Temporal Fluctuation of Infection with Different Trypanosoma cruzi Genotypes in the Wild Rodent Octodon degus

We identified and followed-up for two years Octodon degus rodents infected with Trypanosoma cruzi genotypes by using xenodiagnosis with two vector species (Mepraia spinolai and Triatoma infestans), polymerase chain reaction DNA-based detection of insect dejections, Southern blot analysis, and minicircle hybridization with genotype-specific probes. Results show temporal fluctuations of infection with four parasite lineages (TCI, TCII, TCV, and TCVI) in one co-infected O. degus. Results are discussed in the context of parasitemia level and infection control in mammal hosts.Chagas disease is a vector-borne zoonosis caused by the protozoa Trypanosoma cruzi. This taxon had been described as composed of two lineages (TCI and TCII) and five subgroups (IIa–IIe), but a recent study reported six lineages or discrete typing units (DTUs) (T. cruzi I–VI).¹ These lineages are defined as sets of stocks that are genetically more related to each other than to any other stock and are identifiable by common genetic molecular and immunologic markers.²Trypanosoma cruzi populations circulate in nature in multiple T. cruzi genotypes that coexist in different hosts, including Octodon degus rodents.^3–5 After a short acute or primary infection, the mammal host sustains subclinical infections, which are microscopically undetectable in peripheral blood during the undetermined and chronic phases. Conversely, parasitemia in those phases is detected only by polymerase chain reaction (PCR). The classic parasitologic diagnostic method for Chagas disease xenodiagnosis, which can amplify T. cruzi after feeding on infected hosts.⁶ Although xenodiagnosis is specific, it lacks sensitivity and is limited to high levels of parasitemia.⁷ The epidemiology of Chagas disease and clinical symptoms are associated with the infective T. cruzi genotypes.⁸ Therefore, would be useful to know the dynamics of these genotypes.In the present study, we assess the occurrence of temporal fluctuations of T. cruzi DTUs in peripheral blood of two naturally infected wild reservoir specimens of O. degus by using a combination of two diagnosis methods: 1) xenodiagnosis with domestic and sylvatic vectors (Triatoma infectans and Mepraia spinolai), respectively, and 2) PCR DNA-based detection specific for minicircles and hybridization analyses with T. cruzi genotype-specific probes.Ten nymphs (stages II and III) of each vector species were allowed to feed simultaneously on anesthetized O. degus rodents for 30 minutes or until engorgement on the rodent (mean ± SD weight of ingested blood = 0.2 ± 0.05 mg). After 30 days, feces and intestinal contents of the triatomines were observed under a light microscope. The minimal theoretical parasitemia detected under these conditions is approximately 5 parasites/mL (1 parasite/0.2 mL). However, because several but not all insects (2–5) were parasite positive by visual examination, the estimated parasitemia would be > 10–25 parasites/mL.After microscopic inspection, the intestinal contents of each vector species pool was collected and PCR was performed as reported.⁹ Amplicons were subjected to electrophoresis on an agarose gel and transferred to nylon membranes. Copies of these membranes were hybridized separately with each probe under high stringency conditions.³ Construction of genotype-specific probes was performed as described.¹⁰ Different T. cruzi clones were used as templates to generate DNA probes to determine parasite genotypes. The probes were P³²-labeled.⁴A total of 35 O. degus were captured at the field and analyzed. Overall, only two O. degus showed infection with both vector species and six were positive only for M. spinolai.⁹ The two O. degus samples positive for both vector species were subjected to serial xenodiagnosis to determine the genotype of the T. cruzi population circulating at different times: time 0, one year, two years, and two and a half years. Results for O. degus sample 5, which was infected with a one genotype (TCI), are shown in Figure 1. This result was confirmed with both vector species at different times. Results obtained with O. degus sample 8 showed mixed infection with DTUs TCI, TCII, and TCVI at time zero for M. spinolai, but only TCII for T. infestans. However, one year later, both vectors showed mixed infections with lineages TCI and TCV. After two years, both vectors contained only genotype TCII. After two and a half years, vectors were still infected with TCII.Open in a separate windowFigure 1.Hybridization patterns of xenodiagnosis samples from Mepraia spinolai (sp) and Triatoma infestans (i) and staining with ethidium bromide (EB) and Southern blot analyses with specific probes (TCI, TCII, TCV, and TCVI) for xenodiagnosis samples at A, time 0 (i.e., immediately after capture); B, one year later, C, two years later, and D, two and a half years later. Numbers 5 and 18 correspond to identification numbers for Octodon degus rodents.Trypanosoma cruzi colonizes several tissues and evades the immune response by a concomitant low parasitemia level not detectable by several diagnosis methods.¹¹ Parasites circulate as mixed infections. This finding is common for T. cruzi because several mammals and vectors are infected with more than one T. cruzi genotype,^4,5 which results in recombination and hybrid genotypes.⁸We report that infection of rodents can show temporal fluctuations with different T. cruzi genotypes, which is probably the result of fluctuation of relative proportions of parasite loads of different genotypes in peripheral blood. We detected infections in this O. degus with at least three of the four T. cruzi genotypes during the complete follow-up (xenodiagnosis at time 0). Two genotypes (TCII and TCVI) disappeared, and another one (TCV) appeared one year later. During the second year, only one genotype (TCII) was detected and maintained. A different scenario was detected for O. degus sample 5, which showed infection with only TCI during the entire sampling period.In this study, we preferentially detected genotype TCII in both vector species. This genotype was likely circulating at high parasitemia levels in O. degus sample 18 because experimental infections in T. infestans with different T. cruzi DTUs indicated that genotype TCII is transmitted at a low rate; genotype TCI is transmitted at a high rate.¹² Our results for T. cruzi genotypes in these two animals are consistent with local prevalence in the study area.⁴ Recent studies of T. cruzi genotypes circulating in the wild vector in this disease-endemic area showed that TCI and TCII are the most prevalent genotypes.⁵We suggest that both rodent species showed moderate or high levels of parasitemia. We used xenodiagnosis with two triatomine species because insect vectors amplify T. cruzi in the midgut, which enables easy detection. Our results indicate fluctuation in specific genotype infections in a T. cruzi-infected sylvatic rodent.The temporal fluctuation of the four T. cruzi genotypes could be explained by at least two hyptheses that are not mutually exclusive. First, colonization of different tissues with T. cruzi described in patients and experimentally infected animals with organ damage^11,13 releases T. cruzi into the vascular system; these parasites then colonize other tissues. Second, infection is controlled by the immune system. Both processes might reach an equilibrium and explain the low parasitemia levels observed in immunocompetent patients in the chronic phase of Chagas disease. Future parasitologic studies of molecular pathogenesis may be necessary to understand the mechanisms underlying infection control in naturally infected hosts.     

Loss of BAP1 Expression Occurs Frequently in Intrahepatic Cholangiocarcinoma

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor gene. Double hit BAP1 inactivation has been reported in a range of tumor types, including intrahepatic cholangiocarcinoma (ICC), sometimes in association with germline mutation.We performed immunohistochemistry for BAP1 on a well-characterized cohort of 211 ICC patients undergoing surgical resection with curative intent at 3 institutions based in 3 different countries. The median age at diagnosis was 65 years (range, 36.5–86) and 108 (51%) were men. Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in nonneoplastic cells) occurred in 55 ICCs (26%). BAP1 loss predicted a strong trend toward improved median survival of 40.80 months (95% CI, 28.14–53.46) versus 24.87 months (95% CI, 18.73–31.01), P = 0.059). In a multivariate model including age, sex, BAP1 status, tumor stage, tumor grade, lymphovascular invasion, and tumor size, female sex was associated with improved survival (hazard ratio [HR] 0.54; 95% CI, 0.34–0.85), while advanced tumor stage and lymphovascular invasion (HR 1.89; 95% CI, 1.09–3.28) correlated with decreased survival. In a multivariate analysis, high grade tumors were associated with BAP1 loss (odds ratio [OR] 3.32; 95% CI, 1.29–8.55), while lymphatic invasion was inversely associated with BAP1 loss (OR 0.36; 95% CI, 0.13–0.99).In conclusion, we observed a trend toward improved prognosis in ICC associated with absent expression of BAP1 and an association of BAP1 loss with higher histological grade and absent lymphatic invasion. Female sex was associated with improved survival while advanced tumor stage and lymphatic invasion were associated with decreased survival.     

Comprehensive review of telbivudine in pregnant women with chronic hepatitis B

AIM:To achieve an evidence-based conclusion regarding the safety and efficacy of telbivudine during pregnancy.METHODS:A pooled analysis of data from a literature search reported 1739 pregnancy outcomes(1673 live births)from 1725 non-overlapping pregnant women treated with telbivudine.The prevalence of live birth defects(3.6/1000)was similar to that of the nonantiviral controls(3.0/1000)and not increased as compared with overall prevalence(14.5 to 60/1000).No target organ toxicity was identified.The prevalence of spontaneous abortion in pregnant women treated with telbivudine(4.2/1000)was not increased compared with the overall prevalence(16/1000).The mother-to-child transmission rate was significantly reduced in pregnant women treated with telbivudine(0.70%)compared to those treated with the non-antiviral controls(11.9%;P0.0001)or compared to the historical rates of hepatitis B virus(HBV)-infected population without antiviral treatment(10%-15%).RESULTS:Cumulatively 489 pregnancy cases have been reported in the telbivudine pharmacovigilance database(with a cut-off date 31 August 2014),of those,308 had known pregnancy outcomes with 249 cases of live births(239 cases of live birth without congenital anomaly and 10 cases of live birth with congenital anomaly).In the latest antiretroviral pregnancy registry report(1 January 1989 through 31 January 2015)of27 patients exposed to telbivudine during pregnancy(18,6 and 3 during first,second and third trimester,respectively)19 live births were reported and there were no cases of birth defects reported.CONCLUSION:Telbivudine treatment during pregnancy presents a favorable safety profile without increased rates of live birth defects,spontaneous abortion or elective termination,or fetal/neonatal toxicity.Exposure to telbivudine in the first,second and third trimester of pregnancy has been shown to significantly reduce the risk of HBV transmission from mother to child on the basis of standard immune prophylaxis procedure.     

Influence of Tooth-Brushing on Early Healing after Access Flap Surgery: A Randomized Controlled Preliminary Study

In the present study, the clinical outcomes obtained using three different protocols of post-operative plaque control for the 4 weeks after surgery were compared. Thirty healthy subjects, presenting at least one periodontal pocket requiring resective surgery, were selected and randomly distributed to three different groups corresponding to respective post-surgical protocols: (A) toothbrushes + chlorhexidine + anti-discoloration system (ADS + CHX); (B) toothbrushes + chlorhexidine (CHX); (C) only toothbrushes. The full-mouth plaque score (FMPS), full-mouth bleeding score (FMBS), probing pocket depth (PPD), recession depth (REC), clinical attachment level (CAL), and bleeding on probing (BoP) were measured in six aspects per tooth (mesio-buccal (MB), buccal (B), disto-buccal (DB), disto-lingual (DL), lingual (L), and mesio-lingual (ML)) at baseline, 3 months, and 6 months after surgery. FMPS and FMBS did not significantly change (p > 0.05), whereas PPD and CAL significantly decreased, and REC significantly increased in all groups during the study (p < 0.05). Clinical results were satisfactory in all cases, with no significant differences between groups 3 months after surgery. Six months after surgery, only PPD-MB was significantly different in the three groups (p < 0.05). Nevertheless, this value was not clinically relevant because the value of PPD-B (about 2 mm) in group C was physiologic. The mechanical plaque control was proven to be fundamental and sufficient in all the six aspects per tooth to guarantee an excellent clinical outcome without the need of chemical plaque control.     

Adiponectin plasma levels decrease after surgery in pediatric patients with congenital heart disease

ObjectiveAdiponectin is a protein secreted by adipose tissue and involved in inflammatory process as well as in metabolic regulation. The aim of this study was to examine the response of plasma adiponectin to cardiac surgery in children with congenital defects to determine whether its measurement is associated to the response to injury.Design and methodsTwenty-five pediatric patients undergoing heart surgery for correction of congenital defects were studied. Adiponectin plasma levels, obtained pre- and three times postoperatively, were determined by dedicated ELISA. Brain natriuretic peptide (BNP) plasma levels were also determined.ResultsAdiponectin levels are highest in the first month of life (p = 0.004 newborns vs. children) with a progressive fall in the next few years. After surgery, adiponectin increased slowly over a 1-month period, following an initial decrease in the first 3 days.ConclusionsAdiponectin could be involved in the acute response to injury although further investigation into the relationship between adiponectin, glucose regulation and inflammatory process is necessary to examine the issue of the adiponectin decrease after surgery from a more integrated prospective.     

Pharmacological LXR activation reduces presence of SR-B1 in liver membranes contributing to LXR-mediated induction of HDL-cholesterol

ObjectivePharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated.MethodsWe investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317.ResultsHDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1 week treatment. T0901317 treatment induced fecal neutral sterol secretion by ～300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body.ConclusionInduction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion.     

The impact of chronic obstructive pulmonary disease in patients hospitalized for worsening heart failure with reduced ejection fraction: an analysis of the EVEREST Trial

BackgroundChronic obstructive pulmonary disease (COPD) is prevalent in heart failure (HF) patients, yet these patients are poorly characterized. We aimed to describe the characteristics and outcomes of patients with systolic dysfunction and COPD in a contemporary HF randomized trial.Methods and ResultsEVEREST investigated 4,133 patients hospitalized with worsening HF and an ejection fraction (EF) ≤40%. We analyzed the characteristics and outcomes (all-cause mortality and cardiovascular mortality/HF hospitalization) of patients according to baseline COPD status. COPD was present in 10% (n = 416) of patients. Patients with COPD had a higher prevalence of comorbidities and were less likely to receive a β-blocker, angiotensin-converting enzyme inhibitor, or aldosterone antagonist. On univariate analysis, COPD was associated with increased all-cause mortality (HR 1.41, 95% CI 1.18–1.67) and cardiovascular mortality/HF hospitalization (HR 1.29, 95% CI 1.11–1.49). After adjusting for potential confounders, the risk associated with COPD remained increased, but was not statistically significant.ConclusionThe presence of COPD in HF patients is associated with an increased burden of comorbidities, lower use of HF therapies, and a trend toward worse outcomes. These findings provide a starting point for prospective investigations of the treatment of HF comorbidities to reduce the high postdischarge event rates.     

Beneficial effect of sulphate-bicarbonate-calcium water on gallstone risk and weight control

AIM: To investigate the effect of drinking sulphate-bicarbonate-calcium thermal water (TW) on risk factors for atherosclerosis and cholesterol gallstone disease.METHODS: Postmenopausal women with functional dyspepsia and/or constipation underwent a 12 d cycle of thermal (n = 20) or tap (n = 20) water controlled drinking. Gallbladder fasting volume at ultrasound, blood vitamin E, oxysterols (7-β-hydroxycholesterol and 7-ketocholesterol), bile acid (BA), triglycerides, total/low density lipoprotein and high density lipoprotein cholesterol were measured at baseline and at the end of the study. Food consumption, stool frequency and body weight were recorded daily.RESULTS: Blood lipids, oxysterols and vitamin E were not affected by either thermal or tap water consumption. Fasting gallbladder volume was significantly (P < 0.005) smaller at the end of the study than at baseline in the TW (15.7 ± 1.1 mL vs 20.1 ± 1.7 mL) but not in the tap water group (19.0 ± 1.4 mL vs 19.4 ± 1.5 mL). Total serum BA concentration was significantly (P < 0.05) higher at the end of the study than at baseline in the TW (5.83 ± 1.24 μmol vs 4.25 ± 1.00 μmol) but not in the tap water group (3.41 ± 0.46 μmol vs 2.91 ± 0.56 μmol). The increased BA concentration after TW consumption was mainly accounted for by glycochenodeoxycholic acid. The number of pasta (P < 0.001), meat (P < 0.001) and vegetable (P < 0.005) portions consumed during the study and of bowel movements per day (P < 0.05) were significantly higher in the TW than in the tap water group. Body weight did not change at the end of the study as compared to baseline in both groups.CONCLUSION: Sulphate-bicarbonate-calcium water consumption has a positive effect on lithogenic risk and intestinal transit and allows maintenance of a stable body weight despite a high food intake.     

Low triiodothyronine and exercise capacity in heart failure

Background

Cardiopulmonary exercise test (CPT) has a prominent value in assessing clinical severity in chronic heart failure (HF) patients. Reduced free triiodothyronine (fT3) plasma level is associated with a more severe disease and prognosis. The aim of this study was to evaluate the relationship between low fT3 plasma level and reduced exercise capacity in chronic HF, and to determine the influence of a low T3 status in subsets of patients with different functional impairment.

Methods and results

240 HF patients (79% males; age 62 ± 12 years, mean ± standard deviation; left ventricular ejection fraction, EF, 30 ± 9%) underwent a CPT, clinical and neurohormonal characterization (assay for plasma brain natriuretic peptide, BNP, norepinephrine, aldosterone, renin activity, fT3, free T4, thyroid-stimulating hormone). At multivariate analysis in the whole population, age, gender and BNP level were independently associated with peak VO2, whereas in patients with severe functional impairment (peak VO2 < 14 ml/min/kg) fT3 resulted independently related to peak VO2, together with gender and BNP. When patients with peak VO2 < 14 ml/min/kg were divided according to fT3 levels, patients with low T3 syndrome showed reduced exercise capacity and worse ventilatory efficiency.

Conclusions

BNP and fT3 are independently associated with exercise capacity in severely compromised HF patients.     

Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

A high incidence of cytomegalovirus (CMV) infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB) gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6%) patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/microg DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/microg DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients.