微板核酸杂交-酶联显色检测性病沙眼衣原体DNA

目的 建立微板核酸杂交—ELISA方法，检测性病沙眼衣原体。方法 采用PCR、核酸杂交和ELISA三大生物技术相结合，建立微板核酸杂交ELISA法，并用此法分别同McCoy细胞培养和免疫学方法检测307份性传播疾病标本进行比较。结果 微板核酸杂交—ELISA法检测的阳性率为40．39％(122／307)，高于细胞培养的9.77％(30／307)和免疫诊断的12．05％(37／307)。结论 微板核酸杂交—ELISA技术检测性病沙眼衣原体灵敏度高、特异性好、简便快速。因此，该方法具有很强的实用价值和发展前景。     

Comparative evaluation of various direct and culture methods for the diagnosis of genital Chlamydia trachomatis infections

Commercially produced fluorescein labelled monoclonal antibodies for Chlamydia trachomatis detection have been recently become available. We have compared the data obtained using two monoclonal antibodies, one for detecting inclusion on cell cultures (culture confirmation) and the other for detecting C. trachomatis in smears from urethral and cervical swabs, with our routine isolation method which utilizes Giemsa staining of cycloheximide treated McCoy cell cultures. We also evaluated an enzyme immunoassay for detecting C. trachomatis antigens in urethral and cervical specimens. The culture confirmation system was slightly more sensitive and simpler than Giemsa staining. Between the results of immunofluorescence direct test and culture there was 96.3% agreement. Sensitivity, specificity and predictive positive and negative value were 72.2, 98.4, 80 and 97.6%. Between results of culture and enzyme immunoassay there was 97.2% agreement. The immunoassay sensitivity, specificity predictive positive and negative value were, in women, 100, 97.1, 63.6 and 100%; in men, 100, 95.7, 81.8, 100%.     

Pneumonia due to Chlamydia trachomatis in Japanese infants

Sera from 109 Japanese infants with pneumonia were tested for antibody to Chlamydia trachomatis (C. trachomatis) L2 strain by an indirect immunofluorescence (IF) technique. Nasopharyngeal swabs were also collected to isolate C. trachomatis. Clinical specimens were inoculated onto cycloheximide-treated McCoy cells and DEAE-dextran-treated HeLa 229 cells. Of 109 patients, 32 (29%) were positive for IgM antibodies (titer, greater than or equal to 1:16) to C. trachomatis. C. trachomatis was isolated from 21 (66%) of 32 IgM antibody-positive infants as compared with 5 (7%) of 77 IgM antibody-negative infants. Detectable levels of IgM antibody were common in infants during the first four months of life. Clinical characteristics of pneumonia of these IgM antibody-positive patients were also described. This is the first report of serology and clinical characteristics of C. trachomatis pneumonitis from Asian countries including Japan.     

Evaluation of two ELISA's for detecting Chlamydia trachomatis from endocervical swabs.

Two enzyme immunoassays (EIAs) detecting Chlamydia trachomatis from endocervical swabs, Syva MicroTrak (MT) and Abbott Chlamydiazyme (CZ), were compared with a tissue culture (TC) standard. Initially, 8% (100 of 1250) of specimens were TC positive, yielding sensitivities of 94% (94 of 100) for MT and 79% (79 of 100) for CZ with identical 98% specificities (1129 of 1150 for MT and 1130 of 1150 for CZ). Discrepant specimens were retested by both EIAs and assayed for elementary bodies (EBs) by a fluorescent antibody test. After discrepancy analysis, 9.5% (118) of 1240 patients were either TC or EB positive, yielding sensitivities of 94.1% for MT (111 of 118) and 79.7% for CZ (94 of 118) with identical specificities of 100% (1122 of 1122). These results indicate that the MT is significantly more sensitive (p less than 0.05, McNemar test) than CZ in detecting C. trachomatis from endocervical swabs.     

Serological evidence of Mycoplasma pneumoniae infection in acute exacerbation of COPD

It is a well-known fact that tubal stenosis and/or peritubal adhesion are often associated with Chlamydia trachomatis infection. Although tubal pregnancy may be attributed to this infection, there are only extremely rare cases in which the presence of C. trachomatis has been confirmed by immumo-histochemical examination on tissues isolated from patients with tubal pregnancy. We thus tried to confirm the presence of C. trachomatis infection by detecting DNA of the organism in tissues surgically isolated from patients with tubal pregnancy.Two detection methods, a ligase chain reaction (LCR) method and an immuno-histochemical staining which detects an antigen of C. trachomatis, were compared using paraffin-embedded tissue samples which were surgically isolated from patients with tubal pregnancy or hydrosalpinx. The LCR method was capable of detecting DNA of C. trachomatis in tissue samples in which the C. trachomatis-specific antigen could not be detected using immuno-histochemical staining. This was due to the fact that immuno-histochemical staining methods are applicable to the analysis of sequences the length of which range from 200 to 400 bp (base pairs), while the LCR method used here allows the analysis of sequences as small as 48 bp. This fact makes the LCR method a very convenient tool, as compared with immuno-histochemical methods, for analysis of the paraffin embedded tissue samples where by effects of formalin--used for fixation for pathologic diagnosis--the risk of fragmenting the DNA samples is relatively high. Presence of C. trachomatis DNA as detected by LCR method in surgically isolated samples from patients with tubal pregnancy supports the involvement of Chlamydia trachomatis infection in the occurrence of tubal pregnancy. Accordingly the LCR method is capable of detecting DNA of C. trachomatis in paraffin-embedded samples of tubal tissue in which presence of C. trachomatis could not be confirmed by an immuno-histochemical staining method.     

Comparison of Buffalo green monkey kidney cells and McCoy cells for the isolation of Chlamydia trachomatis in shell vial centrifugation culture.

A total of 745 clinical specimens from patients attending hospitals and clinics in an area with a low prevalence of Chlamydia trachomatis were inoculated in parallel into shell vial cultures containing Buffalo green monkey kidney (BGM) and McCoy cells. The shell vial cultures were prepared in-house and were less than 5 days after seeding when inoculated. A total of 38 specimens (5%) were positive for C. trachomatis. In 36 of the cases, C. trachomatis was detected in both the BGM and McCoy vials. In two cases, C. trachomatis was detected in only the BGM vial. The BGM cells were more resistant to cytotoxicity and seemed to show more and larger inclusions than the McCoy cells. Furthermore, because the BGM cells displayed contact inhibition without the rounding and piling of cells that was encountered with the McCoy cells, they could be stored, ready to use, with an optical monolayer of cells, at 35 degrees C for at least 10 days.     

Evaluation of a DNA probe of plasmid origin for the detection of Chlamydia trachomatis in cultures and clinical specimens

This study evaluates five cryptic plasmid-derived DNA probes in a 4-h slot-blot hybridization assay for the detection of Chlamydia trachomatis in cultures and clinical specimens. The probes, consisting of either the entire cloned 7.5 kbp cryptic plasmid pSE8 or one of four Hin dIII/Eco RI fragments measuring 710, 1055, 710, and 500 bp, respectively, were labelled with Photoprobe biotin. The probe was detected using a streptavidin-alkaline phosphatase conjugate followed by addition of BCIP and NBT. The sensitivity of the assay, using 25 ng of probe DNA, ranged from 50 pg (with the entire plasmid as probe) to 5 ng (with the 500 bp fragment as probe). A total of 103 reference strains of Chlamydia trachomatis and other bacteria were tested for reactivity with the probes. All 18 reference strains of C. trachomatis hybridized with the probes. None of the DNA from the heterologous organisms tested was found to hybridize with any of the probes. A total of 174 samples taken from patients visiting the STD clinic at the University Hospital, University of Seville were used in the study. The overall sensitivity of the assay, using the 710 bp biotinylated probe was 94.5% compared to culture while the specificity was 97.5%. Positive and negative predictive values were 96.5% and 97.5%, respectively. It appears that the plasmid-derived probes used in this study could serve as useful tools for the rapid and specific detection of Chlamydia trachomatis in cell cultures and clinical specimens.     

Evaluation of a HSV specific monoclonal antibody reagent for laboratory diagnosis of herpes simplex virus infection

A new FITC-conjugated HSV specific monoclonal antibody reagent (Syva Co., Palo Alto, Ca) was evaluated for the confirmation of HSV clinical isolates. The reagent was also compared to type-specific monoclonal antibodies for the pre-CPE detection of HSV from clinical specimens in centrifugation culture and by direct examination of specimens smears by direct immunofluorescent antibody staining (DFA). HSV was isolated from 75 of 232 specimens (32%). All 75 isolates were confirmed with both the type-specific antibodies and the HSV-specific reagent. In centrifugation culture HSV was detected in 36 of 105 (34%) specimens. The HSV specific reagent detected all 36 specimens that were positive with the type-specific reagents. HSV infection was diagnosed by DFA in 31 of 50 (62%) specimen smears. The HSV-specific reagent detected all 31 positive specimens. This reagent confirmed and detected all HSV positive specimens that were positive by the type-specific monoclonal antibody reagents. The reagent contains monoclonal antibodies specific for both HSV1 and HSV2 in a single mixture, which produces a highly sensitive HSV FA staining reagent.     

Diagnosis of cytomegalovirus hepatitis by histopathology and in situ hybridization in liver transplantation.

Hematoxylin-eosin staining (H&E) of cytomegalovirus (CMV) inclusion bodies was compared with in situ hybridization using a biotinylated DNA probe for the detection of CMV. Of 29 biopsy specimens selected from 23 patients with clinical CMV hepatitis and typical CMV inclusions on histopathologic examination, 23 (79%) were positive by DNA probe and 17 (59%) were detected in cell cultures. The mean number of CMV foci per tissue section was higher by DNA probe (12) compared with H&E (5). We do not recommend in situ hybridization in microbiology laboratories as a replacement for histopathology for the diagnosis of CMV in tissue specimens.     

Correlation of the percent of positive Chlamydia trachomatis direct fluorescent antibody detection tests with the adequacy of specimen collection

Chlamydia trachomatis is an obligate, intracellular parasite infecting the columnar and transitional cells lining the endocervix, uterus, fallopian tubes, rectum, urethra, and epididymis. We determined if the percent of specimens positive for C. trachomatis in the Microtrak Direct Specimen Test depended on the quality of specimens obtained. Female genital slides (649) were evaluated by the direct fluorescent antibody (DFA) test for the presence and numbers of (a) C. trachomatis elementary bodies and (b) columnar, transitional and squamous epithelial cells, and polymorphonuclear neutrophils (PMNs). Only 138 (21.3%) of the 649 slides were considered to be adequately taken, that is, containing columnar/transitional cells either alone or in conjunction with squamous cells and/or PMNs. Of the 138 adequate slides, 10 (7.2%) were C. trachomatis positive. However, 511 (78.7%) of the 649 slides were judged inadequate; 395 contained only squamous cells and/or PMNs, 19 were too thick to determine cell types, 46 contained only cell debris, and 51 contained neither cells nor debris. Only four (0.78%) of 511 were C. trachomatis positive. Thus adequate specimens containing columnar/transitional cells for C. trachomatis detection had a tenfold increase in the percent of positive results compared to inadequately collected specimens. By using the DFA test, one has the advantage of determining the adequacy of the specimens obtained as well as the presence of chlamydiae.     

A prospective study of Chlamydia trachomatis in first trimester abortion

Chlamydia trachomatis was isolated from 34 (17.9%) of 190 unselected women applying for first trimester abortion and from 27 (15.9%) of the 170 women who actually had the operation. C. trachomatis was more common among the younger women. Neisseria gonorrhoeae was isolated from 3 (1.6%) of 190 women. Culture positive patients and partners were given antibiotic treatment for 10 days, usually postoperatively. Early postoperative genital infection developed in 2 (7.4%) of the 27 chlamydia-positive and in 3 (2.0%) of the 143 chlamydia-negative women. There were no late infections. The antibiotic treatment significantly reduced the rate of postoperative pelvic inflammatory disease. At examination after 4-7 weeks all cultures were negative. Significantly more women with chlamydia-positive cultures were sero-positive preoperatively: with a microimmunofluorescence method, IgG titres greater than or equal to 1:160 were found in 74.1% of culture positive and in 47.6% of culture negative patients (p = 0.01). However, serological IgG screening does not identify individual high risk patients and so is of little clinical use in this context. We recommend preoperative screening for C. trachomatis in all women requesting an abortion and 10 days antibiotic treatment for all carriers.     

Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Genitourinary infections in males by the Amplicor PCR assay of urine

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.     

Analysis of the repertoire of human B-lymphocytes specific for type A and type B blood group terminal trisaccharide epitopes

Naturally occurring serum antibodies specific for the A and B blood group isoantigens are of great importance in medicine. By using A-type terminal trisaccharide (ATS) or B-type terminal trisaccharide (BTS) coupled to albumin as coating antigens, an enzyme-linked immunosorbent assay capable of detecting all ATS/BTS-binding antibodies was performed. The combination of this enzyme-linked immunosorbent assay with limiting-dilution methodology, using a polyclonal B-lymphocyte activator, permitted a monoclonal analysis of the human antibody repertoire that is specific for ATS and BTS in persons of different blood types. Most (78%) positive supernatants from type O cultures were monospecific for either ATS or BTS, and these were present at roughly equivalent frequencies. Nine supernatants with reactivity toward both ATS and BTS were tested by red cell adsorption; six had properties expected for true dually reactive monoclonal antibodies: adsorption with either A1 or B red cells eliminated both anti-ATS and anti-BTS activity. This finding accords with a monoclonal origin for anti-A,B. The analysis of cultures of peripheral blood lymphocytes from type A and B donors unexpectedly showed significant numbers of clones with apparent autospecificity. However, none of the anti-ATS-positive supernatants from type A cultures or anti-BTS-positive supernatants from type B cultures were adsorbable with A1 or B red cells, respectively. Consideration of only true (adsorbable) positives indicates that the type A and B anti-trisaccharide repertoires differ significantly from the type O repertoire, probably as a result of the action of normal self-tolerance mechanisms.     

Evaluation of selective and enrichment media for isolation of glycopeptide-resistant enterococci from faecal specimens

OBJECTIVES: Vancomycin-resistant enterococcus enrichment broth (VEB) and vancomycin-resistant enterococcus selective agar with vancomycin 6 mg/L (VSA) are novel azide-aesculin agar-based media that contain meropenem as an additional selective agent. The media were compared with enterococcosel broth (EB) and enterococcosel agar with vancomycin 6 mg/L (EA) for the isolation of glycopeptide-resistant enterococci (GRE) from routine faecal screening specimens. METHODS: Two hundred and eighteen routine faecal screening specimens from patients at Addenbrooke's Hospital were examined. The majority were from patients on haematology wards (155) or the intensive therapy unit (ITU) (21). Specimens were inoculated on to VSA and EA directly, and after enrichment in VEB and EB, respectively. RESULTS: One hundred and twenty-eight GRE isolates were recovered from 93 (43%) specimens with enterococci carrying vanA or vanB genes. There were no statistically significant differences between media (specimens positive; numbers of GRE isolates) on direct plating on VSA (87; 104) or EA (86; 97) or following 24 h enrichment in VEB (89; 103) or EB (86; 98). There was no significant advantage to enrichment compared with direct plating. Incubation of enrichment broth cultures for only 6 h appeared detrimental. Enterococci with vanC were isolated significantly less frequently from VEB and VSA than from EB and EA. Growth of organisms other than GRE was more common on VSA than on EA. CONCLUSIONS: VEB and VSA were at least as effective as EB and EA for the recovery of GRE from faecal screening specimens, but substantially more non-GRE grew on VSA than on EA. Enrichment culture offered no significant advantages over direct plating.     

A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis

We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1-VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted.     

In vitro analysis of the change in resistance of Chlamydia trachomatis under exposure to sub-MIC levofloxacin for a therapeutic term

BACKGROUND: While fluoroquinolone-resistant Chlamydia trachomatis strains have not been clinically isolated, they were isolated in an in vitro study recently. METHODS: To determine whether C. trachomatis strains develop resistance under sub-MIC antibacterial exposure in a clinical therapeutic term, C. trachomatis strains were exposed to sub-MIC levofloxacin (LVFX) for about 2 weeks. The MIC of LVFX was measured and DNA fingerprinting was performed every 72 h by PCR using random primers. RESULTS: There was almost no change in the MIC under exposure to 0.125 microg/ml LVFX. However, some mutational changes in DNA fingerprints developed. CONCLUSIONS: In clinical therapeutic terms, resistant strains of C. trachomatis will probably not develop, even if sub-MIC LVFX is employed.     

Evaluation of Syva's enzyme immunoassay for the detection of Chlamydia trachomatis in urogenital specimens.

A newly developed microwell enzyme immunosorbent assay (EIA) system by Syva Company (Palo Alto, CA) can detect Chlamydia trachomatis in < 3 hr. It uses a polyclonal antibody to chlamydial lipopolysaccharide and end points are determined with a spectrophotometer. Three clinical trial sites (University of California Medical Center, San Francisco, CA; University of Washington, Seattle, WA; and Louisiana State University Medical Center, New Orleans, LA), compared this EIA with tissue culture (TC) for identifying Chlamydia in urogenital specimens. Overall prevalence by TC was 10.4% (136 of 1306). When tests were compared with TC (using vials or microtiter plates and a fluorescent antibody stain), we found an EIA sensitivity of 93.4% (127 of 136) and a specificity of 98.1% (1148 of 1170). This EIA has a performance profile that is, at the very least, comparable with other nonculture methods for diagnosing genital tract infections with C. trachomatis.     

Characterization of non-typable strains of Pseudomonas aeruginosa from cystic fibrosis patients by means of monoclonal antibodies and SDS-polyacrylamide gel electrophoresis

Fifty-three non-typable strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined to determine if these bacteria could be classified by means of their interactions with a recently completed panel of serotype specific monoclonal antibodies and by analysis of their lipopolysaccharide (LPS) separation (or banding) patterns in SDS-polyacrylamide gels. Based on agglutination reactions and interaction of LPS of these strains in Western immunoblots with monoclonal antibodies, 24 (45%) of all the strains tested were found to be typable. These include 13 that typed 06, five that typed 05, three that typed 04, two that typed 01, one that typed 03, and one that typed 016. LPS from these “newly” typable strains were isolated and characterized and a correlation was found between the LPS banding pattern of each strain and its O-specificity. Among the group of isolates that agglutinated with the 05-specific monoclonal antibody MF15-2, strain C418M had a different LPS banding pattern when compared to that of the standard serotype 05 strain and the other type 05 clinical isolates. The LPS of this strain was subsequently found to be non-reactive with monoclonal antibody MF15-2 in immunoblots thus confirming that this bacterium did not belong to the 05 serogroup. While there are still some clinical strains that could not be typed, we found that the occurrence of polytypability characteristics among clinical isolates was substantially reduced by the use of monoclonal antibodies.     

The microbicidal agent C31G inhibits Chlamydia trachomatis infectivity in vitro.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.     

Chlamydia DNA extraction for use in PCR: Stability and sensitivity in detection

We evaluated multiple procedures for extracting chlamydial DNA from specimens for detection in PCR tests. Commercial kits and an in-house method were tested for their sensitivity and utility. Quantifiable chlamydial elementary bodies (EB) were used for spiking buffy coats from EDTA-collected blood. EBs of Chlamydia pneumoniae at 2,500 and 25 EB/ml were used as specimens for DNA extraction using seven different procedures. These included either columns (3 procedures), centrifugation (1), glass (1), or patented extraction matrices (2), coupled with either alcohol precipitation (6) or heat-detergent treatment (1). Five procedures required 10–40 minutes manipulation; two required 2–5 hours. PCR results for DNA extracts using chlamydial 16S genus primers were generally more intensely positive with denser bands on electrophoresis gels for the higher concentrations of EB (up to 4+ for stained product on gels) than was PCR with lower EB concentrations (up to 2+). Further, the incidence of procedures with positive results was: 5 of 7 for chlamydial genus primers with 5 EB vs. 6 of 7 with 500 EB. Maximal sensitivity for one of the extractions was in the range of 2.5–5.0 EB/ml of test specimen with 4 of 5 replicates being positive with EB controls or extracts. Extracts were stable up to 2+ weeks at 4°C and were effective in multiplexing with fluorescent-tagged primers. Taking into consideration the time factor and sensitivities, the two procedures with extraction matrices are favored for routine laboratory use. J. Clin. Lab. Anal. 12:47–53, 1998. © 1998 Wiley-Liss, Inc.