| In recent years, it was estimated by WHO that there were about10.6 million new emerging syphilis cases around the world annually,and the incidence of syphilis in China also increased continuously in a straight line. According to the 2015 National Notifiable Disease Situation issued by the Disease Prevention and Control Bureau of National Health and Family Planning Commission, the number of syphilis incidence in 2015 nationwide reached 458,682, which was16% higher than that in 2011. Therefore, effective surveillance and control of syphilis has become the focus of public health at home and abroad.Molecular subtyping of T. pallidum in the local area can not only reveal important information about the spread of syphilis, but also distinguish different strains on the molecular level to determine the infectious status of syphilis patients, and be used to identify strains that are associated with particular clinical outcomes. Currently, the enhanced molecular subtyping method is the most widely applied bymost scholars worldwide. This typing method, which is stable and has strong discernability, is a powerful tool for syphilis epidemiological investigation.Antibiotic resistance is the main factor that causes the failure of clinical treatment of infectious diseases. Azithromycin, a kind of macrolides, is widely used for syphilis treatment. However, it was reported recently azithromycin-resistant Tp strains increased year by year in many countries, including China. Therefore, it is very important to carry out resistance surveillance of azithromycin in local region. Unlike azithromycin, up to date there are no reports on Tp resistance to doxycycline, which is recommended as the second-line antibiotics for the treatment of syphilis. The national CDC has clearly put forward the necessity to monitor doxycycline resistance of Tp.In the book of Planning of Prevention and Control for Syphilis in China(2010-2020), one of the main measures of syphilis control taken in our country is to improve the quality of syphilis detection and strengthen the syphilis screening in the laboratory. At present,seroassay, as the main basis of syphilis diagnosis, is subject to certain limitations. In the past ten years, there were many reports onPCR-based detection methods for syphilis diagnosis, all of which has good sensitivity and specificity in detecting chancre swabs. However,when specimens were replaced by other samples, such as blood, urineand CSF, although these methods had high specificity, the poor sensitivity of them hindered their application clinically. Thus, to establish a rapid, simple, high sensitive and specific detection method for syphilis is very necessary. Recently, a novel nucleic acid-based method named loop-mediated isothermal amplification(LAMP) has been developed. It has the characteristics of simplicity, rapidity, high sensitivity and specificity, and the result could be observed by many ways. Moreover, LAMP can be automated and especially has a broad prospect in the diagnosis of disease and epidemiological investigation.ObjectiveMolecular subtyping and detection of points mutation within 23 S rRNA and 16 S rRNA were performed on circulating Tp strains in various regions of Hunan province, including Hengyang, Changsha,Chenzhou, Changde, Yueyang and Yongzhou, which revealed regional trends in subtype and antibiotic resistance of this spirochete and provided data for the surveillance and control of syphilis in this region. In addition, LAMP was developed and used to detect Tp DNA in whole blood from SS patients. The clinical application of this method was preliminarily evaluated.MethodsWhole-bloods were obtained from untreated patients with suspected secondary or latent syphilis in various regions of Hunan province, China, including Hengyang, Changsha, Chenzhou, Changde,Yueyang and Yongzhou. Whole-blood samples were screened by PCR targeting pol A, tpp47, bmp, and tp0319 respectively. A sample that was PCR-positive for any of the target genes was considered positive for Tp DNA and used for further study.The PCR-positive samples screened above were amplified by PCR using arp/tpr/tp0548 as the target gene respectively. The subtypes of Tp strains in bloods from syphilis patients were determined by combination of the repeats number of arp gene, the RFLP pattern of tpr gene and the variable region of tp0548 gene.23S rRNA gene was amplified by nPCR from PCR-positive samples. After purification the amplicons were digested by Mbo Ⅱand BsaⅠrespectively and sequenced.16S rRNA gene was amplified by nPCR from PCR-positive samples. After purification the amplicons were sequenced and blasted to the reference sequence.Bmp gene was chosen as gene target to develop LAMP method.A serial 10-fold dilution of purified DNA template of T. pallidum Nichols strain was used to determine the detection limit of the LAMP and the specificity of LAMP was evaluated by amplifying non-treponemal bacteria. LAMP was adopted to detect Tp DNA in bloods from secondary syphilis patients and blood donors.Results2,253 specimens were screened by traditional PCR. The positive rate of detection for pol A, tpp47, bmp, and tp0319 genes inPCR assays was 18.8%, 18.4%, 17.5%, and 14.2% respectively. This yielded a total of 455 PCR-positive specimens.The arp gene was successfully amplified by touchdownPCR from 203 of the 455 PCR-positive samples. A total of eight different arp repeat sizes(5, 6, 8, 11, 12, 14, 18, and 22 repeats) were obtained in these samples, with 14 repeats being the most common(62.6%,127/203). We then used nPCR to amplify the tpr gene and identified385 positive samples. When these amplification products were analyzed by digestion with the restriction endonuclease, Mse I, they could be classified into eight RFLP patterns, among which the most prevalent was type d. The tp0548 gene were categorized as type f, a, c,and g for each sample, with type f being the most. 181 of the original455 PCR-positive samples were fully subtyped and revolved into 32 subtyps, among which subtype 14d/f was the most commonly detected. Other major subtypes were as follows: 14a/f, 14e/f, 12e/f,12d/f,; 6d/f, 11d/f, 14j/f, 8d/f, 14d/a, 14p/f, 14d/c, 14l/f, 14e/a, 14a/c,12d/a, 5d/f;14b/f, 6d/a, 11o/f, 12e/a, 14d/g, 14e/c, 14a/a, and 14b/f.23S rRNA and 16 S rRNA genes were amplified by nPCR. The results showed that 390(97.5%, 390/400) Tp DNA harbored the A2058 G mutation, representing 96.3%(260/270) of patients with secondary syphilis and 100%(130/130) of patients with latent syphilis and the A2059 G mutation was not detected. In addition, none of the G1058 C mutation within 16 S rRNA genes have been found.The detection limit of LAMP was within 4.3×100 copies/μL magnitude, but the detection limit of traditional PCR was 4.3×102copies/μL. When LAMP was used to detect the bacteria of control groups and the bloods from healthy population, all the results were negative. Moreover, among the 603 secondary syphilis specimens,503 samples were positive by LAMP. The sensitivity and specificity of LAMP were 83.4% and 100%, respectively.Conclusions1) Arp, tpp47 and bmp genes were suitable to be used as target gene for nucleic acid test(NAT).2) The circulating Tp in Hunan during 2013 and 2015 were revolved into 32 subtypes, among which subtype 14d/f was the most common subtype.3) High frequent mutation of A2058 G within 23 S rRNA has occurred in the circulating Tp strains, in Hunan. It must be cautioned that macrolides antibiotics are used to treat syphilis clinically in the region.4) G1058 C point mutation within 16 S rRNA gene was not observed in Hunan region. Doxycycline may be used as a second-line drug for syphilis treatment and the close follow-up must be conducted.5) LAMP has the potential to be used as a new laboratory addition in combination with seroassay for the diagnosis of secondary syphilis. |
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