A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil

Candida species are an important cause of bloodstream infections (BSI). To evaluate the epidemiological, clinical and microbiological aspects of two cohorts {1994-1999 [period 1 (P1) ]; 2000-2004 [period 2 (P2) ]} of candidaemic patients, we performed a retrospective analysis from a laboratory-based survey. A total of 388 candidaemias were identified, with an incidence of 0.20/1,000 patient-days and a significant increase in P2 vs. P1 (0.25 vs. 0.15, p = 0.04). Cancer and prior antibiotic use were frequent and Candida albicans was the most prevalent species found (42.4%). Resistance to fluconazole was found in 2.47% of the strains. No differences were observed in the species distribution of Candida during the study periods. In the P2 cohort, there were higher prevalence of elderly individuals, cardiac, pulmonary and liver diseases, renal failure, central venous catheters and antibiotic therapy. In P1, there were higher prevalence of neurological diseases and chemotherapy. The crude mortality was 55.4%. In conclusion, our incidence rates remained high. Furthermore, the distribution pattern of Candida species and the fluconazole resistance profile remained unchanged. Moreover, we found a clear trend of higher prevalence of candidaemia among the elderly and among patients with comorbidities. Finally, it is necessary to discuss strategies for the prevention and control of Candida BSI in Brazil.

Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.

Spatial and temporal changes in Lutzomyia longipalpis abundance, a Leishmania infantum vector in an urban area in northeastern Argentina

This study aimed to analyse changes in the spatial distribution of Lutzomyia longipalpis in Posadas, an urban area located in northeastern Argentina. Data were obtained during the summer of 2007 and 2009 through two entomological surveys of peridomiciles distributed around the city. The abundance distribution pattern for 2009 was computed and compared with the previous pattern obtained in 2007, when the first human visceral leishmaniasis cases were reported in the city. Vector abundance was also examined in relation to micro and macrohabitat characteristics. In 2007 and 2009, Lu. longipalpis was distributed among 41.5% and 31% of the households in the study area, respectively. In both years, the abundance rates at most of the trapping sites were below 30 Lu. longipalpis per trap per night; however, for areas exhibiting 30-60 Lu. longipalpis and more than 60 Lu. longipalpis, the areas increased in both size and number from 2007-2009. Lu. longipalpis was more abundant in areas with a higher tree and bush cover (a macrohabitat characteristic) and in peridomiciles with accumulated unused material (a microhabitat characteristic). These results will help to prioritise and focus control efforts by defining which peridomiciles display a potentially high abundance of Lu. longipalpis.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .

Incidence and transmission patterns of tuberculosis among indigenous populations in Brazil

Approximately 10% of the Brazilian indigenous population lives in the state of Mato Grosso do Sul (MS), where a large number of new cases of tuberculosis (TB) are reported. This study was conducted to assess TB occurrence, transmission and the utility of TB diagnosis based on the Ogawa-Kudoh (O-K) culture method in this remote population. The incidence of TB was estimated by a retrospective review of the surveillance data maintained by the Notifiable Diseases Surveillance System for the study region. The TB transmission pattern among indigenous people was assessed by genotyping Mycobacterium tuberculosis isolates using the IS 6110restriction fragment length polymorphism (RFLP) technique. Of the 3,093 cases identified from 1999-2001, 610 (~20%) were indigenous patients (average incidence: 377/100,000/year). The use of the O-K culture method increased the number of diagnosed cases by 34.1%. Of the genotyped isolates from 52 indigenous patients, 33 (63.5%) belonged to cluster RFLP patterns, indicating recently transmitted TB. These results demonstrate high, on-going TB transmission rates among the indigenous people of MS and indicate that new efforts are needed to disrupt these current transmissions.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Epidemiological aspects of influenza A related to climatic conditions during and after a pandemic period in the city of Salvador, northeastern Brazil

During the influenza pandemic of 2009, the A(H1N1)pdm09, A/H3N2 seasonal and influenza B viruses were observed to be co-circulating with other respiratory viruses. To observe the epidemiological pattern of the influenza virus between May 2009-August 2011, 467 nasopharyngeal aspirates were collected from children less than five years of age in the city of Salvador. In addition, data on weather conditions were obtained. Indirect immunofluorescence, real-time transcription reverse polymerase chain reaction (RT-PCR), and sequencing assays were performed for influenza virus detection. Of all 467 samples, 34 (7%) specimens were positive for influenza A and of these, viral characterisation identified Flu A/H3N2 in 25/34 (74%) and A(H1N1)pdm09 in 9/34 (26%). Influenza B accounted for a small proportion (0.8%) and the other respiratory viruses for 27.2% (127/467). No deaths were registered and no pattern of seasonality or expected climatic conditions could be established. These observations are important for predicting the evolution of epidemics and in implementing future anti-pandemic measures.

Influence of the Paracoccidioides brasiliensis14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensisremains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensismolecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensisuses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

The spatiotemporal trajectory of a dengue epidemic in a medium-sized city

Understanding the transmission dynamics of infectious diseases is important to allow for improvements of control measures. To investigate the spatiotemporal pattern of an epidemic dengue occurred at a medium-sized city in the Northeast Region of Brazil in 2009, we conducted an ecological study of the notified dengue cases georeferenced according to epidemiological week (EW) and home address. Kernel density estimation and space-time interaction were analysed using the Knox method. The evolution of the epidemic was analysed using an animated projection technique. The dengue incidence was 6.918.7/100,000 inhabitants; the peak of the epidemic occurred from 8 February-1 March, EWs 6-9 (828.7/100,000 inhabitants). There were cases throughout the city and was identified space-time interaction. Three epicenters were responsible for spreading the disease in an expansion and relocation diffusion pattern. If the health services could detect in real time the epicenters and apply nimbly control measures, may possibly reduce the magnitude of dengue epidemics.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Respiratory syncytial virus seasonality in Brazil: implications for the immunisation policy for at-risk populations

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalisation for respiratory diseases among children under 5 years old. The aim of this study was to analyse RSV seasonality in the five distinct regions of Brazil using time series analysis (wavelet and Fourier series) of the following indicators: monthly positivity of the immunofluorescence reaction for RSV identified by virologic surveillance system, and rate of hospitalisations per bronchiolitis and pneumonia due to RSV in children under 5 years old (codes CID-10 J12.1, J20.5, J21.0 and J21.9). A total of 12,501 samples with 11.6% positivity for RSV (95% confidence interval 11 - 12.2), varying between 7.1 and 21.4% in the five Brazilian regions, was analysed. A strong trend for annual cycles with a stable stationary pattern in the five regions was identified through wavelet analysis of the indicators. The timing of RSV activity by Fourier analysis was similar between the two indicators analysed and showed regional differences. This study reinforces the importance of adjusting the immunisation period for high risk population with the monoclonal antibody palivizumab taking into account regional differences in seasonality of RSV.