Extracción de ADN de Trypanosoma cruzi mediante tratamiento con bromuro de hexadecil-trimetil-amonio

The present work describes a fast, simple and efficient method of isolating pure and easily handling of genomic DNAfrom Trypanosoma cruzi This protocol is based on parasite lysis with SDS and the removal of proteins by digestion with proteinase K, followed by polysaccharides and remaining proteins' selective precipitation with CTAB. Finally, the DNA is extracted with chloroform-isoamyl alcohol and is recovered from the aqueous supernatant by isopropanol precipitation.En el presente trabajo se describe un método rápido, sencillo y eficaz para la obtención de ADN genómico de Trypanosoma cruzi, libre de impurezas y fácil de manipular. Dicho procedimiento se basa en la lisis del parásito con SDS y remoción de proteínas mediante la digestión con proteinasa K, seguida de la precipitación selectiva de carbohidratos y proteínas residuales con bromuro de hexadecil-trimetil-amonio (CTAB). Finalmente, el ADN se extrae con cloroformo: alcohol isoamílico y se recupera de la fase acuosa mediante precipitación con isopropanol

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Safety profile of nifurtimox and treatment interruption for chronic Chagas disease in Colombian adults

Nifurtimox (NFX) is one of the approved drugs used to treat Chagas disease. Safety profile studies and models on risk factors for treatment interruption in adults are scarce in Latin America. This study evaluated retrospectively the medical records of adult Chagas disease patients treated with NFX between 2007 and 2012 in Bogotá, Colombia. An accelerated failure time model was used, and associations were expressed as time ratio (TR). In total, 76 adult patients with NFX were included: 60 (79.0%) completed 60 days of treatment, 61 (80.3%) presented adverse drug reactions (ADRs), and 16 (21.0%) required treatment interruption. The predominant symptoms were epigastric pain (23.7%), nauseas (18.4%), sleep disturbances (18.4%), loss of appetite (17.1%), and temporary loss of memory (15.2%). ADRs were classified as mild (64.5%), moderate (30.4%), and severe (5.1%). Time of treatment was significantly longer when presenting ≤ 3 ADRs (TR: 1.78; 95% CI: 1.04–3.03), presence of non-severe ADRs (TR: 6.52; 95% CI: 3.24–13.1), doses of NFX ≤ 8 mg/kg/day (TR: 1.78; 95% CI: 0.90–3.49), and age < 48 years (TR: 1.57; 95% CI: 0.90–2.74). Treatment with NFX in adults caused a high frequency of ADRs, but most of the cases were mild and did not require treatment interruption. Severity and number of ADRs were the main predictors for treatment interruption

Standardization and evaluation of ELISA for the serodiagnosis of amoebic liver abscess

An ELISA test for the serological diagnosisof amoebic liver abscess (ALA) was standardized and evaluated in sera from three groups of patients: (1) three patients with diagnosis confirmed by isolation of the parasite,(2) thirty seven patients with diagnosis established by clinical findings and ultrasound studies and (3) seven patients whose diagnosis were established by clinical findings and a positive double immunodifusion test. Ninety one serum samples from healthy subjects and 22 from patients with other liver or parasitic diseases were also included in the study. the optimum concentration of Entamoeba histolytica antigen was 1.25 µg/ml and optimum dilutions of serum and anti-human IgG-alkaline phosphatase conjugate were 1:400 and 1:4000 respectively. The cut-off point of the ELISA test in this study was an absorbance value of 0.34. The test parameters were: sensitivity = 95.7 per cent, specificty = 100 per cent, positive predictive value = 100 per cent and negative predictive value = 98.2 per cent.The ELISA test was found to be of great use as a diagnostic tool for the establishment of amoebic etiology in patients with clinical supposition of ALA. The test could also be used for seroepidemiological surveys of the prevalence of invasive amoebiasis in a given population, since it allows the processing of a greater number of samples at a lower cost tahn other serological tests

Follow-up of an asymptomatic chagas disease population of children after treatment with nifurtimox (lampit) in a sylvatic endemic transmission area of Colombia

Background Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi. Two drugs are currently used for the etiological treatment of the disease: Nifurtimox (Lampit) and Benznidazole. This study presents a quasi-experimental trial (non-control group) of sixty-two patients who were treated for Chagas disease with Nifurtimox (Lampit), and were then followed for 30 months post-treatment. The safety of Nifurtimox (Lampit) for Chagas disease in this group of children primarily between 4 and 19 years old was also evaluated. Materials and methods The 62 patients included in the study were selected when resulted seropositive for two out of three fundamentally different serological tests. All children were treated during two months according to protocols established by WHO. Monitoring was performed every twenty days to evaluate treatment safety. In 43 patients, two different serological tests: ELISA and IFAT; and two parasitological tests: blood culture, and real time PCR, (qPCR) were performed to assess therapeutic response, defined as post-treatment serological negativization. Principal findings All patients completed the treatment successfully, and six patients abandoned the post-treatment follow-up. Adverse effects occurred in 74% of patients, but only 4.8% of cases required temporary suspension to achieve 100% adherence to the 60-day treatment, and all symptoms reverted after treatment completion. Both parasite load (measured through qPCR) and antibodies (ELISA absorbance) evidenced a significant median reduction 6 months after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 0.6 to 0.2 absorbance units respectively (p<0.001). Serological negativization by ELISA was evident since 6 months post-treatment, whereas by IFAT only after 18 months. Serological negativization by the two tests (ELISA and IFAT) was 41.9% (95%CI: 26.5–57.3) after 30 months post-treatment. qPCR was positive in 88.3% of patients pre-treatment and only in 12.1% of patients after 30 months. Survival analysis indicated that only 26.3% (95%CI: 15.5–44.8) persisted with negative qPCR during the whole follow-up period. Conclusions Nifurtimox was very well tolerated and successfully reduced parasite load and antibody titers. Re-infection, lysed parasites or a lack of anti-parasitic activity could explain these persistently positive qPCR cases

Risk factors associated with Chagas disease in pregnant women in Santander, a highly endemic Colombian area

Objective To determine the prevalence and risk factors associated with Chagas disease in pregnant women in an endemic area of Santander, Colombia. Methods Cross-sectional study included 23 municipalities of Santander, Colombia. Serological IFAT and ELISA tests were undertaken to detect IgG anti- Trypanosoma cruzi. A questionnaire was conducted for assessing the risk factors of each participant. Newborns were evaluated at birth and followed up to 1 year of age to determine congenital infection. Results An overall prevalence of 3.2% (95% CI 2.4–4.2) among 1518 pregnant women was detected. Prevalences by provinces were as follows: Guanentina: 6.0% (95% CI 4.1–8.5), García Rovira: 2.9% (95% CI: 1.5–4.8) and Comunera: 0.4% (0.4–2.3). The main risk factors identified were age >32 years old (OR: 2.1; 95% CI: 1.1–3.9); currently having a thatched roof (OR: 11.8; CI95% 2.2–63.2) and a thatched roof during childhood (OR: 3.0; 95% CI: 1.4–6.6); having below primary school education level (OR: 4.6; 95% CI: 2.2–9.5); and a history of a close contact with the vector (triatomine bugs) at least once during their lifetime (OR: 6.9; 95% CI: 3.7–12.9). No congenital cases were detected by parasitological or serological techniques. Conclusions Prevalence of Chagas disease in pregnant women is a potential source of infection in this Colombian endemic area. The main risk factors associated with seropositivity were related to conditions favouring the contact with the vector. The results show that it is necessary to continue an active surveillance in order to offer diagnosis and treatment to mothers and their newborns in addition to screening to pregnant women from endemic areas