A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.

Serum Ubiquitin C-Terminal Hydrolase-L1, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain Are Good Entry Points and Biomarker Candidates for Neurosyphilis Diagnosis Among Patients Without Human Immunodeficiency Virus to Avoid Lumbar Puncture.

BACKGROUND: Laboratory tests to diagnose neurosyphilis using cerebrospinal fluid (CSF) are currently disadvantageous as a lumbar puncture is required, which may result in patients with neurosyphilis missing an opportunity for early diagnosis. Thus, blood biomarker candidates that are more convenient and minimally invasive to collect for diagnosing neurosyphilis is urgently needed. METHODS: This observational study aimed to analyze serum ubiquitin C-terminal hydrolase-L1 (UCH-L1), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NF-L) levels in 153 patients without human immunodeficiency virus (HIV) and to evaluate their diagnostic performance in neurosyphilis compared with CSF. RESULTS: Serum UCH-L1, GFAP, and NF-L levels were significantly higher in patients with neurosyphilis compared with patients with uncomplicated syphilis or non-syphilis. For the diagnosis of neurosyphilis, serum UCH-L1, GFAP, and NF-L revealed sensitivities of 90.20%, 80.40%, and 88.24%, and specificities of 92.16%, 78.43%, and 80.39%, respectively, at cutoff levels of 814.50 pg/mL, 442.70 pg/mL, and 45.19 pg/mL, respectively. In patients with syphilis, serum UCH-L1, GFAP, and NF-L levels correlated strongly or moderately with those in the CSF, with similar or better diagnostic performance than those in the CSF. The testing algorithms' sensitivity and specificity increased to 98.04% and 96.08%, respectively, when subjected to parallel and combination testing, respectively. CONCLUSIONS: To avoid lumbar puncture, each serum UCH-L1, GFAP, and NF-L is a good entry point and biomarker candidate for the diagnosis of neurosyphilis among patients without HIV. These proteins used in concerto can further improve the diagnostic sensitivity and specificity.

Recombinant Treponema pallidum protein Tp47 promotes the migration and adherence of THP-1 cells to human dermal vascular smooth muscle cells by inducing MCP-1 and ICAM-1 expression.

Vascular inflammation is a complex and multifactorial pathophysiological process that plays a crucial role in all stages of syphilis and is responsible for tissue damage. Little is known about the interactions of infiltrating immunocytes with human dermal vascular smooth muscle cells (HDVSMCs) in arterioles during the immunopathogenesis of syphilis. The Treponema pallidum subsp. pallidum membrane protein Tp47 is considered a major inducer of inflammation initiation and development. In this study, we demonstrated that Tp47 promoted the migration and adhesion of THP-1 cells to HDVSMCs. Furthermore, Tp47 increased monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression levels in a dose- and time-dependent manner. The migration and adhesion of THP-1 cells to HDVSMCs were significantly suppressed by anti-MCP-1 and anti-ICAM-1 neutralizing antibodies, respectively. Further studies revealed that treatment of HDVSMCs with Tp47 activated the PI3K/Akt, p38 MAPK and NF-κB signalling pathways. Inhibition of PI3K/Akt, p38 MAPK and NF-κB suppressed the MCP-1 and ICAM-1 expression induced by Tp47. In addition, the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs were significantly decreased by pretreatment with PI3K/Akt, p38 MAPK and NF-κB inhibitors. These findings demonstrate that Tp47 promotes the migration and adherence of THP-1 cells to HDVSMCs by inducing MCP-1 and ICAM-1 expression, which is mediated by activation of the PI3K/Akt, p38 MAPK and NF-κB pathways. This study provides a novel potential therapeutic strategy for controlling the vascular inflammatory response in syphilis patients.

Metabolic Disorders in Patients with Central Nervous System Infections: Associations with Neurosyphilis.

INTRODUCTION: Recently, neurosyphilis was found to be associated with diabetes mellitus (DM). Whether the association was specific to neurosyphilis among central nervous system (CNS) infections, and whether neurosyphilis is associated with other prevalent metabolic disorders deserves further study. METHODS: An in-depth cross-sectional study was conducted with 74 neurosyphilis patients and 74 sex- and age-matched patients with other CNS infections. DM-, hypertension-, and dyslipidemia-related factors were compared between patients with neurosyphilis and those with other CNS infections. RESULTS: The prevalence rates of hypertension and hyperlipidemia in neurosyphilis patients were 45.9 and 21.4%, respectively, which were higher than those in patients with other CNS infections (45.9 vs. 28.4%, p = 0.027; 21.4 vs. 8.3%, p = 0.028). In addition, neurosyphilis patients had significantly higher systolic blood pressure (BP; median 139 mm Hg; interquartile range [IQR] 121-151 mm Hg), -diastolic BP (median 83 mm Hg; IQR 76-89 mm Hg), total cholesterol (median 4.86 mmol/L; IQR 3.80-5.51 mmol/L), low-density lipoprotein (median 3.39 mmol/L; IQR 2.52-3.95 mmol/L), and apolipoprotein A1 (apoA1; median 1.31 g/L; IQR 1.06-1.52 g/L) levels and lower apoB/A1 ratios (median 0.67; IQR 0.49-0.99) than patients with other CNS infections (p< 0.05). There were no differences in the DM-related factors between patients with neurosyphilis and those with other CNS infections (p> 0.05). CONCLUSION: Potential association between neurosyphilis and metabolic disorders was found among CNS infections. The results could have important implications for clinical practice, alerting more clinicians to this issue.

Origin of Nontreponemal Antibodies During Treponema pallidum Infection: Evidence From a Rabbit Model.

The origin of nontreponemal antibodies during syphilis infection is hotly debated. Here, we analyzed the immune response in rabbits immunized with various antigens. Inactivated treponemes elicited the production of low-titer nontreponemal antibodies in some rabbits. Cardiolipin combined with bovine serum albumin also induced anticardiolipin antibody production. These findings indicate that Treponema pallidum contained a cardiolipin antigen with weak immunogenicity. However, active T. pallidum induced higher nontreponemal antibody production with strong immunogenicity at an earlier time point, and the antibody titer was consecutive, suggesting the high nontreponemal antibody titer resulted from the combined effects of both the T. pallidum cardiolipin antigen and the damaged host-cell cardiolipin antigen during syphilis infection, the latter of which plays a major role in the induction of nontreponemal antibody production. Our study provides direct animal evidence of the origin of nontreponemal antibodies during T. pallidum infection.

Treponema pallidum promotes macrophage polarization and activates the NLRP3 inflammasome pathway to induce interleukin-1ß production.

BACKGROUND: The involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1ß production and its influence on macrophage polarization triggered by T. pallidum. RESULTS: The results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1ß and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1ß secretion in T. pallidum-treated macrophages, and the observed production of IL-1ß occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1ß by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1ß release from T. pallidum-treated macrophages. CONCLUSIONS: These findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1ß secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1ß production in macrophages in response to T. pallidum infection involves K+ efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.

Assessing effects of different processing procedures on the yield of Treponema pallidum DNA from blood.

In this work, we employed real-time PCR analysis targeting tp0574 to investigate the effects of different processing procedures on the yield of T. pallidum DNA from blood to improve assay sensitivity. The T. pallidum DNA yields following red blood cell lysis pretreatment were 40.4 times greater from whole blood and 32.4 times greater from residual hematocytes than yields without pretreatment. For the simulated whole-blood experiments, the T. pallidum DNA yields from the lower layer were 2.8, 4.6, 7.3, 12.6, 15.24, 16.7, 65.1 and 73.1 times those from the upper layer following centrifugation at 500×, 1000×, 2000×, 4000×, 5000×, 7000×, 10,000× and 20,000 × g, respectively. However, the T. pallidum DNA yields from blood clots were only 1.0% at different centrifugal forces. The experiment with infected rabbit blood showed results similar to those mentioned above. In addition, sample processing time (within 48 h) and storage temperature (4 °C and 25 °C) did not affect T. pallidum DNA extraction efficiency. The T. pallidum DNA yield can be significantly improved by red blood cell lysis pretreatment and appropriate centrifugation. Furthermore, the T. pallidum DNA extraction yield is greater from whole blood or residual hematocytes from anti-coagulated blood than from plasma, serum or blood clots.

Correlation between polymorphisms in toll-like receptor genes and the activity of hepatitis B virus among treatment-naïve patients: a case-control study in a Han Chinese population.

BACKGROUND: Because of the high prevalence and absence of cure for infection, chronic hepatitis B virus (HBV) infection has been acknowledged as a pressing public health issue. Toll-like receptors (TLRs) activate the human innate immune system and the polymorphisms in TLRs may alter their function. The present study aimed to investigate the association between TLR polymorphisms and disease progression of chronic HBV infection. METHODS: During the study period, 211 treatment-naïve patients with chronic HBV infection were recruited, and blood samples were collected from each individual. Matrix-assisted laser desorption/ionization time of flight mass spectrometry was employed to genotype the selected TLR polymorphisms after human genome extraction. In addition, HbsAg, TNF-α, and IL-6 levels were quantified using enzyme linked immunosorbent assay (ELISA). Statistical analyses were conducted to investigate the association between TLR polymorphisms and hepatitis activity, liver function parameters, HbsAg level, and cytokine level. RESULTS: We did not observe any mutations in rs4986790, rs4986791, and rs5743708 among all study subjects. A logistic regression revealed that mutations in rs3804099 and rs4696480 were associated with milder hepatitis activity. Consistent with the logistic regression, improved liver function parameters and reduced level of both HbsAg and cytokines were also correlated with the mutant carriers of rs3804099 and rs4696480. CONCLUSIONS: TLR mutations were significantly associated with milder hepatitis activity among patients with chronic HBV infection. Therefore, we conclude that the activation of TLR pathways may further intensify the inflammation of hepatocytes, and leads to progression of disease.

Novel predictors of neurosyphilis among HIV-negative syphilis patients with neurological symptoms: an observational study.

BACKGROUND: Known predictors of neurosyphilis were mainly drawn from human immunodeficiency virus (HIV)-infected syphilis patients, which may not be applicable to HIV-negative populations as they have different characteristics, particularly those with neurological symptoms. This study aimed to identify novel predictors of HIV-negative symptomatic neurosyphilis (S-NS). METHODS: From June 2005 to June 2015, 370 HIV-negative syphilis patients with neurological symptoms were recruited, consisting of 191 S-NS patients (including 123 confirmed neurosyphilis and 68 probable neurosyphilis patients) and 179 syphilis/non-neurosyphilis (N-NS) patients. Clinical and laboratory characteristics of S-NS were compared with N-NS to identify factors predictive of S-NS. Serum rapid plasma reagin (RPR), Treponema pallidum particle agglutination (TPPA), and their parallel testing format for screening S-NS were evaluated. RESULTS: The likelihood of S-NS was positively associated with the serum RPR and TPPA titers. The serum TPPA titers performed better than the serum RPR titers in screening S-NS. The optimal cut-off points to recognize S-NS were serum RPR titer ≥1:4 and serum TPPA titer ≥1:2560 respectively. A parallel testing format of a serum RPR titer ≥1:2 and serum TPPA titer ≥1:1280 screened out 95.8% of S-NS and all confirmed cases of neurosyphilis. S-NS was independently associated with male sex, serum RPR titer ≥1:4, serum TPPA titer ≥1:2560, and elevated serum creatine kinase. Concurrence of these factors increased the likelihood of S-NS. CONCLUSIONS: Quantitation of serum TPPA is worthwhile and performs better than serum RPR in screening S-NS. Serum RPR, serum TPPA, male sex, and serum creatine kinase can predict S-NS. Moreover, patients with both a serum RPR titer <1:2 and a serum TPPA titer <1:1280 have a low probability of S-NS, suggesting that it is reasonable to reduce lumbar punctures in such individuals.

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing.

The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays.

Evaluation of the boson chemiluminescence immunoassay as a first-line screening test in the ECDC algorithm for syphilis serodiagnosis in a population with a high prevalence of syphilis.

We developed a new Boson chemiluminescence immunoassay (CIA) and evaluated its application with cross-sectional analyses. Our results indicated that the Boson CIA demonstrated strong discriminatory power in diagnosing syphilis and that it can be used as a first-line screening test for syphilis serodiagnosis using the European Centre for Disease Prevention and Control algorithm or as a confirmatory test when combined with a patient's clinical history.

Impact of mannose-binding lectin 2 polymorphism on the risk of hepatocellular carcinoma: a case-control study in Chinese Han population.

BACKGROUND: Mannose-binding lectin2 (MBL2) is implicated in the host immune response, but there are limited data about MBL2 polymorphisms and hepatocellular carcinoma (HCC) risk. This study aimed to investigate the relationship between the MBL2 rs7096206 polymorphism and HCC risk in a Chinese Han population. METHODS: A population-based case-control study of 220 HCC patients and 220 age- and gender-matched healthy control subjects from a Chinese Han population was conducted. Genomic DNA was extracted from blood samples, and the presence of the MBL2 polymorphism rs7096206 was assessed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Conditional logistic regression was performed to assess the risk of HCC by determining odds ratios and 95% confidence intervals (CIs). RESULTS: The odds of HCC among carriers of CG and GG genotypes were 7.33 (95% CI, 2.53-21.29) and 12.48 (95% CI, 2.08-74.90), respectively. In the dominant genetic model, GG+CG carriers had an approximately 8-fold increased risk (95% CI, 2.83-22.62) compared with those with the CC genotype. The G allele was significantly associated with elevated HCC risk, with an odds ratio of 6.83 (95% CI, 2.90-16.10). CONCLUSIONS: Our findings suggest that the MBL2 polymorphism rs7096206 is associated with HCC susceptibility and has the potential to serve as a biomarker to detect populations at increased HCC risk.

Distributions of heavy metals in maternal and cord blood and the association with infant birth weight in China.

OBJECTIVE: To measure serum levels of heavy metals in Chinese pregnant women and their newborns, and to evaluate the association of these metals with infant birth weight. STUDY DESIGN: We measured serum concentrations of lead (Pb), thallium (Tl), cadmium (Cd), selenium (Se), arsenic (As), nickle (Ni), vanadium (V), cobalt (Co), and mercury (Hg) in 81 mother-infant pairs using an inductively coupled plasma mass spectrometry method. Multiple linear regression analyses were used to evaluate the associations of these heavy metals with infant birth weight. RESULTS: Se, Pb, As, and Cd showed the highest detection rates (98.8%) in both the maternal and cord blood, followed by Tl, which was detected in 79.0% and 71.6% of the maternal and cord blood samples, respectively. Pb had the highest concentrations in both the maternal and cord blood samples of all toxic metals detected, with concentrations of 23.1 ng/g and 22.0 ng/g, respectively. No significant associations were observed between any heavy metals and birth weight. However, Tl in the maternal and cord blood was most notably inversely associated with birth weight. CONCLUSION: Se intake was low in Chinese women and their newborns, whereas Pb had the highest concentrations in both the maternal and cord blood samples of all toxic metals detected. Tl was a unique pollution source in this population, and Tl levels were shown to have the largest effect on decreasing infant birth weight in this pilot study. Further research incorporating larger sample sizes is needed to investigate the effects of prenatal exposure to heavy metals--especially Tl and Pb--on birth outcome in Chinese infants.

[The relationship between the KIF1B (rs17401966) single nucleotide polymorphism and the genetic susceptibility to Hepatocellular carcinoma].

OBJECTIVE: To study the relationship between SNP rs17401966 at the KIF1B gene and the genetic susceptibility to Hepatocellular carcinoma (HCC). METHODS: All study objects were recruited from two Grade A hospitals of Amoy from January 2011 to October 2014.They were surveyed in individual matching case-control study. Accepting criterias in the cases: HCC was first diagnosed based on diagnostic basis during the investigations, over 18 years old, present addresses were as same as surveyed areas in the district (county) level range, no past history of cancers; Exclusion criterias: patients with other liver diseases. The tumor patients without HCC, patients with autoimmune hepatitis or toxic hepatitis, patients who refused to be investigated or too ill to be investigated. Accepting criterias in the controls: the control who passed the physical examination matched the case in ages (no more than 3 years old), sex, health screening in the same hospital over the same period and district (county); Exclusion criterias: people with liver disease or any history of cancers. This study consisted of 376 HCC patients and 403 controls, 5 ml morning fasting venous blood of all subjects were obtained to isolate cells and distribute genotype. The differences in general information between cases and controls were tested by χ² test and t-test. The association between SNP rs17401966 and the risk of developing HCC were assessed by using the multiple factors logistic regression. RESULTS: The mean age and standard deviation for case and control groups were (61.7 ± 12.8) years and (60.6 ± 12.7) years (t = 1.15, P = 0.251), respectively. The proportion of family history of cancer [28.7% (108/376)] and the HBsAg positive rate [26.9 % (101/376)] in case group were higher than these in control group [15.9% (64/403), 2.7% (11/403)] (χ² = 18.65, 92.02, P < 0.001). In HBsAg carriers, GG genotype genetic susceptibility to HCC is 0.12 (0.02-0.75) times for AA genotype, and G allele susceptibility to HCC is 0.38 (0.15-0.98) times for A allelc. In HBsAg negative group, it showed no statistical significance in the relationship between SNP rs17401966 and susceptibility to HCC, and compared with the A allele, the risk for HCC of G allele is 0.79 (0.62-1.01). CONCLUSION: The results demonstrated that the presence of the GG genotype, the GA genotype and the G allele at rs17401966 of the KIF1B gene might decrease the risk for HCC.

[Occult hepatitis B virus infection in normal population, Xiamen].

OBJECTIVE: To investigate the prevalence of occult HBV infection in the normal population in Xiamen. METHODS: 4 437 registered permanent residents, aged 1-59 years old, were selected in Xiamen using stratified random sampling method from September to October in 2006. Serum samples were obtained, the basic characteristics, inoculation of HBV vaccine, and liver disease were surveyed. The serum samples were tested five HBV seroimmunological markers. The HBsAg-negative specimens were subjected to HBV-DNA detection by nested PCR targeting for multiple gene segments. The amplified products were sequenced and the sequence was used for determination of HBV genotype and mutation analysis of amino acids located in HBsAg "a" epitope. Subjects with serum detectable HBV-DNA and negative result of HBsAg were considered as occult HBV infection. RESULTS: Among the 4 437 subjects, 482 individuals were observed HBsAg positive and 3 944 were observed negative. Of the 3 955 HBsAg- negative specimens, 27 occult HBV infections were determined with the positive rate of 0.68% (27/3 955). There were 16 samples with genotype B and 11 with genotype C. 3 types of amino acid (AA) mutation (M133T, T140I, G145R) that influence "a" epitope conformation were observed in 9 subjects with occult HBV infection. S region was successfully sequenced in 312 of the 482 HBsAg positive samples. In subjects with occult HBV infection, the infection rate of genotype C HBV (40.74%, 11/27), inoculation rate of HBV vaccine (62.96%, 17/27), positive rate of HBsAb (51.85%, 14/27), and mutation rate of critical amino acid of "a" epitope (33.33%, 9/27) were higher than HBsAg positive individuals (22.76% (71/312), 13.78% (43/312),0.32% (1/312),0.99% (31/312), respectively), and all the difference were significant (χ(2) = 4.29, 41.26, 156.00, 13.07, respectively, and P value = 0.038, <0.001, <0.001, <0.001, respectively). While the average age in subjects with occult HBV infection (18.3 ± 16.2) were lower than that in HBsAg positive infection (34.4 ± 11.6), and the difference was significant (t = 6.67, P < 0.001). The reactive rate of HBeAb (11.11%, 3/27) and HBcAb (62.96%, 17/27) in subjects with occult HBV infection were lower than that in HBsAg positive infection (74.36% (232/312), 98.40% (307/312)), and the difference were significant (χ(2) = 46.74, 73.78, respectively, and P value <0.001, <0.001, respectively). CONCLUSION: In normal population in Xiamen, the infection rate of genotype C, the positive rate of HBsAb, the HBV vaccination rate, and the key AA mutation rate in "a" epitope are significantly higher in occult HBV infection than in HBsAg positive infection, and the age, the positive rate of HBeAb and HBcAb are significantly lower.

Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.

The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions -17 to -8), inhA position 94, and the ahpC promoter (positions -44 to -30 and -15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2×10(3) to 2×10(4) bacilli/ml), reproducibility (standard deviation [SD], <0.4°C), and the lowest heteroresistance level (40%) all met the parameters preset by the kit. The assay could be run on five types of real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC→ACC), inhA -15C→T, katG S315N (AGC→AAC), and ahpC promoter -10C→T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.

A survey of five broad-host-range plasmids in gram-negative bacilli isolated from patients.

OBJECTIVES: To learn the prevalence of the primary classical broad-host-range (BHR) IncA/C, IncN, IncP, IncQ, and IncW plasmids in dominant gram-negative bacilli from inpatients in a teaching hospital in southern China. METHODS: A multiplex polymerase chain reaction based on the replicons of BHR IncA/C, IncN, IncP, IncQ, and IncW plasmids was developed and used to determine these BHR plasmids. The difference in prevalence rates among the different species from three specimens was evaluated by a binary logistic regression model and the differences between multidrug-resistant organisms (MDRO) and non-MDRO were assessed using a chi-square test. RESULTS: The average positive detection percentages of the replicons were 4.3%, 3.7%, 3.0%, 2.6%, and 1.9%, respectively, for IncN, IncP, IncQ, IncW, and IncA/C in descending order. The distribution of all five BHR plasmids did not differ significantly between specimens collected from wounds and urine, although both were significantly higher than those of sputum. The prevalence rates of all five BHR plasmids in MDROs were significantly higher than those in non-MDRO for Enterobacteriaceae; however, no significant difference was seen in non-fermenting gram-negative bacilli (NFGNB). CONCLUSIONS: BHR IncA/C, IncN, IncP, IncQ, and IncW plasmids, which occur more often in bacilli from wound and urine specimens than those of sputum, are widespread in Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii strains isolated from inpatients. The prevalence rates in MDRO are higher than those in non-MDRO for Enterobacteriaceae but not significantly different for NFGNB.

Identification of pneumococcal serotypes with individual recognition of vaccine types by a highly multiplexed real-time PCR-based MeltArray approach.

BACKGROUND: Pneumococcus serotyping is important for monitoring serotype epidemiology, vaccine-induced serotypes replacement and emerging pathogenic serotypes. However, the lack of high-resolution serotyping tools has hindered its widespread implementation. METHODS: We devised a single-step, multiplex real-time polymerase chain reaction (PCR)-based MeltArray approach termed PneumoSero that can identify 92 serotypes with individual recognition of 54 serotypes, including all 24 currently available vaccine types. The limit of detection (LOD) and the ability to coexisting serotypes were studied, followed by analytical evaluation using 92 reference pneumococcal strains and 125 non-pneumococcal strains, and clinical evaluation using 471 pneumococcus isolates and 46 pneumococcus-positive clinical samples. RESULTS: The LODs varied with serotypes from 50 to 100 copies per reaction and 10 % of the minor serotypes were detectable in samples containing two mixed serotypes. Analytical evaluation presented 100 % accuracy in both 92 reference pneumococcal strains and 125 non-pneumococcal strains. Clinical evaluation of 471 pneumococcus isolates displayed full concordance with Sanger sequencing results. The 46 clinical specimens yielded 45 typeable results and one untypeable result. Of the 45 typeable samples, 41 were of a single serotype and four were of mixed serotypes, all of which were confirmed by Sanger sequencing or separate PCR assays. CONCLUSION: We conclude that the PneumoSero assay can be implemented as a routine tool for pneumococcal serotyping in standard microbiology laboratories and even in clinical settings.