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Objective To analyze the characteristics of genotyping and gene polymorphism of Neisseria gonorrhoeae(N.go) with azithromycin(AZM)-resistance(AZM-R) and decreased susceptibility to ceftriaxone(CROD).Methods The minimum inhibitory concentration(MIC) of AZM and CRO were determined.AZM-R isolates were detected for mutations in 23S rRNA,mtrR and penA genes.Genotypes were analyzed by using N.go multi-antigen sequence typing(NG-MAST).Results All total of 485 isolates of N.go were detected.77(15.9%) strains were AZM-R(MIC≥1 mg/L),including 33(6.8%) isolates of AZM low-level resistant(AZM-LLR,MIC=1 mg/L) strains and 44(9.1%) isolates of AZM middle-level resistant(AZM-MLR,MIC≥2 mg/L) strains.There were more CROD(MIC≥0.125 mg/L) strains in AZM-MLR isolates(43.2%),compared with those in AZM-LLR isolates(18.2%,P0.05).Similar results were found between combined AZM-LLR/CROD isolates and combined AZM-MLR/CROD isolates(P>0.05).No mutation of A2059G and AZM high-level resistant(AZM-HLR,MIC≥256 mg/L) isolate were found.Among 77 AZM-R isolates,67 sequence types(ST) were identified by NG-MAST,of which 30 types were novel.Most ST were represented by a single isolate.Conclusion AZM-R and CROD isolates,presented in this area,might be deserved continuous surveillance to identify the mechanism of concurrent resistance.
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Objective To investigate the feasibility and prospects of nested real-time PCR(NR-PCR)technique for Treponema palladium(Tp)detection in various samples of different stages of syphilis from patients preliminarily diagnosed as syphilis. Methods Targeting the Tp polA gene, NR-PCR was performed to detect Tp DNA in various samples from the patients with various stages of syphilis at the first clinic visit, including skin tissue fluid swabs, serum, whole blood, cerebrospinal fluid(CSF)and earlobe blood. Data were analyzed with SPSS software version 13. Results A total of 368 clinical samples were collected from 200 patients with syphilis. With a detection limit of 2 Tp/ml, NR-PCR showed that the total positive rate for Tp DNA was 71.7%(264/368). The Tp DNA positive rate was highest in earlobe blood samples (92.0%, 23/25), followed by CSF samples(90.2%, 46/51), skin tissue fluid swabs(74.3%, 26/35), serum samples(66.9%, 99/148)and whole blood samples(64.2%, 70/109). There was good agreement between NR-PCR results and serologic test results, with a consistency rate of 76.0%(152/200). Furthermore, the Tp DNA positive rate did not differ between patients with primary(12/19)and secondary syphilis(14/16)in skin tissue fluid swabs(χ2 = 2.62, P > 0.05), and was slightly but insignificantly higher in patients with secondary syphilis than those with primary syphilis in the serum samples(χ2=3.6, P=0.06). The Tp DNA positive rate of whole blood samples was also higher in patients with secondary syphilis than those with any other types of syphilis. Among patients with neurosyphilis, no significant difference was observed in the Tp DNA positive rate between earlobe blood samples and CSF samples(P=0.06). Among patients with latent syphilis, the Tp DNA positive rate was significantly higher in serum samples with an RPR titer of ≥ 1:8 than those with an RPR titer of≤1:4. Conclusion NR-PCR is feasible for detecting Tp DNA in various kinds of samples, and the Tp DNA positive rate is influenced by stages of syphilis and types of samples, as well as RPR titers.
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Objective To investigate genetic characteristics of Neisseria gonorrhoeae (N.gonorrhoeae) isolates from Guangzhou city in 2014,and to analyze the relationship of N.gonorrhoeae multi-antigen sequence typing (NGMAST) sequence types (STs) with ciprofloxacin resistance.Methods An agar dilution method was used to determine the minimal inhibitory concentration (MIC) of ciprofloxacin in 97 N.gonorrhoeae isolates from Guangzhou city.PCR was performed to amplify the gyrA,parC,porB and tbpB genes from these isolates,followed by gene sequencing and determination of NG-MAST STs.Results Of the 97 N.gonorrhoeae isolates,95 (97.9％) were resistant to ciprofloxacin,including 41 high-level (MIC ≥ 16 mg/L) and 54 low-level (1 mg/L ≤ MIC ＜ 16 mg/L) resistant strains.Mutations were detected at codons 91 and 95 encoding serine in the gyrA gene of all the 95 ciprofloxacin-resistant strains,and in the parC gene of 93 resistant strains.The frequency of the mutation at codon 87 in the parC gene was 85.4％ (35/41) in high-level resistant strains,significantly higher than that in low-level resistant strains (59.3％[32/54],x2 =7.64,P ＜ 0.05).MAST STs were successfully determined for all the 97 N.gonorrhoeae isolates except 1 isolate with incorrect PCR amplicons.Of the 96 genotyped isolates,50 were assigned to 35 known STs by using the NG-MAST website (www.ngmast.net),among which,10 STs each contained 2 to 4 isolates.The most common ST was ST5309.Phylogenetic tree analysis revealed that the 96 genotyped N.gonorrhoeae isolates could be classified into 2 groups,and the proportion of isolates with MIC ≥ 16 mg/L is 46.4％ (39/84) in group 1,but only 1/12 in group 2 (x2 =6.27,P=0.012).Conclusions High-level resistance of N.gonorrhoeae to ciprofloxacin may be mainly associated with the mutation at codon 87 in the parC gene.NG-MAST STs may be related to the degree of ciprofloxacin resistance.
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Objective To determine the prevalence of penicillinase-producing Neisseria gonorrhoeae(PPNG) and the distribution of blaTEM-135 gene variants in PPNG at several gonococcal antimicrobial surveillance sites in China, to compare N. gonorrhoeae multi-antigen sequence typing(NG-MAST)types of PPNG and its blaTEM-135 gene variants, and to assess the difference and association in NG-MAST types of blaTEM-135 gene variants among different regions. Methods A total of 572 N. gonorrhoeae isolates were collected at 6 gonococcal antimicrobial surveillance sites from Jiangsu, Shanghai, Zhejiang, Tianjin, Guangdong and Guangxi in 2012. After isolation, purification, and identification, cefalotin paper discs were used for detection of PPNG. DNA was extracted by QIAxtractor DX kits after cultivation of the PPNG strains. Then, mismatch amplification mutation assay (MAMA) PCR was performed to identify blaTEM-135 variants, and NG-MAST analysis to determine N. gonorrhoeae genotypes. Results Among the 572 N. gonorrhoeae strains, 38.1%(218/572) were identified as PPNG, and of the PPNG strains, 52.3% (114/218) were blaTEM-135 variants. The detection rate of PPNG at these surveillance sites from high to low was as follows: 51.7% (45/87, Zhejiang), 45.6%(36/79, Shanghai), 38.0% (78/205, Guangdong), 37.5% (12/32, Guangxi), 31.2% (24/77, Jiangsu) and 25.0%(23/92, Tianjin), and that of blaTEM-135 variants was as follows: 68.9%(31/45, Zhejiang), 58.3%(14/24, Jiangsu), 50.0%(39/78, Guangdong), 47.2%(17/36, Shanghai), 39.1%(9/23, Tianjin)and 33.3%(4/12, Guangxi). NG-MAST analysis showed that the ST2318, ST1768, ST1866, ST1053 and ST8726 types predominated among these bla TEM-135 variants, and a strong correlation was found between blaTEM-135 variants and some NG-MAST types, such as ST1768, ST1053 and ST8726 types. The distribution of NG-MAST types was significantly different between the surveillance site in Tianjin (in the Northern part of China) and the other sites (in the Southern part of China), but highly similar among the surveillance sites in Jiangsu, Zhejiang and Shanghai regions. Conclusions There is a high prevalence of PPNG and its blaTEM-135 variants at several gonococcal antimicrobial surveillance sites in China, with significant differences in NG-MAST genotype distribution of PPNG and its blaTEM-135 variants among different regions.
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Objective To compare the sensitivity and specificity of venereal disease research laboratory (VDRL) test versus several other laboratory tests in the diagnosis of neurosyphilis. Methods Lumber puncture was conducted to obtain cerebrospinal fluid (CSF) from untreated outpatients with latent syphilis (LS) or serofast outpatients with LS. Then, VDRL test, rapid plasma regain (RPR) test, Treponema pallidum particle agglutination (TPPA) assay, fluorescent treponemal antibody-absorption (FTA-ABS) test and protein quantification were performed on these CSF samples. The sensitivity, specificity, positive predictive value and negative predictive value were compared between VDRL test and four other laboratory tests in the diagnosis of neurosyphilis. Results Totally, 61 cases of latent syphilis were included in this study. The sensitivity, specificity,positive predictive value and negative predictive value were 93.44% (57/61), 99.32%(293/295), 96.61%(57/59), 98.65% (293/297)for CSF-RPR, respectively, 91.80% (56/61), 82.71% (244/295), 52.34% (56/107),97.99 (244/249) for CSF-TPPA, respectively, 93.44% (57/61), 82.71% (244/295), 52.78%(57/108), 98.39%(244/248) for CSF-FTA-ABS, respectively, and 49.18%(30/61), 97.29% (287/295), 78.95% (30/38),90.25% (287/318) for CSF protein quantification, respectively. Conclusions CSF-VDRL cannot be replaced by CSF-RPR, -TPPA, -FTA-ABS, or CSF protein quantification in the diagnosis of neurosyphilis. CSF-RPR shows a high sensitivity and specificity in the diagnosis of neurosyphilis, with an increased diagnostic capability (area under the receiver operating characteristic curve) compared with CSF-TPPA, CSF-FTA-ABS or CSF protein quantification.
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Objective To investigate the prevalence of resistant Neisseria gon orrhoeae and plasmid-mediated resistant strains in Guangzhou from 1996 to 2001. Methods The resistant N.gonorrhoeae and plasmid-mediated resistant strains to tetracycline (TRNG) were determined using agar dilution method, and penicillinas e-producing N. gonorrhoeae (PPNG) by acidometric method. Results A total of 793 gonococcal isolates were tested from 1996 to 2001. The resistant rate for penic illin increased from 57.2%to 81.8%and PPNG from 2%to 27.2%, respectively, du ring the six years. Resistance to tetracycline remained high and stable over the years, while the rates of TRNG were increased from 1.5%to 27.2%. Conclusions The prevalence of plasmid-mediated resistant strains of N. gonorrhoeae increase s year by year in Guangzhou. These results suggest that the clinical isolates of gonococcal strains in this city are highly resistant to penicillin and tetracyc line.