In June 2006, a 74-year-old woman residing in a house in rural New Orleans was bothered by a considerable number (>50) of insect bites. The woman observed many bugs in the house and showed them to a fumigator, who identified them as triatomines. An internet search showed the potential for transmission of Chagas disease, and the woman sought help from a local health sciences center.
Serum samples from both residents of the house were tested for antibodies to T
at the Centers for Disease Control and Prevention (CDC) by an indirect fluorescent antibody (IFA) test. Samples were also tested at Loyola University (New Orleans, LA, USA) and then at CDC. by using an experimental dipstick assay (Trypanosoma
Detect; InBios International Inc., Seattle, WA, USA). The woman resident was positive for antibodies to T
by IFA at dilutions of 1:128 (≈4 weeks after being bitten) and 1:64 (≈10 weeks after being bitten) and by dipstick assay. She was positive for trypanosomes by hemoculture testing with ≈10 mL blood and coculture in macrophages (13
) ≈4 months after being bitten. Trypanosomes consistent with T
were observed in culture beginning on day 46 of culture, and amplification of a T
–specific 24Sα rRNA gene target confirmed that the isolate was T
. The other resident was negative by both serologic tests.
The index resident had a history of 5 trips to areas endemic for Chagas disease: Zacatecas, Mexico (1970); Cozumel, Mexico (1990); Belize (1991); Guatemala (19988); and Costa Rica (1998), each of <2 weeks duration, with stays in improved tourist hotels (less likely to harbor triatomines) except for the Belize trip, which included an ≈1-week stay in a palm thatch-roofed cabin. She had not used intravenous drugs or had a blood transfusion or organ transplant, and she is not the daughter of Latin American immigrants. Except for fatigue, the index patient had no symptoms and had an active lifestyle. Cardiac evaluation that included an electrocardiogram showed normal results, and she decided not to take medication.
Her residence of 29 years was located on 7.66 acres of bottomland hardwood forest, with many gaps that provided ready access for insects. A house inspection showed fecal streaks characteristic of triatomines on walls, which were identical to what the patient reported seeing on her nightgown. Twenty dead adult triatomines were collected in the house (after fumigation) and in another building on the property that contained a bed. No nymphs or eggs were found, which suggests that the house had not been colonized. One live second-stage nymph was collected in a nearby armadillo burrow ≈50 m from the house. All triatomines collected were identified as T
according to the key of Lent and Wygodzinsky (6
Male Triatoma sanguisuga.
Because all triatomines except the nymph were dead, PCR was used to determine T
infection status (14
). The last 2 segments of the abdomen were removed from each insect, placed in 200 μL 1× PCR buffer (Applied Biosystems, Foster City, CA, USA), boiled for 15 min, and centrifuged. A total of 5 μL of supernatant was amplified in a 50-μL reaction (3.5 mmol/L MgCl2
and 2 U Taq DNA polymerase). The primers used anneal to the T
minicircle DNA and were TC3: 5′-TTGAACGCCCCTCCCAAAAC-3′ and TC4: 5′-GATTGGGGTTGGTGTAATATA-3′. The cycling parameters were an initial denaturation step at 94°C for 3 min; 35 cycles at 94°C, 55°C, and 72°C, each for 1 min; and a 10-min extension at 72°C (programmable thermal controller; MJ Research, Watertown, MA, USA). Twenty percent of the PCR product was subjected to electrophoresis on a 1.8% agarose gel and visualized by UV transillumination after staining with ethidium bromide. A positive control of 5 μL of T
parasites boiled in 1× PCR buffer and a negative control without the DNA template were included with every PCR. Samples that failed to amplify were spiked with 5 μL of T
parasites boiled in 1× PCR buffer and reamplified to ensure that the lack of product was not caused by inhibition of the PCR. More than half of the triatomines were positive for T
(56%, 10/18; 3 failed to amplify), with more positive females (73%, 8/11) than males (50%, 3/6). Plasma from the resident dog and 7 other dogs living ≈1 mile away all tested negative by IFA at CDC.