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In utero detection of fetal infection

As mentioned in Section, Possible transmission routes of Trypanosoma cruzi parasites, T. cruzi DNA is rarely detected in AF and amniocentesis is currently not recommended for the prenatal diagnosis of T. cruzi congenital infection.27

Fetal blood sampling (cordocentesis to be performed by experienced clinical practitioners) for standard parasitological or molecular testing has been rarely used for the prenatal diagnosis of congenital T. cruzi infections.202

Detection of neonatal infection (0—4 weeks after birth)

Blood samples can be collected either at birth, from the umbilical cord (easy to collect without trauma for newborns and mothers) or later in neonates by peripheral venipuncture (from heel, arm, or finger). In case of symptoms suggesting meningoencephalitis, cerebrospinal fluid can also be collected in neonates.

Parasitological tests: Live T. cruzi trypomastigotes can be detected in blood samples by microscopic examination of buffy coat from centrifuged heparinized microhematocrit tubes. The latter method is rapid, cheap, affordable, one of both currently recommended gold standards, since detecting around 40 p/mL using four to six tubes (more sensitive than the other parasitological methods, such as microscopic examination of fresh blood samples, fixed blood smears, or thick smear) and, overall, because detection of parasites in blood definitively confirms congenital infection.105 The sample has to be examined within 24 h to avoid a decrease of sensitivity due to parasite lysis. Examination of parasites can be done after cutting the tube.203,204 However, to avoid possible contamination of the examiner, buffy coat can be also examined in the tube without previous rupture using immersion oil , or by rotating it. ,

The alternative Strout concentration method (so-called microstrout using Eppendorf tubes),207,208 as well as indirect parasitological methods, such as hemo- culture (more expensive and needing weeks before obtaining results), can also be used for detecting blood parasites in congenital T. cruzi infection.55,204 All these parasitological methods, which indisputably confirm infection by showing live parasites, need well-skilled personnel and regular quality controls.

PCR assays: Molecular tests are able to detect low amounts of T. cruzi DNA in blood samples of neonates born to infected mothers.121,204,209 —211 T. cruzi PCR protocols have been recently compared and efforts have been done to standardize them.212 Despite of its high sensitivity, PCR does not detect 100% of congenital cases and a negative PCR does not completely exclude a possible congenital infection.

The main question about molecular tests concerns the association between the detection of parasitic DNA and the real presence of live parasites in biological fluids (see section: From maternal—fetal transmission of Trypanosoma cruzi to congenital Chagas disease: definitions and limits). As mentioned in section, Main biorelevant transferred molecules, ccfDNA released by T. cruzi (dead or live) transferred from mother to uninfected fetuses might be detected by PCR, complicating the interpretation of results, particularly in the case of low intensity amplicons susceptible to correspond to trace amounts of parasitic DNA instead of live parasites. So, an early positive PCR is not absolutely indicative of a congenital infection and even less for its treatment (see section: Treatment of congenital infection with T. cruzi). It might be considered that blood sampling later after birth might provide more reliable PCR positive results. However, critical information is still lacking on the stability and duration of parasitic DNA in maternal and umbilical cord blood, and ccfTcDNA might be also produced in some fetuses that have naturally auto-cured their congenital infection (see section: Interpretation attempt of fetal/neonatal immune responses to Trypanosoma cruzi).

This probably explains the apparent high maternal—fetal transmission rates reported from human studies considering only results of PCR tests close to birth,204,209,213,214 as well as in experimental infections,163,164 whereas reports confirming congenital infection by parasitological or late serological tests display lower rates.6,110

Quantitative (real time) PCR that estimates parasite DNA levels, might be useful in completing the results of PCR if protocols are more standardized and a discriminant cut-off value to predict parasite infection is determined.66,72,82,215,216 New molecular tests targeting exclusively live parasites (e.g., detecting mRNA by reverse transcriptase PCR) or other markers are urgently needed to improve such diagnosis.

Detection of T. cruzi-specific total IgM and IgA antibodies: Antibody isotypes not transferred by mothers are not present in all infected newborns and are also detected in uninfected newborns of infected mothers (see section: Immune responses in newborns of Trypanosoma cruzi-infected mothers).1,101,128,129,217,218 Such detection is presently not recommended for the diagnosis of congenital T. cruzi infection.

Detection of IgG/IgM antibodies recognizing Shed Acute Phase Antigen (SAPA): A dot-blot assay detecting IgG antibodies recognizing SAPA has been developed in order to diagnose congenital cases, considering such antibodies as being synthesized by acutely infected fetus/neonates and not transferred from mother.127 However, further studies indicated that the SAPA-specific antibodies are also detected in chronic patients (including around 50% of infected mothers) and could also be transferred.219

A TESA blot (see section: Detection of infection in pregnant women) allows detecting IgM antibodies recognizing a band at 130—200 kDa corresponding to

SAPA in blood of congenital cases.220 The specificity of this TESA (SAPA) blot-

103,220

IgM is close to 100%, but its sensitivity varies according to the studies. ,

Detection of parasite soluble antigens: Detection of T. cruzi soluble antigens in urine and serum by capture ELISA has been proposed for diagnosis of congenital cases. However, these tests did not detect all infected cases.221,222 Recently, a new test has been developed using nanoparticles to capture, concentrate (by 100-fold), and preserve such antigens in urine (so-called “Chunap”). The antigens are eluted and detected by Western Blot using a monoclonal antibody against T. cruzi lipopho- sphoglycan.223 This method remains to be validated at a larger scale for the diagnosis of congenital infection.

Placental histopathology: Standard histopathological or immunoenzymatic studies, PCR analyses or in vitro cultures of placental biopsies have been also considered for the diagnosis of congenital T. cruzi infection. However, placental parasitism can be either not detected in congenitally infected neonates, or, conversely, observed in uninfected newborns.22,49,51,70,176,224 So, placental analysis is presently not recommended for the diagnosis of congenital T. cruzi infection.

 
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