Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip.

BACKGROUND: Female reproductive tract infection (RTI) is the common source of varied diseases, especially as an important risk factor for pregnancy outcomes, therefore the rapid, accurate and simultaneous detection of multiple pathogens is in urgent need for assisting the diagnosis and treatment of RTI in pregnant women. Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Gardnerella vaginalis (G. vaginalis), Candida albicans (C. albicans) and Chlamydia trachomatis (C. trachomatis) are five main pathogens in lower genital tract with high risk, serious consequences and clinical demands. The combination of loop-mediated isothermal amplification (LAMP) and microfluidic technology was used to develop the LAMP-microfluidic chip for rapid, simple, sensitive and simultaneous detection of the five target pathogens above. RESULTS: Standard strains and clinical isolates were used for the establishment of the novel LAMP method in tube and LAMP-microfluidic chip, followed by the chip detection on 103 clinical samples and PCR verification partially. The sensitivities of LAMP of S. agalactiae, E. faecalis, G. vaginalis, and C. albicans in tube were 22.0, 76.0, 13.2, 1.11 CFU/µL, respectively, and C. trachomatis was 41.3 copies/µL; on LAMP-microfluidic chip they were 260, 154, 3.9 and 7.53 CFU/µL, respectively, and C. trachomatis was 120 copies/µL. The positive coincidence rates of clinical stains in tube and on chip experiments were 100%. Compared with the classic culture method performed in hospitals, the positive coincidence rate of the 103 clinical samples detected by LAMP-microfluidic chip were 100%. For the six inconsistent ones, including four G. vaginalis and two C. albicans positive samples tested by LAMP-microfluidic chip and verified by PCR were negative by culturing method in hospitals, indicating the lack of efficient detection by the classic culturing method. CONCLUSION: Our study suggested that the LAMP-microfluidic chips could simultaneously, efficiently, and accurately detect multiple main pathogens, including S. agalactiae, E. faecalis, G. vaginalis, C. albicans and C. trachomatis, in clinical samples of female RTI to give a great clinical value. Accordingly, this novel method has the potential to provide a valuable reference for female RTI screening and early diagnosis during pregnancy.

Pathogen-Specific Bactericidal Method Mediated by Conjugative Delivery of CRISPR-Cas13a Targeting Bacterial Endogenous Transcripts.

The emergence of antibiotic-resistant bacteria threatens public health, and the use of broad-spectrum antibiotics often leads to unintended consequences, including disturbing the beneficial gut microbiota and resulting in secondary diseases. Therefore, developing a novel strategy that specifically kills pathogens without affecting the residential microbiota is desirable and urgently needed. Here, we report the development of a precise bactericidal system by taking advantage of CRISPR-Cas13a targeting endogenous transcripts of Salmonella enterica serovar Typhimurium delivered through a conjugative vehicle. In vitro, the CRISPR-Cas13a system exhibited specific killing, growth inhibition, and clearance of S. Typhimurium in mixed microbial flora. In a mouse infection model, the CRISPR-Cas13a system, when delivered by a donor Escherichia coli strain, significantly reduced S. Typhimurium colonization in the intestinal tract. Overall, the results demonstrate the feasibility and efficacy of the designed CRISPR-Cas13a system in selective killing of pathogens and broaden the utility of conjugation-based delivery of bactericidal approaches. IMPORTANCE Antibiotics with broad-spectrum activities are known to disturb both pathogens and beneficial gut microbiota and cause many undesired side effects, prompting increased interest in developing therapies that specifically eliminate pathogenic bacteria without damaging gut resident flora. To achieve this goal, we developed a strategy utilizing bacterial conjugation to deliver CRISPR-Cas13a programmed to specifically kill S. Typhimurium. This system produced pathogen-specific killing based on CRISPR RNA (crRNAs) targeting endogenous transcripts in pathogens and was shown to be effective in both in vitro and in vivo experiments. Additionally, the system can be readily delivered by conjugation and is adaptable for targeting different pathogens. With further optimization and improvement, the system has the potential to be used for biotherapy and microbial community modification.

Effects of active and latent H. pylori infection coupled with chronic alcohol ingestion on cytokine profiles and markers of oxidative balance in men seropositive for H. pylori CagA Ab: An observational study.

This study aimed to explore the effects of active and latent Helicobacter pylori infection coupled with alcohol consumption on cytokine profiles and markers of oxidative balance in men seropositive for H. pylori CagA Ab.The 100 male subjects were divided into groups with active H. pylori infection and H. pylori CagA Ab coupled with chronic alcohol ingestion (group A, n = 38), latent H. pylori infection with H. pylori CagA Ab coupled with chronic alcohol ingestion (group B, n = 30), and latent H. pylori infection with H. pylori CagA Ab without chronic alcohol ingestion (group C, n = 32).No differences in serum levels of CRP, IL-10, ADP, E-selectin, MDA, or SOD were detected between the 3 groups or between any 2 groups (all P > .05). The serum IL-6 and TNF-α concentrations in groups A and B were significantly lower than those in group C (P = .004, P = .005, P = .009, and P = .023). However, there were no differences in serum IL-6 and TNF-α between group A and group B (all P > .05).In conclusion, active or latent H. pylori infection coupled with chronic alcohol ingestion may decrease certain cytokines, that is, IL-6 and TNF-α, in men with H. pylori CagA Ab seropositivity. However, there was no difference in the detected cytokine profile between active and latent H. pylori infection coupled with chronic alcohol ingestion, and no changes were detected in markers of oxidative balance in men with H. pylori CagA Ab.

Association between circulating inflammatory molecules and alcoholic liver disease in men.

The association between alcoholic liver disease (ALD) and the inflammatory response remains controversial. The aim of this study was to explore this association between ALD and inflammation. We enrolled 214 male participants, who were divided into three age-matched groups: ALD (n = 135), chronic alcohol ingestion without ALD (non-ALD; n = 42), and control (n = 37). The BMI was significantly higher in the ALD group than in the non-ALD and control groups (all P = 0.000). Further, the constituent ratio of the liver inflammatory level was significantly higher in the ALD group than in the non-ALD and control groups (P = 0.002 and P = 0.000, respectively). In addition, the median serum ALT, AST, and GGT levels were significantly higher in the ALD group than in the control group (P = 0.023, P = 0.008, and P = 0.000, respectively); these levels were also significantly higher in the ALD group than in the non-ALD group (P = 0.013, P = 0.010, and P = 0.000, respectively). The median serum CRP level was significantly higher in the ALD group than in the non-ALD and control groups (P = 0.006 and P = 0.000, respectively). Further, the median serum TNF-α level was significantly lower in the ALD group than in the non-ALD and control groups (P = 0.004 and P = 0.000, respectively). The median serum sOX40L and HSP70 levels were significantly lower in the ALD group than in the control group (P = 0.008 and P = 0.018, respectively). In addition, the ALT, AST, and GGT levels were positively correlated with the CRP level (r = 0.211, P = 0.002; r = 0.220, P = 0.001 and r = 0.295, P = 0.000, respectively), and the GGT level was negatively correlated with the TNF-α (r = -0.225, P = 0.001), sOX40L (r = -0.165, P = 0.016), and HSP70 levels (r = -0.178, P = 0.009). Further, the Cr level was negatively correlated with the IL-10 level (r = -0.166, P = 0.015). Logistic regression analysis verified that the BMI (OR  =  1.637, 95%CI: 1.374-1.951, P  =  0.000) and GGT level were significantly higher (OR  =  1.039, 95%CI: 1.020-1.059, P  =  0.000) and that the TNF-α (OR  =  0.998, 95%CI: 0.996-1.000, P  =  0.030) and HSP70 levels were significantly lower (OR  =  1.017, 95%CI: 1.003-1.031, P  =  0.029) in the ALD group than in the non-ALD group. Further, the moderate-to-severe ALD patients had a significantly higher serum CRP level (Or =   1.349, 95%CI: 1.066-1.702, P  =  0.013) and significantly lower HSP60 (OR  =  0.965, 95%CI: 0.938-0.993, P  =  0.014) and HSP70 levels (OR  =  0.978, 95%CI: 0.962-0.995, P  =  0.010) than the mild ALD patients. These results suggest that ALD patients may present with obesity, liver damage, and an imbalanced inflammatory immune response, mainly manifesting as decreased levels of immune inflammatory cytokines. In addition, they suggest that certain liver and kidney function parameters and ALD severity are either positively or negatively correlated with certain inflammatory cytokines. Hence, ALD patients may be at increased risks of obesity- and inflammation-related diseases. Accordingly, to control the inflammatory response, preventative measures for patients with this disease should include weight control and protection of liver and kidney function.

Risk Factors Associated With Increased Carotid Intima-Media Thickness in a Male Population With Chronic Alcohol Consumption: A Prospective Observational Study.

Previous studies have reported a relationship between alcohol consumption and carotid intima-media thickness (CIMT). However, the exact associations between different severities of CIMT and dyslipidemia, dyslipoproteinemia, inflammatory immune markers, and oxidative markers associated with chronic alcohol consumption remain unknown. The aim of this study was to explore whether there are associations between different severities of CIMT and dyslipidemia, dyslipoproteinemia, inflammatory immune markers, and oxidative markers associated with chronic alcohol consumption. We enrolled 173 males with chronic alcohol consumption and categorized them into 2 groups: 104 chronic alcohol consumers with normal CIMT (group A) and 69 chronic alcohol consumers with increased CIMT (group B). Nonparametric statistics showed that age, body mass index (BMI), and serum TC, TG, Apo A1, and ApoB levels were significantly higher in group B than in group A (P = 0.002, 0.019, 0.021, 0.023, 0.001, and 0.001, respectively). Additionally, tumor necrosis factor alpha (TNFα) and HSP70 serum levels were significantly lower in group B than in group A (P = 0.023 and 0.017, respectively). A binary logistic regression analysis showed that age (OR: 1.077, 95% CI: 1.024-1.13, P = 0.004), ApoB (OR: 6.828, 95% CI: 1.506-30.956, P = 0.013), and TNF-α (OR: 0.999, 95% CI: 0.998-1.00) were independent risk factors associated with CIMT. The present study demonstrated that age, ApoB, and TNFα are independent risk factors associated with CIMT. Thus, older subjects with increased serum ApoB levels are more likely to present with increased CIMT, suggesting that age and ApoB promote such thickening and that TNFα downregulation might play a protective role against the progression of subclinical atherosclerosis in subjects with chronic alcohol consumption.

PRISMA-Compliant Article: Clinical Characteristics and Factors Influencing Prognosis of Patients With Hepatoid Adenocarcinoma of the Stomach in China.

Most previous studies have been single case reports, and studies with large samples are presently lacking. In addition, no studies have investigated the associations between the clinical characteristics and prognosis of hepatoid adenocarcinoma of the stomach (HAS). The aim of this study was to explore the associations of different clinical characteristics with the ages, serum alpha-fetoprotein (AFP) levels, and survival times of HAS patients. The present study was conducted using the CBM disc, HowNet, Wanfang and VIP data resource systems, and PubMed. According to the PRISMA Flow Diagram, certain case reports from the same center, those that did not provide patient age or sex, and those that did not report serum AFP levels or AFP immunohistochemistry results were excluded. A total of 131 relevant articles, including 124 case reports, 5 reviews, and 2 postgraduate Master's theses, were reported in the above-mentioned five databases. We applied inclusion criteria to case reports on the clinical characteristics and prognosis of HAS, which resulted in the ultimate inclusion of 180 patients from 62 case reports for statistical analyses. The main finding was that the age of the men was significantly higher than that of the women (P = 0.004). In addition, the serum AFP levels of the participants with antral disease were significantly higher than those with nonantral disease (P = 0.001). The median serum AFP levels and survival times significantly differed among the patients with the three lesion types (P = 0.001 and 0.019, respectively). The serum AFP levels of the participants with ulcerative-upheaval-type tumors and purely ulcerative tumors were significantly higher than those with upheaval-type tumors (P = 0.000 and 0.017, respectively). In addition, the serum AFP levels of the participants with ulcerative-upheaval-type tumors were significantly higher than those with ulcerative-type tumors (P = 0.019), and their survival time was also significantly higher (P = 0.000). The serum AFP levels of the participants without metastasis or liver metastasis were significantly lower than those with metastasis or liver metastasis (P = 0.000 and 0.000, respectively), and their survival time was significantly longer (P = 0.000 and 0.001, respectively). Finally, the survival time of the participants treated with surgery was significantly longer than those treated using nonsurgical methods (P = 0.046). However, survival analysis revealed that the survival time was only significantly associated with the presence of metastasis (P = 0.002) and liver metastasis (P = 0.036). The main limitations of this study are as follows: it was a retrospective analysis of published case reports, the clinical data were incomplete, and the cases included in subgroup analyses were different. Our study results have demonstrated that the prognosis of HAS patients is poor. In addition, the survival time is significantly negatively correlated with the presence of metastasis and liver metastasis.

Influence of H. pylori CagA Coupled with Alcohol Consumption on Cytokine Profiles in Men.

The aim of this study was to evaluate the effect of Helicobacter pylori (H pylori) cytotoxin-associated gene A (CagA) coupled with chronic alcohol ingestion on cytokine profiles.A total of 215 male subjects were divided into the following 4 groups: 130 alcohol H pylori CagA-negative consumers (CagA-) (group A), 50 alcohol H pylori CagA-positive consumers (CagA+) (group B), 24 nonalcohol H pylori CagA-negative consumers (group C), and 11 nonalcohol H pylori CagA-positive consumers (group D). The serum CagA, C-reactive protein (CRP), interleukin (IL)-6, IL-10, E-selectin, adiponectin (ADP), and tumor necrosis factor-α (TNF-α) levels were measured through enzyme-linked immunosorbent assays (ELISAs).After adjusting for age and mean alcohol drinking history, a multivariable linear regression analysis revealed that the mean daily alcohol consumption, IL-6, TNF-α, and ADP levels were significantly increased with increases in the serum CagA concentrations (P = 0.008, P = 0.000, P = 0.000, and P = 0.006, respectively). The serum IL-6 and IL-10 levels of group A were significantly lower than those of group B (all P = 0.000). Furthermore, the serum IL-6 and IL-10 levels of groups A and C were significantly lower than those of group D (all P = 0.000), and the serum IL-6 and IL-10 levels of group C were significantly lower than those of group B (all P = 0.000). The serum ADP and E-selectin levels of groups B and D were significantly higher than those of group A (P = 0.000). The serum ADP levels of group B were significantly higher than those of group C (P = 0.000), and the serum ADP and E-selectin levels of group C were significantly lower than those of group D (P = 0.000 and P = 0.005, respectively). Finally, the serum TNF-α levels of groups B, C, and D were significantly higher than those of group A (all P = 0.000), and the serum TNF-α levels of group C were significantly higher than those of group D (P = 0.005).In conclusion, H pylori CagA may result in significantly higher levels of several inflammatory markers in both alcohol consumers and nonalcohol consumers. However, chronic alcohol ingestion coupled with H pylori CagA positivity does not result in significant changes in cytokine profiles.

The detection and role of heat shock protein 70 in various nondisease conditions and disease conditions: a literature review.

As an intracellular polypeptide, heat shock protein 70 (HSP70) can be exposed on the plasma membrane and/or released into the circulation. However, the role of HSP70 in various nondisease and disease conditions remains unknown. Quantitative methods for the detection of HSP70 have been used in clinical studies, revealing that an increase in circulating HSP70 is associated with various types of exercise, elderly patients presenting with inflammation, mobile phones, inflammation, sepsis, chronic obstructive pulmonary disease, asthma, carotid intima-media thickness, glutamine-treated ill patients, mortality, diabetes mellitus, active chronic glomerulonephritis, and cancers. Circulating HSP70 decreases with age in humans and in obstructive sleep apnea, arteriosclerosis, atrial fibrillation (AF) following coronary artery bypass surgery, nonalcoholic fatty liver disease, moderate-to-severe alcoholic fatty liver disease, hepatic steatosis, and Helicobacter pylori infection. In conclusion, quantitative methods can be used to detect HSP70, particularly in determining circulating HSP70 levels, using more convenient and rapid screening methods. Studies have shown that changes in HSP70 are associated with various nondisease and disease conditions; thus, HSP70 might be a novel potential biomarker reflecting various nondisease conditions and also the severity of disease conditions. However, the reliability and accuracy, as well as the underlying mechanism, of this relationship remain poorly understood, and large-sample clinical research must be performed to verify the role.

Causes of Thickening of Carotid Intima-Media Thickness in Patients With Alcoholic Liver Disease: A Prospective Observational Study.

Several studies have reported the relationship between alcoholic liver disease (ALD) and carotid intima-media thickness (CIMT). Few studies, however, have investigated the causes of CIMT thickening in patients with ALD. The authors explored the causes of CIMT thickening in patients with ALD. The authors enrolled 152 patients who were stratified into groups: nonthickening CIMT with ALD (group A); thickening CIMT with ALD (group B); nonthickening CIMT without ALD (group C); and thickening CIMT without ALD (group D). The CIMT was significantly different between patients with and without ALD (χ 2= 3.875, P = 0.049). The patients in groups A, B, and C were significantly younger than group D (P = 0.001, 0.036, and 0.001, respectively). The body mass indexes (BMI) in groups A and B were significantly higher than in group C (P = 0.000 and 0.007, respectively). The blood glucose levels in groups B and D were significantly higher than in group C (P = 0.016 and 0.018, respectively). The blood uric acid levels in group B were significantly higher than in groups A, C, and D (P = 0.009, 0.000, and 0.003, respectively). The blood uric acid in group A was significantly higher than in group C (P = 0.002). The serum total cholesterol (TC) levels of patients in group B were significantly higher than in groups A and C (P = 0.027 and 0.000, respectively) and the serum TC level in group A was significantly higher than in group C (P = 0.048). The serum triglyceride (TG) levels in groups A and B were significantly higher than in group C (P = 0.027 and 0.000, respectively). The serum of very low-density lipoprotein (VLDL) levels in group B were significantly higher than in group C (P = 0.000). Although a comparison of the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) serum levels among the 4 groups indicated no changes. The serum LDL levels in group B were significantly higher than in group A (P = 0.008). No significant differences were observed among the groups with respect to serum homocysteine, C-reactive protein (CRP), interleukin-6 (IL-6), malondialdehyde (MDA), superoxide dismutase (SOD), soluble OX40 ligand (sOX40L), or heat shock protein (HSP) 60 or 70. Alcoholic liver disease may result in CIMT thickening. Carotid intima-media thickness is associated with age and metabolic factors in patients with ALD. In addition, ALD might promote the premature occurrence of CIMT thickening. The thickening of carotid artery intima thickness, however, is not associated with cytokine profiles, oxidative balance, or immune responses in patients with ALD.

Effect of H. pylori Infection on Cytokine Profiles and Oxidative Balance in Subjects with Chronic Alcohol Ingestion.

Different amounts of ingested alcohol can have distinct effects on the human body. However, there is limited research on chronic alcohol consumption with Helicobacter pylori infection. We sought to investigate the relationship between the cytokine profile, oxidative balance and H. pylori infection in subjects with chronic alcohol consumption. A total of 142 subjects were divided into three groups: 59 subjects with chronic alcohol ingestion and H. pylori infection (group A); 53 subjects with chronic alcohol ingestion without H. pylori infection (group B); and 30 control subjects (group C). The serum levels of CagA, interleukin (IL)-10, E-selectin, TNF-α, malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured by enzyme-linked immunosorbent assay (ELISA). We found that the ages and serum H. pylori CagA levels among the three groups, as well as both the mean drinking age and the mean daily alcohol consumption between groups A and B, were matched and comparable. Comparing the BMIs among the three groups, the BMI differences were found to be statistically significant (F=3.921, P<0.05). Compared with group C, the BMIs in groups A and B were significantly higher (P<0.001 and P<0.01, respectively); however, the BMI differences between group A and group B were not statistically significant (P>0.05). Additionally, no differences in the serum CagA levels were found in comparisons among the groups (all P>0.05). The serum IL-10 and E-selectin levels in group A were significantly lower than those in group B (serum IL-10: P<0.05; E-selectin: P<0.05). The serum IL-10 in group A was significantly higher than that in group C (P<0.01); the serum E-selectin levels in group A did not significantly differ compared with those in group C (P>0.05). Furthermore, the serum IL-10 and E-selectin levels in group B were significantly higher than those in group C (serum IL-10: P<0.001; E-selectin: P<0.05); however, the serum TNF-α levels did not differ among groups (all P>0.05). Although the serum levels of MDA and SOD in groups A and B were slightly lower than those in group C, there were no significant differences among groups (all P>0.05). In conclusion, we believe that H. pylori infection might cause a significant inhibition of certain cytokine profiles in subjects with chronic alcohol ingestion. Moreover, chronically ingested alcohol may exert an adjusted inflammatory effect, but there was no association between H. pylori infection, chronic alcohol consumption and oxidative balance.

Changes in tumor necrosis factor-α, heat shock protein 70, malondialdehyde, and superoxide dismutase in patients with different severities of alcoholic fatty liver disease: a prospective observational study.

The relationships among inflammation, oxidative balance, and the severity of alcoholic fatty liver disease (AFLD) remain unknown. The aim of this study is to explore the relationships among tumor necrosis factor alpha (TNF-α), heat shock protein 70 (HSP70), malondialdehyde (MDA), superoxide dismutase (SOD), and the severity of AFLD.From January 2012 to December 2013, 162 participants were enrolled in this study and divided into 4 groups: 44 cases of mild AFLD (group A), 55 cases of moderate-to-severe AFLD (group B), 44 cases of alcohol consumption without AFLD (group C), and 20 cases of no alcohol consumption without AFLD (group D). A cross-sectional study was conducted by detecting the serum levels of TNF-α, HSP70, MDA, and SOD by enzyme-linked immunosorbent assay.The median serum levels of TNF-α and HSP70 among the 4 groups were statistically significant (P = 0.000 and 0.001, respectively). The median serum levels of TNF-α in groups A and B were significantly lower than in group C (P = 0.002 and 0.000, respectively), and the median serum level of TNF-α in group B was significantly lower than in group D (P = 0.023). In addition, the median serum level of HSP70 in group B was significantly lower than in groups A and C (P = 0.002 and 0.000, respectively), and the median serum level of HSP70 in group C was significantly higher than in group D (P = 0.044). However, the median serum level of MDA in group B was significantly lower than only group C (P = 0.008).Chronic alcohol ingestion without AFLD may result in a significant increase in the circulation of certain inflammatory markers; the severity of AFLD is associated with circulating inflammatory markers, and moderate-to-severe AFLD may result in a more significant reduction of these markers. However, moderate-to-severe AFLD may also result in a significant downregulation of oxidative stress products.