Claudia Nagel

Usefulness of PCR Strategies For Early Diagnosis of Chagas' Disease Reactivation and Treatment Follow-Up in Heart Transplantation

Heart transplantation (HTx) is a useful therapy for end‐stage Chagaś cardiomyopathy; however, Chagas reactivation remains a mayor complication. Parasitological methods offer poor diagnostic sensitivity, and use of more sensitive tools such as the Polymerase chain reaction (PCR) is usually necessary. In the present study, reactivation incidence and PCR usefulness for early reactivation diagnosis, as well as for treatment response evaluation during follow‐up, were analyzed using Strout parasite detection test, in 10 of 222 consecutive HTx patients suffering Chagas cardiomyopathy. PCR strategies targeted to minicircle sequences (kDNA, detection limit 1 parasite/ 10 mL blood) and miniexon genes (SL‐DNA, 200 parasite/10 mL) were performed to compare parasite burdens between samples. No patients received prophylactic antiprotozoal therapy (benznidazole). Five patients (50%) exhibited clinical reactivation within a mean period of 71.6 days; positive Strout results were observed in most cases presenting clinical manifestations. kDNA‐PCR was positive 38–85 days before reactivation, whereas SLDNA‐PCR became positive only 7–21 days later, revealing post‐HTx parasitic load enhancement present prior to clinical reactivation development. Reactivations were successfully treated with benznidazole and generated negative PCR results. Results observed in this study indicate the value of PCR testing for an early diagnosis of Chagas reactivation as well as for monitoring treatment efficacy.

Organ Transplantation and Chagas Disease

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Predictores de mortalidad hospitalaria en 186 episodios de endocarditis infecciosa activa en un centro de tercer nivel (1992-2001)

Introduction and objectives. The aim of this study was to describe the predictors of hospital mortality found in patients admitted for infective endocarditis (IE) to a cardiovascular surgery ward. Patients and method. Prospective study of 186 patients with IE treated in our hospital between 1992 and 2001. Results. One hundred fourteen patients (61.3%) had native valve endocarditis and 72 (38.7%) had prosthetic valve endocarditis (early in 28 patients [up to 12 months after surgery] and late in 44 [later than 12 months]). Blood cultures were positive in 82%. The predominant organism was Streptococcus viridans (36%) in native valve endocarditis and Staphylococcus aureus (33%) in prosthetic valve endocarditis. The hospital mortality was 22.6%. Severe sepsis (4.8%) produced a high mortality rate (88%) and was caused by Staphylococcus aureus in 60%. One hundred nineteen patients (64%) required surgery, 79 (66.4%) of them urgently. Negative blood cultures predicted need for surgery in native valve endocarditis (p < 0.05). The surgical mortality was 21.8% and was related to NYHA III-IV class (p = 0.014) and emergency surgery (p = 0.009) in patients with native valve endocarditis. This last factor also predicted higher surgical mortality in patients with early prosthetic valve endocarditis (p < 0.001). Conclusions. The hospital mortality of this group of patients with infective endocarditis treated in a tertiary medical center was high. The presence of severe sepsis, although infrequent, had a somber prognosis. Severe heart failure in native valve endocarditis and urgent surgery in native and prosthetic valve endocarditis increased surgical mortality.

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.

Pandemic influenza A/H1N1 virus infection in solid organ transplant recipients: a multicenter study.

Background. The 2009 novel influenza A/H1N1 virus pandemic did not spare solid organ transplant (SOT) recipients. We aimed to describe the behavior of pandemic influenza infection in a group of SOT recipients in Argentina. Methods. Data from 10 transplant (Tx) centers were retrospectively collected for SOT that presented with a respiratory illness compatible with pandemic influenza A infection, between May and September 2009. Cases were defined as suspected, probable, or confirmed according to diagnostic method. Results. Seventy-seven cases were included. No significant differences in presenting symptoms, pulmonary infiltrates, and graft involvement were found among 35 suspected, 19 probable, and 23 confirmed cases. The 33 ambulatory cases had significantly more sore throat and headache when compared with 34 cases admitted to medical ward (MW) and 10 admitted to intensive care unit (ICU), 9 of whom required ventilatory support. MW and ICU cases had significantly more dyspnea, hypoxemia, pulmonary infiltrates, and graft dysfunction. Time from onset of symptoms to first visit and to treatment was significantly longer in MW and ICU cases (P=0.008). Coinfections were found in six cases. Most cases received oseltamivir for 5 to 10 days. Six patients (7.8%) died from viral infection at a median of 15 days from admission. No differences in outcome were seen related to the transplanted organ, the immunosuppressive regimen, time from Tx, or confirmation of diagnosis. Conclusions. Mortality is higher in Tx recipients than in the general population. Poor outcome seems to be related to a delay in the beginning of treatment.

Early molecular diagnosis of acute Chagas disease after transplantation with organs from Trypanosoma cruzi-infected donors

Organ transplantation (TX) is a novel transmission modality of Chagas disease. The results of molecular diagnosis and characterization of Trypanosoma cruzi acute infection in naïve 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.

Subcutaneous infection by Graphium basitruncatum in a heart transplant patient

Graphium basitruncatum, a synanamorph of Pseudoallescheria has been rarely reported in human infections. We report a case of subcutaneous phaeohyphomycosis caused by this fungus in a heart transplant recipient. We also describe the phenotypic, molecular methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) used to achieve isolate identification.

Phenotypic and molecular identification of Coccidioides posadasii in a patient evaluated for bilateral lung transplantation

BACKGROUND Coccidioidomycosis is an endemic fungal infection caused by Coccidioides immitis and Coccidioides posadasii. It can be particularly severe in transplant recipients that have a current or a previous coccidioidal infection. Fatal case of coccidioidomycosis has been described in this group of patients. AIMS We report a severe case of pneumonia caused by C. posadassi in a 29 year-old white woman that had been admitted to hospital as part of the evaluation for bilateral lung transplantation. The patient was a native and resident of Catamarca, Argentina. Molecular methodologies contributed to the species identification. METHODS Clinical, laboratory records and microbiological tests were carried out to diagnose the infection and to identify C. posadasii. RESULTS A fungus was isolated from BAL culture. Phenotypic characterization, specific PCR and experimental animal inoculation demonstrated the presence of C. posadasii. The patient responded well to amphotericin B deoxycholate. Lung transplantation was postponed. CONCLUSIONS Specific PCR can be an important alternative for the correct identification of C. immitis or C. posadasii in laboratories with implemented molecular biology tools. This case emphasizes the need for a systematic assessment in organ transplant units of patients inhabiting endemic areas of coccidioidomycosis.