The presence of Fasciola hepatica (Liver-fluke) in humans and cattle from a 4,500 Year old archaeological site in the Saale-Unstrut Valley, Germany

During an excavation of a site of the corded ware culture in the Saale-Unstrut-Valley (ca. 3000 BC) in Germany, a soil sample from the pelvis of a human skeleton was studied under palaeoparasitological aspects. Eggs of the trematode Fasciola hepatica and of the nematode genus Capillaria were found. This is the first case of a direct association of a F. hepatica-infestation to both a prehistoric human skeleton and domesticated animal remains. Sheep and cattle bones were present at the same site and F. hepatica eggs were found in bovine samples. This strongly points toward an existing infection cycle, involving humans as a final host.

Ecological analysis of Acari recovered from coprolites from archaeological site of Northeast Brazil

Coprolite samples of human and animal origin from the excavations performed at the archaeological site of Furna do Estrago, at Brejo da Madre de Deus in the state of Pernambuco, Brazil and sent to the Paleoparasitology Laboratory at Escola Nacional de Saúde Pública-Fiocruz, Rio de Janeiro, were analyzed for mites. After rehydratation and sedimentation of the coprolites, the alimentary contents and the sediments were examined and the mites collected and prepared in definitive whole mounts, using Hoyer's medium. Mites of the following suborders and orders were recovered: suborder Acaridia; order Gamasida; order Ixodida with the familiy Ixodidae (Ixodes sp. and Amblyomma sp. larvae, scutum, idiosoma, gnathosoma); order Oribatida (Aphelacarus sp., Apolohmannia sp., Eophypochthonius sp., Cosmochthonius sp., Pterobates sp., Poronoticae with pteromorphae not auriculate); order Astigmata with the families Atopomelidae (Chirodiscoides caviae), Anoetidae hypopus, Acaridae (Suidasia pontifica), Glycyphagidae (Blomia tropicalis), Pyroglyphidae (Hirstia passericola); order Actinedida with the family Tarsonemidae (Iponemus radiatae). The present work discusses the possibility of the preservation of the mite groups found up to the present day. We also discuss their relationship with the environment and their importance to present populations.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

New finding of Giardia intestinalis (Eukaryote, Metamonad) in Old World archaeological site using immunofluorescence and enzyme-linked immunosorbent assays

In this study, nine organic sediment samples from a medieval archaeological site at Pineuilh, France, were examined for Giardia intestinalis using two commercially available immunological kits [enzyme-linked immuno sorbent and immunofluorescence (IFA) assays]. Both techniques detected G. intestinalis in one sample, dated to 1,000 Anno Domini. This is the first time IFA was successfully used to detect protozoa in Old World archaeological samples. Such immunological techniques offer important perspectives concerning ancient protozoa detection and identification.

Parasites in rodent coprolites from the historical archaeological site Alero Mazquiarán, Chubut Province, Argentina

The aim of this study was to examine the parasitic remains that were found in rodent coprolites collected from the archaeological site Alero Mazquiarán (Chubut Province, 45º44'15"S, 70°25'9"W), which is assigned to the interface of the Araucanian and Tehuelche cultures, dated at 212 ± 35 years B.P. The faecal material from two unidentified rodent species (X-10 and X-11) was collected from one human pelvic cavity found in a multiple burial. The faecal samples were processed and examined using paleoparasitological procedures. The X-10 coprolites were positive for eggs of Monoecocestus sp. (Cestoda: Anoplocephalidae) and the X-11 faeces were positive for Pterygodermatites sp. (Nematoda: Rictulariidae), Trichosomoides sp. (Nematoda: Trichosomoididae) and Monoecocestus sp. In this study, we discuss parasitic life cycles, the zoonotic importance of parasites and the behaviour of the aboriginal people.

Development of physiological resistance and its stage specificity in Culex quinquefasciatus after selection with deltamethrin in Assam, India

The study investigated the development and stage specificity of physiological resistance to insecticides in a colony of Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes, which are vectors of bancroftian filariasis in India, after selection with deltamethrin. Resistance was selected by exposing the larvae to the concentration of deltamethrin that caused 50% mortality in the tested population (i.e., LC50). Under continuous selection pressure, the LC50 increased steadily in subsequent generations. The estimated LC50 for the F0 generation was 0.409 μg/L; the LC50 first displayed a substantial increase in the F5 generation (5.616 μg/L) and reached 121.902 μg/L in the F10 generation. The objective of this study was to establish a deltamethrin-resistant colony to develop a research programme that will study the evolution of physiological resistance patterns and stage-specific resistance responses in Cx. quinquefasciatus larvae and adults under laboratory conditions. An approximately 298-fold increase in resistance was recorded after 10 generations, as evidenced by the resistance ratio (RR50). The progress and effect of the selection pressure in the adult stage was monitored with the World Health Organisation (WHO) diagnostic test. The mortality, as observed using the WHO diagnostic test, declined significantly from the F5 generation (85%) onwards and the highest rate of survival (65%) was observed in the F10 generation.

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.

Trichuris sp. from 1,040 +/- 50-year-old Cervidae coprolites from the archaeological site Furna do Estrago, Pernambuco, Brazil

We present results of the paleoparasitological analysis of Cervidae coprolites that were recovered from the archaeological site Furna do Estrago, Pernambuco, Brazil. Trichuris sp. eggs were recovered from the coprolite samples dated 1,040 ± 50 years before present. This is the first record of Trichuris sp. in semiarid Cervidae, unexpectedly recorded in archaeological material.

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

A palaeoparasitological analysis of rodent coprolites from the Cueva Huenul 1 archaeological site in Patagonia (Argentina)

The aim of the present study was to examine the parasite fauna present in rodent coprolites collected from Cueva Huenul 1 (CH1), northern Neuquén (Patagonia, Argentina), an archaeological site that provides stratified sequences of archaeological and palaeontological remains dating from the Late Pleistocene/Early Holocene Transition to the Late Holocene period. Twenty rodent coprolites collected from different sedimentary units from the site, with ages ranging from 13.844 ± 75-1.416 ± 37 years BP, were examined for parasites. Each coprolite was processed as a whole: rehydrated, homogenised, spontaneously sedimented and examined using light microscopy. The coprolites and the eggs of any parasites present were described, measured and photographed. In all, 158 parasite eggs were found in 10 coprolites. The faeces were positive for Viscachataenia quadrata Denegri, Dopchiz, Elissondo & Beveridge and Monoecocestus sp. Beddard (Cestoda: Anoplocephalidae) and for Heteroxynema (Cavioxyura) viscaciae Sutton & Hugot (Nematoda: Oxyuridae). The coprolites examined were tentatively attributed to Lagidium viscacia Molina (Mammalia, Rodentia, Caviomorpha, Chinchillidae). The life cycles of these parasites are discussed.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Culicidae (Diptera) selection of humans, chickens and rabbits in three different environments in the province of Chaco, Argentina

Studies were conducted to determine the selection of humans, chickens and rabbits by Culicidae in three different environments in the province of Chaco, Argentina. Mosquitoes were collected fortnightly using cylindrical metal traps containing animal bait (chickens and rabbits). The mosquitoes were collected between June 2001-May 2002. During the same period and with the same frequency, mosquitoes biting the human operators of the traps were collected during the first 15 min of exposure within different time intervals: from 09:00 am-11:00 am, 01:00 pm-03:00 pm, 05:00 pm-07:00 pm and 09:00 pm-10:00 pm. A total of 19,430 mosquitoes of 49 species belonging to 10 genera were collected. Culex species mainly selected chicken bait and Wyeomyia species selected rabbit bait. Ochlerotatus and Psorophora species were more abundant in rabbit-baited traps. Anopheles triannulatus, Coquillettidia nigricans, Ochlerotatus scapularis, Mansonia titillans and Psorophora albigenu showed a strong attraction for human bait. The Anopheles, Coquillettidia, Culex and Mansonia species were more active between 05:00 pm-09:00 pm, while Ochlerotatus, Psorophora, Haemagogus and Wyeomyia were most active from 09:00 am-07:00 pm. This study provides additional information about the biology and ecology of arbovirus vectors in Chaco.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.