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.

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.