Demonstration of tuberculostearic acid in sputum from patients with pulmonary tuberculosis by selected ion monitoring.

Selected ion monitoring was used to detect tuberculostearic acid (10-methyloctadecanoic acid) in sputum from patients with pulmonary tuberculosis. The specimens were autoclaved, lyophilized, extracted, and methanolysed before being subjected to thin-layer chromatography and injected into the gas chromatograph/mass spectrometer. Tuberculostearic acid could be detected in five of six tuberculous sputum specimens containing acid-fast rods detectable by light microscopy of Ziehl-Neelsen stained smears. After the sputum specimens had been cultured for five days on Löwenstein-Jensen medium, when still no colonies could be observed visually, the presence of tuberculostearic acid was demonstrated in all six cases of tuberculosis. In corresponding analyses of sputum from eight patients with non-tuberculous pneumonia, tuberculostearic acid was not found. This fatty acid, the presence of which was also demonstrated in cultures of various mycobacterial and nocardial species, is characteristic of organisms of the order Actinomycetales. The demonstration of tuberculostearic acid in sputum specimens may constitute a rapid and sensitive way of diagnosing pulmonary tuberculosis.

Evaluation of chlamydial tests in early trachoma.

An enzyme linked immunosorbent assay (ELISA) and an immunofluorescence (IF) test were compared with culture for their value in the diagnosis of chlamydial infection in early trachoma. Conjunctival swabs and scrapings from 81 school children with active trachoma from Khartoum, Sudan, were tested. Fourteen (17.3%) of the children with signs of active trachoma were positive by isolation. The sensitivities of the ELISA and IF tests, compared to cultures, were 85.0% and 68.2%, while the specificities for the ELISA and IF tests were 99.2% and 99.3%, respectively. Chlamydial IgM and IgG antibodies reactive with serotypes A-C and D-K of Chlamydia trachomatis were detected in sera of 28 (71.2%) of the 39 children from whom serum was obtained. Only eight of the 14 isolation-positive children were tested for chlamydial antibodies, and all of them had IgM and IgG antibodies at a titer of greater than or equal to 1:16. An IgG antibody titer of 1:16 or greater was detected in 20 (65.0%) of the 31 isolation-negative patients tested for chlamydial antibodies. The geometric mean titers of IgG antibodies to the antigen pools A-C and D-K were 132 and 42, respectively. Our study suggests that trachoma is prevalent in the child population studied. Clinical signs of active trachoma were found in 47.0% of the altogether 172 children investigated. ELISA can be recommended as a diagnostic tool in trachoma field surveys.

Hemagglutination by Staphylococcus aureus. Studies on strains isolated from bovine mastitis.

Staphylococcus aureus strains isolated from bovine mastitis exhibited hemagglutinating activity when cultivated on nutrient-poor, solid medium. Out of 56 strains, 41 (73%) hemagglutinated. S. aureus agglutinated sheep erythrocytes to a greater extent than it agglutinated either human, bovine or equine erythrocytes. Hemagglutination was more conspicuous with erythrocytes from certain sheep individuals, indicating blood-group specificity. Receptors on sheep erythrocytes were apparently of carbohydrate nature since periodate-treated erythrocytes were not agglutinated. Hemagglutinating properties were associated with the ability to adhere to human buccal cells. Crude staphylococcal extracts were able to partially inhibit bacterial adherence to epithelial cells.

Antibiosis between bacteria isolated from the vagina of women with and without signs of bacterial vaginosis.

Lactobacilli from women with and without bacterial vaginosis (BV) were tested for H2O2 production. Thirty-seven (79%) of the 47 strains of lactobacilli isolated from the women without BV produced H2O2, while only nine (23%) of the 39 strains of lactobacilli obtained from women with BV did so. Five of 20 H2O2-producing and two of 26 non-producing strains of Lactobacillus exhibited antibiosis against four of 12 strains of peptostreptococci and two of 10 strains of Mobiluncus spp. None of a further 41 different anaerobic and facultative anaerobic bacterial strains were inhibited by any of the isolates of lactobacilli tested. Some strains of Gardnerella vaginalis, Bacteriodes spp., Mobiluncus spp. and Peptostreptococcus spp. inhibited the growth of three strains of lactobacilli belonging to different species. When the pH of the culture medium was increased from 6.0 to 6.5 this led to a decrease in the number of strains inhibited and/or the size of the growth-inhibitory zones. Different concentrations of H2O2 did not inhibit any of the strains tested. The growth-inhibitory effect of lactobacilli could not be related to their bacteriocin production. Increasing the iron content of the medium by adding FeCl3 (0.01 mM-1 mM) decreased or completely abolished the antibiosis.

Immune response in chlamydial conjunctivitis among neonates and adults with special reference to tear IgA.

Tear and serum samples from 128 neonates and 122 adults with conjunctivitis were examined for antibodies to Chlamydia trachomatis with a micro-immunofluorescence (MIF) technique and the results compared to antigen detection by culture, enzyme immunoassay (EIA) (Chlamydiazyme, Abbott) and direct immunofluorescence (IF) (MicroTrak, Syva and Chlamyset, Orion) tests. From the 52 culture-positive adults, chlamydial IgA (titre greater than or equal to 1:8) antibodies were detected in 81% of the tear and in 62% of the serum samples, while 88% had such serum IgG antibodies (titre greater than or equal to 1:32). The persistence of chlamydial IgA in tears and sera was related to the duration of symptoms of conjunctivitis and the antibody titres declined after institution of antibiotic treatment. In the adults, the sensitivity of the MIF tear IgA antibody test (81%) was higher than that of the EIA (71%) and the IF (MicroTrak 71% and Chlamyset 62%) tests. The specificity for the MIF test was 79%, while it was 100% for the EIA and the two IF tests. Of the 67 chlamydia-infected neonates, 36% had chlamydial tear IgA antibodies, while such antibodies were only found in 15% of the sera. No neonates with chlamydia-negative conjunctivitis had chlamydial IgA antibodies. The MIF test may be used as a diagnostic method complementary to culture, EIA and IF tests in the diagnosis of chlamydial conjunctivitis in adults, but is not applicable in neonates.

Detection of Chlamydia trachomatis antigens in first-void urine to identify asymptomatic male carriers.

Early morning first-void urine collected from 279 sexually active Swedish male recruits (mean age 19.5 years) was tested by two commercial enzyme immunoassay (EIA) kits, MicroTrak and IDEIA III, and by MicroTrak direct fluorescence assay (DFA), to detect Chlamydia trachomatis antigens. A result was assumed to be true-positive when any of the two non-culture tests were positive for the same specimen. In one case where only DFA was positive, confirmatory chlamydial testing was performed by isolating the organism from a urethral swab. On these premises, the number of true-positive men was 26 (9.3% of all men studied). The sensitivity, specificity, positive predictive value and negative predictive value for MicroTrak EIA were 85%, 98%, 85%, and 98%, respectively. IDEIA III was less sensitive than MicroTrak EIA (42% vs 85%). In conclusion, the diagnosis of asymptomatic chlamydial infections in men can be established with reasonable accuracy by the detection of Chlamydia antigens in urine samples using MicroTrak EIA.

Detection of Chlamydia trachomatis in first-void urine collected from men and women attending a venereal clinic.

Cervical, urethral and first-void urine (FVU) specimens from 196 men and 245 women attending a venereal outpatient clinic were studied by culture and a commercial enzyme immunoassay (EIA) (CHLAMYDIAZYME). Confirmatory chlamydial testing by a direct fluorescence assay (DFA) (MICROTRAK) was performed on the sediments of the positive EIA samples from culture-negative patients. Chlamydia trachomatis was isolated from 11% of the men and 12% of the women. Of the women, 67% were positive in both sampling sites and 33% in the cervix only. No further cases were found when a female urethral swab was cultured. All the chlamydia-positive urine samples were obtained from women who were positive in the urethra. The denominator used to calculate sensitivities was the combination of patients with culture- and EIA-positive results which could be confirmed by DFA. The sensitivity of our culture method was 85% for men and 77% for women. In men, the sensitivity of EIA was greater on urine than on urethral specimens (77% vs 62%; p less than 0.1). In women, the sensitivity of EIA on urine was significantly poorer than that on cervical specimens (54% vs 85%, p less than 0.001). The specificity of EIA ranged between 94 and 100%. Our study suggests that it may be worth using FVU in a trial for the diagnosis of genital chlamydial infections in symptomatic men, but not in symptomatic women.

Characterization of glycoproteins from Chlamydia trachomatis using lectins.

Glycoproteins in Chlamydia trachomatis, serotype L1, elementary bodies were studied by lectin blotting. A panel of 23 lectins representing a variety of sugar specificities was used. The pattern of lectin-binding specificities at a peptide band was studied in order to determine the type and structure of its glycoconjugate. To establish chlamydial origin of the glycopeptide bands in the blot, control samples from non-infected host cell membranes were run in parallel. Terminal mannosidic structures were demonstrated in a 72 kDa glycopeptide (gp72) by its selective binding of Galanthus nivalis lectin (GNA). Sialic acids were found in two chlamydial glycopeptides, gp40 and gp64, which appear to carry O-linked glycoconjugates as they bound the peanut agglutinin (PNA, both gp40 and gp64) and jackfruit lectin (Jac, only gp40). Such structures were also present in other chlamydial glycopeptides. Lectins with specificities for fucose in different links, galactose and N-acetyl glucosamine bound to several chlamydial peptides. On the basis of our results we suggest an alternative mechanism for uptake of chlamydial elementary bodies into host cells, namely phagocytosis mediated by eukaryotic cell surface lectins.

Age-dependent antichlamydial activity of human saliva. A study of infants, children and adults.

Saliva samples from 51 children ranging from 1 month to 12 years of age were studied for their effect on the capacity of Chlamydia trachomatis, serotypes I and L2 elementary bodies (EB), to form inclusions in cycloheximide-treated McCoy cell cultures. The results were compared to those of tests using saliva from adults. No antibodies against C. trachomatis or Chlamydia pneumoniae could be detected by microimmunofluorescence tests in either group. Saliva of children between 1-4 years of age showed an age-related decrease in the chlamydial inclusion count (i.c.). Saliva from children older than 4 years of age, like saliva from healthy adults, showed a pronounced reduction of the i.c. (up to 70%). The study indicates that children between 1-4 years gradually develop a natural antichlamydial activity against C. trachomatis, and above that age they exhibit the same level of antichlamydial activity as adults. The inhibitory activity was heat-resistant.

Culture, ELISA and immunofluorescence tests for the diagnosis of conjunctivitis caused by Chlamydia trachomatis in neonates and adults.

The relative value of culture, direct specimen antigen detection tests, i.e., enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) tests in the diagnosis of Chlamydia trachomatis infection was studied in 125 newborns and 121 adults with signs of conjunctivitis. Eye and nasopharyngeal samples were tested by culture using cycloheximide-treated or irradiated McCoy cells, ELISA (i.e., Chlamydiazyme, Abbott) and IF tests (i.e., Chlamyset, Orion and MicroTrak, Syva). Of the neonates, 70 (35 boys and 35 girls) and 54 (33 males and 21 females) of the adults were positive in one or both eyes in one or more tests: 191 (39%) in cultures, 173 (35%) in ELISA and 160 (33%), 176 (36%) in each of the IF tests. Using culture as standard reference, the sensitivities of ELISA and the IF tests were 88%, 81% and 87%, while the corresponding specificities were 99%, 98% and 97%, respectively. The predictive values for a negative test (PVN) were 93%, 89% and 92% and for a positive test (PVP) 98%, 96% and 94%. Of the 124 cases chlamydia-positive in the eyes, 67 (54%), 76 (61%), 64 (52%) and 70 (57%) were positive in nasopharyngeal samples in one or more of culture, ELISA and the two IF tests, respectively. The sensitivities of ELISA and the IF tests in nasopharyngeal samples were 87%, 78% and 81%, while the corresponding specificities were 90%, 93% and 91%, respectively. The predictive values for a negative (PVN) test were 95%, 92% and 93%, and for a positive test (PVP) 76%, 81%, and 77%. Nasopharyngeal swabs were more often positive in cases with 2 or more weeks' duration of symptoms than in those with shorter duration.

Antichlamydial activity of saliva.

Saliva collected from adults with no antichlamydial antibodies in their serum or saliva, was tested for its capacity to inhibit the formation of inclusions of Chlamydia trachomatis in McCoy cell cultures. Pooled saliva, diluted in tissue culture medium and sterilized by filtration, was found to reduce the inclusion count by up to about 40%. However, the pretreatment of the chlamydial organisms for 2 hours with diluted saliva caused a 75% decrease in the number of inclusions. The inhibitory activity, which was concentration-dependent, seems to affect the attachment of the chlamydial elementary body to the host cell by acting on both the chlamydiae and the McCoy cells. Saliva did not seem to affect the intracellular development of the chlamydiae. The inhibitory activity was not affected by trypsin treatment, while absorbtion with a gel of a chelating agent caused total loss of the antichlamydial effect. The purpose of our study was to test saliva for its possible antichlamydial activity and to partially characterize the active principle.

Influence of whole human milk, and fractions thereof, on inclusion-formation of Chlamydia trachomatis in McCoy cells.

A study of the effect of human breast milk, and components thereof, on the capacity of Chlamydia trachomatis to form inclusions in cycloheximide-treated McCoy cells, was undertaken. Pooled whole milk collected during the first week of breast feeding caused a concentration-dependent inhibition of the chlamydial inclusion-formation. The activity resided in the fat and fat globule membrane (FGM) components of the milk. The active principle in the FGM fraction is heat-stable and pronase-sensitive, but resistant to both neuraminidase and periodate. Immunoglobulins was not responsible for the inhibition. Whey and casein fractions of milk increased the chlamydial inclusion-formation. The activity of the whey was heat-stable, dose-related, and had a mol. wt. of greater than or equal to 12,000. The casein fraction was still active after heat treatment. Whey samples collected up to 28 days after delivery varied slightly in their stimulatory activity, with an optimum between the 7th and 14th days. The present study demonstrated a multieffect of breast milk on chlamydial inclusion-formation: an inhibitory activity due to a protein compound as well as another factor in the fat fraction and an enhancing effect due to a heat-stable factor(s).

Binding of Galanthus nivalis lectin to Chlamydia trachomatis and inhibition of in vitro infection.

A glycoprotein present in Chlamydia trachomatis, serotype L1, elementary bodies (EBs) was earlier found to bind the lectin from Galanthus nivalis (GNA). In the present paper we investigate the interaction of GNA with chlamydial EBs and its effect on in vitro infectivity. The binding affinity was studied with 125I-GNA lectin. Within 15 min about 80% maximal binding was obtained. The chlamydia-GNA interaction was inhibited by alpha-methylmannoside, causing a decrease of about 50% at 1 mM. Curve fit analyses indicated two types of binding sites for GNA on the EBs. The affinity to these differed by a factor of 15. The influence of the lectin on the ability of C. trachomatis to infect McCoy cells was also investigated. There was a GNA-dependent inhibition with a 50% reduction in the number of intracellular inclusions at 0.2 microM of the lectin. The findings indicate the presence of terminal mannose structures on the chlamydial surface at or in the proximity of the cell-binding domains. Mannose-binding proteins of eukaryotic cells could be important for the initial uptake of EBs.

In vitro effect of fibrinogen on Candida albicans germ tube formation.

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.

Antibodies to microbial, leukocyte and organ antigens.

Hemagglutinating antibodies to Mycoplasma hominis were present in 30 of 83 infertile, 15 of 40 pregnant and 5 of 20 post-partum females and 20 of 82 infertile males in contrast to only 2 of 21 fertile females and 5 of 25 fertile males. Their presence correlated with sperm antibody detection by TAT in Lab. 4, the immunobead-binding assay of Lab. 1 and the SIT of Lab. 11, but not with other sperm antibody assays. Immunofluorescent antibodies to Chlamydia trachomatis, on the other hand, did not correlate with the incidence of sperm antibodies. Among 305 serum samples tested, 12 were positive for testicular antibodies, 8 had antibodies to kidney, 7 to ovary and 15 to endometrium. A majority of serum samples positive for antibodies to testis and ovary, but not endometrium, reacted against sperm in different assays. Eight of 135 samples tested had antibodies to human leukocyte antigenic HLA-Aw19 (Aw19, A28, A29, A30 and A32) and/or B35 (B35, B5 and B15) complexes. Six of these samples were also positive for sperm antibodies by one or more antibody assays. Cross-reactive antigens may be present in sperm, M. hominis, testis, ovary and leukocytes.

Diagnosis of bacteraemia by automated head-space capillary gas chromatography.

Blood cultures from 196 patients with suspected bacteraemia or septicaemia were analysed by automated head-space gas chromatography, using a 25 m fused silica capillary column, when turbidity indicated growth. Gas chromatography correctly identified 105 cultures as positive and 71 correctly as negative. No false-positive results were obtained. Of the 20 false-negative chromatographic results, Staphylococcus spp accounted for 14. Automated head-space gas chromatography is quicker, easier and more efficient than other gas chromatographic techniques for the evaluation of blood cultures.

Chlamydia screening--yes, but of whom, when, by whom, and with what?

The importance of screening programs in reducing the prevalence of genital chlamydial infections is stressed by the fact that the majority of infected persons are more or less asymptomatic. The use of oral contraceptives may mask infections affecting the upper genital tract. This imposes selective screening and rescreening of women with a history of pelvic inflammatory disease. The recent knowledge that vaginal introital samples will provide a detection rate equal to or even higher than that of cervical samples collected in the same women opens up the possibility of screening women in health units lacking a gynecological examination chair. It also opens up the possibility of outpatient screening programs, for example, home sampling and mailing samples to laboratories that will perform analyses. The use of nucleic acid-based assays means increased sensitivity and specificity compared with earlier used techniques such as ELISA. These former methods can also be used in low-prevalence populations with acceptable positive predictable value, but may be misleading if used in post-therapy check-ups because the antigen may persist in microbiologically cured cases.

Chorioamnionitis and the isolation of microorganisms from the placenta.

Specimens for the isolation of microorganisms were obtained from the cervical canal in 83 and from the placenta in 86 of 90 women delivered by cesarean section. In addition, the placentas from 87 of these women were examined histopathologically. No correlation was found between the isolation of a given microorganism from the cervical canal and infectious disease in mothers and/or neonates. Isolation of microorganisms from the placenta was correlated with rupture of the membranes occurring six or more hours before surgery and, in women with intact fetal membranes, was correlated with uterine activity. Chorioamnionitis histopathologically was associated with maternal and/or neonatal infections and prematurity. Microorganisms were not isolated significantly more often from the placenta in cases with chorioamnionitis than without. The value of cervical and placental cultures in predicting intrauterine infection is questionable.

Chlamydia trachomatis cervicitis in gynecologic outpatients.

Symptoms suggestive of a lower genital tract infection (LGTI) are common complaints in women who consult gynecologists. Sexually transmitted microorganisms, such as Chlamydia trachomatis. Neisseria gonorrhoeae, and Trichomonas vaginalis, are responsible for a substantial proportion of LGTI. This study was performed to establish the frequency of LGTI caused by C trachomatis in women attending a gynecologic outpatient clinic. Of 170 women with LGTI, 32.9% harbored one or more of these organisms: C trachomatis was found in 19.3%, N gonorrhoeae in 4.7%, and T vaginalis in 25.9%. The results of the isolation studies were correlated with clinical signs and symptoms. In women under 25 years of age, chlamydial cervicitis was found significantly more often in users of oral contraceptives than in nonusers. In women not taking such drugs, cervical erosion was found more often in Chlamydia-positive than Chlamydia-negative women. Since clinical examination failed to reveal any characteristic signs in cases of chlamydial infection, it was not possible to distinguish a chlamydial from a gonococcal infection. This study also reports the successful treatment of 15 women affected by chlamydial cervicitis with doxycycline or trimethoprim sulphamethoxazole.

Pelvic infection after elective abortion associated with Chlamydia trachomatis.

Two groups in Denmark and Sweden, totalling 943 healthy women in the first trimester of pregnancy who were undergoing elective abortion, were examined in a prospective study. Group 1 consisted of 432 women from Denmark, all examined prior to and 2 weeks after the operation. At the first examination, samples were taken from the cervix for the isolation of chlamydiae and gonococci. Chlamydia trachomatis was isolated from 23 (5.3%) of the women. Postoperatively, 15 patients developed acute pelvic inflammatory disease (PID). Five of these patients harbored C trachomatis before the operation, and all 5 developed a significant change in the titer of immunoglobulin G microimmunofluorescence antibodies to the organism. In group 2, 25 (4.9%) of the 511 women from Sweden harbored C trachomatis before the intervention. Patients with chlamydiae were treated, before or in conjunction with the operation, with antibiotics active against chlamydiae. None of the women with cultures positive for chlamydiae showed signs of PID postoperatively. The difference in the postoperative frequency of PID in chlamydia-positive patients between groups 1 and 2 was significant (2P = .0393, Fisher exact test). Gonorrhea was diagnosed in 1% of the women in both groups. Only 1 of these patients developed PID postoperatively, but no pili antibodies to Neisseria gonorrhoeae were found during the course of the disease. It is concluded that women applying for abortion should be examined and treated not only for gonorrhea but also for infection with C trachomatis either before or, at the latest, in conjunction with the abortion.