Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)‍-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens.
Humans ; CRISPR-Cas Systems ; Communicable Diseases ; Gene Editing/methods*

Objective:To explore the clinical application value of group A Streptococcus (GAS) antigen rapid detection method in children suffering from GAS infection.Methods:A total of 44 733 children with suspected GAS infection who were admitted to the Outpatient and Inpatient Departments of Shenzhen Children′s Hospital from January 2015 to December 2019.Throat swab specimens from all children were collected, and BinaxNOW Strep A Test reagent was used for GAS antigen rapid detection.Among them, the throat swabs of 346 children were inoculated with blood culture medium for traditional bacterial culture, and then the GAS antigen rapid detection was tested.The sensitivity and specificity of the two methods were compared, and according to the result of the GAS antigen rapid detection, its age, gender and seasonal trends were analyzed.SPSS 19.0 software was applied for statistical analysis of the data.Results:Among the 346 children tested by both methods, the results of bacterial culture were adopted as the reference method, the sensitivity of the rapid detection method for GAS antigen was 89.41%(152/170 cases), and the specificity was 94.32%(166/176 cases) compared with culture methods.A total of 44 733 cases GAS antigen were tested in children in Shenzhen, of which 10 024 cases were positive, with the positive detection rate of 22.41%.The trend of GAS antigen rapid detection was consistent with the five-year trend, with the high positive rate of 3-8 years, of which 4-6 years of positive rate was the highest.The two seasonal peaks were evident each year, with peaks occurring in April-June, and November and January of next year.The detection rate ratio of male and female was 1.74∶1, and the gender difference was significant ( χ2=27.93, P<0.000 1). GAS antigen rapid detection rate in different clinical departments from high to low in order are as follows: dermatology outpatient (52.34%), emergency clinic (47.74%), internal medicine outpatient (37.36%), infectious disease area (19.71%), five-level disease area (10.27%), internal medicine area (8.63%), surgical areas (7.34%) and neonatal areas (0). Conclusions:GAS antigen rapid detection method and bacterial culture method have high coincidence rate, and high sensitivity and specificity, and can be popularized and applied in the diagnosis of GAS infectious diseases in children.GAS detection rate is higher in outpatient emergency department and dermatology clinics.There are obvious differences from seasonal and population (age and gender) in the positive detection of GAS antigen.No neonates were found.

Objective:To construct a core competency evaluation index system for ophthalmic specialist nurses so as to provide theoretical reference for cultivation and evaluation of ophthalmic specialized nurses in China.Methods:A research group was set up in October 2020, and the core competency evaluation index system for ophthalmic specialist nurses was initially formed by means of literature review, semi-structured interview and group discussion. From December 2020 to January 2021, an online questionnaire was used to conduct Delphi letter consultation among 23 experts in related fields nationwide, and the items were screened and modified. The weight of indicators was determined by the proportional distribution method.Results:Two rounds of consultation were conducted. The positive coefficients of experts were respectively 91.30% and 100.00%, Kendall's concordance coefficients were respectively 0.172 and 0. 192 ( P<0.05) , and the expert authority coefficient was 0.90. Finally, an index system consisting of 5 first-level indicators, 12 second-level indicators and 58 third-level indicators was formed. Conclusions:The core competence evaluation index system of ophthalmic specialist nurses constructed in this study has a high degree of enthusiasm and authority of experts, which reflects the characteristics of the ophthalmic nursing specialty and provides theoretical reference for cultivation and ability evaluation of ophthalmic specialist nurses in China.

Objective To understand the epidemiological characteristics, genomic variations and macrolide resistance of Bordetella pertussis ( B. pertussis) strains circulating in Shenzhen with clinical data analysis, genotype profiling, phylogenetic analysis and antimicrobial susceptibility test. Methods Clinical data of patients with pertussis in Shenzhen Children's Hospital were collected from the electronic medical re-cord system. Genome sequences of 31 B. pertussis isolates were analyzed with next-generation sequencing and de novo assembled. Multilocus sequence typing (MLST) was performed to identify their sequences types. Sequence alignment by BLASTn was used to identify virulence genotypes and mutations in 23S rRNA gene. A phylogenetic tree was constructed to analyze the relationships among them. E-test was used to identify ma-crolide resistance. Results All of the 31 B. pertussis strains were identified as sequence type-2 (ST-2) by MLST with diverse virulence genotypes. Two were prn-deficient strains. Based on the phylogenetic tree, all of the isolates were distant from vaccine strains. Nineteen isolates were resistant to erythromycin with A2047G mutation in 23S rRNA. Conclusions The virulence genotypes of B. pertussis strains in Shenzhen were diverse with increasing non-vaccine genotypes. Macrolide-resistant strains were prevalent. This study might provide reference for improving the prevention, management and vaccination strategy of pertussis.

Objective To explore the dynamic change and relationship between post-traumatic stress disorder (PTSD) and cognition in patients transferred from ICU. Methods Participants patients were selected from Critical Medical Department of the First Affiliated Hospital of Xi'an Jiaotong University by convenience sampling method between October 2016 to February 2017. The Montreal Cognitive Assessment (MoCA) and the PTSD Cheeklist-Vivilian Version (PCL-C) were used to collect data at 3 days, 3 months and 6 months after transferring from ICU. Results The incidence of cognitive disorder at 3 time points were 29.4％(30/102), 20.0％(18/90), 17.8％(13/73)respectively, and MoCA scores was 25.83 ± 6.29,28.57 ± 5.43,28.86 ± 5.11, the difference was significant (F=6.204,P<0.01). The incidence of PTSD symptoms were 42.2％(43/102), 23.3％(21/90), 19.2％(14/73)respectively, and PCL-C scores was 35.24 ± 5.94, 28.68 ± 5.13, 26.92 ± 4.85, the difference was significant (F=10.125,P<0.01). There were significant relationship between cognition and PTSD level (r =0.299-0.543,P <0.05). The PTSD level in cognitive disorder patients was 37.52 ± 5.88, 31.15 ± 5.12, 29.84 ± 4.82, and that in non-cognitive disorder patients was 34.32 ± 5.76, 27.68 ± 4.91, 25.74 ± 4.59 the difference was significant (t =2.117, 2.651, 3.334,P<0.05). Conclusions Health workers should pay attention to the mental status and cognitive impairment of patients transferred from ICU, implement psychological and cognitive interventions early, which could improve the cognitive status and PTSD progression of patients, and improve their quality of life.

Objective This study was to analysis the epidemiologic characteristics of hand-foot-month (HFMD) in Shenzhen during 2015-2016,to provide reference for the prevention and treatment of HFMD.Methods 7 758 statistical data from Shenzhen children's hospital clinical lab during 2015-2016 was included.We used real time fluorescent reverse transcription polymerase chain reaction (RT-PCR) to detect enterovirus general (EV),enterovirus 71 (EV71) and coxsackievirus A group 16 (CoxA16),and analyzed the age,sex and epidemic time of the patients.Results In 2015 and 2016,the positive rate of EV was 67.19％ (2679/3987) and 52.61％ (1 984/3 771) respectively,with statistically significant difference (x2 =71.84,P ＜ 0.05).The radio of male to female children was 1.91∶1 (1 757/922) and 1.83∶1 (l 283/701) in 2015 and 2016,with statistically significant difference (P ＜ 0.05).The age of the children was ＜ 5 years old,accounting for more than 90％ of the total number of patients.April to July and September were the two peaks of HFMD.The enterovirus of hand foot and mouth disease in Shenzhen was dominated by other enteroviruses (more than 82％ of the total number of patients).With the increase of age,the proportion of EV71 in children with severe hand and foot was increasing,and the proportion of other enteroviruses was decreasing.Conclusions Vaccination is one of the important measures of HFMD control.It's beneficial for the diagnosis and treatment of HFMD to collected epidemiologic characteristics data about HFMD in Shenzhen.

Objective To analyze the difference of serum levels of anti-Mullenan hormone (AMH) in chil-dren with different ages and different types of cryptorchidism,and to explore its role in the evaluation of tes-ticular development.Methods 60 children with simple cryptorchidism were selected as case group and 52 healthy children were selected as control group.The levels of serum AMH in two groups of children were measured and the differences were compared.Results (1)The level of AMH in the case group was lower than that in control group (P < 0.05),and there was no statistical significance between two subgroups of >6 to 11 years old children with cryptorchidism and healthy children (P>0.05).(2)The level of AMH in bi-lateral cryptorchidism group was lower than that in unilateral cryptorchidism group (P<0.05),and there was no significant difference between two subgroups of >6 to 11 years old children with bilateral cryptorchidism and unilateral cryptorchidism (P>0.05).(3)The level of AMH in the high level cryptorchidism group was lower than that of the low level cryptorchidism group (P<0.05),and there was no statistical difference be-tween between two subgroups of 3~11 year old children with cryptorchidism and low level cryptorchidism (P>0.05).(4)AMH level was negatively correlated with age,and positively correlated with testicular devel-opment.Conclusion AMH can be used as an important indicator of testicular development in children with cryptorchidism.

Objective To evaluate the creditability of warning message of white differential count (WDF) and white precursor cell (WPC) channels in Sysmex XN-3000 hematology analyzer,and verify its optimal threshold and adjust the alarm threshold.Methods A total of 61 EDTA-K2 anticoagulated blood samples without abnormal warning and 521 EDTA-K2 anticoagulated blood samples with abnormal warning were simultaneously detected in WDF and WPC channels.After the smear specimens of blood sample were automatically prepared by the instrument,microscopic examinations were performed manually.The results of microscopic examination were considered as the gold standard to determine the reliability of the warning message from the instrument and verify the reasonability of initial warning threshold value provided by the manufacture.Consequently,the threshold values were adjusted based on the requirements in practical work.Results The warning messages of atypical lymphocytes and blasts/abnormal lymphocytes in WDF channel were higher sensitive (95.8％ and 100％ respectively),but lower specific (34.7％ and 23.5％ respectively) compared with microscopic examination.The warning messages of atypical lymphocyte,blasts and abnormal lymphocytes in WPC channel were lower sensitive (81.3％,66.7％,and 76.5％ respectively) but higher specific (61.9％,55.5％ and 88.3 ％ respectively) compared with microscopic examination.According to the ROC curve analysis,the prognostic values of warning message of microscopic examination were of medium level,except the warning message for abnormal lymphocytes was poor compared with WPC channel.Combining the practical retest rules,the optimal critical threshold values of atypical lymphocytes and blasts/Abn lymph in WDF channel were adjusted as 120,and they were adjusted as 140 in WPC channel.Conclusion The high sensitive WDF channel should first be used for screening,and the detectable warning message could be retested by using high specific WPC channel to shorten the turnaround time of the test results and improve the working efficiency.The initial critical warning threshold provided by the manufacture should be verified and adjusted to the optimum critical threshold in order to ensure the accuracy of test results.

Objective To establish the pipeline and evaluate the feasibility of high-throughput sequencing method used in the detection of severe pneumonia pathogens.Methods Clinical control study was used.Bronchi alveolar lavage fluids (BALF) samples from 76 patients with severe pneumonia (treatment group) and 18 patients with tracheal malacia (control group) and without clinical detected pathogens were collected during March 2015 to December 2016 in Shenzhen Children′s Hospital.The pathogens in the samples were detected using clinical tests and high-throughput sequencing respectively.The results of high-throughput sequencing were confirmed by real-time quantitative PCR and the high-throughput sequencing method used in the detection of severe pneumonia pathogens was evaluated.The χ2 test was used to analyze the correlation of detection rate between the high-throughput sequencing group and the non high-throughput sequencing group.Results The pipeline and method of high-throughput sequencing used in the severe pneumonia pathogens detection was established.The pipeline included sample collection, DNA extraction, library construction, sequencing, and bioinformatic analysis.In 76 cases of patients with severe pneumonia, the results of high-throughput sequencing in 66 cases of bronchoalveolar lavage fluid specimens were positive.The sensitivity was 86.84%, which was significantly higher than the total sensitivity of traditional clinical detection methods including bacterial culture, immunofluorescence and quantitative PCR(68.42%,52/76),χ2=7.426,P<0.001.A total of 13 pathogens were detected in 66 positive samples of high-throughput sequencing, including Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae and adenovirus, etc.Nine kinds of pathogens were detected in these samples through non-high-throughput sequencing.In the experimental group, the results obtained by clinical test and high-throughput (80.26%) were entirely consistent in 61 samples and not completely consistent in 15 samples (19.74%) specimens.These inconsistent results were mainly concentrated in the detection of adenovirus, Streptococcus pneumoniae and Haemophilus influenzae through high-throughput sequencing, whereas clinical cultures and immunofluorescence methods were not able to detect these pathogens.PCR validation showed that there was no significant difference between the results of high-throughput sequencing and the PCR tests (χ2=0.517,P=0.472), and the difference between the results of high-throughput sequencing and traditional clinical detection methods was statistically significant (χ2=11.671,P<0.001).Conclusion The method for the detection of severe pneumonia pathogens based on high-throughput sequencing is highly sensitive and can detect unknown pathogens, which is superior to those used in traditional clinical detection.

Objective To investigate the effects of p300/CBP on histone acetylation of Foxp3 gene and its roles in the immunological pathogenesis of Kawasaki disease (KD).Methods Forty-six children with KD and twenty-eight age-matched health children were consented to participate in this study.Co-immunoprecipitation and real-time PCR were performed to detect Foxp3-associated acetylation levels of histone H4 and binding abilities of p300, CBP, pSmad3 (phosphorylated mothers against decapentaplegic homolog 3) and NF-AT (nuclear factor of activated T cells) with Foxp3 gene in CD4+ T cells.The percentages of CD4+CD25high Foxp3+ cells (Treg) and the expression of Foxp3, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), p300, CBP, TGF-βRⅡ (transforming growth factor β receptor Ⅱ) and pLAT1 at protein level were analyzed by flow cytometry.Quantitative real-time PCR was used to measure the expression of Foxp3, IL-10, TGF-β, TGF-βRⅠ, Egr-1 (early growth response protein 1), RARα (retinoic acid receptor α) and PLCγ1 (phospholipase C-γ1) in Treg cells at mRNA level.Plasma concentrations of TGF-β and retinol acid (RA) were measured by enzyme-linked immunosorbent assay.Results (1) The percentages of Treg cells, levels of Foxp3 and molecules associated with suppressive function of Treg cells (TGF-β, IL-10 and CTLA4), acetylation levels of histone H4 associated with promoter, conserved non-coding DNA sequence 1 (CNS1) and CNS2 of Foxp3 gene decreased remarkably during acute KD (P<0.05), but were restored after IVIG therapy (P<0.05).Meanwhile, all of the aforementioned items in KD patients with coronary artery lesions (KD-CAL+) were lower than those without coronary artery lesions (KD-CAL-) (P<0.05).No significant differences in histone H4 acetylation associated with CNS3 were found among different groups (P>0.05).(2) The levels of p300 and CBP in Treg cells and their binding abilities with Foxp3 gene were down-regulated significantly during acute KD (P<0.05), but were restored to some extent after IVIG treatment (P<0.05).The Foxp3-associated histone acetylation was positively correlated with the expression of p300 and CBP at mRNA level during acute KD (r=0.65, 0.42, P<0.05).Furthermore, the expression of p300 and CBP and their binding abilities with Foxp3 gene in KD-CAL+ group were lower than those in KD-CAL-group (P<0.05).(3) Compared with healthy subjects, plasma concentrations of TGF-β and RA and the expression of TGF-βRⅠ/Ⅱ/Egr-1, RARα and pLAT1/PLCγ1 were down-regulated during acute KD (P<0.05);the binding abilities of pSmad3 and NFAT with Foxp3 gene were reduced remarkably in patients with acute KD (P<0.05).All the items mentioned above were restored after IVIG treatment (P<0.05).Moreover, the ten items aforementioned in KD-CAL+ group were lower than those in KD-CAL-group (P<0.05).(4) Higher acetylation levels of histone H4 associated with promoter, CNS1 and CNS2, and enhanced binding abilities of p300 and CBP with Foxp3 gene were found in CD4+ T cells isolated from patients with acute KD after co-stimulation with TGF-β, RA and anti-CD3/CD28 antibodies as compared with those in CD4+ T cells without stimulation (P<0.05).However, no statistical difference in the acetylation level of histone H4 associated with CNS3 was found between the two groups (P>0.05).Conclusion Hypoacetylation of histone H4 associated with Foxp3 gene caused by insufficient expression of p300/CBP and their impaired binding abilities might be involved with immune dysfunction in KD.IVIG therapy regulates the expression of p300/CBP and their binding abilities with Foxp3 gene through up-regulating TGF-β signal.