Evolution of the newest diagnostic methods for COVID-19: a Chinese perspective / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Coronavirus disease 2019 (COVID-19) has continued to spread globally since late 2019, representing a formidable challenge to the world's healthcare systems, wreaking havoc, and spreading rapidly through human contact. With fever, fatigue, and a persistent dry cough being the hallmark symptoms, this disease threatened to destabilize the delicate balance of our global community. Rapid and accurate diagnosis of COVID-19 is a prerequisite for understanding the number of confirmed cases in the world or a region, and an important factor in epidemic assessment and the development of control measures. It also plays a crucial role in ensuring that patients receive the appropriate medical treatment, leading to optimal patient care. Reverse transcription-polymerase chain reaction (RT-PCR) technology is currently the most mature method for detecting viral nucleic acids, but it has many drawbacks. Meanwhile, a variety of COVID-19 detection methods, including molecular biological diagnostic, immunodiagnostic, imaging, and artificial intelligence methods have been developed and applied in clinical practice to meet diverse scenarios and needs. These methods can help clinicians diagnose and treat COVID-19 patients. This review describes the variety of such methods used in China, providing an important reference in the field of the clinical diagnosis of COVID-19.

Previously unrecognized non-reproducible antibody-antigen interactions and their implications for diagnosis of viral infections including COVID-19

Antibody-antigen (Ab-Ag) interactions are canonically described by a model which exclusively accommodates non-interaction (0) or reproducible-interaction (RI) states, yet this model is inadequate to explain often-encountered non-reproducible signals. Here, by monitoring diverse experimental systems and confirmed COVID-19 clinical sera using a peptide microarray, we observed that non-specific interactions (NSI) comprise a substantial proportion of non-reproducible antibody-based results. This enabled our discovery and capacity to reliably identify non-reproducible Ab-Ag interactions (NRI), as well as our development of a powerful explanatory model ("0-RI-NRI-Hook four-state model") that is [mAb]-dependent, regardless of specificity, which ultimately shows that both NSI and NRI are not predictable yet certain-to-happen. In experiments using seven FDA-approved mAb drugs, we demonstrated the use of NSI counts in predicting epitope type. Beyond challenging the centrality of Ab-Ag interaction specificity data in serology and immunology, our discoveries also facilitated the rapid development of a serological test with uniquely informative COVID-19 diagnosis performance.

A diagnostic decision-making protocol combines a new-generation of serological assay and PCR to fully resolve ambiguity in COVID-19 diagnosis

The capacity to accurately diagnosis COVID-19 is essential for effective public health measures to manage the ongoing global pandemic, yet no presently available diagnostic technologies or clinical protocols can achieve full positive predictive value (PPV) and negative predictive value (NPV) performance. Two factors prevent accurate diagnosis: the failure of sampling methods (e.g., 40% false negatives from PCR testing of nasopharyngeal swabs) and sampling-time-dependent failures reflecting individual humoral responses of patients (e.g., serological testing outside of the sero-positive stage). Here, we report development of a diagnostic protocol that achieves full PPV and NPV based on a cohort of 500 confirmed COVID-19 cases, and present several discoveries about the sero-conversion dynamics throughout the disease course of COVID-19. The fundamental enabling technology for our study and diagnostic protocol--termed SANE, for Symptom (dpo)-Antibody-Nucleic acid-Epidemiological history--is our development of a peptide-protein hybrid microarray (PPHM) for COVID-19. The peptides comprising PPHMO_SCPLOWCOVIDC_SCPLOWO_SCPCAP-19C_SCPCAP were selected based on clinical sample data, and give our technology the unique capacity to monitor a patients humoral response throughout the disease course. Among other assay-development related and clinically relevant findings, our use of PPHMO_SCPLOWCOVIDC_SCPLOWO_SCPCAP-19C_SCPCAP revealed that 5% of COVID-19 patients are from an "early sero-reversion" subpopulation, thus explaining many of the mis-diagnoses we found in our comparative testing using PCR, CLIA, and PPHMO_SCPLOWCOVIDC_SCPLOWO_SCPCAP-19C_SCPCAP. Accordingly, the full SANE protocol incorporates orthogonal technologies to account for these patient variations, and successfully overcomes both the sampling method and sampling time limitations that have previously prevented doctors from achieving unambiguous, accurate diagnosis of COVID-19.

Clinical significance of KL-6 detection in the diagnosis, monitoring and evaluation of interstitial lung disease / 中华检验医学杂志

In recent years, Krebs von den Lungen-6 (KL-6) has attracted clinical attention as a serum non-invasive biomarker of interstitial lung disease (ILD), which are complex and diverse.The level change of KL-6 is related to the activity and therapeutic effect of ILD.The clinical significance of KL-6 in the diagnosis, monitoring and evaluation of interstitial lung disease is summarized.

Desmoglein 3 gene mediates epidermal growth factor/epidermal growth factor receptor signaling pathway involved in inflammatory response and immune function of anaphylactic rhinitis.

OBJECTIVE: To investigate the effects of desmoglein 3 (DSG3) gene mediating epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling pathway on inflammatory response and immune function of anaphylactic rhinitis (AR). METHODS: Ten of the seventy male BALB/c mice were randomly selected as the normal control group, and the remaining 60 were used to construct the AR mice model. AR model mice were divided into 6 groups: model group (instilled with 5 µL saline), empty vector group (instilled with 5 µL of liposome and empty vector mixture), siRNA-DSG3 group (instilled with 5 µL of liposome and siRNA-DSG3 carrier mixture), AG1478 group (instilled with 5 µL of EGF/EGFR inhibitor AG1478), siRNA-DSG3+AG1478 group (instilled with 5 µL of liposome and siRNA-DSG3 carrier and EGF/EGFR inhibitor AG1478 mixture) and oe-DSG3 group, 10 in each group. After taking serum, each group of mice was sacrificed to get nasal mucosa tissues. HE staining was used to observe the pathological changes of nasal mucosa tissues in each group. The expression levels of DSG3, EGF and EGFR in nasal mucosa tissues of mice in each group were detected by qRT-PCR and western blot methods respectively. TUNEL staining was used to observe the apoptosis of nasal mucosa cells in mice. The expression of IgE, INF-γ, TNF-α, IL-2, IL-4 and IL-6 in serum of mice was determined by ELISA method. The immune adhesion function of red blood cells was detected by complement sensitization yeast hemagglutination method. RESULTS: All the mice with AR showed different degrees of nasal mucosa injury and inflammatory cell infiltration, and silencing DSG3 or inhibiting the activity of EGF signaling pathway could alleviate the nasal mucosa injury. Compared with control group, the INF-γ and IL-2 levels of serum in AR model mice were significantly decreased; IgE, TNF-α, IL-4 and IL-6 levels were significantly increased (all P < 0.05); the mRNA expression levels and protein levels of DSG3, EGF and EGFR were significantly increased (all P < 0.05); C3b receptor rosette rate and Ic rosette rate were significantly decreased (all P < 0.05). Detected by ELISA method, the expression levels of IgE, TNF-α, IL-4 and IL-6 were increased, while the expression levels of INF-γ and IL-2 were decreased after DSG3 silencing or using AG1478. Detected by qRT-PCR and western blot methods, the expression of DSG3, EGF and EGFR did decrease after DSG3 silencing. There was no significant difference in the EGF and EGFR expression between DSG3 silencing and using AG1478, and the expression decreased even more under the double effect. The mRNA and protein expression levels of DSG3, EGF and EGFR in the nasal mucosa tissues of mice with overexpression of DSG3 plasmid were significantly higher than those of normal mice (all P < 0.05). CONCLUSION: Silencing DSG3 gene can inhibit the activation of EGF signaling pathway, alleviate the inflammation of AR nasal mucosa, and enhance red blood cells immune adherence function.

Radon single-pixel imaging with projective sampling.

A novel technique for Radon single-pixel imaging with projective sampling, which is based on the theorem of the Radon transform, is proposed. In contrast to current patterns in conventional single-pixel imaging systems, candy-striped patterns called Radon basis patterns, which are produced by projecting the 1D Hadamard functions along different angles, are employed in our proposed technique. Here, the patterns are loaded into a projection system and then illuminated onto an object. The light reflected from the object is detected by a single-pixel detector. An iterative reconstruction method is used to restore the object's 1D projection functions by summing the 1D Hadamard functions and detected intensities. Next, the Radon spectrum of the object is recovered by arranging the 1D projection functions along the projection angle. Finally, the image of the object can be recovered using a filtered back-projection algorithm with the Radon spectrum. Experiments demonstrate that the proposed technique can obtain the information of the Radon spectrum and image of the object. Recognition directly in the Radon spectrum domain, rather than in the image domain, is fast and yields robust and high classification rates. A recognition experiment is performed by detecting the lines in one scene by searching the singular peaks in the Radon spectrum domain. According to the results, the lines in the scene can be easily detected in the Radon spectrum domain. Other shapes can also be detected by the characteristics of those shapes in the Radon spectrum domain.

The diagnostic value of combined specific IgG and specific IgE of Aspergillus fumigatus in allergic broncho pulmonary aspergillosis and severe asthma with fungal sensitization / 中华内科杂志

This study aims to explore the diagnostic value of specific immunoglobulin E (sIgE) and specific immunoglobulin G (sIgG) of Aspergillus fumigatus in the diagnosis of allergic broncho-pulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). A total of 17 ABPA patients and 14 SAFS patients were enrolled. The levels of sIgG [2 294.00 (1 527.00, 14 170.00) U/ml vs. 972.60 (650.90, 1 792.00) U/ml] and sIgE [8.77 (1.64, 16.85) kU/L vs. 1.04 (0.70, 2.05) kU/L] in ABPA patients were significantly higher than those in SAFS patients (P<0.05). Aspergillus fumigatus sIgG was strongly correlated with Aspergillus fumigatus sIgE (rs=0.797, P<0.001) in ABPA patients. When combined with Aspergillus fumigatus sIgG (>1 000.00 U/mL) and Aspergillus fumigatus sIgE (>1.00 kU/L), the sensitivity was 82.3% and specificity 78.6% for the differential diagnosis of ABPA and SAFS. It demonstrates the diagnostic value of Aspergillus fumigatus sIgG and sIgE.

The sensitization pattern of Blomia tropicalis in allergic respiratory disease in Guangzhou / 实用医学杂志

Objective To investigate the sensitization pattern of Blomia tropicalis in patients with aller-gic respiratory disease and the association between Blomia tropicalis with allergic rhinitis and / or asthma. Meth-ods 330 allergic rhinitis and / or asthma patients sensitized with mites were selected in Guangzhou,and sIgE lev-el in the serum of all recruited patients of Blomia tropical,Dermatophagoide pteronyssinus and Dermatophagoide fa-rina was measured. Results The sensitization rate of Blomia tropicalis was significantly lower than that of Derma-tophagoide pteronyssinus and Dermatophagoide farina(P < 0.001,both). 80.0% Patients were sensitized with both three mites,and only 0.3% patients were of monosensitization to Blomia tropicalis. The sIgE of these three mites were significantly positive correlated with each other(P < 0.001),Dermatophagoide pteronyssinus and Derma-tophagoide farina have strong correlation(r = 0.906),Blomia tropicalis has moderate correlation with Dermatopha-goide pteronyssinus and Dermatophagoide farina(r = 0.540 and r = 0.512,respectively). With the increase of Blomia tropicalis sIgE,the severe sensitization rate(sIgE level:class 5~6)of patients sensitized combined Der-matophagoide pteronyssinus or Dermatophagoide farina significantly increased(P < 0.001). The sIgE level of Der-matophagoide pteronyssinus and Dermatophagoide farina in allergic asthma and rhinitis patients were significantly higher than that of patients with rhinitis or asthma alone(P < 0.001),and the sIgE level of Blomia tropicalis in pa-tients with both allergic asthma and rhinitis or with asthma alone were also significantly higher than that of patients with rhinitis alone(P = 0.006 and P = 0.020,respectively). Conclusion The Blomia tropicalis sensitized pa-tients usually sensitize together with Dermatophagoide pteronyssinus or Dermatophagoide farina,and the degree of Blomia tropicalis sensitized in asthma patients are higher.

The correlation between serum total IgE,blood eosinophil,degree of sensitization to allergens and frac-tional exhaled nitric oxide in children with asthma and allergic rhinitis / 实用医学杂志

Objective To analyze the correlation among serum total IgE(TIgE),blood eosinophil(EOS) counts,the degree of sensitization for skin prick test(SPT),and fractional exhaled nitric oxide(FeNO)in chil-dren with asthma and allergic rhinitis. Methods We collected 121 children with asthma and allergic rhinitis andtheir serum TIgE,blood EOS,FeNO,and SPT for 14 common allergens. Results There were 75.2%patients sensitizing to at least two allergens.TIgE levels in poly-sensitized subjects were higher than that of mono-sensitized subjects(P < 0.05)The number of SPT sensitized allergens was positively correlated with the TIgE levels(P <0.05)FeNO levels were correlated with TIgE levels and EOS percentage(P < 0.05). TIgE levels in subjects with class 2 or 3 in SPT for house dust mite increased significantly ,compared with class 0 or 1. Conclusion There were some correlations among the four detections ,providing important reference for clinical diagnosis and treatment of respiratory allergic disease

Distributions of sIgE and tIgE in patents with allergic diseases in Guangdong / 实用医学杂志

Objective To investigate the types and distributions of allergens, and the responsiveness to these allergens SIgE and tIgE as related to allergic disorders in patients with allergies in Guangdong. Methods Serum samples were obtained from patients with allergic disorders (n = 7 144) who visited our hospital between 2009 and 2014. The sera were subjected to analysis of 15 common allergens. Results The positive rates of sIgE and tIgE were 62.4%and 54.6%, respectively. Dermatophagoides pteronyssinus(Der p) had the highest prevalence of aeroallergen-specific IgE and milk the highest prevalence of food allergen-specific IgE. Other aeroallergens and food allergens produced mild responses except Der p and Der f. The sensitization peak of Der p and Der f appeared at the age of 10 to 12 years. The sensitization peak of milk appeared at the age of less than 3 years and that of eggs did at the aged of 4 to 6 years. The averaged tIgE positive rate went up with the increase in the number of allergen sensitization. Conclusion Der p, Der f, milk and eggs are major sensitizers responsible for common allergic disorders in Guangdong. Knowledge concerning allergen characteristics at various age groups may be helpful for early diagnosis and intervention for allergies.

Distribution of 112 allergen in multi-sensitized patients with respiratory symptoms / 中华检验医学杂志

Objective To investigate the role of molecular allergen diagnosis in respiratory allergic diseases, and measure the prevalence and the allergen molecular mapping of the asthma and allergic rhinitic patients.Methods Totally 80 cases of asthma or allergic rhinitis patients with two or more inhaled allergens from First Affiliated Hospital of Guangzhou Medical University during 2013 January to 2014 December were tested for serum specific immunoglobulin E ( sIgE ) against 112 allergen components by ImmunoCAP ISAC.Results About 78.8% ( 63/80 ) patients is three or more allergen components sensitization, the most prevalence allergen components were house dust mites′Der f 1 67.5%(54/80), Der f 2 66.2%(53/80), Der p 1 and Der p 2 were 63.8%(51/80) and 61.2%(49/80), Der p 10 was only 12.5%(10/80), follewed by Fel d 1 22.5%(18/80), Cyn d 1 16.2%(13/80), Phl p 4 15.0%(12/80), Pen m 1,Pla a 2,Jug r 2 and Can f 1were all 11.2%(9/80), Lep d 2, Blo t 5, Bla g 7 and Ani s 3 10.0%(8/80).Other allergen components positive rates were low.Conclusions Molecular allergen diagnosis can provide more comprehensive diagnostic information for allergic patients, by providing allergen map.Allergen microarray can discover the neglected allergens such as bermuda grass, timothy grass and plane tree pollen allergens in southen China.

Analysis of food allergen spectrums in food anaphylaxis in Guangzhou / 国际检验医学杂志

Objective To analyse distribution of age and gender characteristics of specific IgG(sIgG)antibodies and specific IgE (sIgE)antibodies of 13 types of food allergens in patients with food anaphylaxis,and to explore the relationship between sIgG and sIgE in food anaphylaxis.Methods 314 cases of patients from 2009 to 2012 were selected as subjects,and divided into underage group(1 63 cases)and adult group(1 5 1 cases).Serum sIgG of 13 types of food allergens were detected by using enzyme linked immu-nosorbent assay,serum sIgE of these food allergens were detected by using immune capture.Results 80.25% of the patients were sIgG-positive,and no obvious gender differences were found;while the positive rates of sIgG in the underage group(94.48%)were higher than that in the adult group(64.90%),there were statistically significant differences(P < 0.05 ).34.39% of the patients were sIgE-positive.The positive rates of sIgE in male patients(40.68%)were higher than that in female patients(26.28%),and that in the underage group(55.21%)were also higher than that in the adults group(1 1.92%),there were statistically significant differences(P <0.05).Conclusion The total positive rates and its distribution characteristics of sIgG and sIgE of same food aller-gens were obviously different.Food anaphylaxis might be associated with age,gender,food types and individual diversity.

Cultivation of laboratory physicians in China：current situation and reform measures / 中华医学教育探索杂志

Cultivation of laboratory physicians in accordance with the needs of discipline should be a priority of education on laboratory medicine in China. Unfortunately, the professional career of Chinese laboratory physicians is presently entrapped in a dilemma owing to unfavorable settings, in-cluding the late start of laboratory physician training in this country, unmatched installation between higher school faculty and curriculum, faulty programs of in-hospital training, disparity in management policies, low social esteem, uncertainty in personal intention of employment, and poor knowledge of position awareness. This paper puts forward a lot of measures such as the development trend of the university curriculum, which is in compliance with the subdivision and refinement of clinical medicine, the establishment of a medical degree for a professional laboratory physician, constantly improving the standardization of laboratory physician residency training and continuing education system for the in-spection of doctors , setting up physicians inspection posts and giving a clear mandate to examine physicians inside the hospital, while outside of the hospital continually improving awareness and accep-tance of the laboratory physicians, giving more educational resources to laboratory physicians, so as to improve the overall level of the laboratory medicine.

Establishment and evaluation of time resolved fluoroimmuno assay for quantification of total immuno globulin E / 重庆医学

Objective To establish a method for quantitative detection of total immunoglobulin E(TIgE) by Time resolved Flu‐oroimmunoassay(TRFIA ) .Methods The method for quantitative detection of TIgE by TRFIA was established on the basis of solidphase double sandwich enzyme linked immunosorbent assay(ELISA) .The methodology was evaluated .Results The TIgE TR‐FIA intra assay and inter assay coefficients of variation (CV) were 1 .59% -1 .68% and 5 .23% -7 .33% ,respectively .The lower limit of detection was 0 .25 IU/mL .The linear range was 1 .47-1 510 .00 IU/mL .The accuracy was within the allowable deviation ( ± 10% ) .The recovery rate was 97 .00% -106 .75% .The cross reaction test and interference experiment could meet the testing requirements .The TIgE TRFIA showed no HOOK effect at least up to 15 000 IU/mL TIgE ,compared with EUROIMMUN ELISA ,the correlation coefficient(r) was 0 .999 2(P<0 .01)for 40 blood specimens in the range of 14 .43-518 .81 IU/mL ,and the expected bias was in the range of acceptable bias (± 12 .50% ) .The reference value 100 IU/mL could be used for a normal ,allergy free adult sample TIgE level detected by TRFIA .Conclusion The established TRFIA for TIgE detection meets the demand of clini‐cal application with good precision ,high sensitivity ,wide linear range ,high accuracy ,specificity and other advantages .

Measurement and analysis of the common food allergens specific IgE / 中华检验医学杂志

Objective To explore the positive distribution characteristics and analyse the correlation of common food allergen-specific immunoglobulin E (sIgE) with suspected food allergy in the First Affiliated Hospital of Guangzhou Medical University.Methods Using fluorescence enzyme-linked immunosorbent assay to detect the serum sIgE antibody of 854 patients,including 7 kinds of food allergens (milk,egg white,egg yolk,shrimp,crab,peanut and soybean) from July 2006 to January 2013.Data were processed by statistical software SPSS19.0.Results The positive rates of the 7 kinds of food allergen were 39.3％ (283/720),36.3％ (216/595),9.8％ (28/285),21.2％ (36/170),24.3％ (17/70),14.8％ (9/61) and 10.0％ (5/50).Two hundred and eighty-two patients were detected egg white and egg yolk sIgE,the positive rate of egg white sIgE 59.6％ (168/282) was much higher than the egg yolk sIgE 9.2％ (26/282).The positive rate of the milk and egg white sIgE were different between the age groups and decreased with the age(milk:x2 =792.88;egg white:x2 =658.21,P ＜ 0.01).The degree in level 4 or above of milk and egg white sIgE were 1.08％ (2/186) and 1.12％ (2/178) in positive patients.Five hundred and twenty-eight patients were detected milk and egg white sIgE,26.9％ (142/528) were both positive,sIgE levels in serum of them was moderately correlated (rs =0.758,P ＜ 0.01).Serum sIgE of shrimp and crab was detected in 64 patients,16 cases was positive of the shrimp,17 cases of the crab was positive,both positive in 16 cases,sIgE levels in serum of them were highly correlated (rs =0.973,P ＜0.01).In the simultaneous detection of 34 patients' serum sIgE of peanut and soybean,peanut was positive in 4 cases,soybean was positive in 3 cases,both positive in 2 cases,sIgE levels in serum of them were highly correlated (rs =0.879,P ＜ 0.01).Conclusions Egg white is the major allergen of egg allergy.The correlations of shrimp and crab,peanut and soybean are extremely high,probably because of their homology; egg white and milk are not homologous food,but the levels of sIgE between them display a moderately correlation,indicating the existing of synergistic reaction.

Discussion on Medical Laboratory Experiment Teaching of Higher Profession / 中华医学教育探索杂志

The article introduces the training of basic skill,renovating of the experiment contents,the diversifi cation and abundance in interest of experiment teaching methods of the students of medical laboratory speciality of higher profession.

Potential polypeptides recognized by interleukin(IL)-5 through screening phage displayed random peptide library / 中国免疫学杂志

Objective:To find related polypeptides which could bind interleukin (IL) 5 with high affinity.Methods:The recombinant human IL 5 was biotinylated with NSH LC Biotin,then biotinylated IL 5 was used to react with random peptide library displaying 7 amino acids fused on protein III of M13 Phage for three rounds biopanning.The selected clones were assayed by sandwich enzyme linked immunosorbent assay(ELISA)and competition ELISA.The positive clones with high affinity were used for automated sequencing with dye labeled dideoxynucleotides,and the amino acid sequence of polypeptide displayed on phage was deduced.Results:The enrichment was shown by ELISA after 3 rounds of biopanning.9 positive clones could bind to IL 5 with high affinity.Sequencing of the genes encoding these peptides on 9 positive clones showed some conserved epitope information such as SX 1 2 AS,ALAS.Conclusion:Potential polypeptides binding IL 5 with high affinity could be selected from phage display peptide library,SX 1 2 AS may be the motifs recognized by IL 5.

Effect of Anti-Midgut-Protein-Ingredient Antibodies of Anopheles stephensi on the Oocysts of Plasmodium yoelii / 中国寄生虫学与寄生虫病杂志

Objective To observe the inhibitory effect of the antibodies against midgut-protein-ingredient of Anopheles stephensi on the oocysts of Plasmodium yoelii.Methods Female An.stephensi mosquitoes raised in laboratory were dissected and the midguts were collected.Eight BALB/c mice were immunized using midgut-protein(100 ?g/mouse,4 times with an interval of 7～10day).Ten days after the last immunization,blood was taken from mice armpit artery and serum separated.The immune active antigen of the midgut protein was analyzed by Western blotting.Protein with Mr 38 000～50 000 was separated by sephadex filtering and used to immunize 12 BALB/c mice(100 ?g/mouse,4 times with interval of 7～10 days).PBS control group was established.Seven days after the last immunization,serum antibody was detected by ELISA.When the antibody titer in immunized mice reached ≥1:2 560,mice in both groups were infected by P.yoelii(about 2?107 plasmodium-infected RBC) by abdominal injection.The mosquitoes were fed on the infected mice when the number of female gametes was higher than 2 per 10 microscopical fields 3 days later.After 9 days,the mosquitoes were dissected and the amount of oocysts in midgut was counted.Results Eight protein bands were shown in midgut-protein of An.stephensi by Western blotting and the band of Mr 38 000～50 000-midgut-protein appeared clearer.The infection rate of oocysts in the experiment and control groups were 28.70%(62/216) and 51.09%(47/92) respectively(P