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The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between A660s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A660 of >2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an A660 of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zoneretesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test. The COBAS AMPLICOR CT/NG test provides a powerful diagnostic tool for screening for both chlamydial and gonococcal infections. We and others have observed that the NG portion of the test (COBAS AMPLICOR NG) cross-reacts with some isolates of certain nonpathogenic Neisseria species (4). When the original, recommended cutoff (A660 of 0.2) is used, the COBAS AMPLICOR NG test can produce false-positive results for Neisseria gonorrhoeae, presumably due to the presence of cross-reactive Neisseria species in some urogenital specimens. In one population, approximately 26% of COBAS AMPLICOR NG-positive results were false positives, which corresponded to approximately 3% of the total population (4). In contrast, the same laboratory observed fewer than 1% falsepositive results among urogenital specimens from a second population (4). In this paper, we confirm that the test does cross-react with some isolates of Neisseria subflava (4) and Neisseria cinerea and compare the target sequences in these cross-reactive species with that of N. gonorrhoeae. Using data from a multicenter trial (n 4,173 patients; prevalence 13.1%) conducted at six sites in the United States (8), we show how test sensitivity and specificity vary with the cutoff value used. We then assess whether both sensitivity and specificity can be optimized by establishing a large equivocal zone and using an algorithm that involves additional testing of specimens yielding equivocal results. Implementation of the equivocal zone-retesting algorithm identified by this analysis produced good specificity (98.8 to 99.9%) without sacrificing sensitivity in the multicenter trial (8).

To determine the etiology of genital ulcers and to assess the prevalence of human immunodeficiency virus (HIV) infection in ulcer patients in 10 US cities, ulcer and serum specimens were collected from approximately 50 ulcer patients at a sexually transmitted disease clinic in each city. Ulcer specimens were tested using a multiplex polymerase chain reaction assay to detect Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV); sera were tested for antibody to HIV. H. ducreyi was detected in ulcer specimens from patients in Memphis (20% of specimens) and Chicago (12%). T. pallidum was detected in ulcer specimens from every city except Los Angeles (median, 9% of specimens; range, 0%-46%). HSV was detected in >/=50% of specimens from all cities except Memphis (42%). HIV seroprevalence in ulcer patients was 6% (range by city, 0%-18%). These data suggest that chancroid is prevalent in some US cities and that persons with genital ulcers should be a focus of HIV prevention activities.

In 1994, an apparent outbreak of atypical genital ulcers was noted by clinicians at the sexually transmitted disease clinic in Jackson, Mississippi. Of 143 patients with ulcers tested with a multiplex polymerase chain reaction (PCR) assay, 56 (39%) were positive for Haemophilus ducreyi, 44 (31%) for herpes simplex virus, and 27 (19%) for Treponema pallidum; 12 (8%) were positive for > 1 organism. Of 136 patients tested for human immunodeficiency virus (HIV) by serology, 14 (10%) were HIV-seropositive, compared with none of 200 patients without ulcers (P < .001). HIV-1 DNA was detected by PCR in ulcers of 6 (50%) of 12 HIV-positive patients. Multivariate analysis indicated that men with chancroid were significantly more likely than male patients without ulcers to report sex with a crack cocaine user, exchange of money or drugs for sex, and multiple sex partners. The strong association between genital ulcers and HIV infection in this population highlights the urgency of preventing genital ulcers in the southern United States.

A multiplex polymerase chain reaction (M-PCR) assay that simultaneously detects the three major causes of genital ulcer disease (GUD), Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus, was used to evaluate swab specimens for 38 sequential patients with GUD at a Thai sexually transmitted disease clinic. Subjects received clinical diagnoses and syndromic treatment. Swab specimens for H. ducreyi cultures and M-PCR were obtained. No H. ducreyi cultures were positive. Of 38 M-PCR specimens, 31 (81.6%) were positive for HSV, 1 (2.3%) for both HSV and T. pallidum, and none for H. ducreyi or T. pallidum alone; 6 (15.8%) were negative for all 3 pathogens. Clinical diagnoses corresponded poorly to M-PCR findings; none of 5 suspected cases of chancroid were positive by M-PCR and none of 1 for syphilis, but 21 of 24 suspected herpes lesions were confirmed by M-PCR. Human immunodeficiency virus infection status was known for 24 of 38 subjects; 11 (45.8%) were seropositive, and all 11 had HSV by M-PCR. HSV appeared to be the most common pathogen overall.

Background: PCR assays for detection of herpes simplex virus DNA sequences in clinical specimens are more sensitive than cell culture. Objective: A non-isotopic PCR assay (glycoprotein B HSV-2 assay, Roche Molecular Systems) for detection of HSV-2 DNA sequences was evaluated on 234 clinical specimens. Study design: A total of 125 patients (73 women, 40 men, 12 unknown) provided 162 samples contained in viral transport medium. Samples were first inoculated in cell culture, centrifuged at 12 000 × g, lyzed and kept frozen. A total of 77 women provided 42 cervicovaginal lavages and 30 vaginal tampons that were lyzed, tested with two isotopic HSV-2 PCR assays and kept frozen. All these samples were subsequently thawed and amplified with the glycoprotein B HSV-2 assay using generic primers for HSV glycoprotein B gene. Amplicons were captured on microplates with a HSV-2-specific probe and were detected with avidin-peroxidase and substrate. Results: Of the 162 samples submitted to viral culture, HSV-2 was isolated from 73 while 89 did not contain HSV-2. All the 73 specimens with culture-proven HSV-2 infections tested positive with glycoprotein B HSV-2 assay (sensitivity of 100%). Herpesviruses other than HSV-2 were isolated from 34 samples that were negative with glycoprotein B HSV-2 assay. Two culture-negative samples tested positive in the glycoprotein B HSV-2 assay (specificity of 98.7%). The latter samples could not be retested in confirmatory isotopic HSV-2 PCR tests. HSV-2 DNA sequences could also be detected directly in cervical lavages or vaginal tampons from 13 women with the glycoprotein B HSV-2 assay. Conclusion: Detection and typing of HSV-2 in clinical samples, including those collected in viral transport medium, can be accomplished with PCR assays using the AMPLICOR format.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified.

The polymerase chain reaction (PCR) was used to specifically detect toxin A gene sequences of Clostridium difficile in DNA isolated from human faeces. A set of oligonucleotide primers derived from the non-repetitive region of the toxin A gene was developed to amplify a 634-bp DNA fragment. All 28 cytotoxic strains of C. difficile, previously characterized by a toxin B-PCR assay, were positive for the presence of toxin A gene sequences. No amplification products were obtained from DNAs extracted from non-toxigenic strains, strains of C. sordellii, or C. bifermentans. In addition, amplification of DNA extracted from C. difficile 8864, a strain which does not produce toxin A, resulted in multiple bands which probed negative for toxin A gene sequences. DNAs extracted from nine stool specimens which were positive for toxin B by the cytotoxicity assay and by the toxin B-PCR assay were also positive in this assay. Toxin A gene sequences were detected in DNAs obtained from 4/11 stool specimens which were negative by the toxin B cytotoxicity assay. These four specimens were from patients who had a history of relapses due to C. difficile-associated colitis, and whose stools had previously been found to be positive by the toxin B-PCR test despite no detectable toxin B in the specimens. These data indicate a comparable degree of clinical sensitivity between these two toxin-gene PCR-based assays. This rapid, sensitive and specific assay may be useful not only in the diagnosis of C. difficile infections, but also in molecular studies of the toxin A gene in C. difficile strains.

The striking similarity between the first two human immunodeficiency virus type 1 (HIV-1) isolates Lai/LAV (formerly LAV, isolated at the Pasteur Institute) and Lai/IIIB (formerly HTLV-IIIB, reported to be isolated from a pooled culture at the Laboratory of Tumor Cell Biology (LTCB) of the National Cancer Institute) provoked considerable controversy in light of the high level of variability found among subsequent HIV-1 isolates. In November 1990, the Office of Scientific Integrity at the National Institutes of Health commissioned our group to analyse archival samples established at the Pasteur Institute and LTCB between 1983 and 1985. Retrospective analyses have shown that contamination of a culture derived from patient BRU by one from patient LAI was responsible for the provenance of HIV-1 Lai/LAV; the contaminated culture (M2T-/B) was sent to LTCB in September 1983. Our goals were to determine which HIV-1 variants were present in the samples and the sequence diversity among HIV-1 isolates from the earliest stages of the AIDS epidemic. We examined archival specimens and report here the detection of six novel HIV-1 sequences in the cultures used to establish the pool: none is closely related to HIV-1 Lai/IIIB. A sample derived from patient LAI contained variants of both HIV-1 Lai/IIIB and HIV-1 Lai/LAV, and a sequence identical to a variant of HIV-1 Lai/IIIB was detected in the contaminated M2T-/B culture. We conclude that the pool, and probably another LTCB culture, MoV, were contaminated between October 1983 and early 1984 by variants of HIV-1 Lai from the M2T-/B culture. Therefore, the origin of the HIV-1 Lai/IIIB isolate also was patient LAI.

The three major causes of genital ulcer disease (GUD) are herpes simplex virus (HSV), Treponema pallidum, and Haemophilus ducreyi. Although techniques exist for the laboratory diagnosis of all three organisms, constraints of cost, availability of equipment and expertise, and the lack of sensitivity and specificity of available tests, result in clinical presentation being primarily used for the diagnosis of GUD both in the United States and in developing countries. Due to the overlapping clinical presentation of the three diseases caused by these etiologic agents, and due to coinfection, these diseases are often misdiagnosed (1). It is now recognized that not only is GUD a cofactor in HIV transmission, but also that treatment of sexually transmitted diseases can reduce the incidence of HIV (2-4), thus efficient and early diagnosis and treatment of GUD is of utmost importance.

Clinical diagnosis of genital ulcers is difficult and diagnostic tests are least available in settings where rates of disease are highest. The WHO has developed protocols for the syndromic management of genital ulcers in resource-poor settings. However because risk factors patterns and causes of disease and antimicrobial susceptibilities differ from region to region and over time they must be adapted to local situations. The goal of this study was to determine etiologic factors evaluate syndromic management and compare polymerase chain reaction (PCR) testing with other diagnostic alternatives for genital ulcers among patients attending sexually transmitted disease clinics in the Dominican Republic and Peru. 81 men with genital ulcers in the Dominican Republic and 63 in Peru underwent identical interviews and identical multiplex PCR (M-PCR) tests of genital lesion specimens for etiologic diagnoses. Algorithms for managing genital ulcers were developed. In the Dominican Republic 5% were M-PCR-positive for Treponema pallidum 26% for Hemophilis ducreyi and 43% for herpes simplex virus (HSV); in Peru 10% 5% and 43% respectively were positive. The WHO algorithm for treating syphilis and chancroid had a sensitivity of 100% a positive predictive value (PPV) of 24% and an overtreatment rate of 76%. A modified algorithm for treating only those without vesicular lesions had 88% sensitivity and a 27% PPV and the overtreatment rate was reduced to 58%. HSV caused 43% of genital ulcers in these populations. The modified algorithm had lower sensitivity but a reduced overtreatment rate. M-PCR testing was more sensitive than standard tests and more specific and sensitive than clinical diagnosis. (authors)

Chlamydia trachomatis and Neisseria gonorrhoeae are universally acknowledged as urethral pathogens, yet the etiology in the majority of cases of urethritis is unclear. Our case-control study assessed the association of Mycoplasma genitalium, Ureaplasma urealyticum, and other potential pathogens with acute nongonococcal urethritis (NGU) in heterosexual men presenting to an urban sexually transmitted diseases clinic. M. genitalium was detected in 27 (22%) of 121 NGU case patients and in 5 (4%) of 117 control subjects (P

While genital ulcers are a risk factor in HIV infection, the association of specific agents of genital ulcer disease (GUD) with HIV infection may vary.To determine the etiology of GUD in HIV-infected and HIV-uninfected men attending sexually transmitted disease (STD) clinics in Durban, Johannesburg, and Cape Town, South Africa, and the association of previous and current sexually transmitted infections with HIV infection in men with ulcerative and nonulcerative STDs.A cross-sectional study of 558 men with genital ulcers and 602 men with urethritis.Patients with GUD were more likely to be infected with HIV than patients with urethritis (39.4% versus 21.4%, Por =0.001). Herpes simplex virus 2 (HSV-2) was the most common agent identified in ulcer specimens (35.9%), and was detected in a significantly higher proportion of ulcer specimens from HIV-infected patients than in specimens from HIV-uninfected patients (47.4% versus 28.2%, Por =0.001). Patients infected with HIV-1 were significantly more likely to have HSV-2 infection, as measured by the presence of the antibody to glycoprotein G-2, than patients not infected with HIV (63.1% versus 38.5%, Por =0.001). Patients infected with HIV-1 were also significantly more likely to have initial HSV-2 infection than HIV-uninfected patients with GUD (50.0% versus 31.6%, P = 0.007). Haemophilus ducreyi was detected in 31.7% of ulcer specimens; prevalence did not vary by HIV-infection status. Treponema pallidum DNA was detected significantly less frequently in ulcer specimens from patients infected with HIV than in specimens from patients not infected with HIV (10.2% versus 26%, Por =0.001); no association was found between HIV-infection status and fluorescent treponemal antibody absorption test seroreactivity, even when men with M-PCR-positive syphilis lesions were excluded from the analyses.The authors found that HSV-2 is a more common etiology of GUD than has been suggested by previous studies conducted in South Africa; serologic evidence of HSV-2 infection and current cases of genital herpes are strongly associated with HIV infection among men who present to STD clinics with GUD or urethritis.

Ulcer material from consecutive patients attending clinics in Antananarivo, Madagascar, was tested using multiplex polymerase chain reaction (M-PCR) to detect Treponema pallidum, Haemophilus ducreyi, and herpes simplex virus. Sera were tested for syphilis and for IgG and IgM antibodies to Chlamydia trachomatis by microimmunofluorescence testing (MIF). By M-PCR, 33% of 196 patients had chancroid, 29% had syphilitic ulcers, and 10% had genital herpes; 32% of the ulcer specimens were M-PCR negative. Compared with M-PCR, syphilis serology was 72% sensitive and 83% specific. The sensitivity of clinical diagnosis of syphilis, chancroid, and genital herpes was 93%, 53%, and 0% and specificity was 20%, 52%, and 99%, respectively. Less schooling was associated with increased prevalence of syphilitic ulcers (P=.001). Sixteen patients (8%) were clinically diagnosed with lymphogranuloma venereum (LGV); 1 plausible case of LGV was found by MIF. In Madagascar, primary care of genital ulcers should include syndromic treatment for syphilis and chancroid.

A multiplex polymerase chain reaction (M-PCR) assay for Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) was compared with clinical and standard laboratory methods for the diagnosis of genital ulcer disease (GUD) in 105 patients; 36% were human immunodeficiency virus (HIV)-seropositive. Chancroid (80%), syphilis (8%), and genital herpes (8%) were the most frequent diagnoses. H. ducreyi and HSV were isolated from ulcers of 43% and 18% of patients, respectively; in 35%, all cultures were negative and the laboratory diagnosis indeterminate. M-PCR detected H. ducreyi, T. pallidum, and HSV in 56%, 23%, and 26% of patients, respectively; (no definitive diagnosis, 6%). The proportion of patients with more than one agent was 4% by culture and 17% by M-PCR (P = .002). Resolved sensitivities of M-PCR for H. ducreyi and HSV cultures were 95% and 93%, respectively. The sensitivities of H. ducreyi and HSV cultures were 75% and 60%, respectively. HSV, detected in 47% of specimens from HIV-infected versus 16% from HIV-uninfected patients (P < .001), may be emerging as a more frequent cause of GUD.

A rapid and sensitive PCR-based microwell plate assay (PCR-MWP) system to detect the 16 S ribosome RNA gene of Helicobacter pylori was developed. Analytical sensitivity, evaluated with purified recombinant plasmid DNA and genomic DNA of H. pylori, was one copy of DNA per PCR. Specificity was validated with a panel of DNA from 75 kinds of microorganisms including Helicobacter showed weak positives, when 1 pg of DNA was input. Other microorganisms gave negative signals even when 100 pg of DNA was used for PCR. When compared with a Nested-PCR system to detect the urease A-subunit gene performed by a commercial reference laboratory, the results obtained (sensitivity 93.3% and specificity 73.3%) was almost equivalent. The PCR-MWP was rapid and easy for the detection of H. pylori DNA in gastric juice specimen.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among travelers and residents of developing countries. ETEC produces either a heat-stable toxin or a heat-labile toxin, or both, encoded by plasmid-borne ST and LT genes, respectively. Diagnosis of infection with this subclass of E. coli can be performed with oligonucleotide hybridization probes; however, the sensitivity and specificity of this method are insufficient. A nonradioactive multiplex PCR assay that provides a sensitive and specific method for detecting the presence of either or both toxin genes has been developed. A simple procedure that removed inhibitors of the PCR while efficiently releasing ETEC DNA from stool specimens for subsequent amplification was used. The results for samples from a human volunteer study of ETEC infection indicated that this method of sample preparation results in greater clinical sensitivity than conventional total nucleic acid extraction and ethanol precipitation. Detection of ETEC by a multiplex PCR assay in stool specimens directly processed with a glass matrix and chaotropic solution had greater sensitivity than culture.

A sensitive and specific PCR-based assay to detect the Helicobacter pylori 16S rRNA gene present in formalin-fixed paraffin-embedded gastric biopsy specimens has been developed. A total of 95 patients with dyspepsia were evaluated for the presence of chronic active gastritis and an infection with H. pylori through the use of diagnostic assays based on biopsy specimens and serology. The "gold standard" for the presence of the bacteria was direct detection in histological sections of biopsy specimens by Giemsa stain. The results obtained with the PCR assay performed on the biopsy specimens (94% sensitivity and 100% specificity) were equivalent to the detection of H. pylori immunoglobulin G antibodies by the commercially available second-generation Cobas Core anti-H. pylori immunoglobulin G enzyme immunoassay (94% sensitivity and 98% specificity) for the diagnosis of H. pylori infection. Urease testing and bacterial culture of the biopsy specimens were inferior (88 and 70% sensitivity and 96% and 98% specificity, respectively). A Western blot (immunoblot) analysis had slightly greater sensitivity (96%), although specificity was reduced to 93%. This research prototype PCR assay was shown to be highly reliable for the detection of infection with H. pylori and the presence of chronic active gastritis in the population studied.

Protein antigens present in the hepatic lesions of mice infected with Schistosoma mansoni were identified by use of fluorochrome-conjugated monoclonal antibodies. All 26 monoclonal antibodies used in these experiments recognized antigenic determinants expressed by both cercariae and adult worms; 23 of these epitopes were also expressed by S. mansoni eggs. These results suggest considerable antigenic conservation during schistosome development. Two antibodies recognizing determinants absent from the egg nevertheless bound to schistosome antigens in hepatic granulomata; this result suggests that circulating worm antigens were also cleared in these lesions. Fifteen antigens were detected in perivascular spaces 5 weeks after infection, before the appearance of eggs in the liver.

A major surface antigen of Schistosoma mansoni has been identified and characterized as a glycoprotein of 66,000 molecular weight (Mr) and isoelectric point of 6.2-6.1 (SM66-GP) by use of a monoclonal antibody. The antigen was expressed by schistosome eggs, cercariae, larvae, and adults, and was recognized by sera of schistosome infected hosts. Direct immunofluorescence microscopy showed the antigen was distributed in a uniform pattern on the entire worm surface. Indirect immunofluorescence microscopy revealed that it was present in the parenchymal tissue of immature and mature Fasciola hepatica, in the gut of the mature fluke, and in embryonated fasciola eggs. The cross-reactive F. hepatica epitope recognized was expressed on a polypeptide of Mr 220,000.

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.

The efficient export of a subset of Escherichia coli envelope proteins is dependent upon the product of the secB gene. Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form. In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits. SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles. The addition of purified SecB to an in vitro system prepared from SecB- cells significantly enhanced MBP translocation. The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes. Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.

Evidence is presented that the E. coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form. The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability. The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.

A subset of Schistosoma mansoni egg glycoproteins that share a common carbohydrate epitope recognized by monoclonal antibody 128C3 was shown to induce formation of hepatic granulomata when conjugated to Sepharose beads and injected into the portal circulation of naive mice. Concanavalin-binding egg glycoproteins exhibited more granuloma-inducing activity than did total egg extract, although deglycosylated egg proteins also induced granulomata; thus, both amino acid and carbohydrate epitopes appeared to be involved. Glycoproteins derived from adult male worms also were active, indicating that immunological processes responsible for granuloma formation may not be absolutely Stage specific.