Detection of drug resistance genes of mycobacterium tuberculosis by rolling circle amplification technique with multicolor fluorescent probes / 中华预防医学杂志

The aim of this study was to construct a simple, rapid and ultra-sensitive optical biosensing technique based on rolling circle amplification (RCA), and to apply it to multiple detection of drug-resistant genes of mycobacterium tuberculosis. The common mutation sites of isoniazid, rifampicin and streptomycin resistance genes are katG315 (AGC➝ACC), rpoB531 (CAC➝TAC) and rpsL43 (AAG➝AGG). For these three gene sites, from February 2020 to May 2021, in the Department of Laboratory Medicine of the First Affiliated Hospital of Army Military Medical University, the padlock probe (PLP), primers and capture probes were designed. And a solid-phase RCA constant temperature amplification reaction system based on magnetic beads was constructed and the experimental parameters were optimized. The RCA products were accurately captured by the multicolor fluorescent probes (Cy3/Cy5/ROX), and the single-tube multiple detection of three mutation genes was realized. The sensitivity, specificity and linear range of this method were further verified. The results showed that the response range of katG315 in the same reaction system ranged from 1.0 pmol/L to 0.1 nmol/L. The response range of rpoB531 and rpsL43 ranged from 1.0 pmol/L to 50.0 pmol/L and 1.0 pmol/L to 20.0 pmol/L, and the method had good specificity and sensitivity, and could accurately identify single base mutations in mixed targets, with the minimum detection limit as low as 1.0 pmol/L. The recoveries of simulated serum samples were 95.0%-105.2%. In conclusion, the constant temperature amplification multiple detection method constructed in this study can quickly realize the single-tube multiple detection of three drug resistance mutation sites. This technology is low-cost, simple and rapid, and does not rely on large equipment, providing a new analysis method for pathogen drug resistance gene detection.

Analysis of distribution characteristics and drug resistance of 2748 strains of pathogens isolated from burn patients / 中华烧伤杂志

<p><b>OBJECTIVE</b>To provide epidemiological data of the distribution characteristics and drug resistance of the pathogens isolated from burn patients in recent years for guiding rational use of antibiotics in clinic.</p><p><b>METHODS</b>Totally 2748 strains of pathogens were isolated from 1977 specimens (blood, catheter, wound excretion, etc.) collected from 478 patients hospitalized in Institute of Burn Research of Southwest Hospital from March 2003 to June 2011. After being identified by API strips, drug resistance of the 2748 isolated pathogens to 55 commonly-used antibiotics including gentamicin, tobramycin, piperacillin, amikacin, etc. was tested by K-B paper disk diffusion method. The WHONET 5.3 software was used to analyze the following subjects: the distribution of the pathogens with different types and different sources each year, the changes in drug-resistant rates of Gram negative bacilli, Gram positive cocci, and fungi to several antibiotics, and the changes in sensitive rates of Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Acinetobacter baumannii (AB), Candida albicans (CA) to several antibiotics.</p><p><b>RESULTS</b>Among 2748 strains of pathogens, 1879 strains of Gram negative bacilli accounted for 68.38%, 628 strains of Gram positive cocci accounted for 22.85%, and 241 strains of fungi accounted for 8.77%. The isolation rate of strains from wound excretion ranked the first (1022 strains accounted for 37.19%), followed by those from respiratory tract (995 strains accounted for 36.21%) and blood (421 strains accounted for 15.32%). Strains isolated from other types of specimens were rare. Isolation rate of PA ranked the first (996 strains accounted for 36.24%), followed by SA (495 strains accounted for 18.01%) and AB (395 strains accounted for 14.37%). Isolation rate of AB showed a trend of increase year by year, but that of SA presented the opposite trend. Isolation rate of PA was quite stable. There were 484 strains of methicillin resistant SA among Staphylococci, accounting for 17.61%. Resistant rates of PA and AB to polymyxin B and polymyxin E were below 30.00%, and those of PA and AB to other antibiotics, such as the third generation cephalosporins, β-lactams, aminoglycosides, and quinolones, were from 57.91% to 100.00%. Resistant rate of AB to minocycline was 39.68%. From 2004 to 2011, sensitive rate of PA to quinolone antibiotics showed an increasing trend year by year, but that of AB to minocycline, netilmicin, imipenem, meropenem, tobramycin, and cefoperazone/sulbactam presented the opposite trend. Resistant rates of Enterococcus faecalis, Enterococcus faecium, and SA to teicoplanin and linezolid were less than 10.00%. Resistant rate of SA, Staphylococcus epidermidis and Enterococcus faecium to vancomycin was 0. Resistant rates of SA to quinupristin/dalfopristin, minocycline, fusidic acid, and compound sulfamethoxazole were low, respectively 0.82%, 9.35%, 2.21%, and 31.85%. Sensitive rates of SA to erythromycin, clindamycin, compound sulfamethoxazole, tetracycline, and minocycline showed an increasing trend year by year. Both infection rate and resistant rate of fungi were low. The resistant rates of CA to 5 kinds of antibiotics were less than 15.00%. The sensitive rate of CA to 5-flucytosine declined slightly, and those of CA to the other 4 antibiotics showed an increasing trend year by year.</p><p><b>CONCLUSIONS</b>The three dominant pathogens that cause infection in burn patients hospitalized in Institute of Burn Research of Southwest Hospital in recent years are PA, SA, and AB in order. PA and AB are outstandingly multidrug-resistant among the isolated strains. AB might replace PA as the main pathogenic bacterium that cause the death of burn patients with infection.</p>

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China.</p><p><b>METHODS</b>All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications.</p><p><b>RESULTS</b>The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of β2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS.</p><p><b>CONCLUSIONS</b>Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.</p>

A complete screen for mutations of the rhodopsin gene in a panel of Chinese patients with autosomal dominant retinitis pigmentosa / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing.</p><p><b>METHODS</b>We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families.</p><p><b>RESULTS</b>Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls.</p><p><b>CONCLUSIONS</b>The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.</p>

Prospective study on the gastro-pulmonary infection route of ventilator-associated pneumonia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the role of gastro-pulmonary infection route in the development of ventilator-associated pneumonia (VAP), so as to improve the management of VAP.</p><p><b>METHODS</b>Forty-three patients who received mechanical ventilation (MV) were enrolled in the study. Intra-gastric contents were labeled with (99)mTc-DTPA. Randomized two-period crossover trial was employed to determine the radioactive level in the oropharyngeal and bronchial secretion when patients were in supine or semi-reclining position. Gastric juice, oropharyngeal secretion and tracheal lavage fluid were collected for bacterial culture every other day. Bronchoalveolar lavage fluid (BALF) was harvested from those suspected of VAP for quantitative bacterial culture. Infrequent-restriction site amplification (IRS-PCR) was employed in the identification of the identity of the bacteria from intra-gastric colonization with those causing VAP. The sIgA content in the BALF was determined.</p><p><b>RESULTS</b>The gastroesophageal regurgitation rate was higher (89.7%) with lower aspiration rate (28.5%) in patients receiving MV. Moreover, the aspiration rate and the radioactivity of deep tracheal aspirates in patients in supine position were significantly higher than those in semi-reclining position (P < 0.01). There was high homology of the bacteria isolated from intra-gastric colonization with that causing VAP (55.8%). The sIgA content in BALF in VAP patients was evidently lower than that in non-VAP patients (P < 0.01).</p><p><b>CONCLUSION</b>Regurgitation and aspiration of stomach contents are very common in patients receiving MV. Intra-gastric colonized bacteria might be one of the important origins causing VAP. The lowering of sIgA in BALF in patients with MV could be a risk factor for VAP.</p>

Construction of anti-lipopolysaccharide single-chain phage antibody library in mice / 第三军医大学学报

Objective　To construct an anti-LPS single chain phage antibody library in mice for further biomedical works. Methods　Total RNA was extracted from splenic cells for reverse transcription after BALB/C mice had been immunized with pure LPS for 4 weeks. The designed primers were used to amplify the variable region genes of both heavy and light chain (VH,VL) with polymerase chain reaction. The VH and VL were then conjugated to form a single chain of variable fragment (ScFv) by a linker. The ScFv was cut by NotⅠ and SfiⅠ and then ligated into a pCANTAB5E phagemid vector which was digested with the same restriction enzymes. The ligated vector was then tranfected into the competent E.coli TG1 cells. Four TG1 clones were randomly selected to detect the exotic DNA. Results　The titer of anti-LPS in murine sera was 1∶12 800. The concentration of total RNA was 12.3813 μg/ml. The length of the fragments were 340 bp for VH, 320 bp for VL and 800 bp for ScFv respectively. 1.9×107 clones were grown after transfection and one from the four randomly selected clones was identified to contain the exotic DNA. Conclusion　A 4.75×106 murine anti-LPS single chain phage antibody library was successfully constructed.

Construction of anti-lipopolysaccharide single-chain phage antibody library in mice / 第三军医大学学报

Objective　To construct an anti-LPS single chain phage antibody library in mice for further biomedical works. Methods　Total RNA was extracted from splenic cells for reverse transcription after BALB/C mice had been immunized with pure LPS for 4 weeks. The designed primers were used to amplify the variable region genes of both heavy and light chain (VH,VL) with polymerase chain reaction. The VH and VL were then conjugated to form a single chain of variable fragment (ScFv) by a linker. The ScFv was cut by NotⅠ and SfiⅠ and then ligated into a pCANTAB5E phagemid vector which was digested with the same restriction enzymes. The ligated vector was then tranfected into the competent E.coli TG1 cells. Four TG1 clones were randomly selected to detect the exotic DNA. Results　The titer of anti-LPS in murine sera was 1∶12 800. The concentration of total RNA was 12.3813 μg/ml. The length of the fragments were 340 bp for VH, 320 bp for VL and 800 bp for ScFv respectively. 1.9×107 clones were grown after transfection and one from the four randomly selected clones was identified to contain the exotic DNA. Conclusion　A 4.75×106 murine anti-LPS single chain phage antibody library was successfully constructed.