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Objective:To establish a new molecular typing method for Treponema pallidum (TP) based on TP0136 protein sequence heterogeneity. Methods:The amino acid sequences of TP0136 open reading frame (ORF) of 9 strains of Treponema pallidum ssp. Pallidum (TPA) , 3 strains of Treponema pallidum ssp. Pertenue (TPE) , 1 unclassified simian strain of Treponema Fribourg-Blanc (FB) and 1 strain of Treponema pallidum ssp. Endemicum (TEN) were searched from Genbank, and multiple sequence comparisons were performed to obtain the molecular typing results of TP0136 protein. The TP0136 protein-based molecular typing method was used to classify 23 TPA clinical isolates, which were collected from Dermatology Hospital of Southern Medical University from January 2015 to December 2018, and the typing results were compared with those by the traditional typing method based on the tp0548/Arp/Tpr genes. Results:TP0136 protein was highly heterogeneous in different TP strains. According to the amino acid sequence of TP0136, TPE, FB and TEN strains were divided into 4 subtypes of Ⅰ- Ⅳ, TPA strains were divided into 6 subtypes of Ⅴ-Ⅹ, and TPA clinical strains were classified into 4 subtypes of Ⅶ, Ⅸ, Ⅹ, Ⅺ. Through the traditional typing method described above, 23 TPA clinical strains could be divided into 5 types (13D/d, 14D/f, 14D/g, 15D/f, 16A/e) . By using the TP0136 protein-based typing method combined with traditional typing method, the above clinical strains could be further subdivided into 10 types, and the 14D/f type could be further divided into 3 subtypes by using the TP0136 protein-based typing method.Conclusion:The TP0136 protein-based molecular typing method can be used to distinguish TP species, which is helpful for further improvement of traditional TPA molecular typing.
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Objective To isolate and culture a clinical strain (GDI) of Treponema pallidum (Tp) in Guangdong province,and to investigate the difference in nonsynonymous single nucleotide polymorphisms (nsSNPs) between the GD1 strain and Tp Nichols strain.Methods The GD1 strain was isolated from the hard chancre in a patient with primary syphilis in Guangdong province,and continuously subcultured in the testes of New Zealand white rabbit.The serial subcultivation of GD1 was multi-verified by dark-field microscopy,polymerase chain reaction (PCR) for TP0548 gene,DNA sequencing and genotyping.Meglumine diatrizoate density gradient centrifugation was performed to isolate rabbit tissues and concentrate GD1,and DNA sequencing was used to verify the nsSNPs in the TP0443 and TP0584 genes.Results The GD1 strain was successfully isolated from the lesions of the patient with syphilis,and classified as a subtype f of TP0548.Compared with the American Tp (Nichols strain),there were nsSNP mutations in the GD1 strain.One mutation was located in the TP0443 gene,leading to the the substitution of threonine by alanine at amino acid position 120,and another one was located in the TP0584 gene,which caused a change from alanine to threonine at amino acid position 314.Conclusion The GD1 strain was successfully isolated firstly from the lesions in a patient with syphilis in Guangdong province,and nsSNP mutations were confirmed in the GD 1 strain on the etiology.
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Objective To investigate the expression of TP 0155 mRNA in rabbits with early infection of Treponema pallidum ( T.pallidum ). Methods Three New Zealand white rabbits were subcutaneously injected with T.pallidum Nichols Seattle strains.Each rabbit was inoculated at ten sites with 106 T.pallidum/site.Skin lesions at the primary stage of syphilis were observed at different time points. Biopsy from one of the lesions was obtained from each rabbit every three days for detection of TP 0155 mRNA and house keeping gene TP 0574 mRNA.TP0155 plasmid standard was constructed by molecular cloning technique , and the quantitative PCR was used to continuously detect the expression of TP 0155 mRNA and TP0574 mRNA from lesion at different time points.Kruskal Wallis test and Bonferroni method were used to analyze the data.Results On d6, red papules appeared on the dorsal skins of rabbits ,there were ulcers in the center of the lesions on d19,presenting typical appearance of syphilis chancre.On d24 the scab of ulcer became smaller; on d25 the rabbits showed disseminated secondary syphilis , which became smaller and disappeared on d30.The copy numbers of TP0155 plasmid standards were 7.48 ×109 copies/μL.There were significant differences in expression of both TP 0155 mRNA and TP0574 mRNA at different time points (χ2 =32.756 and 52.344,both P<0.01).The expression levels of TP0155 mRNA and TP0574 mRNA increased in the early stage, and both reached the peak at d15 (both P<0.05), and then rapidly declined. There were significant differences in normalized TP 0155 mRNA ( TP0155 ×1000/TP0574 mRNA ) at different time points(χ2 =19.758,P<0.05),which reached the peak on d24 and d30,respectively (all P<0.05).Conclusion The level of TP0155 mRNA increases with the disappearance of chancre and secondary syphilis lesions , suggesting that TP0155 might be involved in immune escape of T.pallidum.
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Objective To investigate the application of venereal disease research laboratory(VDRL) test and toluidine red unheated serum test(TRUST) in syphilis laboratory diagnosis.Methods Serum,plasma and cerebrospinal fluid (CSF) in syphilis patients and healthy controls were measured by VDRL and TRUST.Results The VDRL detection results in serum and CSF sepcimens were generally higher than the TRUST detection results by 1-2 titers,while in plasma specimen,the VDRL detection results were generally lower than the TRUST detection results by 1-2 titers than TRUST when using plasma specimen.In addition,the VDRL detection in normal control serum and plasma specimens all appeared different degrees of false positive,but in the detection of normal control CSF,the results of TRUST and VDRL were consistent.Conclusion TRUST is more suitable for serum and plasma specimens,and CSF is suitable for the CSF specimen,but not suitable for serum and plasma detection.
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Objective To trace changes in the transcript level of the Treponema pallidum(Tp)protein Tp0751 in skin lesions of a rabbit model of early syphilis. Methods Three New Zealand white rabbits were intracutaneously injected with 0.1 ml of Tp (Nichols Seattle strains)suspensions (107 treponemes/ml)at 10 sites on the shaved back to establish a model of early syphilis. All the rabbits received a single injection with the total amount of treponemes being 107. Then, skin changes at injection sites were observed, and the size of skin rashes was recorded on a daily basis. Skin specimens sized 0.4 cm × 0.4 cm were excised from an injection site and a non-injection site(negative control)separately every 3 days for the detection of Tp0751 and Tp0574 mRNAs. The whole experiment lasted 30 days, and a total of 11 skin biopsies were carried out. Fluorescence-based quantitative PCR was performed to measure the mRNA expressions of Tp0751 and Tp0574 continuously and dynamically during the development of chancre. Results After intracutaneous injection of Tp suspensions, red papules occurred on the back of rabbits on day 6, and reached maximum size on day 19 with the formation of ulcer and chancre. On day 25, disseminated secondary syphilides gradually appeared all over the body surface of the rabbits. The mRNA expression levels of Tp0574 and Tp0751 increased at the early stage, peaked onday 15 (compared with the other time points, all P < 0.05), thereafter rapidly declined, but rose slightly on day 27. The standardized expression level of Tp0751 mRNA increased gradually after day 15, and peaked on day 24 (compared with the other time points, all P < 0.05). Conclusion The transcript level of Tp0751 was high in rabbits at the late stage of Tp clearance when generalized disseminated secondary syphilides had not appeared, suggesting that Tp0751 may be involved in the systemic spread of Tp.
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Objective To assess polymerase chain reaction(PCR)combined with restriction fragment length polymorphism(RFLP)and gene sequencing technologies in the detection and typing of HPV DNA.Methods Tissue specimens were collected from skin diseases and venereal disease in perianal or genitals.PCR was performed with HPV DNA general primers(MY09/11)in tissue samples. Positive fragments of HPV DNA were purified and digested by restriction enzymes.The digested fragments were typed by po]yacrylamide gel electrophoresis(PAGE).The Resultswere verified by direct sequencing.Results In 50 clinical samples there were 35 HPV DNA positive,including 26 from patients with condyloma acuminatum,8 from patients with bowenoid papulosis,and 1 from patients with squamous cell carcinoma.In HPV DNA positive samples,19 were HPV6,3 were HPV11,8 were HPV16,4 were HPV6 and HPV 11,and I was HPV62.Sequencing Resultswere in accordance with the PCR-RFLP Results .Conclusion PCRRFLP method is effective in the detection and typing of HPV DNA.
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Objective To investigate the influence of an anti-integrin α6 monoclonal antibody (GoH3) on the in vitro infection of a human keratinocyte cell line HaCaT with HPV6/11 virus particles (VP). Methods HaCaT cells were infected in vitro with 4 different concentrations of HPV6/11 VP alone, HPV6/11 VP of 106 copies/ml after incubation with 6 different dilutions of GoH3, or 8 clinical isolates of HPV6/11 VP of 106 copies/ml before or after the incubation with 1∶ 100 dilution of GoH3. After additional culture, the infected HaCaT cells were collected and fluorescence quantitative (FQ)-PCR was performed to detect the HPV DNA load in cells. The inhibition rate of CoH3 on the infection was calculated. Results The viral load was different among the HaCaT cells infected with different concentrations of HPV6/11 VP (P < 0.01). The inhibition rate on the infection positively correlated with the concentration of CoH3 when the dilution was more than 1∶ 100; however, when the dilution was less than 1∶ 100, the increase in CoH3 concentration had no influence on the inhibition rate. The average viral load in HaCaT cells infected with clinical isolates of HPV6/11 VP was (5.81 ± 2.51) × 104 copies/ml in the absence of GoH3, (3.02 ± 1.21) × 104 copies/ml with the presence of CoH3, and the average inhibition rate of GoH3 was (46.9 ± 4.7)%. Conclusions GoH3 could partially suppress the adhesion of HPV6/11 VP to HaCaT cells, hinting that integrin a6 is an important HPV6/11 VP receptor on host cells.
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Objective To investigate the expression of integrin α6 mRNA in skin lesions of patients with condyloma acuminata. Methods Sixty outpatients with condyloma acuminate, including 30 primary cases and 30 recurrent cases were enrolled in the study;30 non-condyloma acuminate patients undergoing circumcision were used as normal controls. The expression of integrin α6 mRNA in condyloma acuminata lesions of primary and recurrent cases, as well as in skin samples of normal controls were determined by RTPCR. ANOVA was used to compare the differences among the groups, and Student-Newman-Keuls test(q test)was used for pairwise comparisons. Results Relative contents of integrin α6 mRNA(integrin α6/GAPDH)in normal controls, primary and recurrent condyloma acuminata lesions were 0.25±0.10, 0.79±0.16 and 1.07±0.29, respectively, and the difference was of statistical significance(F=127.687, P=0.001). Conclusion Integrin α6 may be associated with the pathogenesis and recurrence of condyloma acuminate, which may provide a new target for prevention and treatment of the disease.