The investigation of congenital infection by Trypanosoma cruzi in an endemic area of Chile: three protocols explored in a pilot project

Abstract

Given the increasing travel of pregnant women from areas were Trypanosoma cruzi is endemic, the congenital transmission of the parasite has become a global public-health problem. In a recent pilot study, which ran in Chile from 2006 to 2010, three strategies for exploring and managing T. cruzi-infected mothers and their infected or uninfected neonates were investigated. Any protocols applied to the investigation of such mother-and-child pairs need to include the detection of infection in pregnant women, the detection of infection, if any, in the children born to the women, the appropriate treatment of the infected neonates, and the serological–parasitological follow-up of all of the neonates until their medical discharge.

The success of attempts to prevent and control human American trypanosomiasis (Chagas disease) via reductions in vector numbers and the transfusion-related transmission of the causative parasite, Trypanosoma cruzi, has increased the relative public-health importance of congenital infection. Congenital transmission represents the only active route of transmission for which no kind of control exists in many endemic countries, and, since pregnant women infected with Try. cruzi are increasingly likely to travel or emigrate to non-endemic regions, it is a form of transmission that may occur anywhere in the world (WHO, 2002; Gascón et al., 2007). Although, if detected, the infections in neonates that result from congenital transmission can usually be treated effectively, without any serious adverse events (Altcheh, 2010), it has been estimated that such transmission will remain a problem for at least the next 30 years — that is, until the number of infected women of fertile age becomes negligible (Dias, 2009). In Latin America there are currently about two million women of fertile age who are infected with Try. cruzi (Anon., 2006), seroprevalence among women of this age ranging from 5%–40% (Altcheh, 2010). Between 1% and 12% of mothers with American trypanosomiasis will each transmit Try. cruzi to their foetus (Carlier and Torrico, 2003) but most of the infected foetuses will be asymptomatic at birth (Yadon and Schmunis, 2009).

In Chile, about 130,000 women of fertile age (15–49 years) live in rural areas where Try. cruzi is endemic. Although it has been estimated that about 122,000 females aged >5 years migrated from such areas between 1997 and 2002, mostly to urban sites within Chile (Anon., 2007a), the importance of such migration to the epidemiology of American trypanosomiasis remains unclear. About 160,000 people in Chile are thought to be infected with Try. cruzi, of whom about 34,000 are women of fertile age (Anon., 2006). These numbers, together with estimates of birth rates and the incidence of congenital transmission, indicate that about 445 new cases of congenital American trypanosomiasis should be expected each year in Chile (Anon., 2006). In their recent study in the Chilean province of Choapa (in region IV), Apt et al. (2010b) estimated that there were about 20,130 women of fertile age in the province and found that 3·7% of the women who had their babies while in the care of the provincial maternity services had American trypanosomiasis and that, at parturition, about 4% of the pregnant women with the disease had transmitted Try. cruzi to their foetuses. If these values are similar across region IV (i.e. the provinces of Elqui, Limarí and Choapa), there should be about 10 congenital cases in the region each year.

Between 2006 and 2010, in a pilot study based in region IV, three different protocols were used, in parallel, to investigate and manage the Try. cruzi-infected mothers and their children (i.e. those who were in utero when their mothers were infected), in the hope that the results would lead to recommendations that would complement other, relevant, national (Apt et al., 2010a) and international guidelines (Carlier and Torrico, 2003; Gascón et al., 2007).

PROTOCOLS

Protocol 1. Serological Examination of Pregnant Women from the Endemic Zone and Interaction with the Women and their Families

All pregnant women who live in a known endemic area or have migrated from such an area should be asked to submit to a serological examination to see if they are infected with Try. cruzi (Carlier and Torrico, 2003). Such serology should preferably be performed during the first prenatal examination, at the same time as routine serological tests for several other infectious agents (such as HIV and Treponema pallidum). Unfortunately, 1·6%–2·5% of individuals infected with Try. cruzi appear false-negative in the serological tests that are frequently used (WHO, 2002). Adequate quality control should be established in the laboratories in which the diagnostic tests for Chagas disease are performed (WHO, 2002) and, according to international consensus, all positive results should be confirmed using at least two different serological techniques (Luquetti et al., 2005), such as indirect haemagglutination (IHA), indirect immunofluorescence (IIF) and/or ELISA (Luquetti et al., 2005; Apt et al., 2010a). Rapid detection tests, based on serology, are being developed, primarily for testing mothers with chronic infections (Gomes and Virginia, 2009).

Serological testing should be repeated in each pregnancy (Dias, 2005) and the results of such investigation should be recorded on the mother's hospital card and in her general clinical record and pregnancy record, so that they can be seen by any maternity service that cares for the woman. Under current Chilean law, Chagas disease is notifiable, with any new cases of human infection with Try. cruzi recorded on a standard form — the Formulario de Notificación de Enfermedades de Declaración Obligatoria — and reported daily to the regional health service and weekly to the national Ministry of Health. It is, however, generally assumed that only a proportion of the cases detected, and perhaps only a small proportion, appears in any national database (Apt et al., 2010a).

A strategy of information, education and communication (IEC) should be used to enhance levels of knowledge about congenital Chagas disease among all pregnant women (and their partners), to improve the detection and management of the disease and social attitudes towards it (Guillén and Cuentas, 2003; Anon., 2007b). As part of this IEC strategy, women of fertile age should be told the place where, and the manner in which, they and their neonates will be tested (assuming consent), the actions that should to be taken if mother and/or child is found seropositive (including chemotherapy of the child), the danger of re-infection from contact with the faeces of infected vinchucas (i.e. the triatomine vectors), and the wisdom of having their older children, especially those aged <5 years, checked for Try. cruzi and, if found positive, immediately started on a course of treatment. If such advice and information can be given, in part, by health staff seeing women (individually or in small groups) at their prenatal checkups, this tends to improve maternal/parental compliance in the serological investigation of the women and their neonates and the follow-up of both the seropositive neonates who are treated and their seronegative counterparts (who should also be followed-up until their discharge). Currently, the serological testing of neonates born to women who are known (or, at least, know themselves) to be seropositive for Try. cruzi, so that infected neonates can be identified and treated early, is the greatest challenge in the control of congenital Chagas disease in non-endemic countries (Gascón and Pinazo, 2008).

Protocol 2. Serological and Parasitological Examination of the Try. cruzi-infected Mothers and their Neonates, at Parturition and over a Prolonged Follow-up

The serological and parasitological investigation of infected mothers (in the pre-partum room) and their neonates (in the delivery room), using peripheral and umbilical-cord blood, respectively, is recommended. In a child aged <9 months, it is currently impossible to distinguish anti-Try. cruzi antibodies produced by the child from those produced by his or her infected mother. Some neonates born to infected mothers are seronegative (Szarfman et al., 1975; Freilij et al., 2004; Apt et al., 2010a) but all children born to infected mothers should be carefully followed-up and checked again for anti-Try. cruzi antibodies at an age (e.g. 1 year) when maternal antibodies will have cleared from their bloodstream. Compared with other infected mothers, those found positive for Try. cruzi by blood culture are particularly likely to give birth to infected neonates (Hermann et al., 2004). Although transmission may occur at any stage of the pregnancy (Freilij et al., 2004), there is some evidence (elevations in parasitaemia, as detected by xenodiagnosis, and the detection of specific IgM) indicating that the risk of congenital transmission may be highest during the final stages of gestation (Szarfman et al., 1975; Storni and Bolsi, 1979). Congenital transmission may occur in one or more pregnancies of an infected woman (Freilij et al., 2004). The identification of mobile trypomastigote forms in umbilical-cord blood at birth or, later, in the peripheral blood of the neonate unequivocally confirms congenital infection and indicates the need for immediate treatment (Carlier and Torrico, 2003; Freilij et al., 2004; Rodríguez Coura and Pinto Dias, 2009; Apt et al., 2010a).

The direct parasitological technique that is most frequently recommended for the detection of Try. cruzi in blood is the microhaematocrit. In the detection of congenital infection (which is often associated with high parasitaemia), this method can offer a sensitivity of at least 90% (Freilij et al., 2004). Although, in the absence of adequate training, the sensitivity of the microhaematocrit method may be much reduced (as seen in the recent study in region IV; unpubl. obs.), the method remains useful for the early diagnosis of congenital infection. It is recommended that blood from the umbilical cord of the neonate is collected in a heparinized tube, with microhaematocrit capillaries only filled with this blood in the health-unit's laboratory (to reduce the exposure of delivery-room personnel to blood that may be infected with Try. cruzi). If the neonate's blood cannot be tested at birth, a sample of peripheral blood should be taken at an age of 15–30 days and similarly investigated (Azogue and Darras, 1995).

Another parasitological technique whose utility has already been demonstrated in several studies of congenital American trypanosomiasis is the PCR-based assay, in which specific segments of the DNA of Try. cruzi are amplified (Schijman et al., 2003; Virreira et al., 2003). The serial parasitological study of neonates with strong clinical indications of congenital infection by Try. cruzi (e.g. prolonged fever, hepato–splenomegaly, acute myocarditis and/or and premature birth) is recommended (Rodríguez Coura and Pinto Dias, 2009). In such serial investigation, Diez et al. (2008) found a PCR-based assay to be more sensitive than direct parasitological tests such as the microhaematocrit. If such an assay is used to test a neonate at birth, it should be repeated after 15 and 30 days (irrespective of any earlier results), especially when a neonate has been found PCR-positive but negative in direct parasitological tests (since PCR positivity may be caused by cell-free Try. cruzi DNA from the infected mother, that has passed the placental barrier).

Rapid diagnostic tests for neonates, such as the T. cruzi OligoC-TesT (Deborggraeve et al., 2009), could be very useful when they become available commercially and, also in the future, real-time PCR may replace conventional PCR (Duffy et al., 2009).

Based on experience gained in the recent study in region IV, it seems reasonable to set three laboratory criteria, each of which can be considered to confirm a congenital infection:

1. at least four positive results in direct parasitological tests of umbilical-cord blood of the neonate and/or peripheral blood of the neonate collected 1–2, 15 and 30 days after the birth (irrespective of the results of any PCR-based assay);

2. consistently positive results in serial PCR-based assays performed until the child is aged at least 15 days (irrespective of the results of any direct parasitological tests); and/or

3. if the post-partum infection of the child can be discounted (e.g. because the child has always lived in areas where the triatomine vectors do not occur), the presence in a child of 9–12 months of anti-Try. cruzi antibodies, confirmed by two conventional serological techniques (e.g. IIF and ELISA).

Protocol 3. Treatment and Prolonged Post-treatment Follow-up of Children with Confirmed Congenital Infection

Once congenital infection by Try. cruzi has been confirmed, the prevention of cardiac and digestive damage and neurological lesions depends upon adequate specific treatment (Abad-Franch et al., 2010). Such treatment should be obligatory for all confirmed cases (Carlier and Torrico, 2003; Apt et al., 2010a; Altcheh, 2010), since frequencies of cure approaching 100% can be obtained — when nifurtimox or benznidazole are given early (WHO, 2002; Carlier and Torrico, 2003; Schijman et al., 2003; Yadon and Schmunis, 2009). The recommended treatment, as agreed upon by the relevant experts, is to give either benznidazole (at 5–10 mg/kg.day) or nifurtimox (at 5–15 mg/kg.day) for 30–60 days. Nifurtimox should be given at 5 mg/kg on each of the first 3 days of treatment and only increased to 10 mg/kg.day if no adverse events have then been observed (Apt, 2010; Apt et al., 2010a). As paediatric preparations of benznidazole and nifurtimox are not currently available, a pharmacist will have to prepare the prescribed doses.

It is recommended that any drug be given under the direct supervision of a responsible family member or health worker, to assure compliance, avoid over-dosage and allow any adverse effects to be monitored (Carlier and Torrico, 2003). There should be clinical follow-up (with weekly clinical examinations and measurements of weight and height during treatment), general laboratory examinations (haemograms, liver-function tests and two different serological tests for anti-Try. cruzi antibodies during and at the end of treatment) and parasitological examinations (PCR recommended; every 6 months for 2 years after the therapy). Only after at least 2 years of negative results in serological and serial parasitological tests should the patient be declared cured (Molinedo et al., 2005). Persistent seropositivity indicates therapeutic failure, when a second course of treatment with prolonged follow-up should be considered (Rodríguez Coura and Pinto Dias, 2009).

CONCLUSIONS

All neonates with Try. cruzi-infected mothers should be studied until their congenital infection can be confirmed or discounted (Anon., 2007b). The first step in this process is the identification of the infected mothers (Yadon and Schmunis, 2009). Most congenital infections are asymptomatic in infanthood, and most infected babies have normal birthweights. Premature infants infected with Try. cruzi may have a variety of clinical manifestations, such as low birthweights for their gestational age, jaundice, anaemia, hepatomegaly, splenomegaly, perinatal hypoxia, meningo-encephalitis and/or myocarditis, and may soon die (Freilij et al., 2004; Rodríguez-Coura and Borges-Pereira, 2010). The mortality and morbidity still associated with Chagas disease, and the relative ease with which the disease can be cured in most young children, justify the establishment of protocols for the multidisciplinary care of infected-mother/neonate pairs in Chile. The training of health teams is fundamental, especially in endemic areas without control programmes and in non-endemic areas with female migrants from endemic areas (Buekens et al., 2008; Gascón et al., 2009; Yadon and Schmunis, 2009). American trypanosomiasis is currently a global parasitosis and congenital transmission is one of its manifestations. The protocols presented here are intended to be a contribution to the strategies for the control of this disease.