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 FindingsThe 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/SignificanceTyping 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.

Local levels of vancomycin were 35 times greater than the highest levels reported with vancomycin-loaded PMMA. A synergistic effect was observed between vancomycin released from impacted allografts and aminoglycoside-loaded PMMA.

Chagas' disease is an endemic zoonosis of South America caused by a protozoan parasite Trypanosoma cruzi. About 30% of infected people develop the disease. This disease is known to reactivate in immunocompromised hosts, such as patients with acquired immunodeficiency syndrome, leukemia, and transplantation. There is some experience with transplantation of infected renal grafts into negative recipients, resulting in an index of transmission of 35%. No cases have been reported involving other organ transplants up to 2002, when the Centers for Disease Control and Prevention reported 3 cases of Chagas' disease transmission to 3 recipients (liver, kidney, and pancreas‐kidney) from a single chagas infected donor. Here we report on a case of orthotopic liver transplant from a chagas infected donor into a negative recipient in clinical emergency status. The recipient was monitored by direct parasitological Strout method and serological tests with detection of transmission on the 84th day by both studies, without clinical signs. The patient was put on benznidazole with rapid clearance of the parasitemia. However, we propose that chagas infected donors may be accepted for liver transplant recipients only in emergency status. (Liver Transpl 2005;11:1112–1116.)

Organ transplantation (TX) is a novel transmission modality of Chagas disease. The results of molecular diagnosis and characterization of Trypanosoma cruzi acute infection in na€ ıve TX recipients transplanted with organs from infected deceased donors are reported. Peripheral blood and cerebrospinal fluid samples from the TX recipients of organs from infected donors were prospectively and sequentially studied for detection of T. cruzi by means of kinetoplastid DNA polymerase chain reaction (kDNA-PCR). In positive blood samples, a PCR algorithm for identification of T. cruzi Discrete Typing Units (DTUs) and quantitative real-time PCR (qPCR) to quantify parasitic loads were performed. Minicircle signatures of T. cruzi infecting populations were also analyzed using restriction fragment length polymorphism (RFLP)-PCR. Eight seronegative TX recipients from four infected donors were studied. In five, the infection was detected at 68.4 days post-TX (36-98 days). In one case, it was transmitted to two of three TX recipients. The comparison of the minicircle signatures revealed nearly identical RFLP-PCR profiles, confirming a common source of infection. The five cases were infected by DTU TcV. This report reveals the relevance of systematic monitoring of TX recipients using PCR strategies in order to provide an early diagnosis allowing timely anti-trypanosomal treatment.