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Parasitological tests

Overview

The natural course of Chagas infection starts with an acute phase, whose manifestations, if present, will arise some days after the entrance of the parasites. Parasites multiply exponentially until the antibody response starts, which becomes evident 10—20 days later. As a consequence, the number of parasites begins to decline, and, at the end of the first month, they are scarce. This phase lasts for 2 months, and is defined by the easy detection of the parasite, which becomes more difficult to find after the first weeks. For diagnosis of this phase, parasitological tests should be employed.27

At this stage, it is useful to include as acute cases all the following conditions, independent of the mechanism of transmission: acute phase by vector transmission, congenital infection, transfusion or by organ transplantation, oral route, accidental infection, and reactivation by immunosuppression. Even if the finding of parasites is the hallmark, clinical manifestations may be different. In those infected by vector contamination, most of them will not be recognized. Those infected through transfusion may also not have been diagnosed, and the manifestations may appear with a longer incubation period, the reasons for which have not been understood yet.28 Congenital transmission may also remain undiagnosed, because most of the cases are born with normal weight and without any other features, apart from the infected mother. Oral route may have digestive manifestations, probably because of the large inoculum and the multiplication of parasites at the digestive tract. For these reasons, many acute cases are not diagnosed at this phase, but many years later by chance (i.e., during a blood donation).29

After the acute phase, which is estimated to be asymptomatic in more than 95% of the infected individuals, even without diagnosis or treatment, the infected person enters the chronic phase, usually in the indetermined form (no symptoms or signs, no ECG, or X-ray abnormalities) which lasts for the entire life. Spontaneous cure has been reported but is certainly highly unusual.30 Circulating parasites are scarce, never found by direct observation (see section: Chronic phase) and may be absent for circulation for some periods. It has been said27 that parasitemia at the chronic phase is variable, not constant, and present at a given moment only in a few mL of the 5 L of blood that a human being has. This fact explains several observations made in the natural history of the infection: transmission by transfusion is successful only in 20% of the cases, up to 50% in hyperendemic areas.31 —34

When performing several parasitological methods (see section: Chronic phase) at the same time, it is normal to find parasites only with one of them; by using xenodiagnosis (40 bugs), positive bugs are only 1—3 of them, the others remain negative because they did not ingest a single parasite.21 Cerisola et al. observed that each bug is as a “single microhemoculture processed at the digestive tract of each bug,” hence the chances to find positive bugs increase as more bugs are included.21

Indications

For all the earlier reasons, parasitological methods should not be used for diagnosis of chronic phase of Chagas disease because most of the results will be negative. A negative parasitological result has no value in terms of diagnosis. On the other hand, a positive test has tremendous value, even when antibodies are present in low titers. For diagnosis of the chronic phase, parasitological examination should not be used.

Parasites are easily detected only in the acute phase or during the reactivation of the chronic phase because of immune suppression. Actually, this is the definition of acute phase. What does “easily detected” mean? This is when finding of parasites by direct observation or by concentration techniques (see later) is possible. These tests are easy to perform and cheap, but only available in specialized laboratories in the field. There is no need to buy them, because they use simple tools that any laboratory has (glass slides, tubes, centrifuges, microscopes). However, there is a need to have personnel with expertise to search and diagnose T. cruzi and at the same time, avoid being infected by live parasites (see section: Prophylaxis to avoid accidental contamination).

There are methods for the detection of T. cruzi when it is present in low numbers (i.e., the chronic phase). These methods are based on another principle: the multiplication of parasites that are not easily found in the sample (usually peripheral blood). For this multiplication, the conditions offered to the parasite should be optimal.

Methods most used are classic xenodiagnosis and hemoculture, and the relatively recent35—37 polymerase chain reaction (PCR), a molecular test, based on the amplification of DNA or RNA present in T. cruzi through probes. Xenodiagnosis and hemoculture have common features; they are not commercially available, demand some time to get multiplication of the parasites (weeks, months), are timeconsuming, and need expertise from technicians. In addition, the former demands a colony for growing bugs, which is available in less than a handful of laboratories in the world; and the latter needs to be processed in absolute sterility and at 4°C, conditions difficult to find in the field. As both have been in use for decades, they are properly standardized.21,38

PCR may be performed in a few hours, but is more expensive, requires special skills, and attempts for standardization and validation have only recently been made. This technique has been applied for T. cruzi since the early 1990s, with high interest after the first publications indicating 100% positivity.39 Further studies revealed much lower figures, depending on the population group studied. When analyzing the first results, they were performed with patients who previously had positive xenodiagnosis, so a bias was introduced (Borges-Pereira, personal communication). The technique revealed variable sensitivity depending not only on the characteristics of the population analyzed but also from the test itself, the volume and conservation of samples, DNA extraction method, parasite sequences used, primers, reagents, and thermo-cycling conditions.40

The need for a standardization was recently stressed (2011) in a multicenter evaluation sponsored by TDR/WHO and performed in two steps. In the first, 29 research groups were invited to participate and received three sets of samples blind. The first set comprised five 10-serial dilutions of T. cruzi DNA from 3 reference stocks. The second were blood samples spiked with 10-fold dilution of culture parasites and the third was a panel of 40 clinical samples (10 from noninfected persons) from several countries. Each laboratory performed its own method with the same samples. From the 49 different strategies used by the 29 laboratories, only 4 methods were selected, on the basis of their performance with a specificity of 100% and a sensitivity of 56—63%. In the second step, 18 participants, selected by their better performance, participated in a workshop, performing the analysis of the clinical samples with the same reagents and methods. Silica gel column extraction followed by satellite-based PCR was more specific and sensitive than other methods, and a standard operating procedure was defined for the test.41

There are two main questions regarding PCR. The first is because of the natural history of the parasitemia at the chronic phase. As written above, some infected individuals may be free of circulating parasites for long periods of time, so PCR could not find DNA of nonexisting parasites, and this fact may explain why several recent reports42 cannot reach the 100% sensitivity predicted years ago. The other main question relates to the reliability of PCR diagnosis, with a high frequency of false positives due to contamination observed in some studies, as well as false negatives due to inhibitory substances in the lysate.40

Conclusions

For all the reasons exposed, these multiplication methods have several limitations and a short range of applications like the follow-up of treated patients to check treatment failure and, in research, for isolation of parasites. These situations are seen in very specialized laboratories devoted to the study of the disease, but up to now not for routine diagnosis.

Nevertheless, in the best conditions, parasitemia can be found in a number of individuals at the chronic phase. Statistics are different according to geographical area (i.e., lower in Piaui, Brazil),43 age of the patients (higher parasitemia before 11 and after 60 years old), pregnancy (increases),44 and use of specific treatment (decreases). Because of the lower parasitemia, the chance to find parasites increases with the number of examinations. All the studies show that figures are higher in the same individual, if the same method is performed two or more times.42,45 Also, if more than one method is applied, the chance of obtaining parasites increases.43 Recent studies show that a single hemoculture or xenodiagnosis may be positive in 24—52% of the patients. The same sort of studies show that if more examinations are performed, the figures increase to a maximum of 65%.46 It is clear from these studies that some patients are always negative, irrespective of the number of tests applied. On the other side, some positives always turn out that way (around 8—12% depending on the study). These are classified as having high parasitemia (each test applied is positive). Another way to measure the degree of parasitemia is to record the number of positive tubes of hemocultures44,47 or the number of positive bugs, which allow you to classify the degree of parasites present. These studies are particularly useful when a new drug is tested.48

 
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