Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (Tm) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the Tm values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate.

Perinatal Screening for Chagas Disease in Southern Texas.

Perinatal screening for Trypanosoma cruzi in a cohort of 4000 predominantly Hispanic women in southern Texas revealed that Chagas disease occurs with sufficient frequency (0.25%) that targeted perinatal screening should be considered to identify infected mothers and infants at risk for congenital infection.

Chagas disease in Latin American immigrants with dilated cardiomyopathy in New York City.

Chagas disease-associated cardiomyopathy is clinically similar to other causes of cardiomyopathy and, therefore, the diagnosis can be easily overlooked. We found a 13% point prevalence of Chagas disease in a sample of New York City immigrants with dilated cardiomyopathy.

Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach.

BACKGROUND: The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. METHODS/PRINCIPAL FINDINGS: Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. CONCLUSIONS/SIGNIFICANCE: These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.

Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2.

Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach (in vitro culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were Leishmania (Viannia) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. (Leishmania) tropica versus L. (L.) major, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.

Autonomic dysfunction and risk factors associated with Trypanosoma cruzi infection among children in Arequipa, Peru.

Chagas disease affects an estimated 8 million people in Latin America. Infected individuals have 20-30% lifetime risk of developing cardiomyopathy, but more subtle changes in autonomic responses may be more frequent. We conducted a matched case-control study of children in Arequipa, Peru, where triatomine infestation and Trypanosoma cruzi infection are emerging problems. We collected data on home environment, history, physical examination, electrocardiogram, and autonomic testing. Signs of triatomine infestation and/or animals sleeping in the child's room and household members with Chagas disease were associated with increased infection risk. Electrocardiogram findings did not differ between cases and controls. However, compared with control children, infected children had blunted autonomic responses by three different measures, the Valsalva maneuver, the cold pressor test, and the orthostatic test. T. cruzi-infected children show autonomic dysfunction, although the prognostic value of this finding is not clear. Sustained vector control programs are essential to decreasing future T. cruzi infections.

Usefulness of PCR method for detection of Leishmania in Poland.

Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations.

Geographic variation in the sensitivity of recombinant antigen-based rapid tests for chronic Trypanosoma cruzi infection.

Chagas disease affects 8-11 million people throughout the Americas. Early detection is crucial for timely treatment and to prevent non-vectorial transmission. Recombinant antigen-based rapid tests had high sensitivity and specificity in laboratory evaluations, but no Peruvian specimens were included in previous studies. We evaluated Stat-Pak and Trypanosoma Detect rapid tests in specimens from Bolivia and Peru. Specimens positive by three conventional assays were confirmed positives; specimens negative by two or more assays were confirmed negatives. In Bolivian specimens, Stat-Pak and Trypanosoma Detect tests were 87.5% and 90.7% sensitive, respectively; both showed 100% specificity. Sensitivity in Peruvian specimens was much lower: 26.6-33.0% (Stat-Pak) and 54.3-55.2% (Trypanosoma Detect); both had specificities > 98%. Even in Bolivian specimens, these sensitivities are inadequate for stand-alone screening. The low sensitivity in Peru may be related to parasite strain differences. Chagas disease rapid tests should be field tested in each geographic site before widespread implementation for screening.

Chagas disease transmission in periurban communities of Arequipa, Peru.

BACKGROUND: Chagas disease, caused by Trypanosoma cruzi infection, is an urban problem in Arequipa, Peru, and the epidemiology of Chagas disease is likely to be quite different in this area, compared with in rural zones. METHODS: We conducted a serosurvey of 1615 children <18 years old in periurban districts that included hillside shantytowns and slightly more affluent low-lying communities. In addition, 639 adult residents of 1 shantytown were surveyed to provide data across the age spectrum for this community. RESULTS: Of 1615 children, 75 (4.7%) were infected with Trypanosoma cruzi. Infection risk increased by 12% per year of age, and children living in hillside shantytowns were 2.5 times as likely to be infected as were those living in lower-lying communities. However, age-prevalence data from 1 shantytown demonstrated that adults were no more likely to be seropositive than were teenagers; the results of maximum likelihood modeling suggest that T. cruzi transmission began in this community <20 years ago. CONCLUSIONS: The problem of Chagas disease in periurban settings, such as those around Arequipa, must be addressed to achieve elimination of vector-borne T. cruzi transmission. Identification of infected children, vector-control efforts, and education to avoid modifiable risk factors are necessary to decrease the burden of Chagas disease.

Canine visceral leishmaniasis, United States and Canada, 2000-2003.

Visceral leishmaniasis, caused by protozoa of the genus Leishmania donovani complex, is a vectorborne zoonotic infection that infects humans, dogs, and other mammals. In 2000, this infection was implicated as causing high rates of illness and death among foxhounds in a kennel in New York. A serosurvey of >12,000 foxhounds and other canids and 185 persons in 35 states and 4 Canadian provinces was performed to determine geographic extent, prevalence, host range, and modes of transmission within foxhounds, other dogs, and wild canids and to assess possible infections in humans. Foxhounds infected with Leishmania spp. were found in 18 states and 2 Canadian provinces. No evidence of infection was found in humans. The infection in North America appears to be widespread in foxhounds and limited to dog-to-dog mechanisms of transmission; however, if the organism becomes adapted for vector transmission by indigenous phlebotomines, the probability of human exposure will be greatly increased.