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1.
Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion. 相似文献
2.
Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner 总被引:4,自引:0,他引:4
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Rzomp KA Scholtes LD Briggs BJ Whittaker GR Scidmore MA 《Infection and immunity》2003,71(10):5855-5870
Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion. 相似文献
3.
The chlamydiae are obligate intracellular bacteria that occupy a non-acidified vacuole (the inclusion) during their entire developmental cycle. These bacteria produce a set of proteins (Inc proteins) that localize to the surface of the inclusion within infected cells. Chlamydia trachomatis IncA is also commonly found in long fibers that extend away from the inclusion. We used standard and confocal immunofluorescence microscopy to demonstrate that these fibers extend to newly developed inclusions, termed secondary inclusions, within infected cells. Secondary inclusions observed at early time points postinfection were devoid of chlamydial reticulate bodies. Later in the developmental cycle, secondary inclusions containing variable numbers of reticulate bodies were common. Reticulate bodies were also observed within the IncA-laden fibers connecting primary and secondary inclusions. Quantitative differences in secondary inclusion formation were found among clinical isolates, and these differences were associated with serovar. Isolates of serovar G consistently produced secondary inclusions at the highest frequency (P < 0.0001). Similar quantitative studies demonstrated that secondary inclusion formation was associated with segregation of inclusions to daughter cells following cytokinesis. We conclude that the production of secondary inclusions via IncA-laden fibers allows chlamydiae to generate an expanded intracellular niche in which they can grow and may provide a means for continuous infection within progeny cells following cell division. 相似文献
4.
Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies. 总被引:19,自引:22,他引:19
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We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01). Improved sensitivity was most evident in specimens with low numbers of inclusions. Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture. 相似文献
5.
Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies. 总被引:7,自引:3,他引:4
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Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X. 相似文献
6.
Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture. 总被引:9,自引:14,他引:9
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Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 23 isolates on passage 1 and 13 isolates on passage 2 were detected by Giemsa staining. Immunofluorescence staining was significantly more sensitive than Giemsa staining for detecting chlamydial inclusions, particularly from specimens containing low titers of Chlamydia. 相似文献
7.
Kena A. Swanson Lacey D. Taylor Shaun D. Frank Gail L. Sturdevant Elizabeth R. Fischer John H. Carlson William M. Whitmire Harlan D. Caldwell 《Infection and immunity》2009,77(1):508-516
Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.Chlamydia trachomatis is a mucosotropic obligate intracellular gram-negative pathogen that is a leading cause of sexually transmitted and ocular infections. Infection can result in serious sequelae such as infertility and blindness (54, 56) and an increased risk of human immunodeficiency virus infection and transmission (38). The pathophysiology of chlamydial infection is associated with the pathogen''s propensities to cause persistent infection and to suppress host immunity (3). A vaccine is needed to control chlamydial diseases; however, progress toward this goal will not be forthcoming until more is known about the virulence factors that mediate persistence and immune evasion.Chlamydiae are characterized by a unique biphasic developmental cycle that modulates between an extracellular, metabolically inactive, infectious elementary body (EB) and an intracellular, metabolically active, noninfectious reticulate body (RB) (34). Their obligate intracellular niche and the lack of a tractable genetic system present unique challenges in the study of chlamydial biology and pathogenesis. To overcome these hurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomics have shown considerable homology among various chlamydial species and have provided important insights into shared and species-specific virulence factors (7, 24, 41, 42, 46, 49).The type V or autotransporter (AT) secretion pathway is the most widespread secretion mechanism employed by gram-negative bacteria to deliver virulence factors involved in initiating infection, disease progression, and immune evasion (reviewed in references 11 and 21). AT proteins are characterized by three domains, (i) a signal sequence (SS), (ii) a diverse N-terminal passenger domain (PD) that confers effector function, and (iii) a conserved C-terminal translocator domain (TD). The TD inserts into the outer membrane (OM) by assembling into a β-barrel pore that facilitates PD translocation to the bacterial surface. The PD remains tethered to the TD or is cleaved and either is released or remains noncovalently associated with the OM. Well-characterized examples of ATs found on the bacterial cell surface as monomers or oligomers are Neisseria meningitidis NalP (37) and Helicobacter pylori VacA (31), respectively.C. trachomatis has a nine-member AT family (20), termed polymorphic membrane proteins (Pmps), whose role(s) in chlamydial pathogenesis has yet to be defined. The pmp paralogs (pmpA to pmpI) constitute 3.2% of the ∼1-Mb genome and are found at three chromosomal loci composed of two gene clusters (pmpA to pmpC and pmpE to pmpI) and the genetically isolated gene pmpD (46). Notably, PmpD is the second most highly conserved Pmp, exhibiting 99.2% amino acid identity among C. trachomatis serovars (16). Despite relatively low abundance in the chlamydial OM, Pmps are major immunogens and may be important virulence factors (29). C. trachomatis PmpD is a target of broadly cross-reactive neutralizing antibodies (Abs), which makes it an attractive vaccine candidate for the prevention of human infections (10).Previous reports have described proteolytic processing of C. pneumoniae and C. trachomatis PmpD (25, 52). Furthermore, recombinant C. pneumoniae PmpD has been suggested to function as an adhesin capable of inducing proinflammatory cytokine production (35, 52). Nothing is known about the native structure of C. trachomatis PmpD or the potential significance of its structure to chlamydial pathogenesis. Here we show that C. trachomatis PmpD is present on the organism''s surface as an oligomer with a higher-order flower-like structure. Moreover, we describe novel infection-dependent proteolytic processing of PmpD that produces soluble fragments with predicted eukaryotic motifs, implying a multifunctional protein important to chlamydial pathogenesis. 相似文献
8.
D M Magee J G Smith C A Bleicker C J Carter L F Bonewald J Schachter D M Williams 《Infection and immunity》1992,60(3):1217-1220
Cytokine induction during Chlamydia trachomatis pneumonia may alter the pathogenesis or course of disease. We examined interleukin-1 (IL-1) and IL-6 production by measuring mRNA and bioactivity in murine lungs. mRNA and bioactivity for IL-1 alpha, IL-1 beta, and IL-6 increased after Chlamydia infection. These cytokines may be important in regulating host defenses against C. trachomatis. 相似文献
9.
Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies.
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A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous. 相似文献
10.
Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation
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Pannekoek Y van der Ende A Eijk PP van Marle J de Witte MA Ossewaarde JM van den Brule AJ Morré SA Dankert J 《Infection and immunity》2001,69(7):4654-4656
To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype. 相似文献
11.
12.
Eighty six women were studied at the Hospital Chiquinquirá in order to determine the role of Chlamydia Trachomatis in infertility. Endocervical samples were taken in 45 infertile women (IG) who concurred to our Gynecologic Endocrinology Clinic and in 41 (CG) who sought medical attention at our General Gynecology Clinic for different reasons. The Chlamydiazyme Test (CT) was performed in all of them. We obtained 12 positive Chlamydiazymes (13.95%), four of them were in the IG (8.89%) and 8 (19.5%) in the CG; this was not statistically significant (p = 0.3). We also found no correlations when positive tests were correlated with sterility (p = 0.5, tube pathology history (p = 0.2), HSG (p = 0.2) and laparoscopic findings (p = 0.1). We did not find an increase in the occurrence of CT in our infertile women as reported by other authors therefore its significance in our study group at this time is uncertain. 相似文献
13.
The Rab genes have recently been cloned and sequenced in mammals, and their products represent good candidates for low molecular weight guanosine triphosphate-binding proteins involved in the regulation of intracellular transport of vesicles in higher eukaryotes. Remarkably, each of the Rab proteins appears to be associated with a distinct step of either the exocytic or endocytic pathway. In particular, Rab6p has been localized to the outermost Golgi cisternae in normal rat kidney cells, where its function remains unclear. In this work, we have carried out a series of immunocytochemical analyses of the subcellular distribution of Rab6p in a polarized cell, the electrocyte of Torpedo marmorata, to elucidate the molecular mechanisms involved in the sorting and targeting of synaptic proteins. We report that, aside from its Golgi localization, the bulk of Rab6p associates with clusters of post-Golgi vesicles, primarily those located at the cytoplasmic face of the innervated membrane of the electrocyte. Consequently, Rab6p presents a polarized distribution in this cell. Furthermore, we show that this distribution is dependent upon the integrity of the microtubule network of the electrocyte. These data are coherent with the notion that Rab6p is involved in the regulation of membrane traffic from the trans-Golgi network to the innervated plasma membrane, delivering, by way of a microtubule-based organelle transport mechanism, synaptic proteins to their appropriate locations. 相似文献
14.
Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane 总被引:3,自引:0,他引:3
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The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein. 相似文献
15.
目的 将原核表达纯化的衣原体噬菌体phiCPG1衣壳蛋白Vp1作用于沙眼衣原体(Chlamydia trachomatis,Ct),以期发现该Vp1蛋白对Ct生长的影响.方法 原核表达并纯化噬菌体phiCPG1的衣壳蛋白Vp1,将纯化后的蛋白复性后与Ct的标准株或临床野生株(终浓度为53μg/ml)室温孵育3h后接种至单层致密McCoy细胞中,培养过程中均加入了了 Vp1蛋白,48 h后碘染色包涵体计数和透射电镜观察Vp1蛋白对Ct生长的影响;MTT法检测Vp1蛋白对McCoy细胞系的细胞毒性作用;液体培养基稀释法检测Vp1蛋白对大肠杆菌BL21和DH5α的抗菌作用.结果 衣壳蛋白Vp1对Q标准株E和D型的抑制率分别为78%和72%,对Ct临床野生株的抑制率为40%~70%,透射电镜结果显示Vp1蛋白能够抑制Ct的正常发育,使包涵体内出现异常增大的网状体.而该Vp1蛋白对非衣原体的其他生物体如大肠杆菌BL21、DH5α和真核细胞McCoy的生长没有影响.结论 噬菌体衣壳蛋白Vp1对Ct的生长具有明显的抑制作用,为临床上Ct感染的治疗提供了新的思路. 相似文献
16.
J. L. Haworth T. R. Moss S. Riddington 《The British journal of general practice》1982,32(242):562-563
A case of Chlamydia trachomatis diagnosed in general practice is described. It is argued that, in the future, the culture methods used in this study, together with the application of at risk criteria, could identify in general practice most patients suffering from chlamydial disease. 相似文献
17.
Mpiga P Mansour S Morisset R Beaulieu R Ravaoarinoro M 《Scandinavian journal of immunology》2006,63(3):199-207
Chlamydia trachomatis, an intracellular obligate bacterium, remains responsible for a large spectrum of disorders that can progress to chronic diseases, resulting in severe sequelae, such as tubal infertility and blindness. These sequelae may be due to deleterious immune responses induced by repeated or persistent infections. By initiating and regulating inflammation as well as immune responses, pro-inflammatory cytokines secreted by local infected epithelial and immune cells, such as monocytes, may play an essential role in immunity and in the immunopathogenesis of chlamydial diseases. In this study, we mimicked the in vivo interaction between epithelial cells and monocytes by co-culturing epithelial-like HeLa cells with monocyte-like THP-1 cells. Pro-inflammatory cytokines [interleukin-beta (IL-1beta), IL-6, IL-8, IL-10, IL-12p70 and tumour necrosis factor-alpha (TNF-alpha)] were measured by multiplexed cytometric bead array assay over a period of 18 days. We observed that pro-inflammatory cytokine secretion was augmented after C. trachomatis infection in HeLa and THP-1 cells. However, this heightened secretion was subsequently reduced. When infected HeLa cells were co-cultured with THP-1 cells, IL-6 and IL-8 secretion was sustained, IL-1beta expression followed a bell-shaped curve and IL-10, IL-12p70 and TNF-alpha synthesis was down regulated. IL-6 and IL-8 may be involved in the immunopathogenesis of chronic chlamydial infections. We also observed that throughout C. trachomatis persistence induced by doxycycline (Dox) treatment, IL-1beta, IL-6, IL-8 and TNF-alpha expression was reduced, whereas the synthesis of IL-10 and IL-12p70 remained unchanged but not sustained. Thus, during chlamydial persistence infection evoked by treatment with Dox, none of the tested cytokines showed sustained expression. 相似文献
18.
A Role for Interleukin-6 in Host Defense against Murine Chlamydia trachomatis Infection 总被引:3,自引:0,他引:3
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Dwight M. Williams Barry G. Grubbs Toni Darville Kathleen Kelly Roger G. Rank 《Infection and immunity》1998,66(9):4564-4567
Interleukin-6-deficient (IL-6−/−) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F2/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6−/− mice. IL-6 is needed for an optimal early host response to this infection. 相似文献
19.
Vesicles containing Chlamydia trachomatis serovar L2 remain above pH 6 within HEC-1B cells. 总被引:2,自引:1,他引:1
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Chlamydia trachomatis serovar L2 is an obligate intracellular bacterium which is internalized in target epithelial cells by endocytosis and resides within a membrane-bound vesicle. Over the next several hours following entry, individual serovar L2-containing vesicles fuse with one another to form a single membrane-bound vesicle (or inclusion) within which the microcolony develops. The experiments reported here directly examined the pH of vesicles containing chlamydiae. The pH was determined by measuring emission ratios of the fluorescent, pH-sensitive probe SNAFL (5-[and 6-]-carboxyseminaphthofluorescein-1, succinimidyl ester) conjugated to chlamydiae. The pH remained above 6.0 at 2, 4, and 12 h after infection, while the pH of vesicles contained heat-killed organisms fell 5.3. In the presence of amines, which raise the pH of acidic compartments, C. trachomatis inclusion formation was unaffected. Inactivation of Na+,K+ -ATPases, the ion pumps responsible for maintaining a pH above 6 within early endocytic vesicles, inhibited the growth of C. trachomatis within epithelial cells. Preventing vesicular acidification by inhibiting the vacuolar proton ATPase did not affect chlamydial growth. Thus, chlamydiae do not reside within highly acidic vesicles and avoid the pathway leading to lysosomes. 相似文献
20.
《Indian journal of medical microbiology》2010,28(1):45-47
Chlamydia trachomatis is considered a major aetiological agent of conjunctivitis in newborns. The objective of the present study was to determine the aetiology of neonatal conjunctivitis and clinico-epidemiological correlates of chlamydial ophthalmia neonatorum. Fifty-eight newborns with signs and symptoms of conjunctivitis were studied. Conjunctival specimens were subjected to Gram staining, routine bacteriological culture, culture for Neisseria gonorrhoeae and direct fluorescent antibody (DFA) staining for diagnosis of C. trachomatis infection. C. trachomatis was detected in 18 (31%) neonates. Findings suggest that since C. trachomatis is the most common cause of neonatal conjunctivitis, routine screening and treatment of genital C. trachomatis infection in pregnant women and early diagnosis and treatment of neonatal Chlamydial conjunctivitis may be considered for its prevention and control. 相似文献