Risk factors for progression to bacteremia among patients with nosocomial carbapenem-resistant Acinetobacter baumannii pneumonia in the Intensive Care Unit.

Antibiotic-resistant Acinetobacter baumannii (A. baumannii) is a common cause of hospital-acquired infections. This study aimed to identify independent factors associated with progression from nosocomial pneumonia to bacteremia in patients infected with carbapenem-resistant A. baumannii (CR-AB). From 2019 to 2021, we conducted a retrospective anaylsis of the medical records of 159 nosocomial CR-AB pneumonia patients in our Intensive Care Unit (ICU). We employed both univariate and multivariable logistic regression models to identify factors associated with the progression of nosocomial CR-AB pneumonia to bacteremia. Among the 159 patients with nosocomial CR-AB pneumonia, 40 experienced progression to bacteremia and 38 died within 28 days following diagnosis. Patients who developed bacteremia had a significantly higher 28-day mortality rate compared to those without bloodstream infection (47.50% vs. 15.97%). Multivariable logistic regression revealed that higher levels of C-Reactive protein (CRP) (OR = 1.01) and the use of continuous veno-venous hemofiltration (CVVH) treatment (OR = 2.93) were independently associated with an elevated risk of developing bacteremia. Among patients who developed bloodstream infection, those who died within 28 days exhibited significantly higher level of interleukin-6 (IL-6), a greater frequency of antifungal drugs usage, and a longer duration of machanical ventilation compared to survivors. Furthermore, the use of antifungal drugs was the only factor that associated with 28-day mortality (OR = 4.70). In ICU patients with central venous catheters who have CR-AB pneumonia and are on mechanical ventilation, higher CRP levels and CVVH treatment are risk factors for developing bacteremia. Among patients with bacteremia, the use of antifungal drugs is associated with 28-day mortality.

Causal associations between gut microbiota and sepsis: A two-sample Mendelian randomization study.

BACKGROUND: Targeting the gut microbiota may become a new therapeutic to prevent and treat sepsis. Nonetheless, the causal relationship between specific intestinal flora and sepsis is still unclear. METHODS: A two-sample Mendelian randomization study was performed using the summary statistics of gut microbiota from the largest available genome-wide association study (n = 18,340). The summary statistics of sepsis were obtained from the UK Biobank (n = 486,484). Inverse-variance weighted, weighted median and MR-Egger were used to examine the causal association between gut microbiota and sepsis. Cochrane's Q test, MR-Egger intercept test, MR-PRESSO Global test and Rucker's Q'-test were used for sensitivity analyses. The leave-one method was used for testing the stability of MR results, and Bonferroni-corrected was used to test the strength of the causal relationship between exposure and outcome. RESULTS: Nine intestinal microflora were found causally associated with sepsis, and 11 intestinal microflora were causally associated with 28-day death in sepsis. Among them, Order Victivallales had a strong causality with lower risk of sepsis (OR = 0.86, 95% CI: 0.78-0.94, p = .00165) and lower 28-day mortality of sepsis (OR = 0.68, 95% CI: 0.53-0.87, p = .00179) after Bonferroni-corrected test. No pleiotropy was detected. CONCLUSIONS: Through the two-sample MR analysis, we identified the specific intestinal flora that had a causal relationship with the risk and prognosis of sepsis at the level of gene prediction, which may provide helpful biomarkers for early disease diagnosis and potential therapeutic targets for sepsis.

Diagnostic Potential of microRNAs in Extracellular Vesicles Derived from Bronchoalveolar Lavage Fluid for Pneumonia-A Preliminary Report.

Current clinical needs require the development and use of rapid and effective diagnostic indicators to accelerate the identification of pneumonia and the process of microbiological diagnosis. MicroRNAs (miRNAs) in extracellular vesicles (EVs) have become attractive candidates for novel biomarkers to evaluate the presence and progress of many diseases. We assessed their performance as biomarkers of pneumonia. Patients were divided into the pneumonia group (with pneumonia) and the control group (without pneumonia). We identified and compared two upregulated miRNAs in EVs derived from bronchoalveolar lavage fluid (BALF-EVs) between the two groups (PmiR-17-5p = 0.009; PmiR-193a-5p = 0.031). Interestingly, in cell-debris pellets and EVs-free supernatants derived from bronchoalveolar lavage fluid (BALF-cell-debris pellets and BALF-EVs-free supernatants), total plasma, and EVs derived from plasma (plasma-EVs), the expression of miR-17-5p and miR-193a-5p showed no difference between pneumonia group and control group. In vitro experiments revealed that miR-17-5p and miR-193a-5p were strikingly upregulated in EVs derived from macrophages stimulated by lipopolysaccharide. MiR-17-5p (area under the curve, AUC: 0.753) and miR-193a-5p (AUC: 0.692) in BALF-EVs are not inferior to procalcitonin (AUC: 0.685) in the diagnosis of pneumonia. Furthermore, miR-17-5p and miR-193a-5p in BALF-EVs had a significantly higher specificity compared to procalcitonin and could be served as a potential diagnostic marker. MiR-17-5p and miR-193a-5p in EVs may be involved in lung inflammation by influencing the forkhead box O (FoxO) signaling pathway and protein processing in endoplasmic reticulum. This study is one of the few studies which focused on the potential diagnostic role of miRNAs in BALF-EVs for pneumonia and the possibility to use them as new biomarkers for a rapid and early diagnosis.

Severe skin and subcutaneous pythiosis in China: Metagenomic identification and characterization of Pythium insidiosum.

Pythium insidiosum is a rare fungus-like pathogen that is known to cause pythiosis in mammals with high morbidity and mortality. Identification of the pathogen is essential for timely treatment and rational use of antibiotics. However, Pythium insidiosum is difficult to detect via conventional microbiological tests. The current gold standard is polymerase chain reaction, which is lacking in most hospitals since human pythiosis is rare in China. In this study, we used metagenomic Next-Generation Sequencing and identified Pythium insidiosum in a 56-year-old Chinese male who was hospitalized due to severe edema in the right lower limb with scattered darkening indurations. The patient had a history of cirrhosis and occupational exposure to swamp water. Serological level of immune biomarkers indicated immunodeficiency, and Proteinase 3-Anti-Neutrophil Cytoplasmic Antibody was positive. Surgical incision of the lesions revealed radiating and reticular cutaneous ulcers. Microbial infections were suspected but conventional tests failed to discover the etiology. Empirical use of penicillin, vancomycin, and ceftriaxone had no effect. As a result, the peripheral blood and tissue biopsies were sent for metagenomic Next-Generation Sequencing, which reported Pythium insidiosum. This finding was corroborated by pathological staining, whole-genome sequencing, and internal transcribed spacer sequencing. Notably, antifungal treatment was ineffective, but the patient responded well to oral trimethoprim-sulfamethoxazole, which may be due to the folp gene found in Pythium insidiosum genome. Our study prompts future studies to determine the optimal treatment of skin pythiosis.

Weaning critically ill patients from mechanical ventilation: a protocol from a multicenter retrospective cohort study.

BACKGROUND: Mechanical ventilation (MV) is an important lifesaving method in intensive care unit (ICU). Prolonged MV is associated with ventilator associated pneumonia (VAP) and other complications. However, premature weaning from MV may lead to higher risk of reintubation or mortality. Therefore, timely and safe weaning from MV is important. In addition, identification of the right patient and performing a suitable weaning process is necessary. Although several guidelines about weaning have been reported, compliance with these guidelines is unknown. Therefore, the aim of this study is to explore the variation of weaning in China, associations between initial MV reason and clinical outcomes, and factors associated with weaning strategies using a multicenter cohort. METHODS: This multicenter retrospective cohort study will be conducted at 17 adult ICUs in China, that included patients who were admitted in this 17 ICUs between October 2020 and February 2021. Patients under 18 years of age and patients without the possibility for weaning will be excluded. The questionnaire information will be registered by a specific clinician in each center who has been evaluated and qualified to carry out the study. DISCUSSION: In a previous observational study of weaning in 17 ICUs in China, weaning practices varies nationally. Therefore, a multicenter retrospective cohort study is necessary to be conducted to explore the present weaning methods used in China. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) (No. ChiCTR2100044634).

Clinical descriptive analysis of severe Pneumocystis jirovecii pneumonia in renal transplantation recipients.

Pneumocystis jirovecii (P. jirovecii) pneumonia (PJP) is an opportunistic fungal infection after renal transplantation, which is always severe, difficult to diagnose, combined with multiple complications and have poor prognosis. We retrospectively analyzed clinical data, including risk factors, diagnosis, treatment and complications of seven clinical cases suffered with severe PJP after renal transplantation in our department in 2019. All the seven recipients were routinely prescribed with PJP prophylaxis after renal transplantation, and six of them suffered acute graft rejection before the infection. P. jirovecii sequence was identified in blood or broncho-alveolar lavage fluid (BALF) by the metagenomic next-generation sequencing (mNGS) in all patients. All the patients were improved with the therapy trimethoprim-sulfamethoxazole (TMP-SMX) combined with caspofungin for the PJP treatment, but suffered with complications including renal insufficiency, leukopenia, thrombocytopenia, gastrointestinal bleeding, mediastinalemphysema, pulmonary hemorrhage, and hemophagocytic syndrome and other severe infections. Taken together, mNGS is a powerful tool that could be used to diagnose PJP in renal transplantation recipients. And PJP prophylaxis should be prescribed during and after treatment for acute rejection. TMP-SMX is the first-line and effective drug for PJP treatment, but the complications are always life-threatening and lead to poor prognosis. We should pay attention to these life-threatening complications.

Co-Infection Pneumonia with Mycobacterium abscessus and Pneumocystis jiroveci in a Patient without HIV Infection Diagnosed by Metagenomic Next-Generation Sequencing.

INTRODUCTION: Co-infection pneumonia with Mycobacterium abscessus (M. abscessus) and Pneumocystis jirovecii (P. jirovecii) is rarely reported in previously healthy patients without HIV infection. The diagnosis of pneumonia of M. abscessus and P. jirovecii remains challenging due to its nonspecific clinical presentation and the inadequate performance of conventional diagnostic methods. CASE REPORT: We report the case of a 44-year-old previously healthy male transferred to our hospital in February 2020 with a 4-month history of productive cough and one month of intermittent fever. At local hospital, the metagenomic next-generation sequencing(mNGS) detected P. jirovecii sequences in blood; with the antifungal therapy (Caspofungin, trimethoprim-sulfamethoxazole [TMP-SMX] and methylprednisolone [MP]), the patient still had hypoxemia, cough and fever. Then he was transferred to our hospital, the mNGS of bronchoalveolar lavage fluid (BALF) detected the sequences of M. abscessus and P. jirovecii. CD4+ T-lymphocytopenia in the peripheral blood cells was presented and HIV serology was negative. Caspofungin, TMP-SMX, clindamycin and MP were used to treat P. jirovecii pneumonia (PJP). Moxifloxacin, imipenem cilastatin and linezolid were used to treat M. abscessus infection. Clinical progress was satisfactory following antifungal combined with anti-M. abscessus therapy. CONCLUSION: Co-infection pneumonia with M. abscessus and P. jirovecii as reported here is exceptionally rare. mNGS is a powerful tool for pathogen detection. M. abscessus infection could be a risk factor for P. jirovecii infection. This case report supports the value of mNGS in diagnosing of M. abscessus and P. jirovecii, and highlights the inadequacies of conventional diagnostic methods.

Online Clinical Consultation as a Utility Tool for Managing Medical Crisis During a Pandemic: Retrospective Analysis on the Characteristics of Online Clinical Consultations During the COVID-19 Pandemic.

Coronavirus disease 2019 (COVID-19) is a newly-identified infectious diseases that has rapidly spread throughout the world with rising fatalities with declaration by World Health Organization as the pandemic. Online consultations have been shown to alleviate the pandemic with our study aims to demonstrate whether online consultation can be a solution for acute health crisis. Retrospective analysis of the characteristics of online consultations through two primary care online-consultation platforms during COVID-19 pandemic was performed at the Third Affiliated Hospital of Sun Yat-Sen University, which led the assessment of COVID-19-symptoms patients in Guangzhou. The 3473 online consultations were divided into pre-pandemic and pandemic period groups with Chi-square test as statistical analysis method. The number of online consultations has increased with diagnosis of upper respiratory tract infection, psychological conditions, COVID-19-related investigations and interventions. The increased online consultations met the increased demand of the relevant clinical services and reduced the overwhelming hospital presentations, thus decreasing the potential COVID-19 spread inside the major tertiary hospital and sparing the resources for acute crisis management. The epidemiology and disease characteristics of online consultations during the pandemic have been demonstrated with identification of the enabling factors and potential barriers in improving online healthcare in China with online consultation model being a durable solution for pandemic in future.

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E. coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

Older adults are prioritized in terms of waiting time under the emergency triage system in Guangzhou, China.

AIM: To assess whether elderly patients are prioritized under the emergency triage system in Guangzhou, China. METHODS: This was a cross-sectional survey of clinical data from adult visitors to the emergency department of the Third Affiliated Hospital of Sun Yat-sen University between 1 August 2015 and 31 December 2017. The primary end-point was receiving the first medical service within the target waiting time, which varied according to the triage level of the patient. Multivariate logistic regression was used to determine whether age was an independent predictor of a shorter waiting time. RESULTS: Data from 262 282 emergency patients were analyzed. The mean age of patients was 35.97 years, and 7.5% were aged ≥65 years. In total, 88.3% of patients received medical service within the target waiting time, and 87.4% of elderly patients received medical service within the target waiting time. Multivariate logistic regression analysis showed that advanced age was independently associated with receiving medical service within the target waiting time (adjusted odds ratio 1.258, 95% confidence interval 1.198-1.321; P < 0.001). The triage level, type of emergency subdivision, availability of outpatient services and time of day were also associated with receiving medical service within the target waiting time. CONCLUSION: Under the emergency triage system of the hospital, older adults are more likely to receive medical service within the target waiting time than younger patients. Geriatr Gerontol Int 2019; 19: 786-791.

[Effect of age on waiting time of emergency patients: a retrospective analysis of 219 299 patients in consecutive 2 years].

OBJECTIVE: To evaluate whether patients aged over 70 years old is an independent predictor for prolonging the waiting time, and to know whether the population can be prioritized in the emergency grading system. METHODS: A retrospective analysis was conducted. All the clinical data of adult visitors to the emergency department of the Third Affiliated Hospital of Sun Yat-sen University from January 1st in 2016 to December 31st in 2017 were collected. All the patients received four emergency first aid triages and treated according to the emergency classification results. The relevant data of selected patients were retrieved from the hospital information system (HIS) including gender, age, triage level, triage department, outpatient service and initial diagnosis time. According to the conclusion of pre-phase analysis that the patients over 65 years old was not associated with waiting time, all subjects were divided into ≥ 70 years old group and < 70 years old group for further analysis. The waiting time was defined as the difference in registration time and the initial diagnosis time, and the patients were divided into two groups according to whether it exceeded the target waiting time (TWT). TWT was considered as follows according to each emergency triage level: level I instantly, level II 10 minutes, level III 30 minutes, level IV 60 minutes (subacute patients) or 2-4 hours (non-urgent patients). The multivariable Logistic regression analysis was conducted to determine whether the factor that aged over 70 years old was an independent predictor for prolonging waiting time over TWT. RESULTS: There were 272 989 adult visits recorded totally, of which obstetrics and gynecology department visits, incomplete personnel information, invalid diagnosis (or no diagnosis), triage level I, abnormal waiting time (the waiting time exceeded 99% of the average waiting time of all samples) records were excluded. Finally, 219 299 emergency patients were enrolled, with 102 218 male and 117 081 female, which showed an average age of (35.86±15.81) years, 208 082 patients (94.9%) in < 70 years old group, and 11 217 (5.1%) in ≥ 70 years old group. Totally 191 588 patients (87.4%) were diagnosed within the TWT, and the number was 86.5% (9 701/11 217) in the ≥ 70 years old group. Multivariate Logistic regression analysis indicated that age under 70 years old was independently associated with an exceeded TWT [adjusted odds ratio (OR) = 1.106, 95% confidence interval (95%CI) = 1.041-1.176, P = 0.001]. Meanwhile, visits during the peak period or low triage level was also a risk factor of exceeded TWT. According to the Logistic regression analysis of different departments and removing other factors, in the emergency internal medicine with the most elderly patients, the probability of exceeded TWT in ≥ 70 years old group was lessened (adjusted OR = 1.203, 95%CI = 1.121-1.291, P < 0.001). CONCLUSIONS: In the emergency pre-grading system, the elderly aged (≥ 70 years old) was more likely to see a doctor in TWT than those under 70 years old, so they were easy to get priority treatment.

Association between arterial hyperoxia and mortality in critically ill patients: A systematic review and meta-analysis.

PURPOSE: The relationship between arterial hyperoxia exposure and clinical outcome is under increasing scrutiny. We therefore performed an update meta-analysis to evaluate the effect of arterial hyperoxia on hospital mortality in critically ill adults. METHODS: We searched relevant articles for trials that investigated the relationship between arterial hyperoxia and mortality in critically ill adults. The end-point was hospital mortality of critically ill patients. RESULTS: Three RCTs and 26 cohort studies involving 257,223 patients were identified. Hyperoxia exposure was associated with increased mortality in critically ill patients (crude OR 1.42, 95% CI 1.26-1.61; adjusted OR 1.20, 95% CI 1.09-1.32). There was no change in significance for outcome in meta-analysis of RCTs (OR 1.36; 95% CI 1.04-1.77) and sensitivity analysis of the included prospective studies (OR 1.32; 95% CI 1.04-1.67). This association was also established in patients admitted to critical care units following cardiac arrest (adjusted OR 1.32; 95% CI 1.12-1.56), ischemic stroke (crude OR 1.31; 95% CI 1.03-1.65) and intracerebral hemorrhage (crude OR 1.47; 95% CI 1.19-1.81). CONCLUSIONS: The results of current meta-analysis suggest that arterial hyperoxia may be associated with increased hospital mortality in critically ill patients.

Mycobacterium tuberculosis peptide E7/HLA-DRB1 tetramers with different HLA-DR alleles bound CD4+ T cells might share identical CDR3 region.

Human CD4+ T cells play an important role in the immune response to Mycobacterium tuberculosis (MTB). However, little is known about the spectratyping characteristics of the CD4+ T-cell receptor (TCR) α- and ß-chains CDR3 region in tuberculosis (TB) patients. We sorted MTB peptide E7-bound CD4+ T cells by using E7/HLA-DR tetramers constructed with different HLA-DRB1 alleles and extracted the CDR3 amino-acid sequences of TCR α- and ß-chains. The results showed that the CDR3 sequences of E7-bound CD4+ T cells were completely or partially identical in a single patient. The sequences of MTB peptide C5-bound CD4+ T cells shared another, and non-peptide bound CD4+ T cells, as well as unbound CD4+ T cells with tetramers were different from each other. Specifically, diverse CDR3 sequences of E7-bound CD4+ T cells displayed similar protein tertiary structure in one TB patient. In summary, the TCR α- and ß-chains of CDR3 lineage of CD4+ T cells in TB patients apparently drifted, and the predominant CDR3 sequences of TCR α- and ß-chains that recognized the MTB antigen exhibited peptide specificity, and certain HLA-DR restriction was also established. This study elucidates the possible causes and mechanisms of peptide-specific CD4+ T-cell-related presentation against MTB.

A life-threatening duodenal ulcer hemorrhage due to previously unknown primary hyperparathyroidism.

Peptic ulcer bleeding due to primary hyperparathyroidism is extremely rare. We report a case of a 42-year-old male with life-threatening acute upper gastrointestinal bleeding secondary to a duodenal ulcer and a history of kidney stones. Gastroscopic therapy, Billroth II gastrointestinal anastomosis and angiographic embolization were sequentially conducted to arrest the hemorrhage. A complete investigative work-up revealed that the duodenal ulcer bleeding was due to primary hyperparathyroidism coexisting with a parathyroid adenoma. Following this event, the patient developed a severe abdominal cavity infection and sepsis. An elective parathyroidectomy was performed, and the histology was confirmed to be a typical parathyroid adenoma. Postoperatively, the patient's calcium and parathyroid levels were normalized. Attention should be paid to patients with an upper gastrointestinal ulcer, especially when it is accompanied by kidney stones.

Seroprevalence of Toxoplasma gondii Infection in Patients of Intensive Care Unit in China: A Hospital Based Study.

The objective of this study was to estimate the seroprevalence of Toxoplasma gondii infection in 394 patients of intensive care unit (ICU) in a hospital between April 2010 and March 2012 and analyze the association between T. gondii infection and ICU patients according to the species of disease. Toxoplasma serology was evaluated by ELISA method using a commercially available kit. Data of patients were obtained from the patients, informants, and medical examination records. Seventy-four (18.78%) of 394 patients were positive for anti-T. gondii IgG antibodies demonstrating latent infection. Of these, the highest T. gondii seroprevalence was found in the age group of 31-45 years (27.45%), and the lowest was found in the age group of <30 years (12.5%). In addition, females (21.6%) had a higher seroprevalence than males (18.36%). With respect to the species of disease, the patients with kidney diseases (57.14%), lung diseases (27.84%), and brain diseases (24%) had high T. gondii seroprevalence. The present study represents the first survey of T. gondii seroprevalence in ICU patients in China, revealing an 18.78% seropositivity. Considering the particularities of ICU patients, molecular identification, genetic characterization, and diagnosis of T. gondii should be considered in future study.

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics.

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study.

Differences in microglia activation between rats-derived cell and mice-derived cell after stimulating by soluble antigen of IV larva from Angiostrongylus cantonensis in vitro.

Angiostrongylus cantonensis is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats. Humans and mice are accidental hosts or named nonpermissive hosts. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningoencephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of the host to parasite during A. cantonensis invasion and development. Microglia is considered to be the key immune cell in the central nervous system like macrophage. To further understand the reasons for why mice and rats attain different outcomes in A. cantonensis infection, we set up the method to isolate and culture newborn rats' primary microglia and observe the activation of the microglia cells, comparing with mice microglia cell line N9. We treated cells with soluble antigen of the fourth larva of A. cantonensis (L4 larva) and measured mRNA levels of IL-1ß, IL-5, IL-6, IL-13, eotaxin, iNOS, and TNF-α by real-time PCR. The results showed that N9 expressed high mRNA level of IL-6, IL-1ß, TNF-α, iNOS, IL-5, IL-13, and eotaxin, but primary microglia only had IL-5, IL-13, and eotaxin mRNA level. It implies that microglia from rats and mice had different reaction to soluble antigen of A. cantonensis. Therefore, we supposed that microglia may play an immune modulation role during the brain inflammation induced by A. cantonensis.

Relatively low level of antigen-specific monocytes detected in blood from untreated tuberculosis patients using CD4+ T-cell receptor tetramers.

The in vivo kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target Mycobacterium tuberculosis (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4(+) T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14(+) monocytes ranged from 0.26-1.44% and 0.21-0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages in situ via immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3-6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14(+) cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the mechanisms of antigen presentation and its relationship with TB infection and latent TB infection.

The complete mitochondrial genomes of three cestode species of Taenia infecting animals and humans.

Mitochondrial (mt) genome sequences provide useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. Although Taenia multiceps, T. hydatigena, and T. taeniaeformis are common taeniid tapeworms of ruminants, pigs, dogs, or cats, causing significant economic losses, no published study on their mt genomes is available. The complete mt genomes of T. multiceps, T. hydatigena, and T. taeniaeformis were amplified in two overlapping fragments and then sequenced. The sizes of the entire mt genome were 13700 bp for T. multiceps, 13489 bp for T. hydatigena, and 13647 bp for T. taeniaeformis. Each of the three genomes contains 36 genes, consisting of 12 genes for proteins, 2 genes for rRNA, and 22 genes for tRNA, which are the same as the mt genomes of all other cestode species studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T of the complete genomes are 71.3% for T. multiceps, 70.8% for T. hydatigena, and 73.0% for T. taeniaeformis. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. T. multiceps and T. hydatigena had two noncoding regions, but T. taeniaeformis had only one. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes revealed that T. multiceps, T. hydatigena, and T. taeniaeformis were more closely related to the other members of the Taenia genus, consistent with results of previous morphological and molecular studies. The present study determined the complete mt genome sequences for three Taenia species of animal and human health significance, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these cestode parasites of animals and humans.

Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 degrees C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.