognized role of infectious agents as precipitants of human neoplasia and the possibility that novel diagnostic, therapeutic, and chemopreventive strategies may emanate directly from research directed at identifying and understanding these agents.

Polymerase chain reaction (PCR)-based testing of cerebrospinal fluid (CSF) specimens has become standard for confirmatory diagnosis of central nervous system (CNS) infections; however, these tests increase health care costs. We reviewed 3-year data from 974 consecutive CSF specimens submitted for detection of seven pathogens by PCR. In 1997, 237 of 367 specimens (64.6%) were submitted for multiple tests, compared with 203 of 522 (38.9%) in 1996 and 18 of 85 (21.2%) in 1995. In each year the arrival of new house officers coincided with a peak in multiple testing. Among 732 specimens submitted for herpesvirus detection, results were positive for 24 (4.6%) of 523 specimens with increased leukocyte counts or protein levels. None of 209 specimens with normal leukocyte and protein levels were positive for herpesviruses. None of 471 CSF specimens submitted for Borrelia burgdorferi detection were PCR-positive. Use of protein and leukocytes to screen CSF specimens before employing PCR for herpesvirus detection would save almost one-third of costs without reducing sensitivity.

Objective: The TT virus (TTV) is a novel DNA virus that has recently been identified. The clinical significance of TTV infection in patients with chronic hepatitis C has not been determined. The aim of this study was to determine the prevalence and possible role of TTV in a well characterized population with chronic hepatitis C infection., Methods: Ninety patients with chronic HCV and known time of HCV acquisition were selected from approximately 250 patients followed at our institution. Characteristics including age, sex, histology, and length of disease were recorded. Direct sequencing of the NS5 region was used for HCV genotyping. TTV DNA detection was based on PCR., Results: TTV infection was present in 24 of 90 (27%) HCV patients. Patients were divided into four groups based on stage of disease; chronic hepatitis (CH, 29 patients), compensated cirrhosis (CC, 17 patients), decompensated cirrhosis (DC, 28 patients), and hepatocellular carcinoma (HCC, 16 patients). TTV was present in 2/29 (7%), 2/17 (12%), 11/28 (39%), and 9/16 (56%) in those with CAH, CC, DC, and HCC respectively. TTV was significantly more prevalent among those with advanced disease (DC and HCC) compared to those with stable disease (CH and CC; p = 0.001). Mean age, sex, and the time from exposure to HCV to development of complications were similar in TTV-positive and -negative patients. TTV infection was more common in patients infected with HCV genotype 1b. Univariate analysis showed that length of HCV infection, HCV genotype 1b, and TTV infection were important in predicting the stage of liver disease in HCV patients. However, after adjusting for length of HCV infection, TTV but not HCV genotype was important in predicting the stage of liver disease., Conclusions: We conclude that 1) TTV infection is common in patients with chronic HCV; 2) TTV infection is more prevalent among patients with advanced HCV-associated liver disease (DC and HCC) than in those with stable disease (CH and CC); and 3) TTV infection is more common in patients with HCV genotype 1b but is independent from genotype in predicting the stage of HCV-associated liver disease.

Consistent with other members of the family Flaviviridae, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5' untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5'UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5'UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2. 0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5'UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5'UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.

Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.

We describe aortic valve endocarditis caused by Bartonella quintana in a 31-year-old man. The diagnosis was made on the basis of polymerase chain reaction amplification of the B quintana citrate synthase gene from cardiac valve tissue, the compatibility of histochemical stains of cardiac valve tissue, and serologic studies.

Conventional methods for the identification and characterization of clinical isolates of bacterial pathogens sometimes fall short when such isolates exhibit unusual phenotypic profiles. Recent advances in DNA sequencing technology have greatly enhanced the ability of the microbiologist to determine the identity of a bacterial isolate. Given the relative objectivity of DNA sequence information and growing availability of sequence information databases, a significant movement is now afoot to use molecular methods for the identification of clinical pathogens.

Background: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV., Methods: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells., Results: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells., Conclusion: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

Objective: To correlate viral genotypes with the immune manifestations of chronic hepatitis C and evaluate the effect of immune features on disease expression and response to antiviral treatment., Design: We undertook a retrospective analysis of 67 patients with chronic hepatitis C., Material and Methods: Patients were selected for study if they had been screened for autoantibodies and concurrent immune diseases and if viral genotyping had been performed or was possible. Concurrent immune manifestations and responses to interferon therapy were determined., Results: Of the 67 patients, 18 (27%) had one or more immune features. Immune manifestations occurred as commonly in patients with genotype 1 as in those with other genotypes (30% versus 14%; P = 0.3). Concurrent immune features did not distinguish patients, and responses to interferon therapy were similar between patients with and those without immune manifestations. None of the 14 patients with concurrent immune diseases or high-titer autoantibodies (serum titers, 1:320 or more) entered remission during interferon treatment. In contrast, 6 of 53 patients without concurrent immune diseases and no or low-titer autoantibodies had treatment-related remission. These differences, however, were not statistically significant (0% versus 11%; P = 0.3)., Conclusion: Autoantibodies and concurrent immune diseases are not associated with a particular viral genotype, clinical profile, or treatment outcome. Larger studies are necessary for complete assessment of the influence of prominent immune manifestations on treatment response.

Blood smear evaluation of two baboons (Papio cynocephalus) experiencing acute hemolytic crises following experimental stem cell transplantation revealed numerous intraerythrocytic organisms typical of the genus Babesia. Both animals had received whole-blood transfusions from two baboon donors, one of which was subsequently found to display rare trophozoites of Entopolypoides macaci. An investigation was then undertaken to determine the prevalence of hematozoa in baboons held in our primate colony and to determine the relationship, if any, between the involved species. Analysis of thick and thin blood films from 65 healthy baboons (23 originating from our breeding facility, 26 originating from an out-of-state breeding facility, and 16 imported from Africa) for hematozoa revealed rare E. macaci parasites in 31%, with respective prevalences of 39, 35, and 12%. Phylogenetic analysis of nuclear small-subunit rRNA gene sequences amplified from peripheral blood of a baboon chronically infected with E. macaci demonstrated this parasite to be most closely related to Babesia microti (97.9% sequence similarity); sera from infected animals did not react in indirect fluorescent-antibody tests with Babesia microti antigen, however, suggesting that they represent different species. These results support an emerging view that the genus Entopolypoides Mayer 1933 is synonymous with that of the genus Babesia Starcovici 1893 and that the morphological variation noted among intracellular forms is a function of alteration in host immune status. The presence of an underrecognized, but highly enzootic, Babesia sp. in baboons may result in substantial, unanticipated impact on research programs. The similarity of this parasite to the known human pathogen B. microti may also pose risks to humans undergoing xenotransplantation, mandating effective screening of donor animals.

Chronic infection with the hepatitis C virus (HCV) is the most common reason for liver transplantation. We examined the results of laboratory tests for HCV on a cohort of patients who received a liver transplant between 1990 and 1994 at three large centers. Seven hundred twenty-two recipients and 604 donors were tested for antibody to HCV (anti-HCV) using a second-generation enzyme-linked immunoassay (EIA-2), followed by recombinant immunoblot (RIBA-2) and HCV RNA confirmation by reverse-transcription polymerase chain reaction (RT-PCR) (with genotyping and viral quantification). Diagnosis of posttransplantation infection required detection of serum HCV RNA that could be genotyped by sequencing or was repeatedly positive despite being unsequenceable. Twenty-five percent of transplantation candidates were seropositive for anti-HCV. Approximately 86% of anti-HCV-positive, 93% of RIBA-positive, and 97% of HCV RNA-positive candidates developed infection after transplantation. Pretransplantation HCV RNA was superior to RIBA-2 for predicting posttransplantation infection. Whereas HCV genotype was identified in nearly all candidates and changed little after transplantation, serum viral levels rose markedly after transplantation. Fifteen donors were either anti-HCV- or HCV RNA-positive. Recipients of grafts from donors with HCV RNA all developed infection, whereas infection was not detected in recipients of grafts from donors with anti-HCV but without detectable HCV RNA. The rate of new infection fell significantly (P =.02) after the introduction of EIA-2 screening of blood. Donor and candidate markers for HCV predict posttransplantation infection.

We describe 2 patients presenting with polyarthritis in whom the synovial fluid (1 patient) or synovial tissue (1 patient) was positive for Tropheryma whippelii, the Whipple's disease-associated bacillus, when examined by polymerase chain reaction (PCR) and DNA sequencing. Histopathologic findings were consistent with articular Whipple's disease in the synovial fluid of 1 patient and the synovial tissue of the other. In both patients, bowel mucosal specimens were negative for Whipple's disease features by histologic and PCR methods. One patient was positive for T whippelii in the peripheral blood. Control synovial fluid specimens from 40 patients with other arthritides, including Lyme arthritis, were negative. Sequencing of a 284-basepair region of the 16S ribosomal RNA gene confirmed that the sequence is closely related to the known T whippelii sequence. Both patients responded to treatment with antibiotics.

Human cases of infection with a granulocytotropic Ehrlichia species closely related to Ehrlichia equi are now being described with increasing frequency in the United States, especially in areas where Lyme disease is already endemic. We describe a case of fatal pancarditis during the course of human granulocytic ehrlichiosis (HGE) in a 44-year-old outdoor worker who was previously treated for presumptive Lyme disease. Serological and molecular diagnostic tests for Borrelia burgdorferi and Babesia microti infections were negative. Postmortem serum specimens were seroreactive for HGE, and molecular evidence of infection with the HGE agent was obtained. These findings suggest that carditis may be a manifestation of HGE, further complicating the differential diagnosis of tick-borne illness.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.

Currently, one of the major indications for liver transplantation is infection with hepatitis C virus (HCV). Many studies have suggested that recurrent infection with HCV is universal after transplantation. Fastidious techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), have proved to be highly sensitive for detecting HCV RNA in serum and in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) liver tissue. In this study, we wanted to determine whether the identification of HCV RNA in liver tissue by RT-PCR might reflect the detection of circulating HCV RNA in blood within the tissue, rather than implying true tissue infection. We performed RT-PCR for HCV RNA in FFPE liver biopsy specimens taken from 14 donor allografts shortly before and immediately after implantation into recipients. The recipients were known to have HCV RNA in serum and explanted liver tissue, as determined by RT-PCR. We were unable to detect HCV RNA in any of the study samples, either before or after transplantation. In a related study, qualitative and quantitative HCV RNA analyses were performed by RT-PCR and branched DNA (bDNA) amplification, respectively, on serum samples collected pretransplantation and immediately posttransplantation from 10 other patients who underwent transplantation for hepatitis C. HCV RNA was detected in all serum samples before and after transplantation by RT-PCR; however, the bDNA assay detected HCV RNA in only 6 of 10 samples pre-orthotopic liver transplantation (OLT) and in none of the immediately post-OLT samples. In our system, despite the RT-PCR detection of HCV RNA in serum before and after the transplantation, HCV RNA is not detectable in the peripheral blood that accompanies formalin-fixed liver tissue. This implies that RT-PCR detection of HCV RNA in tissue reflects true liver infection, rather than contamination by HCV RNA in accompanying peripheral blood.

High prevalence of hepatitis C (HCV) and hepatitis G (HGV) viruses has been reported among hemodialysis patients with substantial heterogeneity of HCV genotypes throughout the world. We studied HCV prevalence, clinical significance, genotype distribution, and HGV coinfection in hemodialysis patients from Syria. Ninety (75%) of 120 screened patients were HCV antibody positive. Forty-nine (87.5%) of 56 HCV antibody-positive patients had HCV RNA detected by the polymerase chain reaction. The HCV genotyping was possible in 37 of 49 patients (76%). The HCV genotype distribution was genotype 1a, seven (19%); genotype 1b, 10 (27%); genotype 4a, 11 (30%); unmatched sequences, nine (24%). Phylogenetic analysis of unmatched sequences indicated that they represent two distinct and novel subtypes of HCV genotype 4. Hepatitis G virus RNA was detected in 29 (59%) of the HCV RNA-positive patients. No differences were identified between patients infected with HCV alone and those coinfected with HGV. These data demonstrate that HCV infection is common in this population with a genotype distribution predominantly made up of types 1 and 4. Coinfection with HGV had no effect on the outcome of HCV infection.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.

A 62-year-old man developed bilateral granulomatous iridocyclitis after uncomplicated cataract surgery. On ophthalmic examination, we found moderate inflammation in the anterior chamber and vitreous, with granular crystalline deposits on the iris, intraocular lens, and capsular bag. Biopsy of the lens capsule and vitreous revealed periodic acid-Schiff-positive, diastase-resistant bacilli consistent with Tropheryma whippelii. Electron microscopy and polymerase chain reaction confirmed the diagnosis of Whipple disease. A jejunal biopsy specimen also revealed T whippelii. Treatment with trimethoprim-sulfamethoxazole, cefixime, rifampin, and doxycycline resulted in improvement of systemic symptoms, but intraocular inflammation persisted. Intraocular inflammation was eventually reduced with the intravenous administration of ceftriaxone sodium.

Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples (n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (> or = 11,000 versus > or = 30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.

Background: Babesiosis, a zoonosis caused by the protozoan Babesia microti, is usually not treated when the symptoms are mild, because the parasitemia appears to be transient. However, the microscopical methods used to diagnose this infection are insensitive, and few infected people have been followed longitudinally. We compared the duration of parasitemia in people who had received specific antibabesial therapy with that in silently infected people who had not been treated., Methods: Forty-six babesia-infected subjects were identified from 1991 through 1996 in a prospective, community-based study designed to detect episodes of illness and of seroconversion among the residents of southeastern Connecticut and Block Island, Rhode Island. Subjects with acute babesial illness were monitored every 3 months for up to 27 months by means of thin blood smears, Bab. microti polymerase-chain-reaction assays, serologic tests, and questionnaires., Results: Babesial DNA persisted in the blood for a mean of 82 days in 24 infected subjects without specific symptoms who received no specific therapy. Babesial DNA persisted for 16 days in 22 acutely ill subjects who received clindamycin and quinine therapy (P=0.03), of whom 9 had side effects from the treatment. Among the subjects who did not receive specific therapy, symptoms of babesiosis persisted for a mean of 114 days in five subjects with babesial DNA present for 3 or more months and for only 15 days in seven others in whom the DNA was detectable for less than 3 months (P<0.05); one subject had recrudescent disease after two years., Conclusions: When left untreated, silent babesial infection may persist for months or even years. Although treatment with clindamycin and quinine reduces the duration of parasitemia, infection may still persist and recrudesce and side effects are common. Improved treatments are needed.

Since blood is a biologic product, it is unlikely that the risk for transfusion-transmitted infection will ever be reduced to zero. The approach to emerging infections associated with transfusion of blood and blood products includes assessing the transmissibility of the agent by this route; developing effective prevention strategies, including screening tests and donor deferral policies; improving viral and bacterial inactivation procedures; and surveillance for known, as well as emerging and poorly characterized, transfusion-transmitted agents. Vigilance is needed to help ensure proper balance between safety and the availability of blood. Finally, vigilance needs to extend to the developing world, where the basic elements to reduce transfusion-transmitted infections and systems of disease surveillance are often not available.

Objective: To assess the frequency and significance of GB virus-C infection in type 1 autoimmune hepatitis., Material and Methods: Serum specimens from 94 patients with type 1 autoimmune hepatitis were tested for GB virus-C RNA by reverse transcription and polymerase chain reaction. Serum samples from 50 normal subjects were also assessed., Results: Three of the 94 specimens from patients with autoimmune hepatitis were positive for GB virus-C RNA in comparison with none of the 50 control samples (3% versus 0%; P = 0.5). Two patients were seropositive after variceal hemorrhage and blood transfusion, including one patient who clearly acquired the infection in this fashion. One patient had no epidemiologic basis for his seropositivity. Viremia was prolonged in all infected patients (mean duration, 69 +/- 23 months; range, 36 to 113); however, no clinical features suggested a concurrent viral infection, and mortality was similar to that among the uninfected counterparts (33% versus 8%; P = 0.2). Liver transplantation was more common in the infected patients (67% versus 9%; P = 0.03), but the duration of disease was also longer in these patients (277 +/- 29 months versus 106 +/- 9 months; P = 0.0008). Clinical features and immediate responses to corticosteroid therapy were similar in both groups., Conclusion: GB virus-C RNA is found infrequently in type 1 autoimmune hepatitis, and GB virus-C is unlikely to be an important etiologic agent or prognostic determinant.

A 68-year-old woman, who had not traveled outside of western Wisconsin, was hospitalized after 4 weeks of chills, fevers, myalgias, neuralgias in her right arm, and pain in the right upper quadrant of her abdomen. Physical examination revealed hepatosplenomegaly, and laboratory studies showed anemia, thrombocytopenia, increased aspartate transaminase level, and microscopic hematuria. Wright's stain of a blood smear revealed intraerythrocytic organisms consistent with Babesia species. A polymerase chain reaction of whole blood specimens along with an increased serologic titer confirmed the diagnosis of Babesia microti. Indirect immunofluorescent antibody serology and Western blot analysis revealed a simultaneous infection with Borrelia burgdorferi. Coinfection with B. microti and B. burgdorferi may occur in endemic areas where both organisms are carried by the same tick vector, Ixodes scapularis. The intensity and duration of illness seem to be greatest in patients with concurrent infection.

During surveillance for various tickborne pathogens in the upper Midwest during the summer and early fall of 1995, a Bartonella-like agent was detected in the blood of mice that were concurrently infected with Borrelia burgdorferi or Babesia microti (or both). The organism was isolated in pure culture after inoculation of blood from wild-caught mice into C.B-17 scid/scid mice. Phylogenetic analysis of the 16S rRNA and the citrate synthase genes showed that the novel Bartonella species and a Bartonella isolate from a mouse captured on Martha's Vineyard, Massachusetts, were closely related to each other and secondarily related to Bartonella grahamii and Bartonella vinsonii. Further analysis of Peromyscus leucopus blood and tissue samples demonstrated that the novel Bartonella species was exclusively found in conjunction with B. burgdorferi and B. microti. Patent coinfection with these agents may be relatively frequent in naturally infected mice.

In a double-blind study, 655 sputum specimens were obtained from individuals suspected of having tuberculosis and were analyzed for the presence of Mycobacterium tuberculosis and rifampin susceptibility with use of a polymerase chain reaction (PCR)-based universal heteroduplex generator assay (PCR/UHG-Rif). Of the specimens containing viable M. tuberculosis, 100% of the smear-positive (n = 41) and 50% of the smear-negative (n = 6) specimens tested positive for the organism by PCR/UHG-Rif. Nineteen of 537 culture-negative specimens tested positive for M. tuberculosis by PCR/UHG-Rif and were from patients with confirmed tuberculosis who were receiving antituberculosis therapy at the time of specimen collection. Thirty-five specimens contained nontuberculous mycobacteria and were negative by PCR/UHG-Rif. Genotypic evidence of rifampin resistance in five of six culture-confirmed, rifampin-resistant isolates was obtained by PCR/UHG-Rif, yielding a sensitivity and specificity for the assay of 83% and 98.2%, respectively. These results demonstrate the feasibility of using a PCR-based assay directly on sputum specimens for simultaneous detection of M. tuberculosis and rifampin susceptibility, and they suggest that patients with smear-positive, untreated tuberculosis and those presenting with suspected drug-resistant tuberculosis are the most appropriate groups for testing by PCR/UHG-Rif.

We recovered an unusual bacterial strain from blood or sputum of three patients with septicemia, endocarditis, and/or respiratory failure. The three isolates were thin, curved, gram-negative, light brown, pigment-producing bacilli with variable catalase activity. They were asaccharolytic, oxidase-negative, nonmotile, and fastidious. Identification was not possible on the basis of these characteristics alone or in combination with cellular fatty acid profiles. Nucleic acid amplification and sequence analysis of the 16S rRNA gene revealed that all three isolates were identical and most closely related to the emerging pathogen Bordetella holmesii, diverging from the published sequence at three nucleotide positions (99.8% similarity). Isolation of a B. holmesii-like pathogen from sputum suggests that, in addition to producing septicemia, the organism may inhabit the respiratory tract like other Bordetella species.

Background: The significance of hepatitis G (HGV) infection in liver transplant recipients is not known. We set out to determine the pre-orthotopic liver transplantation (OLT) prevalence, the pre- and postoperative viral titers of HGV, and the allograft histology in patients infected with HGV who underwent OLT for cryptogenic cirrhosis., Methods: HGV RNA was measured using a research-based branched DNA assay. The assay used a target-specific probe set that was based on the 5'-untranslated region of the HGV genome. Allograft histology was assessed with protocol liver biopsies in all patients who survived longer than 6 months., Results: The preoperative prevalence of HGV infection in recipients transplanted for cryptogenic cirrhosis was 26%. Thirty-seven percent (12 of 33) of recipients who had serum available in the first postoperative month had HGV infection. Mean HGV RNA levels were 9.8 (+/-4.2) (viral molecular equivalents/ml x 10[6]) before OLT and 37.5 (+/-10.7) at 1 year after OLT. In 4 of the 11 cryptogenic recipients in whom HGV RNA was detectable in the first postoperative month, HGV RNA fell to undetectable levels at the most recent follow-up (mean 70 months). Of the five cryptogenic recipients who continue to have measurable HGV RNA, three have unexplained hepatitis histologically., Conclusions: These findings suggest the following: 1) The prevalence of HGV infection in patients undergoing OLT for cryptogenic cirrhosis is about 25%. 2) In recipients persistently infected with HGV, mean HGV RNA titers increase after OLT. 3) HGV RNA becomes undetectable in about one third of recipients who had detectable HGV RNA in the first month after OLT. 4) Hepatitis of uncertain etiology occurs in 60% (3 of 5) of persistently HGV-infected cryptogenic recipients.

Human Granulocytic Ehrlichiosis (HGE) is a recently described human illness in the US which manifests as fever, myalgia and headache combined with pancytopenia and elevated concentrations of hepatic transaminases. Genetic analyses indicate that the agent of HGE appears to be an Ehrlichia species that is closely related to E. equi and E. phagocytophila. Ixodes dammini and I. scapularis were identified as potential vectors of HGE. Ixodes ticks are also the vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The presence of antibodies against Ehrlichia in 132 sera from Danish patients with definite Lyme neuroborreliosis were examined in order to provide immunoserologic evidence of this infection in Denmark. Patients with Lyme neuroborreliosis were chosen as a test cohort, as these patients had been infested by a tick sufficient for transmission of B. burgdorferi. All had cerebrospinal fluid lymphocytic pleocytosis. As controls, serum samples from 50 healthy Danish blood donors were included. Of the 132 patients with Lyme neuroborreliosis, 5 (3.8%) reacted with the E. equi antigen substrate at titres 1:128. None of the blood donors were found seropositive for E. equi. At least 2 of the patients found seropositive for HGE constituted probable cases of HGE with E. equi antibody titres of at least 80 combined with fever, headache and myalgias. However, in no cases were we able to detect the presence of the HGE agent in the serum by PCR. We conclude that human exposure to granulocytic Ehrlichiae species may also occur in Europe, although further studies will be necessary to document active infection with these potential pathogens.

Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.