Human trichinellosis in Hungary from 1965 to 2009

Human trichinellosis was first documented in Hungary in 1891 and then there were an increased number of reports up to 1964 when the most severe outbreak occurred. After that, no information was available on the international literature on human trichinellosis which occurred from 1965 up to the present years. The aim of this study was to collect all the data available in Hungarian official data-sources on human trichinellosis which occurred from 1965 up to 2009 in Hungary. Furthermore, a comparative analysis was performed on the different serological tests used along the 45 years of investigation. In the period in question, 573 infections were documented in Hungary. Of them, 57 occurred in the years 1965-69, 130 in 1970-79, 302 in 1980-89, 27 in 1990-99, and 57 in 200009. The most common sources of infection were pork from backyard pigs and hunted wild boars. Sporadic cases and small family outbreaks marked the last ten years. The comparison of serological tests shows that the ELISA is a good test for the first screening, but ELISA-positive serum samples should be confirmed by western blot except for clinically clear-cut cases.     

Molecular identification of natural hybrids between Trichinella nativa and Trichinella T6 provides evidence of gene flow and ongoing genetic divergence

To date, there are no data available on the population genetics of Trichinella due to the lack of genetic markers and the difficulty of working with such small parasites. In the Arctic region of North America and along the Rocky Mountains, there exist two genotypes of Trichinella, Trichinella nativa and Trichinella T6, respectively, which are well differentiated by biochemical and molecular characters. However, both are resistant to freezing, show other common biological characters (e.g. low or no infectivity to rodents and swine) and produce fertile F1 offspring upon interbreeding. To data, these two genotypes have been considered allopatric. In this study, we detected both genotypes in wolves of the same wolf packs in Alaska, suggesting sympatry. A single GTT trinucleotide present in the ITS-2 sequence of T. nativa but not in Trichinella T6 was used as a genetic marker to study gene flow for this character in both a murine infection model and in larvae from naturally-infected Alaskan wolves. Only F1 larvae originating from a cross between T. nativa male and Trichinella T6 female were able to produce F2 offspring. Larvae (F1) originating from a cross between Trichinella T6 male and T. nativa female were not reproductively viable. As expected, all F1 larvae showed a heterozygote pattern for the GTT character upon heteroduplex analysis; however, within the F2 population, the number of observed heterozygotes (n=52) was substantially higher than expected (n=39.08), as supported by the F(is) index, and was not in the Hardy-Weinberg equilibrium. Larvae from two of the 16 Trichinella positive Alaskan wolves, showed the Trichinella T6 pattern or the T. nativa/Trichinella T6 hybrid pattern. Our data demonstrate that T. nativa and Trichinella T6 live in sympatry at least in Alaskan wolves, where T. nativa occurs more frequently (69%) than Trichinella T6 (31%). One explanation for this phenomenon is that glacial periods may have caused a geographical relocation, colonisation and independent evolution of T. nativa within the Rocky Mountains, resulting in a bifurcation of the freeze-resistant genotype. Additional studies will be required to test this hypothesis.     

A micrite-dominated norian carbonate platform from Northern Calabria (Southern Italy)

Summary  This paper is focused on the facies associations of an Early-Middle Norian stratigraphic succession cropping outin the Northern Calabria (Buonvicino, Cosenza). These carbonate deposits, pertaining to the Verbicaro Unit, represent a dolomitized platform characterized by a clear dominance of automicrites over skeletal metazoans. On the basis of the chronostatigraphic data and sedimentary evolution, two stratigraphic units have been distinguished: the Lower Unit (object of the present paper) and the Upper Unit. The Lower Unit consists of an Early-Middle Norian high-relief prograding carbonate platform. The Upper Unit is represented by Middle p.p.-Upper Norian basinal deposits. Three main facies associations, indicative of different depositional settings, have been identified. The inner platform facies association is characterized by automicrite (cauliflower-columnar subordinate planar stromatolites) associated with detrital carbonates (intraclastic breccia, bioclastic grainstone and packstone). Apeculiar facies is represented by megalodontid-bearing beds. Mud-cracked horizons and low-developed teepees occur quite frequently. The margin facies association is dominated by automicrite (planar to low relief stromatolites associated with thrombolitic fenestral boundstone) and detrital carbonates with subaerial exposure features. The slope facies association includes detrital carbonates (breccia/megabreccia) associated with serpulid/sphinctozoan bioconstructions and automicrite (planar stromatolites/thrombolitic boundstone). The whole carbonate body is completely dolomitized; nevertheless the morphology and microarchitecture of carbonate components (cements, grains and automicrite) is still clearly recognizable. The dolomite Mg content ranges from 40 to 48 mole%, sometimes reaching the stoichiometric value. Cements, primary and late, represent a minor component of the rock volume; they occur more frequently on the margin and upper slope setting. Quantitative tacies analyses lead to the following conclusions:

Highly Active AntiRetroviral Therapy and opportunistic protozoan infections

Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has been observed in the HAART era, characterised by a high rate of relapses, which could be explained by the increased survival rate resulting from the effective antiretroviral therapy. A 64.8% decrease of the visceral leishmaniasis incidence was detected after HAART began to be used extensively in Spain. In a large cohort study carried out in ten European countries and in Australia, the relative risk to contract cryptosporidiosis as the first AIDS defining disease was reduced by 96% in the HAART era. In Italy, the relative risk of death for cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-positive persons received HAART. In a large study carried out in Italy, isosporiasis was included in the group of opportunistic infections, of which the relative hazards showed a reduction of 95% in the HAART era. Since 1997, there was the evidence that the use of HAART in persons with advanced HIV infection can improve chronic diarrhoea and lead to disappearance of Enterocytozoon bieneusi from the stools. Although the reconstitution of the cellular immunity seems to be the main factor influencing the reduction of OPIs in persons with AIDS who undergo HAART, there are clinical and microbiological evidences, as well as in vitro and in vivo results, which indicate direct effects of HIV-PIs on the proteases of opportunistic parasites. These findings stress the existence of non-conventional unexpected benefits of PIs in HAART against protozoa. In addition, this benefit of PIs has been demonstrated also for Candida albicans secreted aspartyl proteases and for P. carinii acid proteases. In spite of these important results, HIV PIs are still very toxic for humans, specially in cases of very long treatment, and no clinical trial has been carried out for persons at risk, such as children and pregnant women, because the priority was to reduce the severity of HIV and not the evaluation of possible side effects of the therapy. It follows that further researches are needed to establish the non-conventional use of HIV PIs. Furthermore, the study of PIs against specific aspartyl proteases of those opportunistic protozoa that cause severe and intractable diseases, could be considered an alternative way towards the development of new drugs that may prove effective against these infections.     

Detection and counting of Cryptosporidium parvum in HCT-8 cells by flowcytometry

The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14%) than for IFA (i.e., 27-38% when performed by a single operator and 19-22% when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs.     

Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum.

The genetic polymorphism among 57 Cryptosporidium parvum isolates belonging to genotype 'C' was studied by PCR amplification and the sequencing of two microsatellite loci (ML1 and ML2). A comparative analysis of DNA sequences showed the presence of three (ML1-238, ML1-226, and ML1-220) and seven (ML2-231, ML2-229, ML2-227, ML2-213, ML2-193, ML2-191, and ML2-187) different alleles at these two loci. Alleles differed by expansions/contractions of the microsatellite repeats that generated length polymorphisms. Some alleles were found to be associated with infections of all examined hosts (calf, kid, lamb, and human), whereas others were either associated with a single host, or were geographically restricted. When considering the information from both loci, some preferential associations between alleles are apparent. These data confirm the utility of microsatellite markers for the molecular identification of C. parvum, which is of particular relevance in the investigation of the source of infection of outbreaks and single cases, as well as for genetic studies.