Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Identification of bloodmeal sources and Trypanosoma cruzi infection in triatomine bugs (Hemiptera: Reduviidae) from residential settings in Texas, the United States.

The host-vector-parasite interactions in Chagas disease peridomestic transmission cycles in the United States are not yet well understood. Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) infection prevalence and bloodmeal sources were determined for adult and immature triatomine (Hemiptera: Reduviidae) specimens collected from residential settings in central Texas. Sequenced cytochrome b DNA segments obtained from triatomine digestive tract identified nine vertebrate hosts and one invertebrate host in four triatomine species (Triatoma gerstaeckeri, Triatoma indictiva, Triatoma protracta, and Triatoma sanguisuga). The broad range of wild and domestic host species detected in triatomine specimens collected from residential sites indicates high host diversity and potential movement between the sylvatic and peridomestic settings. Domestic dogs appear to be key in the maintenance of the peridomestic transmission cycle as both a blood host for the triatomine vectors and a potential reservoir for the parasite. The high rate of T. cruzi infection among triatomine specimens that were collected from inside houses, outside houses, and dog kennels (69, 81, and 82%, respectively) suggests a current risk for Chagas disease vector-borne transmission for humans and domestic animals in residential settings in Texas because of overlap with the sylvatic cycle.

Elementary school-based influenza vaccination: evaluating impact on respiratory illness absenteeism and laboratory-confirmed influenza.

BACKGROUND: Studies of influenza vaccine effectiveness in schools have assessed all-cause absenteeism rather than laboratory-confirmed influenza. We conducted an observational pilot study to identify absences due to respiratory illness and laboratory-confirmed influenza in schools with and without school-based vaccination. METHODS: A local public health agency initiated school-based influenza vaccination in two Wisconsin elementary schools during October 2010 (exposed schools); two nearby schools served as a comparison group (non-exposed schools). Absences due to fever or cough illness were monitored for 12 weeks. During the 4 weeks of peak influenza activity, parents of absent children with fever/cough illness were contacted and offered influenza testing. RESULTS: Parental consent for sharing absenteeism data was obtained for 937 (57%) of 1,640 students. Fifty-two percent and 28%, respectively, of all students in exposed and non-exposed schools were vaccinated. Absences due to fever or cough illness were significantly lower in the exposed schools during seven of 12 surveillance weeks. Twenty-seven percent of students at exposed schools and 39% at unexposed schools had one or more days of absence due to fever/cough illness (p<0.0001). There was no significant difference in the proportion of students absent for other reasons (p = 0.23). During the 4 week period of influenza testing, respiratory samples were obtained for 68 (42%) of 163 episodes of absence due to fever or cough illness. Influenza was detected in 6 students; 3 attended exposed schools. CONCLUSIONS: Detection of laboratory-confirmed influenza in schools was challenging due to multiple consent requirements, difficulty obtaining samples from absent children, and a mild influenza season. School-based influenza vaccination was associated with reduced absenteeism due to fever or cough illness, but not absenteeism for other reasons. Although nonspecific, absence due to fever or cough illness may be a useful surrogate endpoint in school-based studies if identification of laboratory confirmed influenza is not feasible.

Southern plains woodrats (Neotoma micropus) from southern Texas are important reservoirs of two genotypes of Trypanosoma cruzi and host of a putative novel Trypanosoma species.

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important public health and veterinary pathogen. Although human cases are rare in the United States, infections in wildlife, and in some areas domestic dogs, are common. In 2008 and 2010, we investigated T. cruzi prevalence in possible vertebrate reservoirs in southern Texas, with an emphasis on southern plains woodrats (Neotoma micropus). Infection status was determined using a combination of culture isolation, polymerase chain reaction (PCR), and serologic testing. Based on PCR and/or culture, T. cruzi was detected in 35 of 104 (34%) woodrats, 3 of 4 (75%) striped skunks (Mephitis mephitis), 12 of 20 (60%) raccoons (Procyon lotor), and 5 of 28 (18%) other rodents including a hispid cotton rat (Sigmodon hispidus), rock squirrel (Otospermophilus variegatus), black rat (Rattus rattus), and two house mice (Mus musculus). Additionally, another Trypanosoma species was detected in 41 woodrats, of which 27 were co-infected with T. cruzi. Genetic characterization of T. cruzi revealed that raccoon, rock squirrel, and cotton rat isolates were genotype TcIV, while woodrats and skunks were infected with TcI and TcIV. Based on the Chagas Stat-Pak assay, antibodies were detected in 27 woodrats (26%), 13 raccoons (65%), 4 skunks (100%), and 5 other rodents (18%) (two white-ankled mice [Peromyscus pectoralis laceianus], two house mice, and a rock squirrel). Seroprevalence based on indirect immunofluorescence antibody testing was higher for both woodrats (37%) and raccoons (90%), compared with the Chagas Stat-Pak. This is the first report of T. cruzi in a hispid cotton rat, black rat, rock squirrel, and white-ankled mouse. These data indicate that based on culture and PCR testing, the prevalence of T. cruzi in woodrats is comparable with other common reservoirs (i.e., raccoons and opossums) in the United States. However, unlike raccoons and opossums, which tend to be infected with a particular genotype, southern plains woodrats were infected with TcI and TcIV at near equal frequencies.

Parasites and vector-borne pathogens of southern plains woodrats (Neotoma micropus) from southern Texas.

From 2008 to 2010, southern plains woodrats (Neotoma micropus) from southern Texas, were examined for parasites and selected pathogens. Eight helminth species were recovered from 97 woodrats including, Trichuris neotomae from 78 (prevalence = 80%), Ascarops sp. from 42 (43%), Nematodirus neotoma from 31 (32%), Raillietina sp. from nine (9%), Taenia taeniaeformis larvae from eight (8%), and an unidentified spiurid, a Scaphiostomum sp. and a Zonorchis sp. each from a single woodrat. Besnotia neotomofelis was detected in three (3%) woodrats and microfilaria were detected in seven (7%). Polymerase chain reaction (PCR) testing of blood samples from 104 woodrats detected a novel Babesia sp. in one (1%) and Hepatozoon sp. in 17 (16%) woodrats. Partial 18S rRNA gene sequence of the Babesia was 94% similar to B. conradae. Histologic examination of tissues detected intestinal coccidia in seven of 104 (7%), Sarcocystis neotomafelis in 26 (25%), Hepatozoon sp. in 21 (20%), and Dunnifilaria meningica in four (4%) woodrats. Three woodrats (5%) were seropositive for Toxoplasma gondii. Ectoparasites recovered included fleas (Orchopeas sexdentatus and O. neotomae), ticks (Ixodes woodi and Ornithodoros turicata), mites (Trombicula sp. and Ornithonyssus (Bdellonyssus) bacoti) and bot flies (Cuterebra sp.). The only difference in prevalence related to gender was for N. neotoma (males > females, p = 0.029). Prevalence of T. neotomae and all intestinal parasites combined was significantly higher in adults compared with juveniles (p = 0.0068 and p =0.0004), respectively. Lesions or clinical signs were associated with Cuterebra and B. neotomofelis. Collectively, these data indicate that woodrats from southern Texas harbor several parasites of veterinary and/or medical importance.

Trypanosoma cruzi and Chagas' Disease in the United States.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century.

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Detection of Blastocrithidia spp. (Kinetoplastida: Trypanosomatidae) in Chagas disease vectors from Texas, USA.

Organisms highly similar to Blastocrithidia triatomae, a pathogenic parasite of Chagas disease triatomine bug vectors, were detected using polymerase chain reaction amplification and DNA sequence analysis of a segment of the small subunit rRNA gene in 3 of 203 triatomine specimens collected in Texas from June 2005 to October 2006. The parasite was identified in three species of triatomine bugs (Triatoma gerstaeckeri, T. indictiva, and T. neotomae) collected from three distinct geographic locations. Flagellated organisms indistinguishable from Trypanosoma cruzi were observed by direct microscopy in hindgut material of two of the three specimens. Coinfection with T. cruzi and Blastocrithidia was detected by molecular methods in one of the specimens. Parsimony analysis provided strong support for clustering of the new sequences within a Blastocrithidia group, clearly separated from other flagellated protozoans. Confirmation of Blastocrithidia in U.S. triatomine species complicates microscopic diagnosis of T. cruzi due to the morphologic similarity of the parasites.

Biogeography and Trypanosoma cruzi infection prevalence of Chagas disease vectors in Texas, USA.

Data were pooled from multiple sources including newly collected triatomine specimens, preserved specimens, government reports, and scientific articles to create a biogeographical profile of triatomine vector species found in Texas. Triatomine specimens were documented in 97 of 254 counties, and Trypanosoma cruzi-infected specimens were reported from 48 counties. Triatomine specimens were distributed in 11 of the 12 ecoregions in Texas, with all but one species found in multiple ecoregions. Of the 241 newly collected specimens, 50.74% were infected with T. cruzi. Triatoma gerstaeckeri was the most frequently collected and most geographically dispersed species followed by T. sanguisuga. Three species, T. gerstaeckeri, T. sanguisuga, and T. lecticularia, were associated with human dwellings, and over half of the new specimens found inside or near houses were infected with T. cruzi. Chagas disease vectors in Texas are widely distributed and have adapted to ecologically diverse settings. The high T. cruzi infection prevalence of specimens found in close proximity to human settings suggests the presence of an active peridomestic Chagas disease transmission cycle.

Evaluation of recombinant oocyst protein CP41 for detection of cryptosporidium-specific antibodies.

Cryptosporidium is an important cause of diarrhea in developed and developing countries, and its epidemiology is of interest. The methodologies used in the detection of Cryptosporidium-specific antibodies vary widely, which complicates comparison of results. This study assesses the performance of a Cryptosporidium recombinant protein (rCP41) in a serological assay compared to that of a crude antigen preparation. The 41-kDa protein from the oocyst wall was previously cloned and expressed in Escherichia coli. Sera from 192 healthy adults from the Texas Medical Center (Houston) were tested for anti-Cryptosporidium antibody reactivity using both crude and recombinant antigen preparations in an enzyme-linked immunosorbent assay. Immunoglobulin G reactivity was highly concordant (88%; P < 0.0001) between the two antigen preparations, with 110 positive (57%) and 59 negative (31%) by both tests. Regression analysis revealed a high correlation between the absorbance values generated with both antigen preparations and suggests that the rCP41 may be used in place of crude antigen. These results indicate that the use of the recombinant CP41 antigen in a standardized serodiagnostic assay could provide a reliable and cost-effective method for assessing human exposure to Cryptosporidium.