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Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line– and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models.Significance:The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.See related commentary by Klempner and Hata, p. 20.This article is highlighted in the In This Issue feature, p. 1

Background: Quick and accurate diagnosis of primary cutaneous amyloidosis (PCA) may be difficult because its symptoms are often subtle and nonspecific. Objective: We sought to review the literature on the roles of various staining methods in the diagnosis of amyloidosis and demonstrate added benefits of using rapid 4,6-diamidino-2-phenylindole (DAPI) staining in the diagnosis of PCA. Methods: Three groups of cases, namely, PCA, neurodermatitis, and prurigo nodularis, were retrieved from a computerized pathology database for study, and their paraffin-embedded tissue blocks were cut following standard procedures. The tissue sections were stained with three stains: hematoxylin-eosin (HE), Congo red, and DAPI stains, and examined under the microscope to compare the staining patterns of these three methods. We also performed amyloid keratin and apolipoprotein E (APOE) staining on the sections of PCA in order to further support our conclusion. The PCA sections were read by junior and senior dermatopathologists for comparison. Results: The sensitivity of DAPI staining for PCA was significantly higher than that of Congo red staining and HE staining (p < 0.001). This statement holds true whether the experiment was grouped in one sample or was divided into groups of junior and senior dermatopathologists (p < 0.001). The DAPI-positive staining areas, except for the nuclei, were consistent with the amyloid deposition areas. In this study, DAPI staining had a sensitivity of 98.6% and a specificity of 100%. Conclusion: DAPI staining could serve as a useful technique to establish the diagnosis of PCA, and its high efficacy in diagnosing PCA makes it less dependent on the experience levels of the evaluators. Additionally, the binding of DAPI to the A-T-rich sequence of double-stranded DNA suggests that amyloid may contain DNA or a similarly structured nucleic acid.

The global incidence of genital

Chlamydia trachomatis detection plays a crucial role in early diagnosis and treatment of C. trachomatis infection. In the current study, the capability of sexually transmitted disease (STD) laboratories to detect C. trachomatis was investigated in Guangdong, China.An external quality assessment panel, including 5 positive samples with different C. trachomatis loads and 2 negative samples was distributed to 654 participating laboratories in October 2019, and the test results were analyzed by Guangdong Central STD Laboratory. The use of various C. trachomatis detection methods in Guangdong from 2015 to 2019 was also retrospectively investigated.Of the 654 participating STD laboratories, 559 (85.47%) used immune chromatographic-rapid diagnostic tests (IC-RDTs) to detect C. trachomatis in 2019, and 95 (14.53%) used nucleic acid amplification tests (NAATs). The rate of NAATs use increased approximately 4-fold from 2015 to 2019. The sensitivity of IC-RDTs decreased markedly from 97.32% to 30.89% with decreasing C. trachomatis load, whereas that of NAATs was 97.62% to 100% in all positive samples. With respect to negative samples the specificity of IC-RDTs was 97.13% to 97.30% and that of NAATs was 98.95% to 100%. Laboratories using IC-RDTs were less likely to detect C. trachomatis than those using NAATs in samples with C. trachomatis loads of 20000 copies/mL or less (P0.0001). Further analysis indicated no significant difference (P0.05) in detection rate among the 4 IC-RDT assays commonly used by the participating laboratories.Immune chromatographic-rapid diagnostic tests are commonly used for C. trachomatis detection by many laboratories in Guangdong, but their low sensitivity may lead to missed diagnoses. Nucleic acid amplification tests exhibit high sensitivity and specificity and should be recommended for C. trachomatis detection in STD laboratories.

BACKGROUND We conducted an observational study to determine whether patients with syphilis who do not demonstrate serological cure or lack of seroreversion in nontreponemal (NT) antibody titers after initial therapy benefit from re-treatment and cerebrospinal fluid (CSF) analysis. METHODS We enrolled patients with syphilis from sexually transmitted disease clinics in Guangzhou, China, who had persistent NT titers after therapy. Serological nonresponse was defined as a

Background Chlamydia trachomatis is the most common sexually transmitted infection and the bacterial agent of trachoma globally. C. trachomatis undergoes a biphasic developmental cycle involving an infectious elementary body and a replicative reticulate body. Little is currently known about the gene expression dynamics of host cell mRNAs, lncRNAs, and miRNAs at different stages of C. trachomatis development. Results Here, we performed RNA-seq and miR-seq on HeLa cells infected with C. trachomatis serovar E at 20 h post-infection (hpi) and 44 hpi with or without IFN-γ treatment. Our study identified and validated differentially expressed host cell mRNAs, lncRNAs, and miRNAs during infection. Host cells at 20 hpi showed the most differential upregulation of both coding and non-coding genes while at 44 hpi in the presence of IFN-γ resulted in a dramatic downregulation of a large proportion of host genes. Using RT-qPCR, we validated the top 5 upregulated mRNAs and miRNAs, which are specific for different stages of C. trachomatis development. One of the commonly expressed miRNAs at all three stages of C. trachomatis development, miR-193b-5p, showed significant expression in clinical serum samples of C. trachomatis-infected patients as compared to sera from healthy controls and HIV-1-infected patients. Furthermore, we observed significant upregulation of antigen processing and presentation, and T helper cell differentiation pathways at 20 hpi whereas T cell receptor, mTOR, and Rap1 pathways were modulated at 44 hpi. Treatment with IFN-γ at 44 hpi showed the upregulation of cytokine-cytokine receptor interaction, FoxO signaling, and Ras signaling pathways. Conclusions Our study documented transcriptional manipulation of the host cell genomes and the upregulation of stage-specific signaling pathways necessary for the survival of the pathogen and could serve as potential biomarkers in the diagnosis and management of the disease.

Additional file 3: Supplementary Fig. 3 Scatter plot of miRNAs in cells containing C. trachomatis at 20 hpi (a), 44 hpi (b), and 44 hpi with IFN-γ treatment (c). The criteria used were a fold-change of ≥2 or ≤-2, and a P-value

Additional file 1: Supplementary Fig. 1 Differentially expressed mRNAs in C. trachomatis-infected HeLa cells at 20 hpi (a), 44 hpi (b), and 44 hpi with IFN-γ treatment (c). The cutoff criteria used in the volcano plot were a fold-change of 2 with a P-value of 1 and P-value

To develop an advanced way of designing transonic airfoils for industry, a reliable and user-friendly mesh generator and a robust CFD solver are necessary. Especially, the prediction of unsteady shock buffet phenomenon is always a focus for the CFD simulation of transonic airfoils. In this study, BOXERmesh (an automatic mesh generator) and NEWT (a robust CFD solver), which are developed by CFS (Cambridge Flow Solutions) in collaboration with MHI (Mitsubishi Heavy Industries), are used to perform the CFD simulation of NACA SC2-0714 transonic airfoil. CFD simulation is conducted at two different attack angles with Mach number as 0.74 and Reynold number as 1.5×107 (non-buffet: α=2°; shock buffet: α=3°). A hexahedral dominant mesh is generated by BOXERmesh with the cell number as 5.52 million. Both unsteady RANS (URANS) and LES are performed using the CFD solver NEWT. Specifically, the governing equation is discretized by central differencing scheme with 2nd order accuracy in space by applying Swanson and Turkel type artificial viscosity, and the Adams-Bashford time integration with the dual-time stepping method is applied for temporal discretization in the density-based solver. Results show CFD simulation could reproduce the time averaged chordwise distribution of pressure coefficient at the two conditions. Both URANS and LES successfully capture the unsteady shock buffet phenomenon when increasing attack angle from 2° to 3°. However, the calculated peak oscillation location and the shock buffet frequency are different between URANS and LES. Applying the same mesh resolution, URANS performances better than LES, with the deviation of the shock buffet frequency less than 6% (Exp.: 69 Hz; URANS: 73 Hz). The reason is considered as the wall-normal mesh refinement near the airfoil surface (y+∼66) being not enough for LES to accurately resolve the turbulence scale and capture the boundary layer separation behavior. On the other hand, URANS is thought to be enough to reproduce the periodic moving of the onset of boundary separation and to predict the main characteristics of the shock buffet phenomenon.

Studies have rarely assessed the genotype distribution of Chlamydia trachomatis (CT) in urine among men attending sexually transmitted disease clinics (MSCs) in China. This study was aimed at investigating the prevalence and molecular epidemiology of CT infection by examining urine samples among MSCs from different geographic areas of Guangdong Province, China. A cross-sectional study was conducted among MSCs from 10 human immunodeficiency virus sentinel surveillance sites in Guangdong Province. CT DNA was extracted from male urine samples and analyzed using a Roche cobas 4800 CT/NG. The ompA genes were amplified by nested PCR and sequenced. The leukocyte esterase test was performed by routine urine analysis at local clinics. Of the 1,903 samples, 163 (8.6%, 95% confidence interval [CI] 3.8-16.3%) tested positive for CT. The highest prevalence (10.5%) of CT infection was observed among participants aged between 21 and 30 years. A total of 130 CT-positive samples (79.8%, 130/163) were successfully genotyped by nested PCR, resulting in 8 genotypes. The most prevalent genotypes were D, E, F, and J, with proportions of 20.8%, 20.0%, 17.7%, and 16.9%, respectively. There were no significant correlations between the geographical areas, leukocyte esterase test results and genotype distribution. Promotion of detection and molecular epidemiology research is needed for effective and comprehensive prevention and control programs.

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRAS

Chlamydia trachomatis is the cause of the most common bacterial sexually transmitted infection worldwide. Azithromycin is effective in treating chlamydial infection; however, resistance to this antibiotic is increasing, and it is important that new therapeutic strategies are developed. In this study, we demonstrated that inhibitors targeting each kinase in the extracellular signal–regulated kinase/ribosomal S6 kinase cascade significantly decreased the size and number of inclusions as well as the number of infectious progeny. The suppressive effects of the inhibitors were observed across the Chlamydia serotypes D, E, F, and L1 and across HeLa, McCoy, and Vero host cells. When combined with azithromycin, all the inhibitors exerted a synergistic suppressive effect on chlamydial infection. Knockdown experiments using small interfering RNA demonstrated that extracellular signal–regulated kinase 1/2 and ribosomal S6 kinase 1 were crucial for chlamydial infection. Moreover, BVD-523, a first-in-class extracellular signal–regulated kinase 1/2 inhibitor currently undergoing a phase II clinical trial, suppressed chlamydial infection both in cell culture and in a mouse model. These observations demonstrated not only that the extracellular signal–regulated kinase/ribosomal S6 kinase pathway plays a critical role in chlamydial infection but also that these kinases have potential as targets for host-directed therapy against C. trachomatis.

The ERK signaling pathway is one of the most commonly deregulated pathways in cancer. Assays that accurately measure ERK signaling output in clinical specimens would be extremely helpful not only in determining the pharmacodynamic effects of drug treatment but also in selecting those patients most likely to respond to therapy. Clin Cancer Res; 23(6); 1365–7. ©2016 AACR. See related article by Brant et al., p. 1471

Keloids represent one extreme of aberrant dermal wound healing. One of the important characteristics of keloids is uncontrolled fibroblasts proliferation. However, the mechanism of excessive proliferation of fibroblasts in keloids remains elusive. In this study, we demonstrated that TRAF4 was highly expressed in keloid fibroblasts and promoted fibroproliferation. We investigated the underlying molecular mechanism and found that TRAF4 suppressed the p53 pathway independent of its E3 ubiquitin ligase activity. Specifically, TRAF4 interacted with the deubiquitinase USP10 and blocked the access of p53 to USP10, resulting in p53 destabilization. Knockdown of p53 rescued cell proliferation in TRAF4-knockdown keloid fibroblasts, suggesting that the regulation of proliferation by TRAF4 in keloids relied on p53. Furthermore, in keloid patient samples, TRAF4 expression was inversely correlated with p53–p21 signaling activity. These findings help to elucidate the mechanisms underlying keloid development and indicate that blocking TRAF4 could represent a potential strategy for keloid therapy in the future.

A microdilution method for the antibiotic susceptibility testing of Neisseria gonorrhoeae was established and improved, and the antibiotic resistance of N. gonorrhoeae samples isolated from 8 cities of Guangdong in 2016 was determined. The improved microdilution method was compared with the agar dilution method recommend by the World Health Organization (WHO) Western Pacific Region by testing the susceptibility of 100 clinical N. gonorrhoeae isolates. The essential agreement (EA), categorical agreement (CA), very major error (VME), major error (ME), and minor error (MIE) levels of the two methods were analyzed; the acceptable performance rates were measured as follows: ≥90% for EA or CA, ≤3% for VME or ME, and ≤7% for MIE. The EA, CA, VME, ME, and MIE of each method for 7 antibiotics, penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, cefixime, and azithromycin, were 96%–100%, 94%–100%, 0%–3%, 0%–2%, and 0%–6%, respectively. The Wilcoxon signed-rank test results indicated 94%–100% agreement between the 2 methods after excluding off-scale values (P > 0.05). The susceptibility of 634 N. gonorrhoeae strains to the 7 antibiotics above were tested through the microdilution method. The resistant rates of the isolates against ciprofloxacin, tetracycline, penicillin, and azithromycin were 99.8%, 88.3%, 53.8%, and 11%, and the percentages of the isolates with decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC] ≥0.125 μg/mL) and cefixime (MIC ≥0.25 μg/mL) were 2.1% and 12%, respectively, in Guangdong. Among 8 cities, Shenzhen had the highest rates of resistance against penicillin (77.8%) and decreased susceptibility against ceftriaxone (5.6%). Zhuhai had the highest rates of decreased susceptibility against cefixime (30.1%), and Jiangmen had the highest azithromycin-resistant isolates (16.8%). The findings from this study indicated that the improved microdilution method is an alternative for testing the antimicrobial susceptibility of N. gonorrhoeae. The resistance rates of N. gonorrhoeae against penicillin, tetracycline, and ciprofloxacin were high. While ceftriaxone, cefixime, and spectinomycin remained effective against N. gonorrhoeae, their effectiveness seemed to be decreasing over time. Azithromycin therapy requires timely susceptibility test results.

Introduction Chlamydia is the most common sexually transmitted disease worldwide. Many studies have been evaluated the prevalence of Chlamydia trachomatis (CT) infection while very rare studies assessed the genotype distribution in urine among males attending sexually transmitted diseases (STD) clinics (MSCs)in China. This study aimed to investigate the prevalence and molecular epidemiology of CT infection by urine samples among MSCs from different geographic areas of Guangdong province, China. Methods A cross-sectional study was performed among MSCs from ten HIV surveillance sites of Guangdong province, China. CT DNA in male urines were extracted and detected by using the Roche cobas 4800 CT/NG. The ompA genes were amplified by nested polymerase chain reaction and sequenced. Urine leukocyte esterase test were performed. Results Of the 1926 urine specimens, 1903 urines were successfully validated for detection of CT. Of the 1903 samples, one hundred and sixty-three (8.6%, 95% CI 8.2% to 9.0%) were found to be positive for CT. One hundred and thirty CT positive specimens were successfully genotyped by nested PCR, resulting in eight genotypes. The most prevalent genotypes were D, E, F, and J with proportions of 20.8%, 20.0%, 17.7%, 16.9%, respectively. There was no significant difference between age, geographic area, leukocyte esterase test and genotype distribution. Conclusion There was a high prevalence of CT infection among males attending STD clinics in eastern area of Guangdong province, China. Promoting detection and molecular epidemiology research are needed for effective and comprehensive prevention and control programs.

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAF

Single-dose azithromycin is recommended for treating Chlamydia trachomatis infections. Here, we established an in vitro cell model of azithromycin-induced persistent infection. Azithromycin inhibited the replication of C. trachomatis in a dose-time-dependent manner. Electron microscopy indicated that small inclusions in the induced model contained enlarged, aberrant and non-infectious reticulate bodies. RT-PCR showed that C. trachomatis still has the ability to express the unprocessed 16S rRNA gene in the model and that C. trachomatis recovered after the removal of azithromycin with a peak recovery time of 24 h. The mutations in 23S rRNA, L4 and L22 genes were not found in persistent infection, and qRT-PCR analysis showed that the relative expression level of euo in azithromycin treated infection was upregulated while omcB was downregulated. In summary, this study provides a novel in vitro cell model to examine the characteristics of azithromycin-induced persistent infection and contribute to the development of treatments for C. trachomatis infection.

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

Background Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA) sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA)-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM) and men who have sex with women (MSW) attending a sexually transmitted diseases (STD) clinic in Guangzhou, China. Methods Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA. Results Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA. Conclusions The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine subtypes of C. trachomatis.

Tumors evolve as they adapt to environmental cues. The principles governing evolution of tumors under the selective pressure of targeted therapy are not well understood. We aimed to evaluate the evolution of resistance and to identify therapeutic modalities that prevent this process in BRAFV600E-mutant tumors. We modeled the selection and propagation of BRAFV600E amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor. Single-cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in multiple subclones of the same tumor shortly after treatment. The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for ERK signaling inhibitors, and single-cell sequencing of a melanoma PDX model showed that drugs of the same pathway do not necessarily select for the same subclones. These data suggest that sequential monotherapy is not optimal, but concurrent targeting of RAF, MEK, and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification. Administered on an intermittent schedule, this treatment inhibited tumor growth without apparent toxicity in 11/11-lung cancer and melanoma PDX models with various additional alterations. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose a high fitness threshold, such as our intermittent triple therapy, will likely prevent the evolution of resistance-causing alterations and merit testing in patients. Citation Format: Yaohua Xue, Luciano Martelotto, Timour Baslan, Alberto Vides, Martha Solomon, Kalyani Chadalavada, Elisa DeStanchina, Gouri Nanjangud, Michael Berger, Scott Lowe, Jorge S. Reis-Filho, Neal Rosen, Piro Lito. An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B015.

An oligonucleotide array technology was established for rapidly detecting and genotyping Chlamydia trachomatis in urogenital infections. The VS1-VS2 region of the omp1 gene was used to design oligonucleotide probes. Eleven serovar-specific probes to serovars A, B, C, D, E, F, G, H, I, J, and K, and 3 group-specific probes to group B (B, Ba, D, E, L1, and L2), group C (A, C, H, I, J, K, and L3), and an intermediate group (F and G) were synthesized and spotted onto the nylon membrane. Two pairs of universal primers were designed for the nested polymerase chain reaction (PCR) amplification of the VS1-VS2 gene. Digoxigenin-labeled amplicons of the VS1-VS2 gene of C. trachomatis were hybridized to the membrane array. Hybridization signals were read by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate color development. The assay developed was tested with reference strains of C. trachomatis serovars and clinical samples. The sensitivity was evaluated for 57 samples previously found to be positive for C. trachomatis by using plasmid PCR, and 98.2% (56/57) concordance was obtained. Fourteen oligonucleotide probes were optimized by trying different reaction conditions, showing specific hybridization with the corresponding reference strains, but no cross-reactions with other urogenital microorganisms. Using this procedure, a total of 59 strains were detected from 56 chlamydial samples. Eight genotypes were found, and type D, E, F, and H were the most frequently observed types (77.9%). Three cases (5.4%) had multiple infections with serovars: 1.D/E, 2.D/F, and 3.F/K. To validate the reference strains and confirm the genotype identity as determined by the oligonucleotide array technology, we sequenced all reference strains and 10 selected specimens across variable sequence VS1 and VS2. No discrepancies were found between the array typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. The findings from this study indicated that the oligonucleotide array is a simple, fast, and specific assay for directly detecting and genotyping C. trachomatis from clinical samples.

BACKGROUND The presence of persistent Chlamydia trachomatis infection after treatment does not always correlate with in vitro susceptibility testing. METHODS The in vitro minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of azithromycin, clarithromycin, roxithromycin, doxycycline, tetracycline, ofloxacin, and penicillin were tested against 61 clinical isolates of C. trachomatis on 6 serovars, and the MIC/MBC of azithromycin and ofloxacin at different points in time after antibiotic administration to infected cultures. RESULTS Of the 7 antibiotics tested, clarithromycin showed the greatest activity against C. trachomatis isolates with MIC90 of 0.032 μg/mL and MBC90 of 0.064 μg/mL, followed by doxycycline with MIC90 0.064 μg/mL and MBC90 0.064 μg/mL, and azithromycin with MIC90 0.160 μg/mL and MBC90 0.320 μg/mL. Azithromycin had roughly the same MIC50 values (0.08 μg/mL) as the other serovars isolates tested, and other antibiotics showed a 2- to 4-fold difference in MICs50 between serovars. In addition, an increase in the azithromyin MIC was observed by 8 hours and the ofloxacin MIC by 16 hours. At 24 hours, the azithromycin MICs were greater than 40 μg/mL and ofloxacin MICs were greater than 64 μg/mL. CONCLUSIONS The current data demonstrated that the antimicrobial susceptibility of C. trachomatis was influenced by both the serovar type and the duration of exposure to antibiotics in infected cultures.

Antibiotic susceptibility of Neisseria gonorrhoeae in Guangzhou during 2002-2011 showed that resistance to penicillin and ciprofloxacin was high, while ceftriaxone remained effective although there was a trend towards reduced sensitivity.

Background The continuing spread of drug-resistant gonococci has posed a challenge for successful treatment worldwide. Guangdong Province in South China has one of the highest resistance rates of gonococci in China and a large number of international migrants. We investigated the in vitro antimicrobial susceptibility and genotypes of N. gonorrhoeae isolated in Guangzhou, the capital city, in 2001–11. Methods MICs to penicillin, ceftriaxone, tetracycline, ciprofloxacin, and spectinomycin were determined by agar plate dilution and susceptibilities were interpreted according to WHO standards. Penicillinase-producing N. gonorrhoeae (PPNG) was determined by paper acidometric testing and high-level tetracycline resistant N. gonorrhoeae (TRNG) by MIC. The isolates with resistant plasmids were genotyped by PCR. Results Of 1250 consecutive gonococci isolated during 2001–11, no ceftriaxone and spectinomycin resistant strains were found, but the prevalence of less susceptible strains to ceftriaxone rose from 17% to 46.5%. The MIC 90 for ceftriaxone showed intermediate sensitivity (0.06–0.125µg/mL) and spectinomycin near the resistant level (16–32µg/mL). The resistance to penicillin, tetracycline and ciprofloxacin were from 85%, 85% and 78% in 2001 to 81.9%, 100% and 98.4% in 2011, respectively. Of 349 (27.9%) PPNG and 539 (43.1%) TRNG detected. 232 (18.6%) strains were both PPNG and TRNG. PPNG increased from 17.0% to 32.3% and TRNG from 26.0% to 41.7% during 2001–10. Genotyping TEM-1 gene showed 94.8% PPNGs carrying the Asia-type β-lactamase plasmids. The Africa-type PPNG (1, 1.3%) emerged in 2008 and has increased to 8(6.2%) in 2011. Genotyping of tetM gene showed all TRNGs were Dutch variants. Conclusion Gonorrhea resistance continues to be a major public health problem in Guangzhou. The emergence and increase of African-type PPNG may be related to the large African diasporas in Guangzhou, migration of Chinese to Africa, or other migration patterns. More research is needed to determine what practises, systems and behaviours contribute to escalating resistance patterns.

Background Chlamydia trachomatis infection increases above gonorrhoea and syphilis, ranking first among the STDs. Molecular epidemiological researches have shown the predominant genotypes vary between regions, periods and population subgroups. However, serovars E, D and F are the most prevalent serovars. It is unclear whether the epidemiological characteristics were contributed to geography or pathogenicity. We explored the pathogenic diversity of different C. trachomatis serovars in mouse genital infection. Methods One hundred of female BALB/C mice were divided into serovar E, F, H, J and K groups. The mice in study group treated by medroxyprogesterone acetate were inoculated 107 C. trachomatis into genital tract. C. trachomatis was detected by culture, direct immunofluorescence assay (DFA) and PCR in the cervicovaginal secretion. On the days 7 and 35 after inoculation, inflammation of the cervix, uterus and oviduct were examined by HE stain, and expressions of cHSP60 and CPAF in the uterus and fallopian tube were detected by ELISA. Results The inflammatory of the cervical mucosa was more severe in serovar E group compared with J, K and H groups on day 7 post-inoculation. Accordingly, cHSP60 and CPAF expression increased significantly in E group compared with other experimental and control groups. On day 35 post-inoculation, the histo-pathological changes of the genital tract were obvious in J, K and H groups, characterised with uterine swelling, pyometra and effusion, fallopian expansion, hydrops, fibrosis and stenosis. cHSP60 and CPAF expression in H group was superior to that in other groups. Positive correlation between cHSP60 and CPAF expression was present on day 7 and 35 post-inoculation, respectively. Conclusion There existed pathogenic diversity among different C. trachomatis servoars in mouse genital infection. The expression of inflammatory cytokines of cHSP60 and CPAF during Chlamydial infection might partially explain the pathogenic mechanism and the stage of the Chlamidal infection.

Background The continuing spread of drug-resistant gonococci has posed a challenge for successful treatment worldwide. Recently third-generation cephalosporins-resistant strains were isolated in Japan. Guangdong Province in South China has one of the highest gonococcal resistance rates in China and a large number of international migrants. We investigated the invitro antimicrobial susceptibility and genotypes of N gonorrhoeae strains isolated in Guangzhou, the capital city, from 2001 to 2009. Methods MICs to penicillin, ceftriaxone, tetracycline, ciprofloxacin, and spectinomycin were determined by agar plate dilution and susceptibilities were interpreted according to WHO standards. s-lactamase production was determined by paper acidometric testing. The resistant plasmids were determined for penicillinase-producing N gonorrhoeae (PPNG) and high-level tetracycline resistant N gonorrhoeae (TRNG) by PCR and the isolates were genotyped. Results Of 1132 consecutive gonococci isolated from 2001 to 2009, no ceftriaxone and spectinomycin resistant strains were found, but the prevalence of strains less susceptible to ceftriaxone rose from 18.2% to 38.5%. The MIC90 for ceftriaxone showed intermediate sensitivity (0.06–0.125 μg/ml) and spectinomycin near the resistant level (16–32 μg/ml). The resistance to penicillin, tetracycline and ciprofloxacin increased from 81.8%, 84.5% and 72.5% in 2001 to 90.1%, 91.4% and 98.7% in 2009, respectively. A total of 313 (27.7%) PPNG and 486 (42.9%) TRNG strains were detected. 206 (18.2%) strains were both PPNG and TRNG. PPNG rose from 21.8% to 29.8% and TRNG rapidly increased from 27.2% to 56.3%. Genotyping TEM-1 gene showed 267 (99.6%) PPNGs in 2001–2008 carried the Asian-type s-lactamase plasmids and one African-type in 2008. Genotyping of tetM gene showed that all 486 TRNGs were Dutch variants. Conclusion Gonorrhoea resistance continues to be a major public health problem in Guangzhou. The emergence of an African gonorrhoea resistance variant may be related to the large African diaspora in Guangzhou, migration of Chinese to Africa, or other migration patterns. More research is needed to determine what practices, systems, and behaviours contribute to escalating resistance patterns.

Background Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. Methods Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. Results Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ2 = 31.57, p