Polymerase Chain Reaction Detection of Trypanosoma cruzi in Suboptimally Preserved Vectors and Comparative Infection Rates 2007–2010 in Escondido,Southern California

Introduction

T rypanosoma cruzi is the insect-vectored trypanosome (Kinetoplastida: Trypanosomatidae) responsible for American trypanosomiasis or Chagas disease in humans, a condition that is difficult to diagnose and in its chronic phase often results in heart failure. Chagas disease is widespread in Central and South America; and despite only one documented autochthonous case in California (Navin et al. 1985), the disease has recently come into focus due to high percentages of contaminated blood samples that may be connected with migration from Central and South America (Bern et al. 2007). The most widespread and abundant kissing bug in California, Triatoma protracta (Uhler), is currently reported to have infection rates of 19%–36% in Southern California (Klotz et al. 2009, Hwang et al. 2010), a range that corresponds with historical infection rates published in the 1930s–1960s (Wood 1975) for that area.

Trypanosome detection in the insect vectors is an essential component in monitoring the public health risk over time. Optimally preserved specimens (i.e., 95% ethanol and kept cold at all times) that allow for polymerase chain reaction (PCR) amplification and sequence verification are a reliable option, but such samples can be relatively costly to obtain. The Southern Californian public frequently sends ethanol preserved or dried specimens to our lab. These specimens are usually shipped and received in conditions that are suboptimal for the preservation of DNA, that is, dried, in lower percentage (70%) ethanol, or exposed to warm temperatures. Here, we investigate whether such suboptimally preserved samples can be used to reliably deduce infection in a specimen. Our samples are derived from one locality in Southern California in Escondido (San Diego County) for which Hwang et al. (2010) determined a 19% infection rate based on PCR of 139 T. protracta specimens collected in 2008. Our goal in the current article is twofold: we investigate whether there is an increase or decrease in the infection rate of T. protracta at this specific locality in 2010 compared with that in 2007 and 2008 (Klotz et al. 2009, Hwang et al. 2010). Second, we evaluate whether detection of the infection rate is affected by the preservation condition of the kissing bug sample.

Materials and Methods

Two independent PCR-based surveys of T. cruzi prevalence in T. protracta in Escondido, San Diego Co., found infection rates of 19%–20% in fresh or 96% ethanol preserved specimens (Klotz et al. 2009, Hwang et al. 2010). We here use samples from the same locality to (a) determine current (summer 2010) infection rates as a comparison to rates published for 2007 (Klotz et al. 2009) and 2008 (Hwang et al. 2010); and (b) determine the effect of three preservation treatments. The treatments consisted of preservation in (1) 96% ethanol, stored at cool temperatures (fridge or freezer); (2) 70% ethanol, storage at room temperature for 3–4 weeks; and (3) specimens collected in 96% ethanol in 2008 and, subsequently, pinned and dried for >12 months. We sampled 20 specimens per treatment. Specimens were bar coded with Unique Specimen Identifiers and are vouchered in the Entomology Research Museum at University of California Riverside. DNA was extracted from the crushed abdomen (dried specimens) or the dissected gut contents (ethanol specimens) by using the QIAGEN DNeasy Blood and Tissue kit. PCR of a ∼420 bp fragment of 18S rDNA was performed according to the T. cruzi 18S protocol established by Souto et al. (1996) as described in Hwang et al. (2010). PCR products were examined by using gel electrophoresis to detect positive bands for T. cruzi. Subsequently, positive samples were sequenced at the UCR Genomics Core facility; the sequences were edited by using Sequencher 4.9 and blasted against sequences generated by Hwang et al. (2010). A Chi-Square statistical analysis was performed to investigate significance of differences to the 2007 and 2008 infection rates (Klotz et al. 2009, Hwang et al. 2010) and between the three treatments.

Results and Discussion

PCR recovered bands for six of the optimally preserved T. protracta specimens collected in 2010 (n=20), and sequence comparison identified them to be T. cruzi. The same number (6; n=20; 30%) of infected kissing bugs was found for dried specimens that had been collected in 2008; the number for specimens preserved at low ethanol percentage and room temperatures was 5 (n=20; 25%). A Chi-Square test shows that these differences between preservation methods are not significant at the 95% confidence level. Compared with the infection prevalence in 2008 published by Hwang et al. (2010), the infection rate found in the optimally preserved samples (30%) in 2010 is not significantly different.

Conclusions

Our results indicate that the infection rate between 2007 and 2010 has not changed significantly, thus indicating that rates within populations may stay relatively stable over time. A larger sample of T. protracta should be used to further test and corroborate this statement. We further conclude that even suboptimally preserved material (ethanol preserved and then dried; low-percentage ethanol and room temperatures) can be a valuable source for examining T. cruzi infection rates in kissing bug specimen populations.