Fresh Blood Examination Research Articles

Impact of gastrointestinal inoculation and benznidazole treatment on infection by Trypanosoma cruzi (Y strain, DTU TcII) in Swiss mice

In Brazil, where Chagas disease is endemic, the most frequent form of transmission of the parasite is the oral route, associated with greater severity and worse response to benznidazole (BZ), the drug used in its treatment. This study aimed to evaluate the impact of gastrointestinal infection (GI) and BZ treatment on the parasitological and histopathological parameters in mice inoculated with a strain of T. cruzi II. Swiss mice were inoculated by GI and intraperitoneal (IP) routes with 2x106 culture-derived metacyclic trypomastigotes of the Y strain (TcII) of T. cruzi and were treated with BZ in the acute phase of the infection. Fresh blood examination, qPCR, histopathological and biochemical evaluations (enzymatic dosages and oxidative stress-OS) were performed. BZ treatment of uninfected animals caused changes in the liver, increased the activity of aspartate aminotransferase and alanine aminotransferase enzymes and OS, showing that the drug alone affects this organ. Inflammation and necrosis in the cardiac tissue were less intense and deaths occurred later in animals inoculated via the GI route than the animals inoculated via the IP route. BZ reduced the intensity of tissue lesions and avoided lethality in animals inoculated via the GI route, and decreased parasitemia and OS in those inoculated via both routes. Although BZ alone caused liver damage, it was less intense than that caused by both routes of inoculation. Infection with the Y strain of T. cruzi II via the GI route proved to be less virulent and pathogenic and responded better to treatment than the infection acquired via the IP route.

S2673 If It Bleeds, It Leads: Endoscopic Presentation of Hemosuccus Pancreaticus

Introduction: A 65 year old male presented as a transfer for further evaluation of multifactorial shock. Pertinent medical comorbidities included active alcohol use disorder and mild alcohol associated hepatitis. On OSH presentation, he was treated for multifactorial shock in setting of new atrial fibrillation with rapid ventricular response and MSSA bacteremia, and was subsequently transferred to our facility. Case Description/Methods: On arrival, he had been started on systemic anticoagulation in light of his new diagnosis of atrial fibrillation and had downtrending hemoglobin from previously normal baseline ∼15 to a nadir of 7-8. Several episodes of witnessed melena were documented, and the gastroenterology service was consulted. He had no known prior history of GI bleeding or cirrhosis. Endoscopic evaluation noted a small amount of hematin in the stomach without concerning source lesions. Examination of the duodenum was initially only notable for scant fresh red blood and small, clean based ulcers thought secondary to recent hypotension and ischemia. On prolonged examination of the duodenum intermittent fresh blood was observed originating from the ampulla, consistent with a diagnosis of hemosuccus pancreaticus. Follow up dedicated CT angiography showed a thrombosed aneurysm in the pancreatic head, abutting the distal main pancreatic duct, that was concerning for the likely source lesion. The patient and family were offered interventional radiology intervention, but instead opted for hospice placement. Discussion: Hemosuccus pancreaticus is typically associated with pancreatic pathology that ultimately causes luminal bleeding via erosion of pancreatic blood vessels in communication with the pancreatic duct. The source of bleeding is often an arterial aneurysm or pseudoaneurysm and associated bleeding can be severe and life-threatening. Active hemorrhage from the duodenal ampulla is a rare endoscopic finding and bleeding in cases of hemosuccus pancreaticus is frequently intermittent. In patients with obscure upper GI bleeding and the appropriate risk factors, a high index of suspicion is required to ultimately make the diagnosis. (Figure) Given the frequency of associated vascular abnormalities, all patients with suspected or confirmed hemosuccus pancreaticus merit dedicated vascular imaging and if a vascular lesion is identified, angiography and coil embolization are the preferred definitive management.Figure 1.: Intermittent active bleeding from ampulla consistent with endoscopic diagnosis of hemosuccus pancreaticus.

Boid Inclusion Body Disease Is Also a Disease of Wild Boa Constrictors.

ABSTRACTReptarenaviruses cause boid inclusion body disease (BIBD), a potentially fatal disease, occurring in captive constrictor snakes boas and pythons worldwide. Classical BIBD, characterized by the formation of pathognomonic cytoplasmic inclusion bodies (IBs), occurs mainly in boas, whereas in pythons, for example, reptarenavirus infection most often manifests as central nervous system signs with limited IB formation. The natural hosts of reptarenaviruses are unknown, although free-ranging/wild constrictor snakes are among the suspects. Here, we report BIBD with reptarenavirus infection in indigenous captive and wild boid snakes in Costa Rica using histology, immunohistology, transmission electron microscopy, and next-generation sequencing (NGS). The snakes studied represented diagnostic postmortem cases of captive and wild-caught snakes since 1989. The results from NGS on archival paraffin blocks confirm that reptarenaviruses were already present in wild boa constrictors in Costa Rica in the 1980s. Continuous sequences that were de novo assembled from the low-quality RNA obtained from paraffin-embedded tissue allowed the identification of a distinct pair of reptarenavirus S and L segments in all studied animals; in most cases, reference assembly could recover almost complete segments. Sampling of three prospective cases in 2018 allowed an examination of fresh blood or tissues and resulted in the identification of additional reptarenavirus segments and hartmanivirus coinfection. Our results show that BIBD is not only a disease of captive snakes but also occurs in indigenous wild constrictor snakes in Costa Rica, suggesting boa constrictors to play a role in natural reptarenavirus circulation.IMPORTANCE The literature describes cases of boid inclusion body disease (BIBD) in captive snakes since the 1970s, and in the 2010s, others and ourselves identified reptarenaviruses as the causative agent. BIBD affects captive snakes globally, but the origin and the natural host of reptarenaviruses remain unknown. In this report, we show BIBD and reptarenavirus infections in two native Costa Rican constrictor snake species, and by studying archival samples, we show that both the viruses and the disease have been present in free-ranging/wild snakes in Costa Rica at least since the 1980s. The diagnosis of BIBD in wild boa constrictors suggests that this species plays a role in the circulation of reptarenaviruses. Additional sample collection and analysis would help to clarify this role further and the possibility of, e.g., vector transmission from an arthropod host.

Therapeutic effects of benznidazole in Swiss mice that are orally inoculated with Trypanosoma cruzi IV strains from the Western Brazilian Amazon

Strains of Trypanosoma cruzi, etiological agent of Chagas disease, are classified into different discrete typing units that may present distinct dynamics of infection and susceptibility to benznidazole (BZ) treatment. Mice that were orally inoculated with T. cruzi IV strains exhibited a more intense course of infection compared with intraperitoneally inoculated mice, reflected by higher parasite loads. We evaluated the efficacy of BZ treatment in Swiss mice that were inoculated with T. cruzi IV strains from the Western Brazilian Amazon. The mice were orally (OR) or intraperitoneally (IP) inoculated with 2 × 106 culture-derived metacyclic trypomastigotes of the AM14, AM16, AM64, and AM69 strains of T. cruzi that were obtained from two outbreaks of orally acquired acute Chagas disease in the state of Amazonas, Brazil. The animals were treated with BZ (100 mg/kg/day for 20 days). Fresh blood examination, hemoculture, conventional and quantitative real-time polymerase chain reaction were performed to monitor the therapeutic effects of BZ. Significant reductions in five of 24 parameters of parasitemia and parasite load were found in different tissues in the OR group, indicating worse response to BZ treatment compared with the IP group, in which significant reductions in nine of those 24 parameters were observed. The cure rates in the OR groups ranged from 18.2% (1/11) to 75.0% (9/12) and in the IP groups from 58.3% (7/12) to 91.7% (11/12), for the AM14 and AM69 strains, respectively. These findings indicate that treatment with BZ had fewer beneficial effects with regard to reducing parasitemia and parasite load in different tissues of mice that were OR inoculated with four TcIV strains compared with IP inoculation. Therefore, the route of infection with T. cruzi should be considered when evaluating the therapeutic efficacy of BZ in patients with Chagas disease.

Essential oils from Syzygium aromaticum and Zingiber officinale, administered alone or in combination with benznidazole, reduce the parasite load in mice orally inoculated with Trypanosoma cruzi II

BackgroundTrypanosoma cruzi is the etiological agent of Chagas disease (CD) or American trypanosomiasis, an important public health problem in Latin America. Benznidazole (BZ), a drug available for its treatment, has limited efficacy and significant side effects. Essential oils (EOs) have demonstrated trypanocidal activity and may constitute a therapeutic alternative. Our aim was to evaluate the efficacy of the EOs of clove (CEO - Syzygium aromaticum) and ginger (GEO - Zingiber officinale), administered alone and in combination with BZ, in Swiss mice infected with T. cruzi.MethodsThe animals were inoculated with 10,000 blood trypomastigotes of the Y strain of T. cruzi II by gavage and divided into four groups (n = 12 to 15): 1) untreated control (NT); 2) treated with BZ; 3) treated with CEO or GEO; and 4) treated with BZ + CEO or GEO. The treatments consisted of oral administration of 100 mg/kg/day, from the 5th day after parasite inoculation, for 20 consecutive days. All groups were submitted to fresh blood examination (FBE), blood culture (BC), conventional PCR (cPCR) and real-time PCR (qPCR), before and after immunosuppression with cyclophosphamide.ResultsClove and ginger EOs, administered alone and in combination with BZ, promoted suppression of parasitemia (p < 0.0001), except for the animals treated with CEO alone, which presented a parasitemia curve similar to NT animals. However, there was a decrease in the BC positivity rate (p < 0.05) and parasite load (< 0.0001) in this group. Treatment with GEO alone, on the other hand, besides promoting a decrease in the BC positivity rate (p < 0.05) and parasite load (p < 0.01), this EO also resulted in a decrease in mortality rate (p < 0.05) of treated mice.ConclusionsDecreased parasite load, as detected by qPCR, was observed in all treatment groups (BZ, CEO, GEO and BZ + EOs), demonstrating benefits even in the absence of parasitological cure, thus opening perspectives for further studies.

Wild and Domestic Canids and Their Interactions in the Transmission Cycles of Trypanosoma Cruzi and Leishmania spp. in an Area of the Brazilian Cerrado.

Trypanosoma cruzi and Leishmania spp. are parasites that infect multiple hosts including canids, considered bioaccumulators of parasites. Deforestation in the Cerrado biome has resulted in the exposure of wild canids to anthropized areas, where they may establish ecological and epidemiological relationships with domestic dogs. We evaluated the infection by trypanosomatids in canids from a Cerrado agroecosystem between 2013 and 2017. Samples of wild canids (blood, bone marrow and skin) and dogs (blood) were collected for parasitological, serological and molecular diagnosis. A total of 414 samples from wild (n = 131) and domestic (n = 283) canids were collected, including recaptures. We obtained five positive hemocultures from Lycalopex vetulus (n = 2), Cerdocyon thous (n = 1) and dogs (n = 2), all characterized as T. cruzi TcIII/V (18S rDNA) and TcIII/V/VI (gGAPDH); one positive skin fragment for Leishmania sp. (C. thous), one positive skin culture (Chrysocyon brachyurus) and one positive fresh blood examination from a dog. Infection by T. cruzi and Leishmania spp. was serologically confirmed in 18% and 4% of the canids, respectively. Active transmission was attested by seroconversion events and occurred despite the low rate of positive parasitological assays. Wild and domestic canids infected by both parasites were detected sharing the same areas, pointing to a possible spillover of parasites among them.

Trypanosomatids in Small Mammals of an Agroecosystem in Central Brazil: Another Piece in the Puzzle of Parasite Transmission in an Anthropogenic Landscape.

We surveyed infection by Trypanosoma spp. and Leishmania spp. in small wild mammals from Cumari, Goiás State aiming to investigate the diversity of trypanosomatid in a modified landscape of the Brazilian Cerrado (and possible infection overlapping with canids from the same area). Blood, skin, spleen, and liver samples were collected for parasitological, serological, and molecular assays. Gracilinanus agilis was the most abundant species (N = 70; 48.6%) and it was the only one with patent parasitemia. Characterization by mini-exon and 18SrDNA targets were achieved in 7/10 hemocultures with positive fresh blood examination, which confirmed the T. cruzi infection by Discrete Typing Units (DTU) TcI in single (N = 2) and mixed infections with other DTUs (N = 5). T. rangeli and T. dionisii were detected in skin fragments from Didelphis albiventris and Oecomys cleberi, respectively. G. agilis were found to be infected by L. braziliensis and L. guyanensis, while Leishmania sp. DNA was detected in the liver of Oligoryzomys nigripes and Calomys expulsus. Subpatent infection by T. cruzi and Leishmania sp. was serologically detected in 15% and 9% of the small mammal fauna, respectively. Small mammals from Cumari are included in T. cruzi and Leshmania spp. transmission cycles, showing a higher diversity of trypanosomatid species and/or genotypes than that observed in canids of the same agroecosystem.

Isolation and characterization of trypanosomatids, including Crithidia mellificae, in bats from the Atlantic Forest of Rio de Janeiro, Brazil.

We studied infection by Trypanosomatidae in bats captured in two areas with different degradation levels in the Atlantic Forest of Rio de Janeiro state: Reserva Ecológica de Guapiaçu (REGUA) and Estação Fiocruz Mata Atlântica (EFMA). Furthermore, we evaluated whether the diversity of trypanosomatids changes according to bat diversity and the different levels of preservation in the region. The results showed no influence of the level of preservation on bat species richness (15 and 14 species, respectively), with similar chiropterofauna and higher abundance of two common fruit-eating bat species in the tropics: Carollia perspicillata and Artibeus lituratus. Of the 181 bat specimens analyzed by LIT/Schneider hemoculture, we detected 24 infected individuals (13%), including one positive Sturnira lilium individual that was also positive by fresh blood examination. Molecular characterization using nested PCR targeting the 18 SSU rRNA-encoding gene fragment showed similar trypanosomatid infection rates in bats from the two areas: 15% in REGUA and 11% in EFMA (p = 0.46). Trypanosoma dionisii was the most frequently detected parasite (54%), followed by T. cruzi DTUs TcI and TcIV and Trypanosoma sp., in Neotropical phyllostomid bats (RNMO63 and RNMO56); mixed infections by T. dionisii/T. cruzi TcIII and T. dionisii/T. cruzi TcI were also observed. The T. cruzi DTUs TcI and TcIV are the genotypes currently involved in cases of acute Chagas disease in Brazil, and T. dionisii was recently found in the heart tissue of an infected child. Surprisingly, we also describe for the first time Crithidia mellificae, a putative monoxenous parasite from insects, infecting a vertebrate host in the Americas. Bats from the Atlantic Forest of Rio de Janeiro state harbor a great diversity of trypanosomatids, maintaining trypanosomatid diversity in this sylvatic environment.

Trypanosoma sp. diversity in Amazonian bats (Chiroptera; Mammalia) from Acre State, Brazil.

Bats are ancient hosts of Trypanosoma species and their flying ability, longevity and adaptability to distinct environments indicate that they are efficient dispersers of parasites. Bats from Acre state (Amazon Biome) were collected in four expeditions conducted in an urban forest (Parque Zoobotânico) and one relatively more preserved area (Seringal Cahoeira) in Rio Branco and Xapuri municipalities. Trypanosoma sp. infection was detected by hemoculture and fresh blood examination. Isolated parasite species were identified by the similarity of the obtained DNA sequence from 18S rDNA polymerase chain reaction and reference strains. Overall, 367 bats from 23 genera and 32 species were examined. Chiropterofauna composition was specific to each municipality, although Artibeus sp. and Carollia sp. prevailed throughout. Trypanosoma sp. infection was detected in 85 bats (23·2%). The most widely distributed and prevalent genotypes were (in order) Trypanosoma cruzi TcI, T. cruzi marinkellei, Trypanosoma dionisii, T. cruzi TcIV and Trypanosoma rangeli. At least one still-undescribed Trypanosoma species was also detected in this study. The detection of T. cruzi TcI and TcIV (the ones associated with Chagas disease in Amazon biome) demonstrates the putative importance of these mammal hosts in the epidemiology of the disease in the Acre State.

Outcome of oral infection in mice inoculated with Trypanosoma cruzi IV of the Western Brazilian Amazon

A new epidemiological view of American trypanosomiasis or Chagas disease has been formulated in recent decades. Oral transmission of the etiological agent of Chagas disease, Trypanosoma cruzi, has been the most common form of transmission. The T. cruzi discrete typing units TcI and TcIV have been involved in tens outbreaks of acute cases of Chagas disease in the Brazilian Amazon region. We investigated the intensity of infection in mice that were orally inoculated (OR group) with four strains of TcIV that were isolated from two outbreaks of acute Chagas disease that was orally acquired in the state of Amazonas, Brazil. We compared the OR group with mice that were intraperitoneally inoculated (IP group). Blood samples were analyzed by fresh blood examination, hemoculture, and conventional and qualitative real-time polymerase chain reaction (PCR). Samples of different tissues were analyzed by quantitative real-time PCR. The OR group exhibited a higher maximum peak of parasitemia, greater rates of positivity, and higher parasite loads in different tissues during acute infection compared with the IP group, indicating a greater intensity of orally acquired infection. Mice that were orally inoculated with TcIV strains that were obtained from two outbreaks of orally acquired Chagas disease in Amazonas, Brazil, exhibited a more intense course of infection compared with intraperitoneally inoculated mice, reflected by higher levels of parasitemia and parasite loads.

Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous Administration in Murine Experimental Chagas Disease.

The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC-poly(d,l-lactide)-polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC-poly-ε-caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease.

Tetradentate polyamines as efficient metallodrugs for Chagas disease treatment in murine model

A series of tetraamine-based compounds was prepared, and their trypanocidal effects against Trypanosoma cruzi and cytotoxicity were determined through the determination of IC50 values. In vivo assays were performed in mice, where parasitaemia levels were quantified by fresh blood examination and the assignment of a cure was determined by polymerase chain reaction and reactivation of blood parasitaemia levels after immunosuppression. The mechanisms of action were elucidated at metabolic and ultra-structural levels, by 1H NMR, Fe-SOD inhibition and TEM studies. The high-selectivity indexes observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. Compound 6 induced a remarkable decrease in the reactivation of parasitaemia after immunosuppression and curative rates of 33%. The experiments allowed us to select compound 6 as a promising candidate for treating Chagas disease, but a further high-level study should be considered to obtain an improved efficiency.

Differential parasitological, molecular, and serological detection of Trypanosoma cruzi I, II, and IV in blood of experimentally infected mice

Trypanosoma cruzi is the etiological agent of American trypanosomiasis (Chagas' disease), which affects 6–7 million people worldwide, mainly in Latin America. It presents great genetic and biological variability that plays an important role in the clinical and epidemiological features of the disease. Our working hypothesis is that the genetic diversity of T. cruzi has an important impact on detection of the parasite using diagnostic techniques. The present study evaluated the diagnostic performance of parasitological, molecular, and serological techniques for detecting 27 strains of T. cruzi that belonged to discrete typing units (DTUs) TcI (11 strains), TcII (four strains), and TcIV (12 strains) that were obtained from different hosts in the states of Amazonas and Paraná, Brazil. Blood samples were taken from experimentally infected mice and analyzed by fresh blood examination, hemoculture in Liver Infusion Tryptose (LIT) medium, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction presented the best detection of TcI, with 80.4% positivity. For all of the detection methods, the animals that were inoculated with TcII presented the highest positivity rates (94.1–100%). ELISA that was performed 7 months after inoculation presented a higher detection ability (95.4%) for TcIV. Intra-DTU comparisons showed that the reproducibility of the majority of the results that were obtained with the different methods was weak for TcI and good for TcII and TcIV. Our data indicate that the detection capability of different techniques varies with the DTUs of the parasites in mammalian blood. The implications of these findings with regard to the diagnosis of human T. cruzi infection are discussed.

In vitro and in vivo identification of tetradentated polyamine complexes as highly efficient metallodrugs against Trypanosoma cruzi

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels by 1H NMR and TEM studies. Finally, as tetraamines are potentially capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%, respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic activity and low toxicity render these tetraamines appropriate molecules for the development of an affordable anti-Chagas agent.

An in vitro iron superoxide dismutase inhibitor decreases the parasitemia levels of Trypanosoma cruzi in BALB/c mouse model during acute phase

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by 1H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.

A comparison of differential leucocyte counts measured by conventional automated venous haematology and darkfield microscopic examination of fresh capillary blood

Analysis of fresh capillary blood using darkfield microscopy (FCB-DM) is a point of care screening tool, used by healthcare practitioners in Australia. However, the relationship between the outcomes of FCB-DM measures and the automated venous haematology measures has not been determined. ObjectivesThe aim of this study was to compare differential white blood cell counts obtained using automated haematology and FCB-DM. MethodsData of 125 individuals were collected either retrospectively (n=74) or from participants specifically recruited for the project (n=51). Retrospective data were collected from active files at a naturopathic clinic. Newly recruited participants provided a fasting capillary blood sample for FCB-DM analysis within 1h of providing a venous blood sample at a commercial laboratory for automated haematologic analysis. ResultsThe mean score of neutrophils was found to be higher, and lymphocytes and basophils to be lower, in FCB-DM analysis (p<0.05). A significant and positive Pearson's correlation coefficient was found between automated haematology and FCB-DM in the cell counts for neutrophils (r=0.60, p<0.05) and lymphocytes (r=0.63, p<0.05), and a significant and positive Spearman's correlation was found for monocytes (rs=0.32, p<0.05) and eosinophils (rs=0.596, p<0.05). Linear regression analysis was also conducted to assess the relationship between the two techniques. The variance explained by the regression model was large for neutrophil (37%), lymphocyte (39%), and eosinophil (37%) scores. ConclusionDespite significant differences in the mean scores of cells counts, significant correlations between the data obtained by the two techniques for neutrophil, lymphocyte, monocyte and eosinophil cells were observed. Given the small amount of blood sample required, FCB-DM would have an advantage in clinical practice, though further research is required to determine the clinical implications of the FCB-DM cell counts.

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease.

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.

Expanding the knowledge of the geographic distribution of Trypanosoma cruzi TcII and TcV/TcVI genotypes in the Brazilian Amazon.

Trypanosoma cruzi infection is a complex sylvatic enzooty involving a wide range of animal species. Six discrete typing units (DTUs) of T. cruzi, named TcI to TcVI, are currently recognized. One unanswered question concerning the epidemiology of T. cruzi is the distribution pattern of TcII and hybrid DTUs in nature, including their virtual absence in the Brazilian Amazon, the current endemic area of Chagas disease in Brazil. Herein, we characterized biological samples that were collected in previous epizootiological studies carried out in the Amazon Basin in Brazil. We performed T. cruzi genotyping using four polymorphic genes to identify T. cruzi DTUs: mini-exon, 1f8, histone 3 and gp72. This analysis was conducted in the following biological samples: (i) two T. cruzi isolates obtained by culturing of stools from the triatomine species Rhodnius picttipes and (ii) five serum samples from dogs in which trypomastigotes were observed during fresh blood examination. We report for the first time the presence of TcII and hybrid DTUs (TcV/TcVI) in the Amazon region in mixed infections with TcI. Furthermore, sequencing of the constitutive gene, gp72, demonstrated diversity in TcII even within the same forest fragment. These data show that TcII is distributed in the five main Brazilian biomes and is likely more prevalent than currently described. It is very probable that there is no biological or ecological barrier to the transmission and establishment of any DTU in any biome in Brazil.

Integration of diagnosis and treatment of sleeping sickness in primary healthcare facilities in the Democratic Republic of the Congo.

Control of human African trypanosomiasis (HAT) in the Democratic Republic of Congo (DRC) has always been a vertical programme, although attempts at integration in general health services were made in recent years. Now that HAT prevalence is declining, the integration question becomes even more crucial. We studied the level of attainment of integration of HAT case detection and management in primary care centres in two high-prevalence districts in the province of Bandundu, DRC. We visited all 43 first-line health centres of Mushie and Kwamouth districts, conducted structured interviews and inspected facilities using a standardised checklist. We focused on: availability of well trained staff - besides HAT, we also tested for knowledge on tuberculosis; availability of equipment, consumables and supplies; and utilisation of the services. All health centres were operating but most were poorly equipped, and attendance rates were very low. We observed a median of 14 outpatient consultations per facility (IQR 8-21) in the week prior to our visit, that is two patients per day. The staff had good knowledge on presenting symptoms, diagnosis and treatment of both HAT and tuberculosis. Nine centres were accredited by the national programme as HAT diagnosis and treatment centres, but the most sensitive diagnostic confirmation test, the mini-anion exchange centrifugation technique (mAECT), was not present in any. Although all nine were performing the CATT screening test, only two had the required cold chain in working order. In these high-prevalence districts in DRC, staff is well-acquainted with HAT but lack the tools required for an adequate diagnostic procedure. Attendance rates of these primary care centres are extremely low, making timely recognition of a resurgence of HAT unlikely in the current state of affairs.

Heterologous expression in Caenorhabditis elegans as an alternative approach to functional studies in Schistosoma mansoni.

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p < 0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory respons.