*3.2.1.3 Immunoassays*

The basis of these tests is to measure the specific union antigen-antibody IgG, by adding a detection complex. The antigen is attached to different supports. That is why


*Var: Variable: Depends on the cost/benefit of the laboratory.*

*Note: Own elaboration.\* Detects late infection.*

*\*\*When the result is low and pregnancy is before the 16th week.*

#### **Table 1.**

*Screening tests. Routine Laboratories [1].*

*The Important Role of Laboratories in the Diagnosis and Prevention of Toxoplasma Infection DOI: http://dx.doi.org/10.5772/intechopen.108313*


*Var: Variable: Depends on the laboratories cost/benefits.*

*Note: Own elaboration.\* Reference reaction.*

*\*\*For paired samples mother-child.*

#### **Table 2.**

*Confirmatory tests. Reference Laboratories [1].*

there are a lot of available types and brands of immunoassays (ELISA, QML, CLIA, IFI), their variety depends on the following:

#### *3.2.1.3.1 The kind of antigen that they use*

There are membrane antigens that appear first (p30 (SAG-1), p22 (SAG-2)) and cytoplasmic antigens that can be detected later (p35 (GRA-8), ROP-1, GRA-6, GRA-7). According to the antigens that the immunoassay uses, the specific IgG can detect them within one or three weeks after infection has started [1]. Deciding which test to choose is one of the most important steps.

#### *3.2.1.3.2 The support that is being used*

Consist on the base where the antigens or antibodies are sustained. They can be microplates (ELISA), microparticles (QML, CLIA), slides (IFI), latex particles (AD), cells (HAI). The smaller they are, the more sensitive the test will be.

#### *3.2.1.3.3 The detection complex is the other ítem to be considered*

Conjugate-enzyme (ELISA), fluorescence (IFI), luminiscencia (CLIA), quimioluminiscencia (QML).

*The sensitivity and specificity of different assays available depend on the combination of all of these variables; therefore, the choice made will depend on the cost/benefit of the laboratories available resources.*

*But the most important after selecting the tests is to follow the insert and do the test respecting it, doing exactly what it recommends and always use quality controls, internal and external ones, to compare the results and be sure that all samples have a certain result.*

#### *3.2.2 Confirmatory tests*

These tests are used in Reference Laboratories (**Table 2**).

#### *3.2.2.1 Sabin Feldman dye test (SF)*

The test is based on complement-mediated cytolysis of antibody-coated live Toxoplasma gondii tachyzoites, which is indicated by their inability to take up methylene blue stain [19–22].

This test deserves a special topic and is a reference method for serodiagnostic *Toxoplasma gondii* infection. Its importance is based on being the only one that detects IgG and IgM together. Therefore, it is the most specific and sensitive of all the indirect methods. There are cases that disagree between values comparing SF and other tests, because it is the first one to render a positive result. This test makes the difference to detect the seroconversion as soon as possible.

We are one of the laboratories that still use it as a routine method. A multicenter study of prevention of congenital toxoplasmosis, based on confirmed equivocal or indeterminate samples from other routine laboratories, gets as result that the 46% of the samples that were serodiagnostic as possible seroconversions in routine laboratories, were defined as cronics in our reference laboratory using SF and ISAGA M method. This means that all of those women would have been treated when not necessary [2].

The problems of most laboratories that stop using SF were maintained live tachyzoites of *Toxoplasma gondii*, each country's animal regulations, trained staff, dangerous to laboratory workers*,* costs, automation, interlaboratory standardization, accessibility of Western blot, better performance of new tests.

Because of them, probably the future of the SF dye test in reference laboratories could be changed for cellular culture, if possible, which is a valid alternative and could be the way to change the SF dye test without losing its advantages and quality.

#### *3.2.2.2 ISAGA*

It consists of capturing the specific antibody (IgM, IgA, or IgE) and then adding a stabilized suspension of *Toxoplasma gondii* to evidence specific Igs with direct agglutination.

#### *3.2.2.3 Avidity Test*

The avidity test measures the power of the union antigen-antibody. When it is recent, the union is weak, but when it is not recent, the union is strong. We could understand it like a traffic light (illustrated in **Figure 3**): When the result is low, the union is weak and it means high probability of seroconversion within the past three months (red light); if it is intermediate, then we must be alert and study the combination of the other different serologies we have (yellow light); and when the result is high, the union is strong and can be reassured that it is a more than four months old infection (green light).

*It is important to consider that this test gives useful information only until the 16th week of pregnancy. Any time after the 16th week, the avidity test loses its importance* [1, 3, 4, 8, 11]*. Another consideration is that there are many brands of avidity tests available. They* 

*The Important Role of Laboratories in the Diagnosis and Prevention of Toxoplasma Infection DOI: http://dx.doi.org/10.5772/intechopen.108313*

*each have their own benchmarks and sometimes the results do not match up. Therefore, it is very important to consider the other confirmatory tests (see Available diagnostic tests) at the time of the final decision.*

#### **4. What should be done in Routine Laboratories for screening?**

Here, the big question to answer what must be done in Routine Laboratories for screening, is: Only IgG? IgG and IgM?

Well, the answer is not an only one, the answer depends on the resources and the cost/benefit analysis.

Do only IgG tests should be done as frequently as possible, which means between every month and every three months, mínimum [1–5, 9, 10]. This is the only way to detect an early SC.

If IgM is added, chances of detecting seroconversion are much better [1–5] because those cases where the SC has just begun will be able to be detected.

This is certainly the best way to work. The decision will depend on the prevalence [2, 5, 6], the patient's knowledge of this disease and their access to primary preventive measures (see Preventive Medicine) and laboratory resources.

#### **5. What should be done in Reference Laboratories?**

As a continuity on the previous topic, here is the great difference.

While Routine Laboratories can choose a lot of brands of kits, and do only IgG depending on their resources and cost/benefit analysis, Reference Laboratories cannot. That is what makes the difference.

Reference Laboratories must do, not only IgG and IgM, but also the most sensitive ones and have the best brands. Besides that, must do ISAGA´s Tests ISAGA M, ISAGA A, and ISAGA E, Western blot, PCR [8, 23], and if possible Sabin Feldman or Cellular Culture and sequence [12–17, 19–23].

This difference is based on accurately diagnosing a seroconversion. Cannot report a doubt. If there is a doubt, the best they can do is to recommend retesting after two or three weeks, to allow the immunological system to mature. This is when a SC has just started and the titer of IgM is not enough to confirm it. Meanwhile, they should suggest doctors in charge start medicating them, as a preventive measure, until the result of the next sample is obtained.

#### **6. Preventive medicine**

Preventive medicine is another very important task to detect SC earlier than anything. There are three ways to do this:

#### **6.1 Primary Preventive Medicine**

There are many things to do to prevent *Toxoplasma gondii* infection. Precautionary measures consist of [1, 3, 4, 7, 8]:

1.Washing hands before handling foods.


There are a lot of things laboratories can do to make pregnant women aware of these precautionary measures and help them prevent *Toxoplasma gondii* infection. There are many ways to do this and it will depend on empathy, enthusiasm, and imagination. Some ideas are as follows:


*Congenital toxoplasmosis can cause miscarriage or stillbirth. In many cases, infants appear healthy at birth but they may develop adverse sequelae of the infection later in life, including decreased vision or blindness, decreased hearing or deafness, and mental and psychomotor retardation.*

*The idea is to focus on these measures and to make pregnant women aware of the Toxoplasma gondii infection and of the consequences they could face if this infection is not taken seriously.*

As an example, we work in a Reference Laboratory Country and we receive samples from many cities in the country, to help them confirm those difficult results, with values near the cut off that routine laboratories cannot resolve because of their limited techniques. We are working on a prevention toxoplasmosis program named "CONTENER," it means "Containing," to expand our services to help more laboratories and concerned patients' get an accurate result. When this happens, our mission

*The Important Role of Laboratories in the Diagnosis and Prevention of Toxoplasma Infection DOI: http://dx.doi.org/10.5772/intechopen.108313*

to help getting a final and accurate diagnosis to apply the right treatment as soon as possible is accomplished. "CONTENER" is also an Spanish acronym for "Contain against Toxoplasmosis in newborns and pregnant women".

#### **6.2 Secondary preventive medicine**

If primary prevention did not work and a SC has happened, this preventive option appears. The secondary prevention is directed to protect the embryo or fetus when the mother has been infected during pregnancy. It is based on antiparasitic treatment until delivery. The most important thing in this situation is to start it as soon as possible. The sooner it starts, the more effective it will be [1, 3, 8].

The next step is to do serologic analysis on the paired mother-child. The ideal test to use is Western blot, but any IgG- and IgM-specific paired serology achieves. Some Reference centers suggest detection of DNA parasites using the blood cord of the newborn as an important complementary fact of great specificity and we also recommend isolation of the parasite in cord blood and placenta if it is possible [12–17].

#### **6.3 Third preventive medicine**

This preventive measure is used when the other two fail. This next step is to make a referral to a specialist doctor who understands this disease and its importance to be medicated as soon as possible to prevent the immediate and late clinical consequences and perform all of the necessary studies and controls.

## **7. Laboratories role in pregnancy and newborn**

After reviewing everything laboratories can do about these topics, it is necessary to highlight the following:


#### **8. Laboratories role in other clinicals group.**

#### **8.1 Immunocompetent patients**

Most toxoplasmosis infections are asymptomatic or have mild nonspecific symptoms, so these patients who have a healthy immune system, probably do not need to be diagnosed. This is the reason why occasional findings of circulating specific antibodies are common. Chronic infections are more frequent than acute ones [24] and patients should not require treatment because it is a self-limited disease.

Mild symptoms could be: high fever, astenia, headache, muscle pain, anorexia, cough, throat pain, lymphadenopathies, and splenomegaly (when there is involvement of the mesenteric nodes) and they must be differentiated with other diseases.

The most characteristic symptom that needs to be differentiated is lymphadenitis: In this case, the role of laboratories gains importance in giving an accurate diagnosis to specialist doctors, not to start treatment, but to rule out other pathologies that do need it.

#### **8.2 Immunosuppressed patients**

These patients are susceptible to develop diseases that may cause severe sequelae depending on the type of infection. Referring to toxoplasmosis, two types of illness can be developed: severe primary infection and reactivation of a latent infection.

For example, immune deficiency conditions such as AIDS and organ transplantation can cause fatal toxoplasmic encephalitis [24–27]. Hence, the role of laboratories in these cases is fundamental to accompany other diagnostics methods such as RMN and TC.

To diagnose, direct methods in samples, such as LCR, lymph nodes, vitreous humor, bone marrow (see Diagnostic available tests), should be used. Even though these tests do not have high sensitivity, they have a high-positive predictive value (VPP) when the result is positive, and when it is negative (negative predictive value (VPN)) it will alert doctors to a risk of a primary infection. Hence, periodic serological controls and preventive measures should be carried out.

#### *8.2.1 VIH patients*

Encephalitis with brain injuries is the most common sequelae when patients are HIV positive and have the disease [24–27]. The role of laboratories is to complement results obtained, with others such as TC and RMN and biopsy.

Direct methods like PCR or isolation can be useful in fluids such as LCR, BAL, biopsies.

Serology results are important to predict risk of infection or reactivation: SF test has a VPN del 99,7% and a VPP of 88% and other serologies, IgG above 1/256 with CD4 lymphocytes less than 150 cel/mm3, ISAGA M 12%, ISAGA A 38% e ISAGA E 25%.

#### *8.2.2 Transplanted patients*

Myocarditis, neumonitis, and SNC compromised could happen in transplanted patients with reactivation of toxoplasmosis. Laboratories importance is based on knowing serologies, donor, and recipient, so that doctors in charge can be alert to any risk of infection or reactivation [24].

*The Important Role of Laboratories in the Diagnosis and Prevention of Toxoplasma Infection DOI: http://dx.doi.org/10.5772/intechopen.108313*

#### *8.2.3 Ocular toxoplasma infection*

Toxoplasmosis is a major cause of retinochoroiditis, especially in individuals with an impaired immune system. It is so important to diagnose a toxoplasma infection when ocular infection occurs, because this acute infection must be treated. The consequences of not doing so, could bring serious complications: When a cyst ruptures, retinochoroiditis presents an intense inflammatory reaction that tends to scarring. In immunodeficiency conditions, necrosis occurs and a retinal detachment, uveitis, and then secondary glaucoma could be presented [24, 26].

The laboratories roles are based on direct detection of the parasite in vitreous humor and confirmatory serology (see Diagnostic available tests). The high VPP of these results with ocular lesions helps treatment as soon as possible to avoid the worse lesions.

## *8.2.3.1 Hodgkin disease and other lymphomas*

Must be also differentiated from toxoplasmosis. Laboratories direct methods in any material like biopsia of nodes is as important as indirect methods, both to rule out or to confirm toxoplasma infection.
