Accurate sequencing by hybridization for DNA diagnostics and individual genomics.

Medical DNA diagnostics will increasingly rely on an accurate and inexpensive identification of mutations that affect the function of a gene. To validate diagnostic sequencing by hybridization (SBH), a number of p53 samples were analyzed with the complete set of 8192 noncomplementary 7-mer oligonucleotides. In four repeated, blind experiments we accurately sequenced 1.1 kb per each of 12 homozygote and heterozygote samples possessing base substitutions, insertions, and deletions. This SBH variant offers a high throughput platform to inexpensively sequence individual gene or pathogen genome samples within the clinical laboratory setting.

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells.

Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.

Analytic performance and contamination control methods of a ligase chain reaction DNA amplification assay for detection of Chlamydia trachomatis in urogenital specimens.

A ligase chain reaction (LCR) DNA amplification assay that targeted the cryptic plasmid of Chlamydia trachomatis was developed to detect C. trachomatis urogenital tract infection. The objectives of this study were to determine the cutoff and analytic performance of the LCR assay and to characterize the effectiveness of its postdetection contamination control method. The assay's cutoff was determined after receiver-operator characteristic (ROC) analysis of 4660 clinical data points. The assay detected one infectious unit per reaction of each of the 15 C. trachomatis serovars and did not cross-react with 13 Chlamydia pneumoniae strains, 13 Chlamydia psittaci strains, and 87 other bacteria, fungi, parasites, or viruses. In addition, the assay did not detect 77 processed urine specimens collected from patients with urinary tract infections caused by yeast or bacteria other than C. trachomatis. The assay was sufficiently precise to detect consistently two infectious units of C. trachomatis per reaction. False-positive assay results attributable to contamination with amplified product were minimized by the use of standard procedures as well as by a postdetection chemical inactivation method that could reduce the amount of amplified LCR product by a factor of > or = 10(7).

Reverse transcriptase PCR and staging prostate cancer.

OBJECTIVE: To present a brief review of the diagnostic dilemma of staging prostate cancer and how a novel diagnostic technique, the reverse transcriptase polymerase chain reaction, is being used as an aid to better stage and manage the disease. DATA SOURCE: Research articles about prostate cancer and the reverse transcriptase polymerase chain reaction published in the last 5 years, as well as data gathered by the authors. STUDY SELECTION: Performed by the authors. DATA EXTRACTION: Performed by the authors. DATA SYNTHESIS: Prostate cancer is the most common cancer among men in the U.S. A wide variety of methods are used for the diagnosis; however, accurate staging of the disease to determine the most effective treatment is a problem. Because metastatic prostate cancer is routinely understaged, the reverse transcriptase polymerase chain reaction to identify prostate cancer cells in the circulatory system is becoming an important diagnostic aid for staging and monitoring the disease. It is analytically and clinically sensitive as well as specific. CONCLUSION: The reverse transcriptase polymerase chain reaction is a highly accurate aid in staging and monitoring prostate cancer. Its prognostic value, particularly when a small number of prostate cancer cells are detected in the circulatory system requires further long-term follow-up studies.

Regulation of membrane-bound enzymes of glycosphingolipid biosynthesis.

The descriptive phase of glycosphingolipid biochemistry has blossomed over the past decade with the application of exquisitely sensitive analytical methods of mass spectrometry, NMR, and monoclonal antibody technology. Consequently, appreciation of the interrelation among the complex carbohydrate structures that are expressed on the cell surface has focused attention on the regulation of the glycosyltransferases responsible for such diversity. This review explores some aspects of several potential molecular mechanisms of regulation--compartmentation of glycosyltransferases and effect of the immediate lipid environment; modulation of enzyme activity by acceptor substrate, divalent cations, and availability of sugar nucleotides; modifications of the enzyme operon elicited by viral transformation integration sites, or to the enzyme by phosphorylation-dephosphorylation--which are presently at the cutting edge of glycosphingolipid science.

Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix.

We performed a multicenter evaluation of ligase chain reaction (LCR) in the diagnosis of Chlamydia trachomatis infection of the cervix. This LCR provides an amplification of target sequences within the chlamydial cryptic plasmid. The LCR results were compared with those of isolation in cell culture. Discrepant (tissue culture-negative and LCR-positive) test results were resolved by the application of a direct immunofluorescent-antibody test to detect chlamydial elementary bodies and by the use of alternate DNA primers that targeted the chlamydial major outer membrane protein gene. A total of 234 of 2,132 specimens (10.9%) could be confirmed as containing C. trachomatis. Of these, 152 were detected by isolation in cell culture and 221 were detected by LCR. The corresponding sensitivities were 94% for LCR and 65% for cell culture. There was greater variability among study site results for cell culture sensitivity (52 to 92%) than for LCR sensitivity (87 to 98%). The specificity of each test was greater than 99.9%. Thus, LCR offers a highly sensitive nonculture method for detecting chlamydial infection of the cervix.

Components responsible for transport between successive Golgi cisternae are highly conserved in evolution.

Transport of a glycoprotein between compartments of the Golgi has been reconstituted in an in vitro system (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416). Cytosolic components and ATP are absolutely required for transport. Here, we have tested the acceptor activity of Golgi fractions and of cytosolic fractions prepared from a variety of organisms. All mammalian Golgi fractions can act as "acceptor" in the in vitro assay. Similarly, the cytosol fractions obtained from plants as well as animals and a lower eukaryote substitute for the homologous CHO cytosol normally used. Moreover, a cytosol subfraction prepared from wheat germ complements a different cytosolic fraction obtained from bovine brain. Apparently, the essential components involved in the post-translational protein transport are remarkably conserved between plants, animals, and lower eukaryotes.

Noninvasive tests for diagnosis of Chlamydia trachomatis infection: application of ligase chain reaction to first-catch urine specimens of women.

Ligase chain reaction (LCR) to diagnose Chlamydia trachomatis infection was evaluated using first-catch urine (FCU) specimens from 4053 women. Results were compared with those of cell culture (TC) isolation from cervix (all) and urethra (2812 women). The reference standard was TC positivity or positive LCR for chlamydial plasmid DNA confirmed by direct fluorescent antibody test or LCR for another chlamydial gene. Compared with cervical culture, LCR was 88.2% sensitive and 100% specific. Adding urethral culture increased TC sensitivity from 67.1% to 74% and reduced LCR sensitivity to 85.9%. The prevalence of chlamydial infection was 5% (142/2812) by the dual culture system and 5.9% (165/2812) by LCR on FCU specimens. LCR on FCU specimens is highly sensitive and specific for diagnosing chlamydial infection. It is more sensitive than TC and may well present public health authorities with a useful noninvasive screening test for chlamydial infection in asymptomatic women.

Phorbol ester-associated changes in ganglioside metabolism.

The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-14C]palmitate and [9-3H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.

Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction.

From April to September 1993, 305 men and 447 women in Hamilton, Canada, consented to the collection of a urethral or cervical swab, respectively, for culture and 20 ml of first-void urine (FVU) for testing by the enzyme immunoassay Chlamydiazyme and by ligase chain reaction (LCR) in the form of a kit from Abbott Laboratories called LCx Chlamydia trachomatis. Evaluation of test performance with each specimen was calculated on the basis of an expanded "gold standard" of a patient found to be positive by culture or by a confirmed nonculture test. By using this expanded standard, the prevalence of infection was determined to be 6% (27/447) for the women and 18.4% (56/305) for the men. LCR testing of FVU in both studies was the most sensitive approach (96%). The performance of Chlamydiazyme was as follows: cervical swab, 78.3% sensitivity; female FVU, 37% sensitivity; and male FVU, 67.9% sensitivity. Culture was the least sensitive approach to diagnosis: female cervix, 55.6%; and male urethra, 37.5%. LCR testing of FVU from men or women diagnosed the greatest number of genitourinary tract infections with no false positives.

Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine.

Genitourinary infection with Chlamydia trachomatis is a common and potentially serious sexually transmitted disease. Diagnosis of C trachomatis infection in women typically relies on culture of endocervical swabs, an invasive and expensive procedure. The ligase chain reaction (LCR) is an in-vitro nucleic acid amplification technique that exponentially amplifies selected DNA sequences. We have compared an LCR-based assay to detect C trachomatis plasmid DNA in first void urine with culture of endocervical swabs for matched specimens from 1937 women from four geographic regions. Discordant specimen pairs were further tested by direct fluorescent antibody staining for elementary bodies and an alternative LCR assay based on the chlamydial outer membrane protein gene. An "expanded gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive women. The sensitivity and specificity of the LCR assay with first void urine samples compared with the expanded gold standard were 93.8% and 99.9%, respectively; the corresponding values for culture were 65.0% and 100%, respectively. Thus, an automated LCR assay of readily obtained urine samples showed a detection rate for infected women almost 30% greater than that of endocervical swab culture. The LCR assay was highly effective for the detection of C trachomatis in urine from women with or without signs or symptoms of chlamydial genitourinary tract infection.

Detection of antibodies to Trypanosoma cruzi among blood donors in the southwestern and western United States. I. Evaluation of the sensitivity and specificity of an enzyme immunoassay for detecting antibodies to T. cruzi.

BACKGROUND: Chagas' disease or American trypanosomiasis, caused by infection with Trypanosoma cruzi, is a significant health problem in Latin America. In the United States, transfusions of T. cruzi-contaminated blood from Latin American immigrants may represent the major source of Chagas' disease. STUDY DESIGN AND METHODS: A new enzyme immunoassay (EIA) for the detection of antibody to T. cruzi was evaluated in the sera of blood donors from the southwestern and western regions of the United States. Serum samples had been screened and were negative for all tests required. Specimens that were repeatedly reactive in the Chagas antibody EIA were analyzed for seroreactivity by a confirmatory EIA and by radioimmunoprecipitation assay. RESULTS: Fourteen of the 13,309 donor samples (0.105%) were confirmed as being positive for antibody to T. cruzi. The Chagas antibody EIA showed improved sensitivity over the Chagas IgG enzyme-linked immunosorbent assay and two indirect hemagglutination assays. The Chagas antibody EIA had a specificity of 99.98 percent with negative samples. The sensitivity of the Chagas antibody EIA was 100 percent (80/80) in xenodiagnosed specimens and 100 percent (50/50) in specimens positive by consensus (i.e., reactive in EIA, indirect hemagglutination assay, and immunofluorescence assays). CONCLUSION: This Chagas antibody EIA meets the need for accurate and rapid identification of seroreactive samples in low-prevalence or endemic populations.

Diagnosis of Chlamydia trachomatis urethral infection in symptomatic and asymptomatic men by testing first-void urine in a ligase chain reaction assay.

A multicenter study compared ligase chain reaction (LCR) of Chlamydia trachomatis plasmid DNA with culture of urethral swab specimens from 542 men (study A); a second study (B) compared LCR of first-void urine (FVU) with urethral swab cultures from 1043 men. Discordant results were resolved with direct fluorescent antibody staining of sediments from the FVU or urethral culture specimen and with a second LCR directed against a fragment of the major outer membrane protein gene. Test performance was calculated on the basis of an expanded reference standard. The LCR plasmid assay had a sensitivity of 98.0% in study A and 93.5% in study B; specificity was 99.8%-100%. The sensitivity of culturing urethral swabs from all study sites was 68.2% (range by sites, 40.0%-84.6%). The presence or absence of urethral symptoms did not alter the results. Use of this LCR test should allow more meaningful investigation and treatment of C. trachomatis infections in men.

Systematic analysis of hMSH2 and hMLH1 in young colon cancer patients and controls.

Germ-line mutations in DNA mismatch-repair genes impart a markedly elevated cancer risk, often presenting as autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC). However, there are no pathognomonic features of HNPCC, not all gene carriers have a family history of the disease, and families fulfilling the Amsterdam criteria are relatively uncommon. Genetic testing of probands with early-onset colorectal cancer, irrespective of family history, is one approach that would allow predictive genetic testing of at-risk relatives. We cloned and sequenced hMSH2 and hMLH1 introns, to optimize genomic sequencing. We then systematically analyzed the entire hMSH2 and hMLH1 genes, by genomic sequencing and in vitro synthesized-protein-truncation assay (IVSP), in 50 colorectal cancer patients <30 years of age at diagnosis. To determine polymorphic variants, 26 anonymous donors also were sequenced. All subjects analyzed had at least 1 of 37 different polymorphic or pathogenic variants. IVSP complemented genomic sequencing, by detection of mutations not identified by genomic analysis. Fourteen cancer patients (28%) had pathogenic mutations, and a number of other variants also may have had a pathogenic significance that remains to be elucidated. Tumor replication-error status was useful in targeting sequencing efforts for this cohort of young patients: sensitivity was 86%, specificity 73%, and positive and negative predictive values 63% and 90%, respectively. These data indicate that an appreciable proportion of young colon cancer probands carry a germ-line mutation in a DNA mismatch-repair gene.

A multicentric seroepidemiological survey of HTLV-I/II in Italy.

BACKGROUND: Several studies carried out in the USA and in Europe have shown the presence of HTLV-I/II antibodies in subjects belonging to high-risk groups for HIV infection as well as blood donors. Concern about the presence of HTLV-I/II markers in the normal population, as well as the efficient transmission of HTLV-I/II by whole blood or infected blood cells have led several countries to include screening for anti-HTLV-I/II among the mandatory serological testing of blood donors. OBJECTIVE: In order to assess the risk of HTLV-I/II infection related to blood transfusions, a multicentric survey for antibodies against HTLV-I and HTLV-II was carried out involving 10 Italian sites during the spring of 1991. STUDY DESIGN: Serum specimens were collected from 14,598 blood donors, 1,411 injecting drug users, 1,015 thalassemics, 142 hemophiliacs and 138 hemodialysis patients. HTLV antibodies were detected by a screening EIA which combines a viral lysate with a recombinant HTLV-I env protein (p21e). The serological confirmation was performed by a semi-automated dot-blot immunoassay that detects gag p19 and p24 and env p21e specific antibodies, while the discrimination of HTLV-I and HTLV-II reactivities was carried out by EIAs employing synthetic peptides of the ENV region specific for each virus. RESULTS: The seroprevalence of confirmed positives was 0.034% among blood donors and 3.61% among IDUs, while no sample of the other categories could be confirmed, although several were indeterminate and one thalassemic reacted against HTLV-I on peptide testing. HTLV-I reactivity was observed in one blood donor, while all 38 of the 51 confirmed seropositive IDU's reacted only to the HTLV-II synthetic peptide. CONCLUSIONS: These data confirm a high prevalence of HTLV-II among Italian IDUs and show an HTLV-I/II seroprevalence among blood donors very similar to that which was found in the USA volunteer blood donors. A surveillance program among blood donors seems advisable in order to establish the possible need of a mandatory screening for HTLV-I/II.