2 English reference Toxoplasma gondii * * * A [WS/T 486-2015 Diagnosis of Toxoplasma gondii]
Overview Toxoplasma gondii (Toxoplasma gondii * * * A) is a protozoan parasitic in human and animals, which can be parasitic in almost all nucleated cells of human body. Toxoplasma gondii is named because its trophozoite is bow-shaped or half-moon-shaped, which can cause Toxoplasma gondii infection or toxoplasmosis. [ 1]
Toxoplasma gondii was first discovered in monocytes of Ctenopharyngodon gondii. It is a protozoan that can parasitize almost all nucleated cells in human body. Cat is its only definite host, and other mammals, birds and humans can be its intermediate hosts. The whole process of its development includes five life history stages: trophozoite (including tachyzoite and bradykinin), cyst, schizont, gametophyte and oocyst, among which tachyzoite (pseudocyst) and oocyst have clinical value. [ 1]
The morphology of Toxoplasma gondii is 4. 1. The tachyzoite is banana-shaped or half-moon-shaped, with a sharp front end and a blunt back end. It is 3 to 7 microns long and 2 to 4 microns wide. After Giemsa staining, the cytoplasm was blue and the nucleus was purplish red, slightly behind the center of the worm. [ 1]
The tachyzoites parasitized in cells proliferate constantly, generally including several to more than ten. The worm aggregates surrounded by the host cell membrane have no real cyst wall, which is called pseudocyst. [ 1]
4.2 The cyst is round with elastic and tough wall, which is secreted by worms and contains dozens to thousands of bradykinins. Bradykinin has a diameter of 5 μ m ~ 100 μ m, which mostly exists in brain, skeletal muscle, retina and other tissues, and can survive in tissues for a long time. [ 1]
The development of life history of Toxoplasma gondii in the final host: after swallowing oocysts, cysts or pseudocysts, cats escape from spores, bradykinin or tachyzoites in the small intestine, invade the epithelial cells of the small intestine to develop and proliferate, and form merozoites. The merozoite is released when it matures, and then invades other intestinal epithelial cells to form the next generation of merozoites. After several generations, some merozoites develop into female gametes and male gametes, and then continue to develop into female gametes and male gametes. After fertilization, the female and male gametes become fertilized eggs, continue to develop and form oocysts, escape from epithelial cells into the intestinal cavity, and are excreted with feces. At the temperature of 25℃ and suitable humidity, it takes 2d ~ 4d days to develop into an infectious mature oocyst. At the same time, Toxoplasma gondii can also reproduce asexually in the extra-intestinal tissues of cats. [ 1]
Development process in intermediate host: When the oocysts in cat feces or the cysts or pseudocysts in animal meat are swallowed by human, sheep, pigs, cattle and other intermediate hosts, spores, bradykinin or tachyzoites escape from the intestine, invade the intestinal wall, enter mononuclear macrophages through blood or lymph, and spread to various tissues and organs of the whole body, such as brain, eyes, liver, heart, lung, muscle and so on. , and enter the cell. When the immune function of the body is normal, some tachyzoites invade the host cells, especially the brain, eyes, skeletal muscle and other tissues, and the proliferation of worms slows down and secretes cystic substances to form cysts. Cysts can survive in the host for months, years or longer. When the immune function of the body is low or defective, it can induce the development and rupture of cysts, release bradykinin, enter the blood stream and invade other tissues and cells to form pseudocysts, and continue to develop and proliferate. [ 1]
Epidemiology of Toxoplasma gondii infection Toxoplasma gondii is an important conditionally pathogenic parasite, which is distributed all over the world. Infection between people is quite common, mostly recessive infection. The National Cooperative Group for Toxoplasma gondii Investigation and Research (1983 ~ 1986) investigated 8 1968 residents in19 provinces (cities, autonomous regions)+0 counties by IHA method. The results showed that the positive rate of Toxoplasma antibody in serum was 0.33%. The national survey of important human parasitic diseases (200 1 ~ 2004) reported that 47444 people were detected in 15 provinces (municipalities and autonomous regions). Results the positive rate of serum antibody was 0.79% ~ 16.8 10/, with an average positive rate of 7.88. [ 1]
6. 1 Cats that excrete Toxoplasma oocysts with feces are the main source of infection, followed by mammals, birds and other warm-blooded animals infected with Toxoplasma gondii. Toxoplasma gondii can infect the fetus through placenta, so the infected mother is also the source of infection. [ 1]
6.2 Transmission route Congenital Toxoplasma infection refers to fetal infection through maternal placenta; Acquired Toxoplasma infection refers to transmission through human digestive tract mucosa, damaged skin, blood transfusion and organ transplantation. [ 1]
6.3 Susceptible population Humans are generally susceptible to Toxoplasma gondii, especially fetuses, infants, people who raise or contact pets such as cats and dogs, animal breeders, butchers and people with low or defective immune function. [ 1]
6.4 Epidemic factors Toxoplasma gondii oocysts have strong resistance to the outside world, and are distributed in cold zone, temperate zone and tropical zone, and there is no strict geographical distribution boundary. People infected with Toxoplasma gondii are related to keeping pets and eating habits. In the process of adding T to food, cross-contamination caused by raw and cooked food will increase the chance of Toxoplasma infection. [ 1]
Clinical manifestations of Toxoplasma gondii infection There are two ways of Toxoplasma gondii infection: congenital and acquired. Most women infected with Toxoplasma gondii during pregnancy can cause fetal congenital infection, and infants usually have no obvious clinical symptoms and signs. When the immune function is low due to various reasons, the central nervous system may be damaged in childhood, and retinal choroiditis may occur in adulthood. A few women infected with Toxoplasma gondii in early pregnancy may have miscarriage, premature delivery, stillbirth or deformity. Infection with Toxoplasma gondii in the second and third trimester of pregnancy may lead to diseases or deformities in the brain, eyes, liver, heart, lung and other organs of the fetus after birth. [ 1]
After acquired infection with Toxoplasma gondii, most people with normal immune function have no obvious clinical symptoms and signs, which belongs to recessive infection. When the immune function is low or lacking, Toxoplasma gondii can invade various organs of human body, causing corresponding serious clinical manifestations, such as Toxoplasma encephalopathy, Toxoplasma gondii eye disease, Toxoplasma gondii liver disease, Toxoplasma gondii myocardial pericarditis, Toxoplasma gondii pneumonia and so on. [ 1]
7. 1 Toxoplasma gondii can parasitize the central nervous system or striated muscle for a long time after Toxoplasma gondii infection, and there are no obvious symptoms and signs in clinic, only Toxoplasma gondii is positive in etiology. [ 1]
7.2 Toxoplasma encephalopathy The clinical manifestations of Toxoplasma encephalopathy include encephalitis, meningitis, meningoencephalitis, epilepsy and mental disorders. May cause headache, dizziness, delirium, myalgia, lymphadenopathy, etc. Toxoplasma gondii tachyzoites can be found in cerebrospinal fluid. [ 1]
7.3 Toxoplasma oculopathy Toxoplasma oculopathy is mainly recurrent, localized and necrotizing choroiditis, and its clinical manifestations are blurred vision, eye pain, photophobia, blind spots and tears. The fundus showed retinal edema and macular exudative lesions in the posterior pole. The boundary of the fresh lesion is vague, bluish gray, slightly raised, and there is retinal hemorrhage around it; Old venereal diseases become satellite-like scattered in white circular plaques and pigment spots, or pigment epithelium falls off in macula. [ 1]
7.4 Toxoplasma gondii liver disease Toxoplasma gondii destroys liver cells, causing inflammatory infiltration and local necrosis of liver parenchyma. The clinical manifestations are anorexia, liver pain, ascites, mild jaundice, liver cirrhosis and splenomegaly. And the course of disease is long and easy to relapse. [ 1]
7.5 Toxoplasma myocardial pericarditis Toxoplasma myocardial pericarditis can cause fever, abdominal pain, tonsillitis and eyelid edema. , often no obvious abnormal heart symptoms, palpitations, jugular vein dilatation, chest pain, dyspnea, etc. , occasionally hear pericardial fricative sound. In severe cases, there may be dull pain and severe pain behind the chest or sternum, and the pain radiates to the neck and shoulders. If not treated in time, heart failure may occur. [ 1]
7.6 The clinical manifestations of Toxoplasma gondii pneumonia include cough, expectoration, chest pain, shortness of breath and wheezing in the lungs. X-ray examination showed inflammatory infiltration. Pulmonary lesions are often complicated with cytomegalovirus and bacterial infection, which are characterized by interstitial and lobular pneumonia. [ 1]
7.7 Other women infected with Toxoplasma gondii in early pregnancy can pass through the placental barrier, which often causes extensive fetal lesions, leading to abortion, premature delivery and stillbirth. , manifested as anencephaly, hydrocephalus, microcephaly and mental retardation, has become one of the most serious diseases in human congenital infection. [ 1]
Laboratory examination of Toxoplasma gondii [1]
8. 1 Detection of Toxoplasma IgG antibody 8. 1. 1 Methods Indirect enzyme-linked immunosorbent assay was used to detect Toxoplasma IgG antibody in human serum, plasma or other body fluids.
8. 1.2 reagent consists of a 96-well plate coated with Toxoplasma gondii antigen, human staphylococcal protein A(SPA) or anti-human IgG enzyme labeling solution, sample diluent (physiological saline containing 0.9% sodium chloride), washing solution (Tween20 buffer), stopping solution (mainly composed of 1 mol/L sulfuric acid solution), positive control and negative control.
8. 1.3 Operation steps: Add 1 mL diluent into a clean test tube, then add 10μL sample solution to be tested and mix well. Add 100μL diluted sample to each well, set 1 well as positive control, 2 wells as negative control (add 2 drops of positive control and negative control to each well respectively), set 1 well as blank control, and incubate at 37℃ for 30 min. Then discard the liquid in the hole, wash it with washing liquid for 5 times, each time at an interval of 1 min, and spin it dry. Except the blank control wells, 50μL of enzyme labeling solution diluted by 1: 50 was added to each well (that is, 20μL of enzyme conjugate was added to the diluted solution of 1 mL) and incubated at 37℃ for 30 minutes. Then discard the liquid in the hole, wash it with washing liquid for 5 times, each time at an interval of 1 min, and spin it dry. Add 1 drop of substrate A solution and substrate B solution to each well in turn, tap the reaction plate to mix them evenly, and keep them away from light for 5 min ~ 10 min at room temperature. Add 1 drop of stop liquid to each well.
8. 1.4 the results were determined by colorimetry, the blank holes were zeroed, and the od value of each hole at 450 nm was determined by enzyme-labeled instrument. If S/N (average value of sample hole OD/negative control hole OD) ≥2. 1, the result is positive.
8.2 Detection of IgM antibody against Toxoplasma gondii 8.2. 1 Methods Antibody capture ELISA was used to detect IgM antibody against Toxoplasma gondii in human serum, plasma or other body fluids.
8.2.2 The reagent consists of a 96-well plate coated with anti-human IgMμ chain antibody, Toxoplasma antigen solution, anti-Toxoplasma antibody enzyme-labeled solution, sample diluent, washing solution, stop solution, positive control, negative control, substrate A solution and substrate B solution.
8.2.3 Operation steps: Add 90μL diluent to each well, then add 10μL sample solution to be tested and mix well. Set 1 well as positive control, 2 wells as negative control (add 2 drops of positive control and negative control to each well), set 1 well as blank control, and incubate at 37℃ for 30 min. Then discard the liquid in the hole, wash it with washing liquid for 5 times, with an interval of 65438±0min each time, and spin it dry. Except for the blank control wells, first add 1 drop antigen solution to each well, then add 50μL of enzyme marker solution diluted by 1: 50 (that is, add 20μL of enzyme conjugate to the diluted solution of 1 mL), tap the reaction plate to mix well, and incubate at 37℃ for 30 min. Then discard the liquid in the hole, wash it with washing liquid for 5 times, with an interval of 65438±0min each time, and spin it dry. Add 1 drop substrate A solution and substrate B solution to each well in turn, tap the reaction plate and keep it away from light at room temperature/10 min ~ 20 min. Add 1 drop of stop liquid to each well.
8.2.4 Determine the results by colorimetry, and zero the blank holes. Determine the wavelength () d value of each hole at 450 nm by enzyme-labeled instrument. If S/N (average value of sample hole OD/negative control hole OD) ≥2. 1, the result is positive.
8.3 Clinical significance and treatment of anti-Toxoplasma IgG antibody and IgM antibody. Toxoplasma antibody detection is the most important auxiliary examination method at present, and it is usually necessary to detect IgG and IgM antibodies in parallel. The clinical significance of the detection results of serum antibodies against Toxoplasma gondii is shown in table D. 1. If pregnant women are suspected to be infected in the near future, samples should be sent to laboratories with experience in toxoplasmosis diagnosis for comparison before taking intervention measures.
Table D. 1? Clinical significance and treatment of IgG antibody and IgM antibody against Toxoplasma gondii
IgG result
IgM result
Clinical significance
job operation
Negative; Negative; Negative; negative
Negative; Negative; Negative; negative
There is no serological evidence of Toxoplasma infection.
If clinical symptoms and signs persist, samples will be collected again after 3 weeks for review.
Negative; Negative; Negative; negative
Suspicious positive
It may be early acute infection or IgM false positive.
After 3 weeks, samples were collected again to detect IgG and IgM. If the two results are the same, the patient may not be infected with Toxoplasma gondii.
Negative; Negative; Negative; negative
active
Recent acute infection or IgM false positive
After 3 weeks, samples were collected again to detect IgG and IgM. If the two results are the same, IgM reaction may be false positive.
Suspicious positive
Negative; Negative; Negative; negative
short-lived
Re-collect samples to detect IgG
Suspicious positive
Suspicious positive
short-lived
Collect samples again to detect IgG and IgM.
Suspicious positive
active
Possible acute Toxoplasma gondii infection in the near future
After 3 weeks, samples were collected again to detect IgG and IgM.
active
Negative; Negative; Negative; negative
Toxoplasma infection generally lasts for more than 6 months.
active
Suspicious positive
Toxoplasma gondii infection, but the IgM result is suspicious, which may be recent infection or false positive IgM.
After 3 weeks, samples were collected again for testing.
active
active
Maybe you will be infected with Toxoplasma gondii in the near future.
8.4 Detection of CAg of Toxoplasma gondii 8.4. 1 Methods CAg of Toxoplasma gondii in human serum samples was detected by double-sandwich one-step ELISA.
8.4.2 The reagent consists of a 96-well plate coated with purified anti-Toxoplasma specific antibody (polyclonal antibody), enzyme-labeled solution of anti-Toxoplasma specific antibody and sample diluent.
Liquid, washing liquid, stop liquid, positive control, negative control, substrate A liquid and substrate B liquid.
8.4.3 Operation steps: Add 40μL diluent to each well, then add 10μL sample solution to be tested, mix well, and set 1 well as positive control, 1 well as negative control, and 1 well as blank control (add 1 to each well respectively Add 50μL diluted enzyme-labeled solution to each well, react and incubate at 37℃ for 65438 0 h, then discard the liquid in the well, wash it with diluent for 5 times, with an interval of 65438±0min each time, and spin-dry. Add 1 drop substrate A solution and substrate B solution into each well in turn, and leave it in the dark at room temperature 10 min ~ 20 min. Add 1 drop of stop liquid to each well.
8.4.4 Determine the results by colorimetry, zero the blank holes, and determine the OD value of each hole at the wavelength of 450 nm by microplate analyzer. If S/N (average value of sample hole OD/negative control hole OD) ≥2. 1, the result is positive.
8.5 Detection of Toxoplasma nucleic acid 8.5. 1 Methods The target gene was screened from Bl gene and AF 146527 sequence by polymerase chain reaction (PCR), and the specific DNA nucleic acid fragment of Toxoplasma gondii was detected by fluorescence PCR.
8.5.2 The reagent consists of nucleic acid extract, Toxoplasma gondii nucleic acid fluorescence PCR detection mixed solution, enzyme (Taq+UNG), water (H2O), internal reference substance and positive reference substance.
8.5.3 Operation steps: First, the nucleic acids of the sample and the reference substance are cracked. If it is blood or body fluid, take 50μL of sample, add 50μL of nucleic acid extract, shake well for 65438±00s, take a dry bath or water bath at 99℃ for 65438 00 min, centrifuge for 2500g 65438±00min, and keep the supernatant for later use. For feces, put the feces with the size of rice grains into a centrifuge tube containing 0.5 mL of normal saline, shake and mix evenly, centrifuge at 2500g for 2 min, and remove the supernatant; Add 65,438+0,000 μ l DNA extract directly into the precipitate and mix thoroughly; After boiling water bath for 65438±00min, 2500g was centrifuged for 5 min, and 4V_L supernatant was taken for PCR reaction, with water (H2O) as negative control. Then, prepare a reagent, take 35μL of Toxoplasma gondii nucleic acid fluorescence PCR detection mixed solution, shake it evenly with 1μL internal standard and 0.4μL of enzyme (Taq+UNG) for a few seconds, 1358 g for a few seconds, take 36μL of the above mixed solution in a PCR reaction tube, and then add 4μL of the treated sample, positive control and water (H2O) respectively. Put it on a quantitative fluorescence PCR instrument, the cycle parameters were set to 37℃ for 2 min, 94℃ for 2 min, 93℃ 15s, 60℃ for 60℃60 s, the single-point fluorescence detection was at 60℃, and the reaction system was 40 μ l. Carboxyfluorescein (FAM) and HEXachlorofluorescein (Hex) channels were selected for fluorescence detection. The principle of threshold setting is that the threshold line just exceeds the highest point of water (H2O) detection fluorescence curve.
Body surface area calculator body mass index calculation and evaluation of female safety period calculator expected date calculator normal weight gain during pregnancy medication safety classification (FDA) five elements and eight characters adult blood pressure evaluation body temperature level evaluation diabetes diet suggestion clinical biochemical common units conversion basal metabolic rate calculation sodium supplement calculator iron supplement calculator prescription common Latin abbreviations quick check pharmacokinetics common symbols quick check effective plasma osmotic pressure calculator alcohol intake calculator.
Encyclopedia of medicine, count now!
8.5.4 When the Ct (intersection point) value is ≤38, the result is positive in FAM channel.
8.6 Etiological examination 8.6. 1 Take peripheral blood or cerebrospinal fluid, subretinal exudate, aqueous humor, pleural effusion, ascites and amniotic fluid directly under the microscope, centrifuge 500g 10min, take sediment smear, dry it, fix it with Giemsa method, and judge if Toxoplasma gondii tachyzoites (pseudocysts) are found under the light microscope.
Biopsy tissue was taken and pathological sections were stained by Giemsa method. If Toxoplasma gondii cysts or tachyzoites (pseudocysts) were detected under the light microscope, it was determined that the pathogen was positive.
8.6.2 Animal Inoculation Peripheral blood or cerebrospinal fluid, subretinal exudate, aqueous humor, pleural effusion, ascites and amniotic fluid were taken and aseptically inoculated into the abdominal cavity of clean healthy mice aged 6-8 weeks, each with 65438±0ml. Observe every day. If jLH mice show symptoms such as loose fur, inactivity, hunchback, eye closure, abdominal distension, tremor or shortness of breath 2-3 weeks after inoculation, they should be slaughtered immediately. The peritoneal fluid, liver, spleen, brain and other tissues of mice were ground, filtered and centrifuged for 65438±00min, and smeared. After Giemsa staining, it was observed under a microscope. If Toxoplasma gondii tachyzoites (pseudocysts) or cysts are found, it is determined that the pathogen is positive.