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The direct agglutination test

The direct agglutination test74 is also a simple test which has been widely used in some countries, but now it is not commercially available. The principle is the agglutination of the parasite by antibodies, if present. It has advantages, like the HA, and the possibility to search for IgM anti-T. cruzi if samples are run in parallel with and without 2-ME. If differences of more than two titers occur, there is a strong possibility that they are due to IgM. This test has been applied with success during the acute phase.75 The disadvantages are mainly related to the reading because of the clear color of epimastigotes. Titers obtained with this test are higher than 1/32 in most infected people. Typical results of serum from acute phase are 1/256 without and 1/16 with 2-ME.76

The indirect immunofluorescence (IIF)

Indirect immunofluorescence is used mainly in research laboratories or diagnostic centers that handle a limited amount of samples per day. One of the advantages for laboratories is that the same conjugate (antihuman IgG) may be used for the diagnosis of several diseases and the ability to use the same equipment (fluorescence microscope). This test is conducted by reacting serum with smear fixed epimasti- gotes and, after washing, incubating with conjugate. The smears are read in the fluorescence microscope. The key advantage of this test is very high sensitivity. It is quite hard to find a serum from an infected individual which does not react. However, a disadvantage is that this same extreme sensitivity may lead to crossreactions with several diseases. These cross-reactions are observed mainly in lower titers (1/40—1/80) and a higher one is quite characteristic of Chagas disease, unless visceral leishmaniasis is present.

This test has several steps and each may be critical, so it should be performed by a skilled technician. Among the variables, the growing phase of the epimasti- gotes, the pH of phosphate buffered saline, the pH of glycerol buffer, the concentration of conjugate, and the life of the microscopic light need to be checked for top performance. The reading is subjective, and after examination of more than 20 smears (200 wells), results may not be reliable because of exhaustion of the technician. Nevertheless, in good hands this is an excellent test. In a recent search, in an analysis of 1302 sera from infected individuals with typical clinical alterations, 92.7% had a titer of 1/320 or higher. More than two-thirds of this group had high titers of 1/1280 or more.77 The cutoff region is between 1/20 and 1/40 and some individuals of the noninfected population may be reactive in these low titers (Table 29.4).

Table 29.4 Titers obtained in 1302 sera from nontreated chronic phase patients, by indirect immunofluorescence1

Titer

Nr

%

1/40

0

0.0

1/80

27

2.1

1/160

67

5.2

1/320

135

10.4

1/640

220

16.9

1/1280

331

25.4

1/2560

315

24.2

1/5120

84

6.5

1/10,240

52

4.0

1/20,480

71

5.5

Total

1,302

100.2

All patients have had cardiopathy (CRBBB), and/or megaesophagus and/or megacolon.77 Source: Laboratory for Chagas disease, Goiania, Brazil.

 
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