MD1390Z - Method for identifying anti-Toxocara IgG marker in blood serum - Google Patents
Method for identifying anti-Toxocara IgG marker in blood serum Download PDFInfo
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Abstract
Description
Invenţia se referă la medicină, şi anume la o metodă de identificare a markerului anti-Toxocara IgG în serul sangvin şi poate fi folosită pentru diagnosticul toxocarozei umane. The invention relates to medicine, namely to a method for identifying the anti-Toxocara IgG marker in blood serum and can be used for the diagnosis of human toxocariasis.
Toxocaroza umană constituie o problemă medico-socială de importanţă majoră cu răspândire globală, dar totodată rămâne a fi una dintre cele mai neînţeleasă invazie parazitară la om cu complexe şi diferite probleme ce ţin de diagnostic, tratament şi profilaxie. Este o infecţie produsă de un parazit din genul Toxocara, familia Ascarididae, contactată prin ingestia de sol contaminat cu ouă embrionate, provenite din fecalele animalului gazdă. Contaminarea mediului ambiant cu ouă de toxocara şi igiena defectuoasă a alimentaţiei asigură răspândirea infecţiei. În cazul copiilor, o sursă importantă de infecţie o reprezintă locurile de joacă în care au acces animalele. În ultima perioadă de timp se constată o extindere continuă a infecţiei, inclusiv în ţările în curs de dezvoltare economică, astfel constituind o problemă prioritară şi pentru Republica Moldova (Plăcintă Gheorghe. Monografie: Toxocaroza - problemă actuală a serviciului medical şi sanitar public, Chişinău, 2017, 240 p.) Human toxocariasis is a major medical and social problem with global spread, but at the same time it remains one of the most misunderstood parasitic invasions in humans with complex and various problems related to diagnosis, treatment and prophylaxis. It is an infection caused by a parasite of the genus Toxocara, family Ascarididae, contracted through the ingestion of soil contaminated with embryonated eggs, originating from the feces of the host animal. Contamination of the environment with toxocara eggs and poor food hygiene ensure the spread of the infection. In the case of children, an important source of infection is represented by playgrounds where animals have access. In recent times, there has been a continuous expansion of the infection, including in economically developing countries, thus constituting a priority problem for the Republic of Moldova (Plăcintă Gheorghe. Monograph: Toxocariasis - current problem of the public medical and sanitary service, Chişinău, 2017, 240 p.)
Tratamentul toxocarozei umane rămâne o chestiune controversă şi nu este în general un consens cu privire la tratamentul specific, în această ordine de idei, actualmente nu există o metodă definitivă de a diagnostica infecţia parazitară cu toxocaroză. Criteriile de vindecare cer a fi stabilite în forme clare clinice, indicatori de imagistică şi de laborator. Strategia optimală de management necesită elaborarea algoritmului de diagnostic, tratament al toxocarozei aplicat în funcţie de activitatea procesului pato-invaziv (Van Den Broucke S., Kanobana K., Polman K., et al. Toxocariasis diagnosed in international travelers at the Institute of Tropical Medicine, Antwerp, Belgium, from 2000 to 2013, in: PLoS Negl.Trop. Dis., 2015; nr. 9). The treatment of human toxocariasis remains a controversial issue and there is no general consensus on the specific treatment; therefore, there is currently no definitive method to diagnose parasitic infection with toxocariasis. The criteria for cure need to be established in clear clinical, imaging and laboratory indicators. The optimal management strategy requires the development of an algorithm for the diagnosis and treatment of toxocariasis, applied depending on the activity of the patho-invasive process (Van Den Broucke S., Kanobana K., Polman K., et al. Toxocariasis diagnosed in international travelers at the Institute of Tropical Medicine, Antwerp, Belgium, from 2000 to 2013, in: PLoS Negl.Trop. Dis., 2015; no. 9).
Medicina clinică contemporană devine vădită prin utilizarea extensivă a tehnologiilor diagnosticului de laborator de înaltă performanţă, care sunt vertiginos implementate în practica medicală (Roldan W.H., Elefant G.R., Ferreira A.W. Deglycosylation of Toxocara excretory-secretory antigens improves the specificity of the serodiagnosis for human toxocariasis. The Journal Parasite Immunology, November 2015, vol. 37, Issue 11, p. 557-567). Contemporary clinical medicine is becoming evident through the extensive use of high-performance laboratory diagnostic technologies, which are rapidly implemented in medical practice (Roldan W.H., Elefant G.R., Ferreira A.W. Deglycosylation of Toxocara excretory-secretory antigens improves the specificity of the serodiagnosis for human toxocariasis. The Journal Parasite Immunology, November 2015, vol. 37, Issue 11, p. 557-567).
Investigaţiile privind nivelul de anticorpi anti-Toxocara IgG una din componentele de bază ale diagnosticului clinic şi realizarea prin metoda de analiză imunoenzimatică pe suport solid, utilizând kiturile de reactivi ai companiei NovaTec (Germania). Complexul imun format din conjugatul legat se vizualizează la adăugarea de tetrametilbenzidină (TMB), substrat care dă un produs de reacţie albastru. Intensitatea produsului obţinut este proporţională cu cantitatea de anticorpi specifici anti-Toxocara canis IgG în specimen. Pentru stoparea reacţiei enzimatice se utilizează stop-reagentul (mai frecvent acidul sulfuric). Detecţia se efectuează fotometric la lungimea de undă 450 sau 620 nm. Investigations regarding the level of anti-Toxocara IgG antibodies are one of the basic components of clinical diagnosis and are carried out by the method of immunoenzymatic analysis on a solid support, using the reagent kits of the NovaTec company (Germany). The immune complex formed by the bound conjugate is visualized upon the addition of tetramethylbenzidine (TMB), a substrate that gives a blue reaction product. The intensity of the product obtained is proportional to the amount of specific anti-Toxocara canis IgG antibodies in the specimen. To stop the enzymatic reaction, a stop reagent (more often sulfuric acid) is used. Detection is performed photometrically at a wavelength of 450 or 620 nm.
Tehnologia testului imunoenzimatic exprimată prin identificarea markerului anti-Toxocara IgG include următoarele etape: The technology of the immunoenzymatic test expressed by the identification of the anti-Toxocara IgG marker includes the following steps:
1. Formarea complexului imunologic, urmare a reacţiei imunologice prin suplimentarea cu reagenţi, unde unul conţine markerul enzimatic. 1. Formation of the immunological complex, following the immunological reaction by supplementing with reagents, where one contains the enzymatic marker.
2. Stoparea fazei solide pentru îndepărtarea componentelor nespecifice. 2. Stopping the solid phase to remove non-specific components.
3. Suplimentarea reacţiei enzimatice prin adăugarea soluţiei cromogen/substrat şi stoparea reacţiei cu stop-reagent. 3. Supplement the enzymatic reaction by adding the chromogen/substrate solution and stopping the reaction with stop reagent.
4. Analiza rezultatelor. 4. Analysis of the results.
5. Evaluarea cu interpretarea rezultatelor obţinute [1]. 5. Evaluation with interpretation of the results obtained [1].
Dezavantajele soluţiei cunoscute constau în aceea, că actualmente nu există o metodă definitivă de a diagnostica infecţiile cu toxocara, iar sensibilitatea şi specificitatea reală a testelor serologice nu poate fi determinată cu acurateţe. Diagnosticul de laborator este şi mai mult complicat de variabilitatea răspunsului imunologic umoral, care depinde de încărcătură şi localizarea infecţiei. Numeroase studii au demonstrat că testele imunoenzimatice care folosesc un antigen excretor, purificat din stratul larval au o sensibilitate şi specificitate sporită comparativ cu alte teste care utilizează antigenul brut. În contextul celor expuse, totuşi unele probe de ser examinate la prezenţa markerului anti-Toxocara IgG în testul imunoenzimatic (ELISA) demonstrează rezultate echivoce (invalide), urmare a prezenţei în seruri a diferitor factori inhibitori nespecifici care influenţează rezultatele finale ale reacţiei, manifestată prin reducerea specificităţii metodei. The disadvantages of the known solution are that currently there is no definitive method to diagnose toxocara infections, and the real sensitivity and specificity of serological tests cannot be accurately determined. Laboratory diagnosis is further complicated by the variability of the humoral immunological response, which depends on the load and location of the infection. Numerous studies have demonstrated that immunoenzymatic tests using an excretory antigen, purified from the larval layer, have increased sensitivity and specificity compared to other tests using the crude antigen. In the context of the above, however, some serum samples examined for the presence of the anti-Toxocara IgG marker in the enzyme-linked immunosorbent assay (ELISA) demonstrate equivocal (invalid) results, due to the presence in the sera of various non-specific inhibitory factors that influence the final results of the reaction, manifested by a reduction in the specificity of the method.
Problema pe care o rezolvă invenţia constă în elaborarea unei noi metode de investigare a serurilor umane la prezenţa markerului anti-Toxocara IgG, pentru sporirea specificităţii şi sensibilităţii testului imunoenzimatic, se efectuează în timp redus de 3,5 ore şi reduce maximal riscul de infectare cu toxocaroză. Aşadar, serurile umane recoltate de la pacienţi cu diagnosticul clinic prezumptiv de toxocaroză, care în testul ELISA au demonstrat rezultate incerte (invalide) se prelucrează cu suspensie de caolin de 25%, pentru înlăturarea inhibitorilor nespecifici, apoi repetat sunt examinate în ELISA. The problem solved by the invention consists in developing a new method for investigating human sera for the presence of the anti-Toxocara IgG marker, to increase the specificity and sensitivity of the immunoenzymatic test, is performed in a reduced time of 3.5 hours and maximally reduces the risk of infection with toxocariasis. Therefore, human sera collected from patients with a presumptive clinical diagnosis of toxocariasis, who in the ELISA test have demonstrated uncertain (invalid) results, are processed with a 25% kaolin suspension, to remove non-specific inhibitors, then are repeatedly examined in ELISA.
Esenţa invenţiei constă în examinarea serului sangvin în testul imunoenzimatic cu utilizarea microplăcii adsorbite cu Ag toxocara canis şi determinarea valorilor densităţii optice a probelor prin metoda fotometrică la lungimea de undă 450 nm, apoi se determină raportul dintre valoarea medie a densităţii optice a serului pacientului x 10 şi valoarea medie a densităţilor optice ale probelor de control negativ, şi dacă raportul este de până la 9 NTU, se consideră că rezultatul este negativ, dacă este mai mare de 11 NTU, rezultatul este pozitiv, iar probele cu rezultatul de 9...11 NTU se prelucrează cu suspensie de caolin de 25%, apoi se repetă testul imunoenzimatic cu determinarea ulterioară a raportului menţionat pentru determinarea rezultatului negativ în cazul când raportul este de până la 9 NTU şi pozitiv dacă este mai mare de 11 NTU. The essence of the invention consists in examining blood serum in the immunoenzymatic test using the microplate adsorbed with Ag toxocara canis and determining the optical density values of the samples by the photometric method at a wavelength of 450 nm, then determining the ratio between the average optical density of the patient's serum x 10 and the average optical density of the negative control samples, and if the ratio is up to 9 NTU, the result is considered negative, if it is higher than 11 NTU, the result is positive, and samples with a result of 9...11 NTU are processed with a 25% kaolin suspension, then repeating the immunoenzymatic test with subsequent determination of the aforementioned ratio to determine the negative result in the case when the ratio is up to 9 NTU and positive if it is higher than 11 NTU.
Rezultatul invenţiei constă în sporirea specificităţii şi sensibilităţii testării imunoenzimatice, se efectuează în timp redus de 3,5 ore şi reducerea maximală a riscului de infectare cu toxocaroză. The result of the invention consists in increasing the specificity and sensitivity of immunoenzymatic testing, performing it in a reduced time of 3.5 hours and maximally reducing the risk of infection with toxocariasis.
Metoda propusă include 3 (trei) etape: The proposed method includes 3 (three) steps:
1. Investigarea primară a serurilor sanguine umane întru identificarea markerului anti-Toxocara IgG prin testul imunoenzimatic ELISA. 1. Primary investigation of human blood sera to identify the anti-Toxocara IgG marker by the ELISA immunoenzymatic test.
2. Identificarea probelor de ser sanguin care au demonstrat rezultatele incerte cu prelucrarea lor cu soluţie de caolin de 25% 2. Identification of blood serum samples that demonstrated uncertain results with their processing with 25% kaolin solution
3. Investigarea repetată a probelor de ser prelucrate cu soluţie de caolin de 25% în testul ELISA cu evaluarea şi interpretarea finală a rezultatelor obţinute. În continuare detaliat se prezintă tehnologiile de realizare, caracteristice pentru fiecare etapă. 3. Repeated investigation of serum samples processed with 25% kaolin solution in the ELISA test with final evaluation and interpretation of the results obtained. The implementation technologies, characteristic for each stage, are presented in detail below.
I etapă Stage I
Procedeu manual. They did it manually.
Iniţial se montează numărul necesar de stripuri cu godeuri care sunt absorbite cu Ag toxocara canis în microplaca din trusa destinată pentru realizarea ELISA conform instrucţiunii anexate NovaTec, Immundiagnostica GmbH, NovaLisa Toxocara canis IgG, Product Number TOCG0450 (96 Determinations). Ulterior în godeurile stripurilor montate se picură reagenţii şi probele investigate cu excepţia blancului (A1), în alte 3 godeuri (B1) se picură 100 µl ser uman (control negativ) care nu conţine anti-Toxocara IgG; C1- 100 µl, ser uman slab pozitiv la anti-Toxocara (cut-off); D1 - 100 µl care conţine ser uman cu anti-Toxocara IgG cu titru mai mare de 1000,0 UI/mL (control pozitiv). Proba investigată în volum de 10 µl se diluează în 1000 µl diluant de probă, care conţine fosfat bufer 10 mM, pH 7,2±0,2. Apoi probele diluate în volum de 100 µl se repartizează în stripuri începând cu E1. Striurile pregătite se sigilează cu peliculă şi se incubează timp de o oră la temperatura de +37ºC, apoi se spală în regim automat de 5 ori cu livrarea şi aspirarea a 300 µl per godeu cu soluţie diluată de fosfat, pregătită anterior (1:20), urmează pipetarea a 100 µl de enzimă conjugată, care conţine globulina conjugată cu peroxidază proteina A în fiecare godeu cu excepţia A1, apoi se incubează timp de 30 min la temperatura de 20…25ºC. Ulterior urmează spălarea stripurilor în regim automat de 5 ori cu livrarea şi aspirarea a 300 µl per godeu cu soluţie diluată de fosfat, pregătită anterior (1:20). În continuare se adaugă câte 100 µl Cromogen substrat, care conţine 3,3; 5,5- tetrametilbenzidin TMB în fiecare godeu şi se incubează la temperatura de 20…25°C, timp de 15 min. Reacţia se stopează prin adăugarea a câte 100 µl de acid sulfuric 0,2 mol/L, apoi se determină valorile densităţii optice la lungimea de undă 450 nm, apoi se determină raportul dintre valoarea medie a densităţii optice a serului pacientului x 10 şi valoarea medie a densităţilor optice ale probelor de control negativ şi, în cazul, în care raportul este de până la 9 NTU, se consideră că rezultatul este negativ, dacă este mai mare de 11 NTU rezultatul este pozitiv, iar probele cu rezultatul de 9...11 NTU sunt cu rezultat incert. Initially, the required number of strips with wells that are absorbed with Ag toxocara canis are mounted in the microplate from the kit intended for performing ELISA according to the attached instructions NovaTec, Immundiagnostica GmbH, NovaLisa Toxocara canis IgG, Product Number TOCG0450 (96 Determinations). Subsequently, the reagents and the investigated samples are dropped into the wells of the mounted strips, except for the blank (A1), in another 3 wells (B1) 100 µl of human serum (negative control) that does not contain anti-Toxocara IgG is dropped; C1- 100 µl, weakly positive human serum for anti-Toxocara (cut-off); D1 - 100 µl containing human serum with anti-Toxocara IgG with a titer higher than 1000.0 IU/mL (positive control). The investigated sample in a volume of 10 µl is diluted in 1000 µl of sample diluent, which contains 10 mM phosphate buffer, pH 7.2±0.2. Then the diluted samples in a volume of 100 µl are distributed into strips starting with E1. The prepared strips are sealed with film and incubated for one hour at a temperature of +37ºC, then washed in automatic mode 5 times with the delivery and aspiration of 300 µl per well with previously prepared diluted phosphate solution (1:20), followed by pipetting of 100 µl of conjugated enzyme, which contains globulin conjugated with peroxidase protein A in each well except A1, then incubated for 30 min at a temperature of 20…25ºC. Subsequently, the strips are washed in automatic mode 5 times with the delivery and aspiration of 300 µl per well with previously prepared diluted phosphate solution (1:20). Next, 100 µl of Chromogen substrate, containing 3,3; 5,5-tetramethylbenzidine TMB, is added to each well and incubated at a temperature of 20…25°C for 15 min. The reaction is stopped by adding 100 µl of 0.2 mol/L sulfuric acid, then the optical density values are determined at a wavelength of 450 nm, then the ratio between the average optical density value of the patient's serum x 10 and the average optical density value of the negative control samples is determined and, if the ratio is up to 9 NTU, the result is considered negative, if it is greater than 11 NTU the result is positive, and samples with a result of 9...11 NTU are of uncertain result.
Etapa II Stage II
Include următoarele proceduri tehnologice. It includes the following technological procedures.
Absorbţia inhibitorilor nespecifici din probele umane incerte şi anume cu rezultatul de 9...11 NTU, se prelucrează cu suspensie de caolin de 25%. Prelucrarea include următoarea procedură: soluţia de caolin în concentraţie de 25,0% se diluează cu soluţie fiziologică (1:5), apoi urmează prelucrarea a 100 µl ser cu 400 µl de soluţie fiziologică. Ulterior volume identice (1:1) de suspensie de caolin şi ser diluat se toarnă într-o eprubetă, care se incubează la temperatura de 18…25ºC, timp de 30 min, fiind amestecate periodic. Urmează centrifugarea (20 min, 2500 rot/min), după care serul se aspiră şi se păstrează la temperatura de 4 ºC. The absorption of non-specific inhibitors from uncertain human samples, namely with a result of 9...11 NTU, is processed with a 25% kaolin suspension. The processing includes the following procedure: the kaolin solution in a concentration of 25.0% is diluted with physiological solution (1:5), then 100 µl of serum is processed with 400 µl of physiological solution. Subsequently, identical volumes (1:1) of kaolin suspension and diluted serum are poured into a test tube, which is incubated at a temperature of 18...25ºC, for 30 min, being mixed periodically. Centrifugation follows (20 min, 2500 rpm), after which the serum is aspirated and stored at a temperature of 4 ºC.
Etapa III Stage III
După inactivare şi prelucrare cu suspensie de caolin de 25% a probelor de ser incerte (echivoce) pentru evitarea influenţei inhibitorilor nespecifici, probele procesate se analizează repetat în testul ELISA cu produsele trusei de diagnostic de laborator pentru infecţii parazitare cu toxocara a companiei NovaTec, Immunodiagnostica GmbH, NovaLisa, Toxocara canis IgG, Product Number : TOCG0450 (96 Determination). After inactivation and processing with 25% kaolin suspension of uncertain (equivocal) serum samples to avoid the influence of non-specific inhibitors, the processed samples are repeatedly analyzed in the ELISA test with the products of the laboratory diagnostic kit for parasitic infections with toxocara of the company NovaTec, Immunodiagnostica GmbH, NovaLisa, Toxocara canis IgG, Product Number: TOCG0450 (96 Determination).
Pentru argumentarea celor expuse prezentăm datele obţinute (Tabel) privind investigarea a 955 seruri sangvine, recoltate de la pacienţii în vârstă de la 2 până la 75 ani din diferite teritorii administrative ale Republicii Moldova cu diagnosticul clinic prezumptiv de toxocaroză, în baza acordului informat, prin metoda cunoscută şi metoda revendicată pentru identificarea markerului anti-Toxacara IgG. Rezultatele obţinute demonstrează că prin metoda cunoscută markerul anti-Toxacara IgG, utilizând testului ELISA, a fost identificat în 469 (49,1%) de probe recoltate de la pacienţii investigaţi. Pentru bărbaţi acest indicator a constituit 208 (48,6%), iar pentru femei 261 (49,5%). Rezultatele negative au demonstrat absenţa markerului anti-Toxacara IgG în 439 (42,4%) din 955 probe investigate, pentru bărbaţi acest indicator a constituit 197 (46,0%), iar pentru femei 242 (45,9%). Este important de menţionat, că din totalul de probe de 955 examinate cu testul ELISA au fost identificate 47 (4,9%) rezultate incerte: 23 (5,4%) cazuri pentru bărbaţi şi 24 (4,6%) la femei. Evaluarea comporativă a rezultatelor obţinute, demonstrează că nu există o diferenţă semnificativă în indicatorii de incidenţă a markerului anti-Toxacara IgG la bărbaţi şi femei, urmare a realizării metodei cunoscute (p˂0,05). To substantiate the above, we present the data obtained (Table) regarding the investigation of 955 blood sera, collected from patients aged 2 to 75 years from different administrative territories of the Republic of Moldova with the presumptive clinical diagnosis of toxocariasis, based on informed consent, by the known method and the claimed method for identifying the anti-Toxacara IgG marker. The results obtained demonstrate that by the known method the anti-Toxacara IgG marker, using the ELISA test, was identified in 469 (49.1%) of samples collected from the investigated patients. For men this indicator was 208 (48.6%), and for women 261 (49.5%). Negative results demonstrated the absence of the anti-Toxacara IgG marker in 439 (42.4%) of 955 investigated samples, for men this indicator was 197 (46.0%), and for women 242 (45.9%). It is important to mention that out of the total of 955 samples examined with the ELISA test, 47 (4.9%) uncertain results were identified: 23 (5.4%) cases for men and 24 (4.6%) in women. The comparative evaluation of the obtained results demonstrates that there is no significant difference in the incidence indicators of the anti-Toxacara IgG marker in men and women, following the implementation of the known method (p˂0.05).
Utilizarea metodei revendicate pentru probele incerte esenţial modifică rezultatele finale privind identificarea repetată a markerului anti-Toxocara IgG prin testul imunoenzimatic. Urmare a utilizării metodei propuse, ponderea probelor pozitive a rămas nemodificată, rezultatele incerte au dispărut, iar incidenţa probelor negative pentru bărbaţi şi femei a constituit respectiv 51,4% şi 50,5%. Este important de menţionat existenţa unei diferenţe semnificative privind incidenţa probelor negative la markerul anti-Toxocara IgG investigate prin metoda cunoscută - 42,4 % şi cea revendicată - 50,9% (p< 0,01) (Tabel). În ansamblu datele obţinute demonstrează că metoda revendicată elimină rezultatele incerte, astfel semnificativ sporind specificitatea şi sensibilitatea testului. The use of the claimed method for uncertain samples essentially changes the final results regarding the repeated identification of the anti-Toxocara IgG marker by the immunoenzymatic test. Following the use of the proposed method, the share of positive samples remained unchanged, uncertain results disappeared, and the incidence of negative samples for men and women was 51.4% and 50.5%, respectively. It is important to mention the existence of a significant difference regarding the incidence of negative samples for the anti-Toxocara IgG marker investigated by the known method - 42.4% and the claimed one - 50.9% (p< 0.01) (Table). Overall, the obtained data demonstrate that the claimed method eliminates uncertain results, thus significantly increasing the specificity and sensitivity of the test.
Tabel Table
Rezultatele identificării şi evaluării prin ELISA a marcherului anti-Toxocara IgG în serurile sanguine ale pacienţilor cu diagnosticul clinic prezumptiv de toxocaroză, obţinute prin metoda cunoscută şi cea revendicată Results of the identification and evaluation by ELISA of the anti-Toxocara IgG marker in the blood sera of patients with a presumptive clinical diagnosis of toxocariasis, obtained by the known and claimed method
Pacienţii în dependenţă de sex Nr. de probe investigate (pacienţi) Identificarea marcherului anti-Toxocara IgG Metoda cunoscută Metoda revendicată Pozitiv Incert Negativ Pozitiv Incert Negativ abs % abs % abs % abs % abs abs % Bărbaţi 428 208 48,6 ± 2,4 23 5,4 ±1,1 197 46,0± 2,4 208 48,6± 2,4 0 220 51,4± 2,4 Femei 527 261 49,5± 2,2 24 4,6 ±0,9 242 45,9± 2,2 261 49,5± 2,2 0 266 50,5± 2,2 Total 955 469 49,1± 1,6 47 4,9 ±0,6 439 42,4± 1,5 469 49,1± 1,6 0 486 50,9 ±1,6Patients with sex addiction No. of investigated samples (patients) Identification of anti-Toxocara IgG marker Known method Claimed method Positive Uncertain Negative Positive Uncertain Negative abs % abs % abs % abs % abs abs % Men 428 208 48.6 ± 2.4 23 5.4 ±1.1 197 46.0± 2.4 208 48.6± 2.4 0 220 51.4± 2.4 Women 527 261 49.5± 2.2 24 4.6 ±0.9 242 45.9± 2.2 261 49.5± 2.2 0 266 50.5± 2.2 Total 955 469 49.1± 1.6 47 4.9 ±0.6 439 42.4± 1.5 469 49.1± 1.6 0 486 50.9 ±1.6
Remarcă: Pacienţii examinaţi din toate teritoriile administrative ale Republicii Moldova au cuprins diapazonul de vârstă de la 2 până la 75 ani. Note: Patients examined from all administrative territories of the Republic of Moldova ranged in age from 2 to 75 years.
1. Instrucţiunea de utilizare a testului: NovaTec, Immundiagnostica GmbH, NovaLisa, Toxocara canis IgG. Product Number: TOCG60450 (96 Determination), Waldstrabe 23A6 - 63128, Dietzenbach, Germany, 25 sept. 2017, Găsit: <http://www.elisakits.co.uk/toxocara-canis-igg-elisa-kit> 1. Instructions for use of the test: NovaTec, Immundiagnostica GmbH, NovaLisa, Toxocara canis IgG. Product Number: TOCG60450 (96 Determination), Waldstrabe 23A6 - 63128, Dietzenbach, Germany, 25 Sept. 2017, Found: <http://www.elisakits.co.uk/toxocara-canis-igg-elisa-kit>
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