key: cord-0034636-ze9fl3se authors: Dai, Jie; Li, Hongjun; Li, Li title: Laboratory Test for Diagnosis of Influenza date: 2016-06-23 journal: Radiology of Influenza DOI: 10.1007/978-94-024-0908-6_7 sha: ab0fa2623abf8c10a47d7818f3ae166c1fddc177 doc_id: 34636 cord_uid: ze9fl3se Laboratory tests for diagnosis of influenza include 4 aspects of examinations, virus culture and isolation, serological test, immunoassay, and molecular biological examination. Virus culture and isolation is the most common and the most reliable way for the diagnosis of infl uenza. The specimens for virus culture and isolation include nasopharyngeal swab and oral gargle. Currently, chicken embryo and Madin-Daby canine kidney cells (MDCK cells) are commonly applied for infl uenza virus culture and isolation. Chicken-embryo culture is one of the commonly used way for virus culture, which can be applied for virus culture and isolation, titration of virulence, neutralization test as well as preparation of antigen and vaccine. Respiratory viruses, such as orthomyxovirus, paramyxovirus and poxvirus, are sensitive to chicken-embryo culture. Specimens can be harvested from patients for culture and isolation of these viruses. For infl uenza virus culture and isolation, dual cavities (chicken-embryonic allantonic and amniotic cavities) inoculation is commonly applied. Generally, chicken embryos aged 9-11 days are applied for amniotic and allantoic cavities inoculation to isolate the virus. Within 24-96 h after inoculation, the fl uid in allantoic cavity of chicken embryo should be collected, and the chicken embryo should be rejected within 24 h after death. The chicken erythrocytes are used to test the hemagglutination activity of allantoic fl uid or cell culture fl uid in order to prove the proliferation and thus the existence of the virus. If the initial virus isolation fails, blind passage for the 2nd generations can be used for another round of test. In recent years, with the development of molecular biology technology, the infl uenza virus isolated via chicken embryo shows different antigenicity from the original specimen, while the antigenicity of infl uenza virus isolated via MDCK cells resembles to the original specimen. Due to the much higher sensitivity of MDCK cells to the O phase virus strain than chicken embryos, MDCK cells constitute an indispensable host system for isolation of infl uenza virus, and have gained wide application. Serological diagnosis is to detect the level of antibody in serum. Serological test for infl uenza virus is based on collection of double sera during the acute stage (within 3 days after onset) and during the convalescent stage (2-4 weeks after onset) for hemagglutination inhibition test, complement binding test, and micro neutralization test for antibody titer. If the antibody titer in the convalescent stage is at least 4 times as high as that in the acute stage, the diagnosis of infl uenza can be made. Single serum is generally inapplicable for the diagnosis. Due to the long time for a cycle of serological test and the complex operations, serological diagnosis of infl uenza is commonly applied in retrospective study, which plays a role in predicting the future prevalence of infl uenza. However, it contributes little to the early diagnosis of infl uenza. Immunofl uorescent assay is based on the basic principle that the fl uorescein labeled antibody binds to the corresponding antigen to form immune complex, which, under a fl uorescence microscope, facilitates the observation of virus antigen in cells and its location. The immunofl uorescent assays mainly include direct fl uorescent antibody (DFA) test and indirect fl uorescent antibody (IFA) test. DFA test adopts monoclonal antibody to target on the antigen of infl uenza virus. Under a microscope, the respiratory epithelial cells are observed. The fi nding of virus antigen demonstrates an infection. The test shows a favorable specifi city but a low sensitivity. Due to the binding of antibody to the corresponding antigen to form immune complex, the fl uorescein labeled globulin antibody is used to detect the immune complex and therefore speculate the existence of antibody or antigen. By IFA, the known antigen can be used to detect unknown antibody and the known antibody can be used to detect unknown antigen. The test shows both high sensitivity and high specifi city, and thus has gained wide application in clinical practice. Immune colloidal gold assay adopts colloidal gold as marker to localize, qualify, and quantify antigen or antibody based on specifi c binding of antibody to antigen. Immune colloidal gold assay mainly includes colloidal gold enhanced immunochromatography assay and rapid dot immuno-gold fi ltration assay. The assay has simple operation with fast result within 10-30 min. In clinic and ICU, it greatly helps in early diagnosis and appropriate treatment. Along with the development of molecular biology, especially the application of polymerase chain reaction technology, molecular biological diagnosis plays an increasingly important role in identifying and typing of infl uenza virus. RT-PCR is a technology integrating reverse transcriptase of RNA and PCR of cDNA. For RT-PCR, the total RNA in tissues or cells can be extracted and the mRNA in it, as template, is reverse transcribed into cDNA by using Oligo (dT) or random primer and reverse transcriptase. The cDNA is then used as template for PCR amplifi cation to obtain the expression of target gene or detected gene. RT-PCR is highly sensitive and specifi c, which shortens the period time consumed in detection. It is a practical way for early rapid diagnosis of infl uenza. For detection of RNA virus, RT-PCR is the most commonly used molecular amplifi cation, being capable of identifying the infl uenza virus and further analyzing its subtype via RNA fragment amplifi cation of the virus genome in the specimen. Multiple RT-PCR is the addition of two or more virus subtypes or specifi c primers of several viruses in the same PCR system. Based on the length discrepancy of the target fragments, multiple virus subtypes or multiple viruses can be simultaneously detected. It is a rapid and sensitive way of detection with low cost. Real-time fl uorescent quantitative RT-PCR is defi ned as the addition of fl uorophores in the RT-PCR system. Based on accumulated fl uorescent signals, the whole process of PCR is real-time monitored and the standard curve is used to quantitatively analyze the unknown template. Compared to conventional RT-PCR, real-time fl uorescent quantitative RT-PCR has the advantages of high sensitivity, high specifi city and rapidity, that enables quantitative analysis of the sample. It has been applied in laboratories specialized in large-scale network surveillance of infl uenza. Multiple realtime PCR is capable of further analyzing the subtype of infl uenza virus. Nucleic acid sequence dependent amplifi cation is a rapid isothermal amplifi cation technology with RNA as template that is independent of reverse transcription. It can be performed in collaborations of reverse transcriptase of birds myeloblastoma virus, RNA polymerase of bacteriophage T7, RNase H, two specially designed specifi c oligonucleotide primers and molecular beacon probe. Its sensitivity is equivalent to currently used virus cultures, and can be applied to accurately detect the virus, especially detection of the virus RNA. Gene chip technology is based on the principle that a large number of known nucleotide sequences, as probes, are integrated onto one chip, followed by hybridization with labeled target nucleotide sequences, and the large quantity of gene information in cells or tissues can be detected and analyzed by detecting the hybridization signal. Gene chip technology has the advantages of high specifi city that consumes short period of time. However, due to its weakness of high cost and requirement of complicated device, its application in the detection of infl uenza virus is still far limited than other examinations. Generally, it presents as infl ammatory responses of the airway, and even lung infection in some severe cases. The main change is primary viral pneumonia, possibly with secondary acute bronchitis, bacterial pneumonia or mixed pneumonia. Autopsies by the CDC of the United States discovered that about 30 % of deaths from infl uenza A (H1N1) shows concurrent bacterial infection, among which, 50 % is induced by pneumococcus. The change in the late stage of the disease is mainly necrotizing bronchitis, diffuse alveolar damage, pulmonary hemorrhage, intrapulmonary formation of hyaline membrane as well as fi brosis and consolidation of different degrees. In the patients with complications, purulent infl ammatory changes are detectable. The main histopathological changes include congestion and edema of the upper respiratory mucosa, degeneration, necrosis and shedding of bronchial epithelium and gland. The lung lesions are mainly serous and hemorrhagic bronchial pneumonia, with interalveolar septal thickening, interstitial congestion, as well as accompanying infi ltration of lymphocytes in a large quantity, serous exudation and focal hemorrhage. In some cases, the condition is accompanied by diffuse alveolar damage, obvious pulmonary edema, apoptosis, necrosis, shedding of the alveolar epithelium, complete alveolar collapse, proliferation of the macrophages and type II alveolar epithelial cells. In the later stage, the pathological changes mainly include necrotizing bronchitis, intrapulmonary formation of hyaline membrane, fl akes of hemorrhage of the lung tissue, fi brosis and consolidation of lung tissues in different degrees. In the cases with concurrent bacterial infection, suppurative bronchiolitis, pleuritis, pleural effusion or empyema are shown. Some studies indicated that infl uenza A (H1N1) may cause high blood coagulation and in some cases, it may cause intrapulmonary minor vascular thrombosis and pulmonary saddle embolism or lobar artery embolism. In literature reports, the pathological changes of the extrapulmonary organs in the cases of infl uenza A (H1N1) are inconsistent. In case reports of some rare severe cases, lymphocytes depletion in bone marrow, spleen and lymph nodes, erythrophagocytosis; acute renal tubular necrosis, myoglobin casts; necrosis, fatty degeneration and cholestasis of the liver; sterile meningitis, myelitis, brain hemorrhage, encephalitis, brain edema, brain herniation; coagulating necrosis of pancreas and spleen; rhabdomyolysis; myocardial infarction, myocarditis, pericarditis and other myocardial damages have been reported. In most literature reports, no obvious abnormalities of extrapulmonary organs have been discovered other than pathological changes of the lungs. Compared to SARS, another emerging infectious disease, deaths from severe infl uenza A (H1N1) showed more serious lung hemorrhage, and more obvious damages to the tracheal and bronchial epithelia and glands. In addition to fi ndings by immunohistochemistry and fl ow cytometry, the virus antigen expression is detected on the pseudostratifi ed ciliated columnar epithelium, tracheal glandular epithelium, bronchiolar epithelial surface, type I and type II alveoli, cytoplasms of vascular endothelial cells and alveolar macrophages. And apoptosis of alveolar epithelial cells is also detected, which may be related to the above mentioned damages. Currently, autopsies of deaths from human infected H5N1 avian infl uenza have been rarely reported both nationally in China and internationally. The main fi ndings include pulmonary hemorrhage and consolidation. The virus infects the nasopharyngeal epithelium and glands, the tonsils, trachea and lung tissue to cause tissue infl ammatory responses, such as congestion, edema, and infi ltration of perivasrcular lymphocytes. Due to the highest effi ciency of virus replication in lungs, the lesions are the most serious there, which are pathologically characterized by pulmonary hemorrhage and necrotizing lesions. The early manifestations include interstitial pneumonia and necrotizing bronchitis; during the progressive stage, with occurrences of diffuse alveolar damage, acute diffuse exudation, accompanying pulmonary edema, multifocal pulmonary hemorrhage, and pulmonary formation of hyaline membrane. During the late stage, the lung tissues are subject to different degrees of fi brosis, organization and consolidation. In the patients with complications, purulent infl ammatory changes are detected, including bronchial pneumonia and empyema. The main histopathological changes include vascular dilation and congestion of alveolar wall in both lungs, fi lling of pale red edema fl uid in the alveolar cavity and different amounts of infl ammatory cells, predominantly lymphocytes and macrophages. In addition, syncytoid cells and foam cells are accompanied by formation of hyaline membrane and multifocal hemorrhage. In some areas, the interalveolar septa are subject to thickening with interstitial fi brosis. Some bronchioles and their surrounding alveoli are subject to epithelial detachment, hyperplasia and squamous metaplasia, with alveolar collapse and emphysema of the surrounding lung tissues. In the cases with secondary bacterial infection, the bronchioles and their surrounding alveoli are subject to damages in some areas, with infi ltration of neutrophils and formation of small abscesses. In the late stage, the lung tissue shows extensive consolidation. H5N1 avian infl uenza virus shows stronger orientation to tissues than infl uenza A H1N1 virus. Autopsies and laboratory tests have demonstrated that H5N1 can replicate and reproduce in lymph nodes and other tissues or heart, liver, brain and other organs to involve multiple organs. In addition to primary lung infection, the virus can also directly or indirectly invade the heart, blood vessels, skeletal muscles, liver, kidney and other organs in severe cases. And the patients may experience the symptoms of myocardial fi brous degeneration and necrosis, myocardial interstitial mononuclear cell infi ltration, interstitial myocarditis; liver congestion, hepatocytic loose cytoplasm, vesicular fatty degeneration; kidney congestion, renal tubular epithelial cells degeneration and necrosis, cellular casts formation; brain hemorrhage and edema; ascites and pleural effusion; rhabdomyolysis; decreased or absent lymphatic hematopoietic tissue in spleen, thymus, tonsils, and lymph nodes, proliferation and erythrophagocytosis of CD68 positive cells. The virus infection can also cause pulmonary hemorrhage, gingival bleeding, gastrointestinal bleeding and bleeding of other sites. So far as we know, apart from the animal models of lung infection, no autopsy has been reported about human infected H7N9 avian infl uenza. Therefore, the clinical management is mainly based on radiological examinations and laboratory tests. The animal experiments have demonstrated that the lung lesions in mice are the most serious in the cases of human infected H5N1 avian infl uenza, followed by infl uenza A H1N1 and human infected H7N9 avian infl uenza. After infection of H7N9 avian infl uenza virus, the mice showed comparatively mild responses, with strong ability of self repair of lung tissues. The main lesions mainly include: 1. Shedding of bronchiolar epithelial cell into the lumen and infl ammatory cells infi ltration around the bronchiolar wall; 2. Interstitial pneumonia, with lung interstitial thickening, congestion and edema of lung, perivascular infi ltration of lymphocytes; 3. Pulmonary vasculitis; 4. Diffuse alveolar damage; 5. Pulmonary hemorrhage; 6. Pulmonary interstitial fi brosis. 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