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Alberto Berardi,1 Viola Trevisani,2 Antonella Di Caprio,2 Jenny Bua,3 Mariachiara China,4 Barbara Perrone,5 Rossella Pagano,6 Laura Lucaccioni,7 Silvia Fanaro,8 Lorenzo Iughetti,2,7 Licia Lugli,1 Roberta Creti9 1Terapia Intensiva Neonatale, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy; 2Scuola di Specializzazione in Pediatria, Università di Modena & Reggio Emilia, Modena, Italy; 3Terapia Intensiva Neonatale, IRCCS Azienda Ospedaliero Universitaria “Burlo Garofalo”, Trieste, Italy; 4Terapia Intensiva Neonatale, Ospedale Infermi, Rimini, Italy; 5Terapia Intensiva Neonatale, Azienda Ospedaliero Universitaria Ospedali Riuniti, Ancona, Italy; 6Unità Operativa di Pediatria, Civile Sassuolo, Sassuolo, Italy; 7Unità Operativa di Pediatria, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy; 8Terapia Intensiva Neonatale, Azienda Ospedaliero-Universitaria S. Anna, Ferrara, Italy; 9Reparto di Antibiotico Resistenza e Patogeni Speciali (AR-PS), Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, ItalyCorrespondence: Alberto Berardi Tel +39 059 4222522Fax +39 059 42223770Email alberto.berardi@unimore.itAbstract: Group B streptococcus (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis in infants. Rates of GBS early onset disease have declined following the widcespread use of intrapartum antibiotic prophylaxis; hence, late-onset infections (LOGBS) are currently a common presentation of neonatal GBS dicsease. The pathogenesis, mode of transmission, and risk factors associated with LOGBS are unclear, which interfere with effective prevention efforts. GBS may be transmitted from the mother to the infant at the time of delivery or during the postpartum period via contaminated breast milk, or as nosocomial or community-acquired infection. Maternal GBS colonization, prematurity, young maternal age, HIV exposure, and ethnicity (Black) are identified as risk factors for LOGBS disease; however, further studies are necessary to confirm additional risk factors, if any, for the implementation of effective prevention strategies. This narrative review discusses current and previous studies that have reported LOGBS. Few well-designed studies have described this condition; therefore, reliable assessment of maternal GBS colonization, breastfeeding, and twin delivery as risk factors for LOGBS remains limited.Keywords: neonatal sepsis, prevention, infant, meningitis, CC17

Abstract Background Geographical inaccessibility, lack of transport, and financial burdens are some of the demand side constraints to maternal health services in Uganda, while supply side problems include poor quality services related to unmotivated health workers and inadequate supplies. Most public health interventions in Uganda have addressed only selected supply side issues, and universities have focused their efforts on providing maternal services at tertiary hospitals. To demonstrate how reforms at Makerere University College of Health Sciences (MakCHS) can lead to making systemic changes that can improve maternal health services, a demand and supply side strategy was developed by working with local communities and national stakeholders. Methods This quasi-experimental trial is conducted in two districts in Eastern Uganda. The supply side component includes health worker refresher training and additions of minimal drugs and supplies, whereas the demand side component involves vouchers given to pregnant women for motorcycle transport and the payment to service providers for antenatal, delivery, and postnatal care. The trial is ongoing, but early analysis from routine health information systems on the number of services used is presented. Results Motorcyclists in the community organized themselves to accept vouchers in exchange for transport for antenatal care, deliveries and postnatal care, and have become actively involved in ensuring that women obtain care. Increases in antenatal, delivery, and postnatal care were demonstrated, with the number of safe deliveries in the intervention area immediately jumping from Conclusions Transport and service vouchers appear to be a viable strategy for rapidly increasing maternal care. MakCHS can design strategies together with stakeholders using a learning-by-doing approach to take advantage of community resources.

Abstract Background An evidence-based Consensus on the treatment of undescended testis (UT) was recently published, recommending to perform orchidopexy between 6 and 12 months of age, or upon diagnosis and to avoid the use of hormones. In Italy, current practices on UT management are little known. Our aim was to describe the current management of UT in a cohort of Italian children in comparison with the Consensus guidelines. As management of retractile testis (RT) differs, RT cases were described separately. Methods Ours is a retrospective, multicenter descriptive study. An online questionnaire was filled in by 140 Italian Family Paediatricians (FP) from Associazione Culturale Pediatri (ACP), a national professional association of FP. The questionnaire requested information on all children with cryptorchidism born between 1/01/2004 and 1/01/2006. Data on 169 children were obtained. Analyses were descriptive. Results Overall 24% of children were diagnosed with RT, 76% with UT. Among the latter, cryptorchidism resolved spontaneously in 10% of cases at a mean age of 21.6 months. Overall 70% of UT cases underwent orchidopexy at a mean age of 22.8 months (SD 10.8, range 1.2-56.4), 13% of whom before 1 year. The intervention was performed by a paediatric surgeon in 90% of cases, with a success rate of 91%. Orchidopexy was the first line treatment in 82% of cases, while preceded by hormonal treatment in the remaining 18%. Hormonal treatment was used as first line therapy in 23% of UT cases with a reported success rate of 25%. Overall, 13 children did not undergo any intervention (mean age at last follow up 39.6 months). We analyzed the data from the 5 Italian Regions with the largest number of children enrolled and found a statistically significant regional difference in the use of hormonal therapy, and in the use of and age at orchidopexy. Conclusions Our study showed an important delay in orchidopexy. A quarter of children with cryptorchidism was treated with hormonal therapy. In line with the Consensus guidelines, surgery was carried out by a paediatric surgeon in the majority of cases, with a high success rate.

Abstract Background Triatoma infestans is the most relevant vector of Chagas disease in the southern cone of South America. Since its genome has not yet been studied, sequencing of Expressed Sequence Tags (ESTs) is one of the most powerful tools for efficiently identifying large numbers of expressed genes in this insect vector. Results In this work, we generated 826 ESTs, resulting in an increase of 47% in the number of ESTs available for T. infestans. These ESTs were assembled in 471 unique sequences, 151 of which represent 136 new genes for the Reduviidae family. Conclusions Among the putative new genes for the Reduviidae family, we identified and described an interesting subset of genes involved in development and reproduction, which constitute potential targets for insecticide development.

Chagas disease is a serious parasitic infection caused by Trypanosoma cruzi. Unfortunately, the current chemotherapeutic tools are not enough to combat the infection. The aim of this study was to evaluate the trypanocidal activity of benznidazole-loaded microparticles during the acute phase of Chagas infection in an experimental murine model. Microparticles were prepared by spray-drying using copolymers derived from esters of acrylic and methacrylic acids as carriers. Dissolution efficiency of the formulations was up to 3.80-fold greater than that of raw benznidazole. Stability assay showed no significant difference (P > 0.05) in the loading capacity of microparticles for 3 years. Cell cultures showed no visible morphological changes or destabilization of the cell membrane nor haemolysis was observed in defibrinated human blood after microparticles treatment. Mice with acute lethal infection survived 100% after 30 days of treatment with benznidazole microparticles (50 mg kg-1 day-1). Furthermore, no detectable parasite load measured by quantitative polymerase chain reaction and lower levels of T. cruzi-specific antibodies by enzyme-linked immunosorbent assay were found in those mice. A significant decrease in the inflammation of heart tissue after treatment with these microparticles was observed, in comparison with the inflammatory damage observed in both infected mice treated with raw benznidazole and untreated infected mice. Therefore, these polymeric formulations are an attractive approach to treat Chagas disease.

To disseminate and colonise tissues in the mammalian host, Trypanosoma cruzi trypomastogotes should cross several biological barriers. How this process occurs or its impact in the outcome of the disease is largely speculative. We examined the in vitro transmigration of trypomastigotes through three-dimensional cultures (spheroids) to understand the tissular dissemination of different T. cruzi strains. Virulent strains were highly invasive: trypomastigotes deeply transmigrate up to 50 μm inside spheroids and were evenly distributed at the spheroid surface. Parasites inside spheroids were systematically observed in the space between cells suggesting a paracellular route of transmigration. On the contrary, poorly virulent strains presented a weak migratory capacity and remained in the external layers of spheroids with a patch-like distribution pattern. The invasiveness-understood as the ability to transmigrate deep into spheroids-was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. Besides, we demonstrated that T. cruzi isolates from children that were born congenitally infected presented a highly migrant phenotype while an isolate from an infected mother (that never transmitted the infection to any of her children) presented significantly less migration. In brief, we demonstrated that in a 3D microenvironment each strain presents a characteristic migration pattern that can be associated to their in vivo behaviour. Altogether, data presented here repositionate spheroids as a valuable tool to study host-pathogen interactions., (© 2020 John Wiley & Sons Ltd.)

This study evaluated the effectiveness of low doses of benznidazole (BNZ) on continuous administration (BNZc), combined with allopurinol (ALO), in C57BL/6J and C3H/HeN mice infected with Trypanosoma cruzi Nicaragua strain and T. cruzi Sylvio-X10/4 clone. TcN-C57BL/6J was also treated with intermittent doses of BNZ (BNZit). The drug therapy started 3 months post infection (pi) in the chronic phase of mice with heart disease progression, followed-up at 6 months pi. TcN-C57BL/6J treated with BNZc was also monitored up to 12 months pi by serology and electrocardiogram. These mice showed severe electrical abnormalities, which were not observed after BNZc or BNZit. ALO only showed positive interaction with the lowest dose of BNZ. A clear parasitic effect, with significant reductions in antibody titres and parasitic loads, was achieved in all models with low doses of BNZ, and a 25% reduction of the conventional dose showed more efficacy to inhibit the development of the pathology. However, BNZ 75 showed partial efficacy in the TcSylvio-X10/4-C3H/HeN model. In our experimental designs, C57BL/6J allowed to clearly define a chronic phase, and through reproducible efficacy indicators, it can be considered a good preclinical model.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Background: It is currently unclear why only a proportion of children born to Trypanosoma cruzi-infected mothers acquire the infection. We have examined the association of 11 single-nucleotide polymorphisms (SNPs) located in genes coding for placental expression enzymes as genetic markers of susceptibility to congenital T. cruzi infection (hereafter, "congenital infection"): rs2014683 and rs1048988 in ALPP; rs11244787 and rs1871054 in ADAM12; rs243866, rs243865, rs17859821, rs243864, and rs2285053 in MMP2; and rs3918242 and rs2234681 in MMP9., Methods: Two groups of children born to mothers seropositive for T. cruzi were compared: 101 had congenital infection, and 116 were uninfected. Novel high-resolution melting and capillary electrophoresis genotyping techniques were designed and used., Results: Logistic regression analysis showed that mutations in rs11244787 and rs1871054 (in ADAM12) and rs243866, rs17859821, and rs2285053 (in MMP2) were associated with susceptibility to congenital infection. Multifactor dimensionality reduction revealed that genotyping results for rs11244787, rs1871054, rs243866, rs17859821 and rs243864 sites would be a good predictor of congenital infection., Conclusions: Our results suggest an important role of human polymorphisms in proteins involved in extracellular matrix remodeling and the immune response during congenital infection. To our knowledge, this is the first study demonstrating the association between mutations in placentally expressed genes and susceptibility to congenital infection., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Oral manifestations in ectodermal dysplasia include oligodontia, alveolar ridges hypoplasia, and others. Due to the special conditions in terms of unhealthy teeth and lack of bone, implant-supported rehabilitation seems to offer the most satisfactory outcome. A 27-year-old male diagnosed with ectodermal dysplasia was referred to our department for oral rehabilitation. Oral manifestations included oligodontia, maxillary and mandibular atrophy, mandibular alveolar ridge with knife-edge morphology, and conical teeth. Treatment planning consisted of a Le Fort I osteotomy with interpositional grafts, bilateral sinus lift, and placement of maxillary and mandibular inlay and onlay corticocancellous grafts, using autologous iliac crest bone. In the second surgery, all remaining teeth were removed and 11 endosteal implants were placed. Six months after implant placement, a bimaxillary fixed implant-supported prosthesis was delivered, maintaining a satisfactory esthetic and functional result after a 2-year follow-up. The use of combined preprosthetic techniques allows the placement of endosteal implants and a fixed implant-supported prosthesis in patients with oligodontia and ectodermal dysplasia, providing an esthetic and functional oral rehabilitation.

Background: Mandible subcondylar fractures may be treated via a traditional visible access incision; however, with the advances in surgical endoscopy surgeons are transitioning to a minimally invasive approach in an effort to reduce surgical morbidity and external facial scarring. We sought to design a clinically applicable teaching tool in a large animal model that would allow the operator to gain experience treating mandible subcondylar fractures via an endoscopic approach., Methods: A large animal model was developed using the Churra sheep. Subcondylar fractures were created, reduced, and internally plated in ten specimens via an extraoral, two-port endoscopic approach. Animals were monitored for surgical success during the intraoperative and immediate postoperative periods., Results: Mandibles were reduced and fixated successfully in each of the animals. Operative time was reduced from 70 to 40 min as the surgeons became more familiar with the surgical procedure. Each of the ten Churra sheep used in the study tolerated the surgeries without postoperative complications., Conclusions: Capitalizing on a mandibular anatomy similar to humans, the Churra sheep successfully demonstrated utility for the extraoral, endoscopic approach in treating mandibular condyle fractures. This model offers surgeons the opportunity to gain surgical endoscopic experience before treating clinical patients.

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., (Copyright © 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Objective: The aim of this comparative double-blind, prospective, randomized, clinical trial was to evaluate two amoxicillin administration patterns. The first was a short prophylactic therapy and the second a long postoperative regimen., Study Design: The study population consisted of 160 patients who underwent mandibular third molar extraction. Patients were randomized into two equal groups. In group 1, 2 grams of amoxicillin were administered 1 hour before the procedure and 1 gram 6 hours after surgery. In group 2, patients received 1 gram of amoxicillin 6 hours after surgery followed by 1 gram every 8 hour for 4 days. All patients received the same number of tablets thanks to the use of placebo pills. A total of 25 variables were evaluated, such as alveolitis, surgical infection, number of analgesic needed, subjective pain scale, post-surgical inflammation, consistency of the diet, axillary temperature and millimeters of mouth opening loss after the surgery., Results: No statistically significant post-operative differences were found within the recorded parameters between the groups., Conclusions: Postoperative 4-days amoxicillin therapy is not justified.

Objective: Cleft lip and palate is a congenital facial malformation with an established treatment protocol. Mixed dentition period is the best moment for correct maxillary bone defect with an alveoloplasty. The aim of this surgical procedure is to facilitate dental eruption, re-establish maxillary arch, close any oro-nasal communication, give support to nasal ala, and in some cases allow dental rehabilitation with osteointegrated implants., Study Design: Twenty cleft patients who underwent secondary alveoloplasty were included. In 10 of them autogenous bone graft were used and in other 10 autogenous bone and platelet-rich plasma (PRP) obtained from autogenous blood. Bone formation was compared by digital orthopantomography made on immediate post-operatory and 3 and 6 months after the surgery., Results: No significant differences were found between both therapeutic groups on bone regeneration., Conclusion: We do not find justified the use of PRP for alveoloplasty in cleft patients' treatment protocol.

Objectives: 1) to compare mandibular bone regeneration by applying autologous bone, platelet-rich plasma and two biomaterials (synthetic calcium hydroxyapatite, and demineralized bone matrix), and thus establish the potential benefits of these biomaterials in the regeneration of postextraction alveolar bone, 2) to identify which of them accelerates more bone regeneration and 3) to determine whether there are differences in the postoperative period (pain, swelling, trismus, infection) depending on the material used., Study Design: It consists in a prospective, controlled (with a split- mouth design) and double blinded study. We use as a model an easily reproducible non-critical bone defect: the defect that remains after extraction of mandibular impacted third molar. The study design is based on the extraction of two mandibular impacted third molars in a patient during the same surgical procedure by the same surgeon. We assessed postoperative clinical data, and short, medium and long term neoformation of alveolar bone after extraction. We compared the two sockets (right and left), which had been grafted in a different way with the various elements mentioned above. In addition, we compared the postoperative inflammatory symptoms between groups., Results: The highest acceleration in bone formation was observed in groups in which we used autologous bone and demineralized bone matrix. There were no statistically significant differences between groups regarding pain, swelling, trismus and infection throughout the postoperative period., Conclusions: According to the results of our study, autologous bone persists as the gold standard material for bone regeneration. Among the assessed biomaterials, demineralized bone matrix has yielded the best results obtained. No significant differences in the postoperative (pain, swelling, trismus and infectious events) were observed, depending on the type of material used as a graft.

Cyclophilins (CyPs) are enzymes involved in protein folding, catalyzing the isomerisation of peptidyl prolyl bonds in proteins and peptides between the cis- and trans-conformations. They are also the major cellular target for the immunosuppressive drug Cyclosporin A (CsA). In Trypanosoma cruzi, the most abundantly expressed CyP is an isoform of 19 kDa, TcCyP19, in which the enzymatic activity is inhibited by CsA. Among a reported set of CsA analogues, two non-immunosuppressive compounds, H-7-94 and F-7-62, proved to be the best inhibitors of TcCyP19 enzymatic activity as well as the most efficient trypanocidal drugs. With the objective of analysing, at the molecular level, how the structural differences between the three above-mentioned inhibitors justify their different inhibitory activity on TcCyP19, three-dimensional molecular modelling structures were generated to computationally simulate behaviours and interactions. An energy-minimized model of each binary complex in water with ions was obtained. These models were then used as starting point for molecular dynamic simulations, performed with GROMOS96 program. With the resulting set of co-ordinates and energies, a comparison of the interaction between CsA and both CsA analogues in T. cruzi and human cyclophilins were performed. Within the different magnitudes analysed, the total potential complex energy exhibited the best correlation with the experimental data. The results obtained in this study support the use of this methodology when designing new lead inhibitor compounds.

The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.

We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC(50) values were 0.82 and 3.41 microM, respectively, compared to CsA IC(50) value 5.39 microM on epimastigote proliferation; and on trypomastigote lysis their IC(50) values were 0.97 and 2.66 microM compared to CsA IC(50) value 7.19 microM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.

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.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).

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.

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.

Several monoclonal antibodies were prepared against the flagellar fraction of Trypanosoma cruzi epimastigotes (Tulahuén strain, stock Tul 2). One of them, FCH-F8-4, has previously shown biologic activity against the parasite (complement-mediated lysis and neutralization of the trypomastigote infectivity). Immunopurified antigens using this monoclonal antibody elicited a protective immune response in mice. Two recombinant cDNA clones were detected with this anti-flagellar fraction monoclonal antibody on a lambda gt11 expression library prepared from T. cruzi epimastigote mRNA. The insert of one of these cDNA clones, lambda(FCH-F8-4)1 (150 bp) coded for a 19-amino acid peptide (PAFLGCSSRFSGSFSGVEP). This insert hybridized with a 5.0-kb mRNA from epimastigotes. The beta-galactosidase fusion protein was produced in lysogenic bacteria. The monoclonal antibody recognized the epitope present in the fusion protein after western blotting of the crude lysate. A synthetic peptide (SP4) containing the complete sequence of lambda(FCH-F8-4)1 was constructed on solid phase. This peptide was able to inhibit the ELISA reactivity (in a range from 13 to 52%) of flagellar fraction immunized mouse sera and when administered (coupled to KLH or alone) to BALB/c mice with Bordetella pertussis as adjuvant, it induced a humoral and cellular immune response which was detected by ELISA, immunofluorescence, blotting, and DTH reactions against T. cruzi antigens. The immune response obtained indicates that this synthetic peptide resembles the parasite antigen conformation and could be useful for diagnosis purposes or be able to elicit immunoprotection against T. cruzi infection.