Diagnosis of Acute Chagas Disease in a Belizean Child with Evidence of a Multiclonal Trypanosoma cruzi Infection

ABSTRACT.

In January 2020, we instituted acute febrile illness surveillance in 11 hospitals and clinics across Belize. Within 3 months, we diagnosed an acute case of Chagas disease by polymerase chain reaction in a 7-year-old child in the northern part of the country. Phylogenetic analyses of the parasite from the acute blood specimen revealed a multiclonal Trypanosoma cruzi infection, including parasites from the TcII (25.0% of haplotypes), TcIV (2.5% of haplotypes), and TcV (72.5% of haplotypes) discrete typing units. The family reported no history of travel, and three Triatoma species vectors were found within the home. The child’s mother was seronegative for antibodies to T. cruzi, ruling out congenital transmission. Convalescent blood samples documented seroconversion and confirmed acute infection. The child was successfully treated with nifurtimox. This is the first known diagnosed case of acute Chagas infection in Belize, highlighting the need for further investigation and public health prevention measures.

Chagas disease (American trypanosomiasis) is a vector-borne infection caused by the parasite Trypanosoma cruzi. Chagas disease is a multistage disease that includes both an acute phase, which typically is asymptomatic or presents with mild symptoms, and an indeterminant chronic phase that will, over time, cause severe cardiac disease in up to 35% of infected individuals.^1,2 Infections are most often acquired from contact with the vector species, the triatomine or “kissing bug.” Triatoma species have a wide natural range throughout the Americas.²

Chagas disease is an important neglected tropical disease with up to 1.6 million people across Latin America infected with T. cruzi.^3,4 In Belize, some of the earliest reports of Chagas disease and its primary vector, Triatoma dimidiata, date back to the 1960s.⁵ Human infections are rarely reported in Belize, and data on seroprevalence are limited. There are no known reports of acute infections diagnosed in Belize.

In January 2020, acute febrile illness (AFI) surveillance was initiated in 11 healthcare facilities across Belize to improve understanding of the incidence of infectious disease pathogens of concern in Central America. As previously described,⁶ participants presenting with AFI are consented, interviewed, and have both serum and whole blood collected for diagnostic workup for seven vector-borne pathogens, including T. cruzi. Samples are tested for T. cruzi by real-time polymerase chain reaction (RT-PCR) using previously published primers and probes.⁷

In late March 2020, a 7-year-old otherwise healthy Latino male presented to a hospital in northern Belize with a 4-day history of fever (38°C), vomiting, diarrhea, sore throat, myalgia, abdominal discomfort, rash on the chest and extremities, headache, and retroorbital pain. Hemoglobin, hematocrit, white blood cell count, and platelets were within normal limits (11.8, 35%, 5,600, and 198,000, respectively). The patient was presumptively diagnosed with dengue. After parental consent and child assent, the patient was enrolled in AFI surveillance, interviewed, and had blood specimens collected for diagnostic testing. The parents of the patient reported no recent travel history and no known exposure to any ill individuals; however, they reported mosquito bites in the 2 weeks before illness onset as well as exposure to domestic and agricultural animals. Diagnostic PCR testing was performed per the AFI study protocol and was found PCR positive for T. cruzi and negative for dengue, chikungunya, Zika, and West Nile viruses. Serum was negative for anti–T. cruzi antibodies by both StatPak (Chembio Diagnostic Systems, Hauppauge, NY) and Hemagen Chagas EIA kit (Hemagen Diagnostics, Columbia, MD), providing evidence for acute infection. The patient was treated with nifurtimox 120 mg twice daily for 60 days.

Convalescent serum and whole blood were collected at 60, 157, and 265 days post-onset of illness. All convalescent samples were negative for T. cruzi by PCR. As anticipated, the convalescent serum collected 60 days post-onset showed evidence of seroconversion with positive anti–T. cruzi antibody results by both StatPak (Figure 1) and Hemagen ELISA, confirming the acute nature of the infection. Seroreversion to negative was evident by 265 days post-onset, representing successful treatment of the child.

Figure 1.

Stat-Pak rapid test findings for the case-patient at 0 (39-A), 60 (39-2), and 157 (39-C) days post-onset.

Four household members were also enrolled in the study, and all were negative for T. cruzi by PCR, Stat-Pak, and Hemagen ELISA. Upon investigation of the case-patient’s home, we identified several risk factors for vector exposure. The family had collective animal housing near their home, woodpiles in the yard, and holes and cracks in the walls of the home structure. Three Triatoma sp. vectors were found within the home of the patient, and one was found positive for T. cruzi by PCR.

Parasite genotyping was performed on the child’s acute blood specimen using a multiplex PCR targeting the mini-exon gene sequence, which produces amplicons of different sizes according to the discrete typing unit (DTU) (TcI-TcVI),⁸ and TrypME3 primers.⁹ PCRs were performed under previously reported conditions.^8,9 Two reference strains were used as positive controls, WB1 (TcI) and ESM (TcII), as well as a negative control with no template DNA (molecular grade water). Amplifications were completed using previously reported cycling conditions.⁸ PCR products were separated on a 2% agarose gel stained with ethidium bromide and then purified using a PureLink Quick PCR Purification Kit (Invitrogen, Waltham, MA).

Amplicons were processed for Nanopore library preparation using the Rapid Barcoding Sequencing (SQK-RBK004) protocol and sequenced using the MinION sequencing platform (Oxford Nanopore Technologies). Raw Fastq reads were mapped to mini-exon reference sequences (Supplemental Table 1) from each parasite DTU as previously described.¹⁰ Sequence variants were identified using the FreeBayes single nucleotide polymorphism/variant tool,¹¹ and only sequences representing ≥ 1% of the total sample were conserved for analysis. Sequences were deposited in Genbank (accession # OM818332–OM818351). Phylogenetic trees based on maximum likelihood were constructed using Phylogeny.fr platform¹² and additional mini-exon sequences from infected vectors, hosts, and reference parasites were included for comparison (Supplemental Table 1).

The genotyping identified a predominance of non-TcI in the child’s blood sample. Phylogenetic analyses indicated the presence of a multiclonal infection with 20 unique haplotypes, including parasites from the TcII (N = 7 [25% of reads]), TcIV (N = 1 [2.5%]), and TcV DTUs (N = 12 [72.5%]) (Figure 2A). Further analysis of haplotype diversity indicated that the TcIV haplotype from the patient was most closely related to TcIV sequences previously identified in Triatoma dimidiata vectors from Toledo district in southern Belize,¹³ within a larger cluster of North and Central American sequences, distinct from the South American CanIII reference sequence (Figure 2B). TcII haplotypes from the patient also formed a well-defined cluster distinct from other TcII sequences from North and South America (Figure 2C). Similarly, TcV haplotypes from the patient formed two to three clusters apart from reference sequences from South American strains (Figure 2C).

Figure 2.

Phylogenetic analysis of Trypanosoma cruzi mini-exon sequences. Mini-exon sequences from the pediatric sample were compared with sequences from reference T. cruzi strains (asterisk) using maximum likelihood. Bootstrap support is indicated only for the main nodes of the tree for clarity. Percentage values indicated within sequence names refer to the relative proportion of each discrete typing unit (DTU)/haplotype in the patient. (A) Global analysis including six T. cruzi DTUs (TcI–TcVI) and (B) comparative analyses of TcIV and (C) TcII, TcV, and TcVI DTUs with triatomine insect vectors and other mammalian hosts.

Through AFI surveillance, we successfully diagnosed the first known acute case of Chagas disease in Belize. Additionally, vectors were positively identified within the home, suggesting risk of ongoing domestic transmission. We also found evidence of multiple DTUs within the acute blood specimen from the child, confirming a multiclonal infection. The rapid identification of this case through surveillance allowed for prompt and successful treatment of the child, along with the implementation of proactive vector control measures within the home.

Although the literature is sparse, previously published reports found limited evidence of autochthonous Chagas disease transmission within Belize. One paper from 1997 reported the seroprevalence of Chagas disease in Belize among three populations and found that 0.5% of blood donors and 6.1% of banana plantation workers were seropositive, yet none of the military personnel tested in the study were positive.¹⁴ The majority of the individuals who tested positive were born outside of Belize in neighboring Chagas-endemic countries. One case from the banana plantation population was in a young child born in Belize who possibly acquired the infection congenitally from their Chagas-positive mother from Honduras.

Human cases are rarely reported in Belize; however, studies on vectors in the southern part of the country found 62% of field collected T. dimidiata vectors positive for T. cruzi.¹³ Blood meal analysis of these vectors identified multiple host species, including humans,¹³ and house infestation by vectors is common.¹⁵ A separate study demonstrated that cave-dwelling T. dimidiata found in the south-central region of Belize had been feeding on humans and other vertebrate hosts opportunistically.¹⁶ These findings demonstrate a higher risk for autochthonous transmission to occur in the southern region of the country. Another study by Polonio et al.¹⁵ found that most people living in the central, western, and southern regions of Belize recognized the T. dimidiata vector; however, few were familiar with T. cruzi or Chagas disease.

A mixed infection with DTUs TcII, TcIV, and TcV is remarkable. TcI is considered the predominant parasite DTU in Central and North America; however, it was not detected in this patient. These results further expand the diversity of strains circulating in Belize, given that TcI and TcIV have been found in T. dimidiata.¹³ Sequencing technology continues to advance, with multiple parasite DTU infections recently reported in U.S. and Mexican patients,^10,17 highlighting the complexity of infections. Implications for diagnosis, disease progression, and treatment are unclear. It is important to note that this patient was effectively treated considering evidence of parasite diversity.

Despite prior literature supporting the potential risk of autochthonous transmission of T. cruzi in Belize, the country was certified free of T. dimidiata transmission of T. cruzi to humans in 2012.¹⁸ This certification may have been influenced by the results of a study carried out in Belize in 2011–2012 in which more than 1,000 young children from 27 schools were tested and surveyed for Chagas disease, with no cases identified. These results led to the conclusion that vector-human transmission cycle of T. cruzi was had been disrupted in Belize. Interrupting vector–human transmission of T. cruzi continues to prove challenging in the Americas.¹⁹

Our identification of an acute case in a child along with evidence of peridomestic infestation of vectors challenges these notions and highlights the need for further investigation into the human disease risk in Belize. Ongoing surveillance for both acute and chronically infected cases, along with vector surveillance and control activities, are needed to establish a better understanding of the risk to public health.

Note: Supplemental materials appear at www.ajtmh.org.