Evaluation of proposed cytomorphologic criteria for the diagnosis of Chlamydia trachomatis in Papanicolaou smears

To evaluate the proposed cytomorphologic criteria for the cervical cytologic diagnosis of Chlamydia trachomatis infection, a study was made of 171 endocervical smears. All cytomorphologic elements that could be ascribed to Chlamydia trachomatis infection were correlated with the diagnostic confirmation of this microorganism by monoclonal antibody (MAb) staining. The presence of Chlamydia trachomatis was detected in 21 samples (12.28%) by MAb staining. Comparing the cytomorphologic results with the MAb results, the sensitivities and specificities of the Papanicolaou smear diagnoses were 19% and 86% using the cytologic criteria proposed by Gupta and coworkers, 38% and 87% using the criteria proposed by Kiviat and coworkers and 23% and 91% using the criteria proposed by Shiina. In view of (1) its low sensitivity, (2) the subjective elements and individual variations in the proposed cytologic criteria, (3) the similarity with Trichomonas vaginalis-produced exudates and (4) the implications of a misdiagnosis of a sexually transmitted disease, it is concluded that cervical cytology is not useful for ascertaining the presence of Chlamydia trachomatis.     

Chlamydia trachomatis infections in asymptomatic women. Results of a study employing different staining techniques

A total of 300 cervical smears randomly collected from asymptomatic women in a mass-screening program for the detection of cervical carcinoma was investigated for Chlamydia trachomatis infection by the use of Papanicolaou and immunofluorescence staining. Features of chlamydial infection detected in 18 cases by Papanicolaou-stained smears were confirmed in 11 cases with immunofluorescence; not a single case that was negative in the Papanicolaou-stained smears was positive by immunofluorescence. The presence of Chlamydia in the Papanicolaou-stained smears in ten cases, including two cases that were negative by immunofluorescence, was also proven by either immunoperoxidase staining or in situ hybridization. On the other hand, either immunoperoxidase or in situ hybridization gave false-negative results in two of the ten cases. Therefore, the combined use of different techniques demonstrated that false-negative results occurred with all techniques, except with Papanicolaou-stained smears, whose sensitivity is apparently the highest.     

Prevalence of chlamydial genital infection and associated risk factors in adolescent females at an urban reproductive health care center in Croatia

The study was undertaken to determine the prevalence of chlamydial genital infection in sexually active, urban adolescent females 15-19 years; to identify behavioral, demographic, and clinical factors associated with chlamydial infections; and to develop criteria for potential screening strategies. 500 adolescent women, median age 17.7 years, who visited gynecological outpatient clinic in Children's Hospital Zagreb for different reasons were enrolled in this study. Gynecological exam, colposcopy, detection of chlamydial infection by the rapid direct immunoassay of endocervical swab (Clearview Chlamydia-Unipath), endocervical cytological examination--Papanicolaou smear, and questionnaire to obtain demographic, social, behavioral and presence of symptoms data were performed. Positive Chlamydia trachomatis test were found in 16.4% of participants, cytologic cervical abnormalities--cervical intraepithelial neoplasia (CIN I-CIN III) were found in 25.2% and cytological signs of Human papilloma virus were found in 11.4%. Stepwise multivariate logistic regression analysis identified five factors associated with infection: the age of menarche < or =13 years, > or =4 lifetime sexual partners, non-use of contraception (rare or never), cervical friability, and abnormal Papanicolaou test. Urban adolescent sexually active women are at high risk for chlamydial infection and other sexually transmitted diseases including HIV infection. Association between chlamydial genital infection and risk-taking sexual and contraceptive behavior was found. Routine Chlamydia trachomatis testing for this population is recommended as well as implementation of school based sexual health education because of their risk-taking sexual behavior.     

Detection of Chlamydia trachomatis in Papanicolaou-stained cervical smears by an indirect immunoperoxidase method

A two-step (indirect) immunoperoxidase method directed against Chlamydia trachomatis was developed. The method was then used to evaluate the specificity of cytologic changes suggestive of C. trachomatis in Papanicolaou smears of cervical specimens from women who were culture-negative for the organism. Positive immunoperoxidase staining was detected in 9 of 21 cases (43%) tested. Technical problems, especially background staining, precluded interpretation in the remainder of the cases. Cervical cytology, as demonstrated by immunoperoxidase staining, may, in some instances, be more sensitive than the culture. However, because the etiology of cytologic changes not specifically identified by immunoperoxidase staining may be due to other organisms or factors, immunoperoxidase procedures, as described, should not replace culture for confirmation of cytologic findings suggestive of C. trachomatis.     

Cytomorphologic and immunocytochemical studies of chlamydial infections in cervical smears

A total of 872 cells in 183 Papanicolaou-stained cervical smears morphologically suspected of harboring chlamydial infections were cytologically investigated in an attempt to differentiate the morphologic features of chlamydial infection from those of mucus vacuoles or bacterial infection. The observed inclusions were classified according to their morphologic appearance and their staining by a Chlamydia-specific peroxidase-antiperoxidase stain and by the periodic acid-Schiff technique. Immunoperoxidase-positive inclusions were detected in 201 cells from 13 cases; 200 (99.5%) of these cells contained "nebular" inclusions while 1 cell (0.5%) contained multiple inclusions with homogeneous central target formation. These findings suggest that nebular-type inclusions may be the key morphologic finding for the identification of chlamydial infection and that the application of an immunoperoxidase staining technique on the destained Papanicolaou preparation may be useful for the diagnosis of equivocal inclusions.     

Infantile chlamydial conjunctivitis. A comparison of Papanicolaou, Giemsa and immunoperoxidase staining methods

The use of conjunctival smears to diagnose infantile Chlamydia trachomatis infection increased sixteen-fold in our hospital between the years 1979 and 1984. The present study was conducted to compare Papanicolaou and Giemsa staining methods with the avidin-biotin technique of immunostaining utilizing a highly specific antichlamydial monoclonal antibody. On retrospective review of 33 patients, chlamydial infection was diagnosed in 61% of the Papanicolaou-stained and 64% of the Giemsa-stained slides. After the Papanicolaou-stained slides were destained and immunostained with the antichlamydial antibody, round particles corresponding in size to elementary and reticulate bodies were readily seen in 79% of the cases. In comparison with the immunoperoxidase method, the sensitivity and specificity of Papanicolaou staining were 73% and 86%, respectively, while the corresponding figures for Giemsa staining were 77% and 100%, respectively. The study established the applicability of the immunoperoxidase method to this clinical condition, confirmed the accuracy of diagnoses with routine stains and highlighted the increasing incidence of chlamydial conjunctivitis in our hospital population.     

Detection of Chlamydia trachomatis in pregnant women by the Papanicolaou technique, enzyme immunoassay and polymerase chain reaction

OBJECTIVE: To compare Papanicolaou staining, enzyme immunoassay (EIA) and the polymerase chain reaction (PCR) techniques for detecting Chlamydia trachomatis in pregnant women. STUDY DESIGN: Endocervical specimens were taken randomly from 125 pregnant women with or without symptoms. These women attended their first medical consultation at the Regional General Ignacio Zaragoza Hospital. Samples were analyzed for detection of C trachomatis. When results differed between tests, specimens were evaluated by direct immunofluorescence staining. RESULTS: The prevalence of chlamydial infection was 2.4%. The characteristics of patients positive for Chlamydia were: average age, 24 years; first sexual encounter at age 21 years, one partner and six to nine months of gestation. The sensitivity, specificity, accuracy, positive predictive values and negative predictive values were 100%, 99.18%, 99.20%, 75% and 100%, respectively, for Papanicolaou staining; 100%, 92.62%, 92%, 25% and 100% for EIA; and 100%, 100%, 100% and 100% for PCR. CONCLUSION: Both Papanicolaou staining and PCR were adequate for diagnosis of C trachomatis infection. EIA was not reliable and therefore is not recommended for use as a diagnostic technique in a pregnant population with low risk and low prevalence.     

Association of human papillomavirus and Chlamydia infections with incidence cervical neoplasia

Incidence cervical neoplasia is defined as disease that becomes manifest during a given period of observation. Association with preceding genital infections having characteristic cytologic findings would seem to be more likely for incidence than for prevalence cases since the usual long latency period of carcinoma in situ (CIS) could allow resolution of infectious processes. For this reason, it was elected to examine the preceding Papanicolaou smears from patients with tissue-confirmed incidence CIS or invasive epidermoid carcinoma. There were 67 women with biopsy-proven CIS or invasive carcinoma of the uterine cervix identified in the files of the University of New Mexico Cytopathology Laboratory from 1966 to 1982 who had two initial negative smears as well as smears at intervals of three years or less. All cytologic smears prior to tissue diagnosis were rescreened for confirmation of cytologic atypia or its absence as well as for morphologic evidence of human papillomavirus (HPV) or chlamydial infections. Control cases matched for age, gravidity, ethnicity and number of smears were reviewed in an identical manner. Koilocytes indicative of HPV infection were found in 17 index cases (25%) and 5 controls (7%) (p = 0.005). Chlamydial infections were identified in 18 index cases (27%) and in 4 controls (6%) (p = 0.001). The times required for conversion from smear negativity to malignancy were determined for each incidence case. The results showed great variability but suggest that the progression to malignancy is not hastened in women with antecedent HPV or chlamydial infections. Our results indicate that the presence of koilocytes and/or chlamydial inclusions in cervical smears serves to identify a group of women with a significantly increased risk of developing cervical carcinoma, even in the absence of concurrent dysplasia.     

Sensitivity and specificity of the Papanicolaou-stained cervical smear in the diagnosis of Chlamydia trachomatis infection

Two hundred young women had simultaneously prepared cultures for Chlamydia trachomatis and cervical smears; they also completed a questionnaire. Twelve of the chlamydial cultures were positive. There was poor correlation between the culture results and the cytologic morphology or symptoms. On initial blind reading, only 10% of the smears cytologically interpreted as positive were actually positive by culture. Under the most favorable (non-blind) interpretation, only 23% of the smears cytologically interpreted as positive for chlamydial infection were also culture positive. Because of the high incidence of false positives, we conclude that routine cytologic examination of Papanicolaou-stained smears is not an acceptable method for the diagnosis of chlamydial infections of the cervix. Immunoperoxidase staining of duplicate smears did not appear to be a successful replacement for culture.     

Detection of sexually transmitted diseases by urethral cytology, the ignored male counterpart of cervical cytology

The detection of sexually transmitted diseases by urethral cytology was investigated in 270 men examined by urethral swabbing smears. Each sample was used to prepare a wet mount smear and smears for staining by the Papanicolaou, Gram and methylene blue techniques. A fifth smear was used for direct staining with fluorescein-conjugated monoclonal antibodies for the detection of Chlamydia trachomatis. The smears were examined for cytoplasmic and nuclear changes as well as for pathogenic organisms and inflammatory changes. Infections with Chlamydia trachomatis, Neisseria gonorrhoeae and human papillomavirus (HPV) produced distinctive cytologic patterns similar to those seen in cervicovaginal smears from women. The patterns in candidiasis, trichomoniasis and herpes simplex virus infection were not as diagnostic. Particularly noteworthy were the nuclear alterations, which appeared to be proplastic in HPV infection but retroplastic in Chlamydia infection. The results of this study indicate that urethral cytology would be an invaluable addition in diagnosing sexually transmitted diseases in men, particularly in the case of Chlamydia and HPV infections. The monomorphic structure of urethral columnar epithelium, as compared to the cervical epithelium, seems to result in a clearer and more constant response to pathogenic infections, as seen in the resulting smears.     

Chlamydia trachomatis infection of human fallopian tube organ cultures

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.     

Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.     

A proposed mouse model for acute epididymitis provoked by genital serovar E, Chlamydia trachomatis

This study was conducted to determine the efficacy of the male mouse as a model for epididymitis caused by human genital serovar E, Chlamydia trachomatis. C. trachomatis was reisolated from all tissues removed on Days 3, 5, and 7 post inoculation (pi). Although some infected epididymides removed on Days 10, 14, and 21 pi were positive, control tissues remained negative. Histopathology of tissues showed a heavy, mixed inflammatory infiltrate consisting of polymorphonuclear cells and lymphocytes. Serum antibody to C. trachomatis was detected in the infected mice only (titer greater than or equal to 1:32). Chlamydial inclusions and individual elementary bodies were confirmed by immunofluorescent and immunoperoxidase staining up to Day 7 pi. These data show that the male mouse is susceptible to C. trachomatis infection and is appropriate for studies dealing with the effect of C. trachomatis on male fertility.     

Fine needle aspiration cytodiagnosis of nasopharyngeal carcinoma in cervical lymph nodes. A study of 40 cases

The cytologic features of fine needle aspiration (FNA) samples of 40 metastatic nasopharyngeal carcinomas in cervical lymph nodes were reviewed. FNA was performed with 21-gauge or 23-gauge needles; the FNA smears were stained with the Papanicolaou stain or with hematoxylin and eosin. Several typical cytologic features were noted. All cases showed the presence of clusters of cohesive tumor cells, most of which were undifferentiated. Medium-sized oval vesicular nuclei were present in 85% of the cases; all cases had prominent nucleoli. The cytoplasm was generally pale, with ill-defined boundaries in 87.5% of the cases. Mitoses were present in 75% of the cases. Mature lymphocytes were intermingled with tumor cells in all cases. The cytologic features correlated with the histologic features in surgical biopsies of the nasopharynx and lymph nodes. The results show that FNA of cervical lymph nodes can aid in the diagnosis of otherwise occult nasopharyngeal carcinoma and in detecting residual or recurrent nodal disease in patients with nasopharyngeal carcinoma who had undergone treatment.     

Chlamydial infection induces host cytokinesis failure at abscission

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.     

Skin imprint cytology in the diagnosis of cutaneous T-cell lymphomas. A preliminary report

A study was undertaken to evaluate the utility of the skin imprint technique and the Papanicolaou stain in the diagnosis of cutaneous T-cell lymphomas (CTCL), to better characterize cytologic findings in CTCL and to establish criteria for the cytologic diagnosis of CTCL. Differential cell counts of 17 specimens, including 12 cases of CTCL and 5 of benign skin infiltrates, were performed. There appear to be some significant differences between the groups in terms of cellularity, number of cerebriform mononuclear cells and presence of mitotic figures. The skin imprint technique, using Papanicolaou staining, seems to facilitate recognition of diagnostically important cells. The usefulness of skin imprint cytology resides in its potential for diagnosing early lesions of CTCL when diagnosis by other methods may be difficult.     

Flow cytometric analysis of Chlamydia trachomatis interaction with L cells

Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p-formaldehyde, stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50-100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion-forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser-based cytometer and gating on low volume.     

Chlamydia trachomatis infection causes mitotic spindle pole defects independently from its effects on centrosome amplification

Chlamydiae are Gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously infected by human papillomavirus (HPV). The inclusion associates very closely with host cell centrosomes, and this association is dependent upon the host motor protein dynein. We have previously reported that this interaction induces supernumerary centrosomes in infected cells, leading to multipolar mitotic spindles and inhibiting accurate chromosome segregation. Our findings demonstrate that chlamydial infection causes mitotic spindle defects independently of its effects on centrosome amplification. We show that chlamydial infection increases centrosome spread and inhibits the spindle assembly checkpoint delay to disrupt centrosome clustering. These data suggest that chlamydial infection exacerbates the consequences of centrosome amplification by inhibiting the cells' ability to suppress the effects of these defects on mitotic spindle organization. We hypothesize that these combined effects on mitotic spindle architecture identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation.     

Prevalence of Chlamydia trachomatis and oncogenic human papillomavirus types in cytologic atypia of the uterine cervix

A history of having substantial Chlamydia trachomatis exposure as detected by serum antibodies is a cofactor of human papillomavirus (HPV) mediated cervical carcinogenesis. In this study, we examined the concurrent C. trachomatis infections in cytologic atypia of the uterine cervix in order to evaluate the impact of C. trachomatis infection in patients with high risk for cervical intraepithelial neoplasia. Cervical scrapes form 707 patients were subjected to PCR amplification with primer sets for HPV and C. trachomatis. Based on negative beta-globin results, 10 specimens were not eligible for further analysis. Oncogenic HPV types were detected in 278 specimens (39.8%). C. trachomatis was found only in six specimens (0.9%). In conclusion, concurrent C. trachomatis infection was uncommon and hence it was an improbable risk factor in cytologic atypia.