Disseminating sexually transmitted infections diagnostics information: the SDI web publication review series.

OBJECTIVES: The World Health Organization Sexually Transmitted Diseases Diagnostics Initiative (SDI) website publication review seeks to provide health care providers in all geographic and economic settings with timely, critical, and concise information concerning new developments in laboratory and field diagnosis of sexually transmitted infections (STI). METHODS: Since 2003, the website (www.who.int/std_diagnostics/literature_reviews) has disseminated information in the form of annotated abstracts and commentaries on articles covering studies of STI laboratory-based and rapid assays that are commercially available or under development. Articles identified through searches of PubMed, specific journals, and by referrals from Editorial Board members are selected for inclusion if they meet pre-specified criteria. The objectives, methods, results, and conclusions for each article are summarised and board members are invited to prepare commentaries addressing study design and applicability of findings to end users. RESULTS: Currently, 91 STI diagnostics experts from 17 countries on six continents serve on the Editorial Board. Twelve quarterly issues have been posted that include summaries of 214 original and 17 review articles published from January 2002 through March 2005, with expert commentaries on 153 articles. Interest in the site has increased every year. In 2005, over 36 700 unique visitors from more than 100 countries viewed over 75,000 pages of information. CONCLUSIONS: The SDI Publication Review series has the potential to contribute to SDI's goal of improving care for patients with STI by increasing knowledge and awareness of STI diagnostics. Given the proliferation of internet-based STI testing services, this website may be broadened to meet the needs of a wider range of users.

An AIDS test that travels well.

PIP: A new dipstick test will enable health workers in remote Third World communities to screen blood for human immunodeficiency virus (HIV) and monitor the spread of HIV infection. The test, developed by the Program for Appropriate Technology in Health (PATH), works without electricity, instrumentation, or cold chain and is locally manufacturable. Results are available in 20 minutes, and the cost is 25 cents per sample. Antibodies to both HIV-1 and HIV-2 are detectable. In the test, a plastic dipstick shaped like a comb with 8 test strips is dipped into blood samples, rinsed, and soaked in a reagent solution. The appearance of a red dot on a dipstick tooth indicates a positive finding. Field trials in Uganda, Kenya, Brazil, China, Indonesia, India, and Thailand indicate the dipstick is as reliable as screening tests already on the market; the false-positive rate was under 2%. India and Indonesia are already manufacturing the new test, and Kenya, Uganda, Cameroon, China, and Thailand have expressed interest. The low cost of the dipstick may enable its use in seroprevalence surveys in previously unstudied regions of the Third World. Such a survey was just conducted among a randomly selected group of pregnant women from across Haiti. Blood samples from simple finger pricks collected on filter paper yielded highly reliable results.^ieng

Inhibition of Trichomonas vaginalis motility by monoclonal antibodies is associated with reduced adherence to HeLa cell monolayers.

Adherence of trichomonads to host epithelial cells appears to be a critical step in the pathogenesis of trichomoniasis. We evaluated the effect of a panel of 10 monoclonal antibodies on attachment of [35S]methionine-radiolabeled Trichomonas vaginalis strains to HeLa cell monolayers. Of 10 monoclonal antibodies, 3 totally eliminated motility of PHS2J strain trichomonads and reduced their adherence to 48 to 60% of control values (P less than 0.001). However, none of the monoclonal antibodies affected motility or adherence of STD13 strain trichomonads. Although the antibodies all reacted with PHS2J trichomonads by immunofluorescence, there was no correlation between inhibition of adherence and findings on either immunofluorescence or radioimmunoprecipitation. Direct microscopic observations showed that incubation with the monoclonal antibodies did not cause cytolysis of T. vaginalis. In quantitative cultures there was no difference in the number of colonies produced by parasites that had been incubated with antibodies that inhibited or had no effect on adherence. We conclude that our monoclonal antibodies reduced adherence not by cytotoxic effects or by competing for specific sites mediating adherence of the protozoa, but by inhibiting motility of T. vaginalis.

Performance and cost-effectiveness of a dual rapid assay system for screening and confirmation of human immunodeficiency virus type 1 seropositivity.

Recent studies have shown that rapid, instrument-free assays for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and specific as enzyme-linked immunosorbent assay (ELISA) for screening of donated blood in developing countries. Currently, however, specimens which test positive on a screening assay must still be confirmed by Western blot (immunoblot), a method which is not feasible in most developing-country laboratories. We examined whether a testing hierarchy which utilizes neither conventional ELISA nor Western blot can be reliably used for screening and confirmation of HIV infection in a high-risk population. In a retrospective analysis of 3,878 specimens which were screened for antibody to HIV in Kinshasa, Zaire, we observed that a testing hierarchy consisting of duplicate HIVCHEK screening assays followed by duplicate Serodia-HIV confirmatory assays resulted in correct confirmation of all ELISA- and Western blot-positive specimens. We conclude that such a testing hierarchy can produce highly accurate results for identification of positive specimens in routine HIV testing and provides a practical alternative to conventional methods of HIV screening and confirmation.

Evaluation of an immunofluorescent-antibody test for rapid identification of Pseudomonas aeruginosa in blood cultures.

An immunofluorescent-antibody test was developed for rapid detection of Pseudomonas aeruginosa in blood cultures. The test uses a murine monoclonal antibody specific for all strains of P. aeruginosa. In initial tests, bright uniform immunofluorescence signals were seen when each of the 17 international serotypes, as well as 14 additional isolates of P. aeruginosa, were examined. No immunofluorescent staining was observed when 37 other gram-negative and 15 gram-positive species were studied. In a clinical study, the assay was applied to broth smears of 86 gram-negative bacilli isolated from 74 bacteremic patients and 28 additional clinical isolates of Pseudomonas sp. and other oxidase-positive gram-negative bacilli recovered from various body sites. Smears were made directly from blood cultures which were positive for gram-negative bacilli by Gram staining. Eleven (15%) of 74 patients with gram-negative bacteremia had a positive test for P. aeruginosa. Including the results of these 11 isolates recovered in a prospective study and an additional 10 isolates from a retrospective study, we obtained a sensitivity and specificity of 100% (21 positive specimens and 103 negative specimens, respectively). These preliminary results suggest that this is a useful reagent for rapid presumptive identification of P. aeruginosa in blood cultures. With the immunofluorescent-antibody test, P. aeruginosa could be identified within 1 h of Gram stain evidence of gram-negative bacteremia.

Antigenic differences in the surface mannoproteins of Candida albicans as revealed by monoclonal antibodies.

Monoclonal antibodies to Candida albicans were prepared with blastoconidia bearing germ tubes used as the immunogen. Four antibodies reacted by immunofluorescence with surfaces of C. albicans as well as Candida stellatoidea, Candida tropicalis, and several strains of C. albicans, but not with Torulopsis glabrata. One antibody reacted with Saccharomyces cerevisiae. In addition, the monoclonal antibodies precipitated material of approximately 200 kilodaltons when tested against metabolically labeled blastoconidia digests. The monoclonal antibodies exhibited heterogeneous staining of C. albicans surfaces, as shown by immunofluorescence. None of the monoclonal antibodies were specific to germ tubes. More importantly, however, two of the monoclonal antibodies reacted with the mannoprotein precipitin arc of C. albicans that was produced by reference rabbit polyclonal antisera by crossed immunoelectrophoresis, thus linking the heterogeneity seen by immunofluorescence to the heterogeneity in mannoproteins. Finally, three of the monoclonal antibodies reacted with a glycan fraction of cell digests, indicating their reactivity with the carbohydrate portion of the mannoprotein.

Diagnosis of trichomoniasis. Comparison of conventional wet-mount examination with cytologic studies, cultures, and monoclonal antibody staining of direct specimens.

The accuracy of (1) conventional wet-mount examination, (2) Papanicolaou-stained gynecologic smears, (3) a direct slide test using fluorescein-conjugated monoclonal antibodies against Trichomonas vaginalis, and (4) two different culture media for the diagnosis of trichomoniasis in a high-risk population of 600 women was compared. Use of Feinberg-Whittington or Diamond's culture medium resulted in a diagnosis of 82 and 78 cases, respectively, and the combination of two cultures identified 88 infected women. In comparison, wet-mount examination detected only 53 (60%) of the cases. Cytologic smears were interpreted as positive for T vaginalis in 49 (56%) of the 88 cases but also resulted in seven false-positive smears, and specimens from 18 women with negative cultures were interpreted as "suspicious" for trichomoniasis. Monoclonal antibody staining detected 76 (86%) of the 88 positive specimens, including 27 (77%) of the 35 cases missed by wet-mount examination. In summary, wet-mount and cytologic studies were insensitive, and cytology study was the least specific method for diagnosis of trichomoniasis. Direct immunofluorescence with monoclonal antibodies holds promise as a sensitive and specific alternative to cultures for rapid detection of T vaginalis in clinical specimens.

[Classification of Gonococcus: auxanologic and serologic aspects using monoclonal antibodies]. / Typisierung des Gonokokkus: Auxotypie und Serotypie mit monoklonalen Antikörpern.

500 N. gonorrhoeae strains from the area of Lübeck/West Germany, collected in 1976-84, were auxologically and serologically classified by means of monoclonal antibodies from the Genetic Systems Corporation, Seattle, Washington (USA), against gonococcal outer membrane protein I. During the first years, auxological classification showed high proportions of AHU-strains (40%), which amounted to 20% over the total observation period. PPNG and DGI strains have characteristic type patterns. Our synopsis of serovars corresponds with the results obtained in a recent world-wide study. Combined auxological and serological classification shows two N. gonorrhoeae pools, 18% each, which cannot be further distinguished: AHU/A1 and prototrophic/B3. The advantages of the system have been proved in answering plain epidemiological questions. For further discrimination, an additional independent classification method might be useful.

Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum.

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent serum from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by IgG isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, an antigenically conserved outer membrane protein, contained most of the blocking activity in IgG. Antibodies specific for gonococcal porin (protein I), the major outer membrane protein, displayed no blocking function. In separate experiments, immune convalescent DGI serum which did not exhibit bactericidal activity was restored to killing by selective depletion of protein III antibodies by immunoabsorption. These studies indicate that protein III antibodies in normal and immune human serum play a role in serum resistance of N. gonorrhoeae.

Use of dot-immunobinding and immunofluorescence assays to investigate clinically suspected cases of chancroid.

In 1984 and 1985, outbreaks of genital ulcers occurred in Florida and New York. Initial investigations for syphilis, herpes simplex, Chlamydia trachomatis, and Haemophilus ducreyi did not implicate any of these organisms as etiologic agents. From the results of dot-immunobinding assays, we found that sera from the patients had higher levels of IgM (30 [47.6%] of 63 patients) and IgG (22 [34.9%] of 63 patients) reactivities with an outer-membrane preparation from H. ducreyi strain CIP542 than with outer-membrane preparations from Haemophilus influenzae ATCC 10211 or Haemophilus parainfluenzae ATCC 7901. In contrast, sera from 35 patients in the control group did not react with any of the outer-membrane preparations (P less than .01 for both IgM and and IgG reactivity), nor did sera from eight individuals with disease caused by H. influenzae (P = .051 for IgM reactivity, P = .02 for IgG reactivity). Indirect immunofluorescence assay using a monoclonal antibody reactive with outer-membrane preparations from H. ducreyi strain CIP542 stained organisms in smears of lesion material from genital ulcers from three of six patients. These results suggested that the cause of both outbreaks of genital ulcers was H. ducreyi which was subsequently isolated in both geographic areas.

Geographic variation among isolates of Trichomonas vaginalis: demonstration of antigenic heterogeneity by using monoclonal antibodies and the indirect immunofluorescence technique.

Although Trichomonas vaginalis causes one of the most common sexually transmitted diseases, little is known about the antigenic variation of the parasite or about differences between strains in epidemiology or virulence. Variation among isolates of T. vaginalis was investigated by using a panel of monoclonal antibodies, each reactive with different antigens, to test 88 isolates from diverse geographic areas of North America. All isolates of T. vaginalis reacted with at least one of the nine monoclonal antibodies; the individual antibodies reacted with 22%-76% of the isolates. A pool of two broadly reactive antibodies identified all isolates in the study. Four of the most narrowly reactive, or "specific," antibodies demonstrated differences in the antigenic composition of trichomonads isolated from patients in Seattle, Baltimore, and Brooklyn, New York (P less than .005 by chi 2 test). Application of these and other monoclonal antibody probes may facilitate epidemiological studies and provide rapid, reliable methods for direct diagnosis of trichomonads in clinical specimens.

Monoclonal antibodies for the diagnosis of sexually transmitted diseases.

Monoclonal antibodies are already being used for the diagnosis of human sexually transmitted diseases. These antibodies can be used to detect a wide range of microorganisms, including bacteria, parasites, and viruses. For both culture and direct tests, monoclonal antibodies showed patterns of specificity and reproducibility that exceeded those available with conventionally prepared antisera. The direct tests for these organisms required less than an hour to perform, representing a major advancement in a diagnosis that previously required 2 to 6 days of culture followed by confirmatory testing. Furthermore, rapid differential diagnosis of infection will now be possible. Because some sexually transmitted diseases may be transmitted simultaneously and share similar clinical manifestations (that is, gonorrhea and chlamydia in cervicitis or urethritis, syphilis or herpes in genital ulcers), it will be possible to differentiate a single from a multiple infection by simultaneous testing of direct samples with the appropriate monoclonal antibody reagents.

Detection of Treponema pallidum in lesion exudate with a pathogen-specific monoclonal antibody.

The diagnosis of early syphilis currently requires dark-field microscopic or serologic demonstration of Treponema pallidum infection. Dark-field microscopy is not widely available and is complicated by the numerous saprophytic spirochetes which are present at oral and rectal mucosal surfaces. Serologic tests are positive in only 70 to 90% of patients with primary syphilis, and several days may be required for results to become available. We used a pathogen-specific, fluorescein-conjugated monoclonal antibody to examine lesion exudates from 61 patients for the presence of T. pallidum and compared the data with results of dark-field microscopy and serologic testing. The direct fluorescent-antibody technique revealed the presence of T. pallidum in 30 of 30 patients with early syphilis, and dark-field microscopy was positive for 29. Serologic tests were reactive for 27 of 30 patients with syphilis; in the 3 patients with nonreactive serologic tests, chancres had been present for 4, 6, and 21 days. Although 7 of 31 patients without syphilis had spiral organisms seen on dark-field microscopy, the direct fluorescent-antibody test was negative for all 31. The presence of nonpathogenic spirochetes was subsequently verified in 5 of 7 patients by using a second monoclonal antibody which reacts with nonpathogenic, as well as pathogenic, treponemes and related spirochetes. The demonstration of T. pallidum by using fluorescein-conjugated monoclonal antibodies is intrinsically specific and is as sensitive as dark-field microscopy for the diagnosis of early syphilis. This method provides a convenient, accurate means for the diagnosis of syphilis by health care providers, many of whom lack access to dark-field microscopy.

New technologies for use in the surveillance and control of yaws.

In the Republic of Ghana, treponemal antigen tests performed on finger-prick blood from patients with yaws proved to be as sensitive as those tests performed on whole sera, and this mode of collection was more economical and acceptable than venipuncture. Under field conditions, dark-field microscopic examination of suspect yaws lesions was difficult as compared with collection of serous exudate in heparinized capillary tubes examined later in a reference laboratory. Direct staining of lesion exudate fixed on microscope slides with fluorescein-conjugated human or mouse monoclonal antibody against Treponema pallidum was more sensitive than dark-field examination. However, these techniques could not distinguish between the early lesions of venereal syphilis and those of yaws. An equally sensitive technique used a cloned segment of the T. pallidum (Nichols strain) genome to detect homologous DNA in lesion exudate fixed on nitrocellulose filter paper. The fixation of lesion exudates on microscope slides or nitrocellulose papers may prove to be the easiest method of collecting and transporting such materials to reference laboratories.

Characterization of monoclonal antibodies to Treponema pallidum.

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.

Direct fluorescent monoclonal antibody stain for rapid detection of infant Chlamydia trachomatis infections.

A method of direct fluorescent antibody staining for rapid diagnosis of Chlamydia trachomatis infections in infants is described. This method utilized a fluorescein-conjugated species-specific monoclonal antibody to C trachomatis for detecting chlamydial elementary bodies in smears of the conjunctiva, nasopharynx, oropharynx, anus, and vagina. The sensitivity of direct fluorescent antibody staining was compared with isolation of the organisms in McCoy cells. Thirty-nine infants with purulent conjunctivitis were studied. Diagnosis of C trachomatis conjunctivitis was correctly made by smear in all 16 infants when inflamed eyes were sampled. Positive smears were obtained from 12/14 culture-positive and 4/16 culture-negative nasopharyngeal specimens from infants with chlamydial conjunctivitis. All nasopharyngeal cultures and smears from infants with nonchlamydial conjunctivitis were negative. These results indicate that the direct smear test is a sensitive and specific test for diagnosing C trachomatis infection of the eye and nasopharynx in infants, and this test can be completed within one hour of specimen collection.

Serological classification of Neisseria gonorrhoeae with use of monoclonal antibodies to gonococcal outer membrane protein I.

Identification of strain-specific markers on Neisseria gonorrhoeae that are capable of differentiating gonococci into a large number of distinct classes could facilitate analysis of patterns of gonorrhea transmission and application of gonorrhea control measures. A panel of 12 monoclonal antibodies to gonococcal outer membrane protein IA (PrIA) and IB (PrIB) was used to classify 1,433 strains serologically in a worldwide survey. Eighteen PrIA and 28 PrIB serovars were identified, and a nomenclature is proposed. Gonococcal strains were classified further by auxotyping. Auxotyping and serotyping served to classify the 1,433 isolates into 107 unique auxotype/serovar classes. Dual classification by auxotype and serovar can be used to identify epidemiologically related gonococcal infections in order to test the effectiveness of innovative, focused measures to control gonorrhea.

Culture-independent diagnosis of Chlamydia trachomatis using monoclonal antibodies.

To simplify the diagnosis of chlamydial genital infection, we used a fluorescein-conjugated monoclonal antibody in immunofluorescence tests on smears prepared from urethral or cervical secretions obtained directly from patients. This direct test, requiring less than 30 minutes to perform, was based on the detection of extracellular chlamydial elementary bodies. A comparison of the direct test with cultures stained with iodine on specimens from 926 patients demonstrated a sensitivity of 93 per cent and a specificity of 96 per cent. The direct test provides a rapid, simple, and sensitive method for the diagnosis of chlamydial infection, which can be performed in laboratories that do not have tissue-culture capability.

Gonococcal protein I-specific opsonic IgG in normal human serum.

Pooled normal human serum (NHS), as well as 10 individual NHS samples, markedly inhibited the reaction between monoclonal antibodies and their cognate epitopes on protein I of serum-sensitive, serum-resistant, and disseminated gonococcal infection-associated strains of Neisseria gonorrhoeae, as determined by ELISA inhibition. IgG was the immunoglobulin class responsible for the inhibition. Only the Fab fragment of IgG was inhibitory, making it likely that the IgG reacted specifically with protein I. After absorption with purified protein I, NHS did not inhibit the binding of a protein III-specific monoclonal antibody, thus excluding the possibility that protein III-specific antibodies in NHS masked epitopes on protein I. In addition, lipopolysaccharide-specific IgG in NHS did not appear to contribute to the inhibition of monoclonal antibody binding to protein I. The IgG from NHS was opsonic; opsonization was prevented by coating gonococci with the Fab fragment of protein I-specific monoclonal antibodies.