Efficacy of continuous versus intermittent administration of nanoformulated benznidazole during the chronic phase of Trypanosoma cruzi Nicaragua infection in mice.

BACKGROUND: Benznidazole and nifurtimox are effective drugs used to treat Chagas' disease; however, their administration in patients in the chronic phase of the disease is still limited, mainly due to their limited efficacy in the later chronic stage of the disease and to the adverse effects related to these drugs. OBJECTIVES: To evaluate the effect of low doses of nanoformulated benznidazole using a chronic model of Trypanosoma cruzi Nicaragua infection in C57BL/6J mice. METHODS: Nanoformulations were administered in two different schemes: one daily dose for 30 days or one dose every 7 days, 13 times. RESULTS: Both treatment schemes showed promising outcomes, such as the elimination of parasitaemia, a reduction in the levels of T. cruzi-specific antibodies and a reduction in T. cruzi-specific IFN-γ-producing cells, as well as an improvement in electrocardiographic alterations and a reduction in inflammation and fibrosis in the heart compared with untreated T. cruzi-infected animals. These results were also compared with those from our previous work on benznidazole administration, which was shown to be effective in the same chronic model. CONCLUSIONS: In this experimental model, intermittently administered benznidazole nanoformulations were as effective as those administered continuously; however, the total dose administered in the intermittent scheme was lower, indicating a promising therapeutic approach to Chagas' disease.

Incidence and risk factors of bacterial sepsis and invasive fungal infection in neonates and infants requiring major surgery: an Italian multicentre prospective study.

BACKGROUND: Limited data are currently available on the incidence rates and risk factors for bacterial sepsis and invasive fungal infections (IFIs) among neonates and infants undergoing major surgery. AIM: To assess the incidence of bacterial sepsis and IFI, fungal colonization, risk factors for sepsis, and mortality in neonates and infants aged <3 months undergoing major surgery. METHODS: A multicentre prospective study was conducted involving 13 level-3 neonatal intensive care units in Italy, enrolling all infants aged ≤3 months undergoing major surgery. FINDINGS: From 2018 to 2021, 541 patients were enrolled. During hospitalization, 248 patients had a bacterial infection, and 23 patients had a fungal infection. Eighty-four patients were colonized by fungal strains. Overall, in-hospital mortality was 2.8%, but this was higher in infected than in uninfected infants (P = 0.034). In multivariate analysis, antibiotic exposure before surgery, ultrasound-guided or surgical placement of vascular catheters, vascular catheterization duration, and gestational age ≤28 weeks were all associated with bacterial sepsis. The risk of IFI was markedly higher in colonized infants (odds ratio (OR): 8.20; P < 0.001) and was linearly associated with the duration of vascular catheterization. Fungal colonization in infants with abdominal surgery increased the probability of IFI 11-fold (OR: 11.1; P < 0.001). CONCLUSION: Preventive strategies such as early removal of vascular catheters and the fluconazole prophylaxis should be considered to prevent bacterial and fungal sepsis in infants undergoing abdominal surgery, and even more so in those with fungal colonization.

Does injection of plasma rich in growth factors after temporomandibular joint arthroscopy improve outcomes in patients with Wilkes stage IV internal derangement? A randomized prospective clinical study.

The aim of this study was to evaluate the efficacy of injection of plasma rich in growth factors (PRGF) after temporomandibular joint (TMJ) arthroscopy in patients with Wilkes stage IV internal derangement. Ninety-two patients were randomized to two experimental groups: group A (42 joints) received injections of PRGF, group B (50 joints) received saline injections. Pain intensity on a visual analogue scale (VAS) and maximum mouth opening (MMO, mm) were measured before and after surgery and compared by analysis of variance (ANOVA). The mean age of patients was 35.8 years (range 17-67 years); 86 were female. Significant reductions in pain were noted in both groups after surgery: VAS 7.9 preoperative and 1.4 at 24 months postoperative. Significantly better clinical results were achieved in group A than in group B only at 6 and 12 months postoperative; no significant difference was noted at 18 or 24 months after the surgical intervention. MMO increased after surgery in both groups: 26.2mm preoperative and 36.8mm at 24 months postoperative. No significant differences in MMO were found when the two groups of patients were compared. In conclusion, the injection of PRGF does not add any significant improvement to clinical outcomes at 2 years after surgery in patients with advanced internal derangement of the TMJ.

Is the infectiousness of dogs naturally infected with Trypanosoma cruzi associated with poly-parasitism?

Interactions among different species of parasites co-infecting the same host could be synergistic or antagonistic. These interactions may modify both the frequency of infected hosts and their infectiousness, and therefore impact on transmission dynamics. This study determined the infectiousness of Trypanosoma cruzi-seropositive dogs (using xenodiagnosis) and their parasite load (quantified by qPCR), and tested the association between both variables and the presence of concomitant endoparasites. A cross-sectional serosurvey conducted in eight rural villages from Pampa del Indio and neighboring municipalities (northeastern Argentina) detected 32 T. cruzi-seropositive dogs out of 217 individuals examined for infection. Both the infectiousness to the vector Triatoma infestans and parasite load of T. cruzi-seropositive dogs examined were heterogeneous. A statistically significant, nine-fold higher mean infectiousness was registered in T. cruzi-seropositive dogs co-infected with Ancylostoma caninum and a trematode than in T. cruzi-seropositive dogs without these infections. The median parasite load of T. cruzi was also significantly higher in dogs co-infected with these helminths. An opposite trend was observed in T. cruzi-seropositive dogs that were serologically positive to Toxoplasma gondii or Neospora caninum relative to dogs seronegative for these parasites. Using multiple logistic regression analysis with random effects, we found a positive and significant association between the infectiousness of T. cruzi-seropositive dogs and co-infections with A. caninum and a trematode. Our results suggest that co-infections may be a modifier of host infectiousness in dogs naturally infected with T. cruzi.

Isolation and characterization of a gene from Trypanosoma cruzi encoding a 46-kilodalton protein with homology to human and rat tyrosine aminotransferase.

The complete sequence of a gene encoding a 46-kDa protein of Trypanosoma cruzi is presented. The first ATG complies with the consensus sequence for initiation of translation. A single band of 2 kb was highlighted by hybridizing a probe from the 46-kDa protein gene to a Northern filter containing total T. cruzi RNA. The gene is present in 50-80 copies per cell and most of them are contained in 2 tandem arrays on large T. cruzi chromosomes (> 2000 kb). A strong homology with rat and human tyrosine aminotransferase was detected. Homology with a Trypanosoma brucei retrotransposon was found in the nonsense strand of the intergenic region.

Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: complement-mediated lytic activity against trypomastigotes and passive immunoprotection in mice.

Trypanosoma cruzi, the causative agent of Chagas' disease, is widely spread in Central and South America. The present report describes three monoclonal antibodies (mAbs) directed against the flagellar fraction of epimastigotes (Ffe) of the parasite, Tulahuén strain. The three mAbs were of IgG1 isotype. Indirect immunofluorescence assays revealed that the three mAbs bind to epimastigotes, the FCH-F8-1 and -3 bind to blood trypomastigotes (BT) and FCH-F8-1 is the only one that binds to amastigotes. Three different proteins of the parasite were recognized by the mAbs in immunoprecipitation assays: an 85 kDa of BT with the FCH-F8-1 mAb, a 40 kDa of Ffe with the FCH-F8-2 mAb, and a 90 kDa of Ffe and of BT with the FCH-F8-3 mAb. Positive complement mediated lysis (CML) of BT and metacyclic forms, obtained from the insect vector feces, were shown by the FCH-F8-1, while FCH-F8-3 only showed CML against the metacyclic trypomastigotes. FCH-F8-2 did not mediate any CML activity. Passive transference of the mAbs to BALB/c mice conferred protection, in terms of survival, ranging from 50 (FCH-F8-2 and -3) to 80% (FCH-F8-1) against a challenge with 1 X 10(3) BT. These results suggest that FCH-F8-1 may be a useful tool to purify proteins in order to investigate their role in immunoprotection experiments.

Polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients.

The feasibility of DNA amplification by the polymerase chain reaction for specific detection of Trypanosoma cruzi in human blood was investigated. We have used primers flanking a 220-bp fragment of highly repetitive elements, the E13 element, in T. cruzi nuclear DNA. Only polymerase chain reaction products from blood samples of chronic chagasic patients showed several amplified fragments in 1.6% agarose gels stained with ethidium bromide. Southern blot analysis demonstrated that the 220-bp amplified fragment is specific for T. cruzi DNA and very useful to detect the presence of the parasite in blood from chronic chagasic patients.

Characterisation of a cyclophilin isoform in Trypanosoma cruzi.

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.

A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities.

Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-transferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.

[Trypanosoma cruzi genome: transcriptional mapping and karyotype correlation; molecular characterization of a surface antigen from the Tc13 family]. / Genoma del Trypanosoma cruzi. Correlación entre el mapa transcripcional y el cariotipo caracterización molecular de un antígeno de superficie de la familia TC13.

We describe herein the genome mapping of Trypanosoma cruzi, the etiological agent of Chagas' disease, by hybridization of a cosmid library gridded in high density filters with cDNA from an epimastigote expression library as probes. We also show the correlation of some cosmid contigs with parasite chromosomal bands. With libraries from the T. cruzi Genome Project we have characterized a new member of the Tc13 family belonging to the superfamily of trypomastigote surface antigens. Starting with a Tulahuén strain (Tul 2 stock) clone, homologous to these antigens, we have sequenced and characterized the complete gene in the CL Strain (CL Brener clone). We have also found homologies with different ESTs which characterization would lead to further knowledge of this antigen family.

[Molecular and immunologic bases for the development of a vaccine against Chagas disease]. / Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas.

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

High levels of Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans support dogs and cats are major sources of parasites for domestic transmission.

The competence of reservoir hosts of vector-borne pathogens is directly linked to its capacity to infect the vector. Domestic dogs and cats are major domestic reservoir hosts of Trypanosoma cruzi, and exhibit a much higher infectiousness to triatomines than seropositive humans. We quantified the concentration of T. cruzi DNA in the peripheral blood of naturally-infected dogs and cats (a surrogate of intensity of parasitemia), and evaluated its association with infectiousness to the vector in a high-risk area of the Argentinean Chaco. To measure infectiousness, 44 infected dogs and 15 infected cats were each exposed to xenodiagnosis with 10-20 uninfected, laboratory-reared Triatoma infestans that blood-fed to repletion and were later individually examined for infection by optical microscopy. Parasite DNA concentration (expressed as equivalent amounts of parasite DNA per mL, Pe/mL) was estimated by real-time PCR amplification of the nuclear satellite DNA. Infectiousness increased steeply with parasite DNA concentration both in dogs and cats. Neither the median parasite load nor the mean infectiousness differed significantly between dogs (8.1Pe/mL and 48%) and cats (9.7Pe/mL and 44%), respectively. The infectiousness of dogs was positively and significantly associated with parasite load and an index of the host's body condition, but not with dog's age, parasite discrete typing unit and exposure to infected bugs in a random-effects multiple logistic regression model. Real-time PCR was more sensitive and less time-consuming than xenodiagnosis, and in conjunction with the body condition index, may be used to identify highly infectious hosts and implement novel control strategies.

Orthognathic surgery in pycnodysostosis: a case report.

Pycnodysostosis is an extremely rare genetic osteosclerosis caused by cathepsin K deficiency. It is a human autosomal recessive genetic disorder characterized mainly by osteosclerosis of the skeleton due to decreased bone turnover. It is characterized by short stature, brachycephaly, short and stubby fingers, open cranial sutures and fontanelle, and diffuse osteosclerosis. Multiple fractures of the long bones and osteomyelitis of the jaw are frequent complications. The authors describe an 18-year-old girl with a clinical and radiological diagnosis of pycnodysostosis and the ortho-surgical treatment undertaken. Bimaxillary orthognathic surgery was carried out using rigid fixation and bone grafts. The authors recommend bimaxillary orthognathic surgery as a choice for treating the dentofacial deformities of pycnodysostosis, emphasizing the good and stable results obtained in terms of facial aesthetics and occlusion.

An unusual intraparotid foreign body simulating a tumour: shrapnel from the Spanish Civil War.

A foreign body lodged for decades in the parotid gland, entering through the oral cavity or the skin, is extremely rare. Even less common is the clinical presentation of a foreign body lodged in the parotid gland simulating a tumour. The authors report the finding of a fragment of shrapnel lodged in the parotid gland for 63 years. The case is unusual owing to the rare clinical presentation, the length of time between the injury and the occurrence of symptoms and the associated mandibular osteolysis in the panoramic radiograph. Initial diagnoses considered were a tumour emerging in the parotid gland, a large odontogenic cyst or an odontogenic tumour with soft tissue extension. Sometimes neither fine needle aspiration nor radiology is capable of providing an accurate diagnosis before surgery.

Angiolipoma in head and neck: report of two cases and review of the literature.

Angiolipomas are either encapsulated or non-encapsulated fatty tumours. They are distinguished from other lipomas by the excessive degree of vascular proliferation and because they are mixed with mature adipocytes. They commonly occur in the trunk and extremities and are rare in the maxillofacial area. Only 36 cases in the head and neck have been reported in the literature. The authors report the first non-infiltrating intramasseterine angiolipoma, and a case of non-infiltrating angiolipoma of the cheek. These tumours appear as homogenous low-density areas on CT with no contrast enhancement. MRI gives better tumour delineation and clear definition of the location and longitudinal extent of the mass. Histopathology demonstrates mature adipose tissue and the proliferation of numerous small branching blood vessels. Management of angiolipomas requires complete surgical excision.

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins.

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

Analysis of the Trypanosoma cruzi cyclophilin gene family and identification of Cyclosporin A binding proteins.

The Trypanosoma cruzi cyclophilin gene family comprises 15 paralogues whose nominal masses vary from 19 to 110 kDa, namely TcCyP19, TcCyP20, TcCyP21, TcCyP22, TcCyP24, TcCyP25, TcCyP26, TcCyP28, TcCyP29, TcCyP30, TcCyP34, TcCyP35, TcCyP40, TcCyP42 and TcCyP110. Under the conditions used, only some of the T. cruzi cyclophilin paralogue products could be isolated by affinity chromatography. The 15 paralogues were aligned with 495 cyclophilins from diverse organisms. Analyses of clusters formed by the T. cruzi cyclophilins with others encoded in various genomes revealed that 8 of them (TcCyP19, TcCyP21, TcCyP22, TcCyP24, TcCyP35, TcCyP40, TcCyP42 and TcCyP110) have orthologues in many different genomes whereas the other 7 display less-defined patterns of their sequence attributes and their classification to a specific group of cyclophilin's orthologues remains uncertain. Seven epimastigote cDNA clones encoding cyclophilin isoforms were further studied. These genes were found dispersed throughout the genome of the parasite. Amastigote and trypomastigote mRNAs encoding these 7 genes were also detected. We isolated 4 cyclosporin A-binding proteins in T. cruzi epimastigote extracts, which were identified by mass spectrometry as TcCyP19, TcCyP22, TcCyP28 and TcCyP40. Cyclosporin A-binding to these cyclophilins might be of importance to the mechanism of action of Cyclosporin A and its non-immunosuppressive analogues, whose trypanocidal effects were previously reported, and therefore, of potential interest in the chemotherapy of Chagas' disease.