[The occurrence of Cryptosporidium in a group of children and adults with diarrhoea of undetermined earlier aetiology]. / Wystepowanie Cryptosporidium u dzieci i osób doroslych z biegunka o nieustalonej etiologii.

In Poland since 2002 it is required to report the cases of human cryptosporidiosis, but so far none has been recorded. The aim of this study was to present some preliminary results of a study on the incidence of Cryptosporidium as a pathogen in children and adults with diarrhoea of undetermined aetiology. In 36 out of 246 stool samples collected from hospitalized patients with diarrhoea of undetermined aetiology, invasive parasite oocysts were detected. All positive results were obtained in a group of children aged up to 4 years (47.5%). The percentage of invasion in this age group was statistically significantly higher than in the group of children aged 12 months and children over 48 months of age, which was respectively 16.2% (p = 0.0018) and 7.7% (p < or = 0.001). On the basis of Restriction Fragment Length Polymorphism (RFLP) it was found that oocysts isolated from stool samples belonged to species of Cryptosporidium parvum and/or Cryptosporidium hominis. In adults with diarrhoea, and among healthy children, no oocysts of Cryptosporidium were found. The analysis of the results demonstrated that Cryptosporidium is one of a cause of diarrhoea in young children in Poland. This is an indication for the introduction of tests for cryptosporidiosis to the schedule of routine diagnostics of gastrointestinal infections in children. This work has been partly founded by Med-Vet-Net. Workpackages 22 Met-Vet-Net is an EU-founded.

[Development of efficient DNA isolation procedures for Cryptosporidium and Trichinella PCR detection in fecal samples]. / Opracowanie wydajnych procedur izolacji DNA Cryptosporidium sp. i Trichinella spiralis z próbek kalu.

PCR detection of genetic material of the parasites present in faeces may be an alternative for microscopic and serological tests routinely used for diagnosing parasitic enteral infections. However, small amount of target DNA combined with low efficiency of total DNA extraction, and presence of PCR inhibitors in the samples to be amplified, may cause false negative detection results. The aim of this work was to evaluate the impact of DNA isolation procedure used on the amplification of DNA fragments from the genomes of protozoan Cryptosporidium parvum and the nematode Trichinella spiralis. Two methods based on different principles of biological material lysis were evaluated; NucliSENS miniMAG employing simultaneously applied chemical lysis and mechanical disruption or mechanical disruption followed by enzymatic lysis in case of QIAamp DNA Stool Mini Kit. Both of the analyzed systems for nucleic acids purification allowed isolation of DNA from purified Cryptosporidium parvum oocysts and Trichinella spiralis muscle larva mixed with mouse or pig faeces. However, sensitivity of PCR detection of DNA obtained by enzymatic lysis based method was lower compared to the one achieved with DNA isolated by chemical lysis. When QIAamp DNA Stool Mini Kit procedure was used detection limit was 5 and 10 fold higher for Cryptosporidium parvum and Trichinella spiralis, respectively. Only NucliSENS miniMAG procedure combined with mechanical disruption of the faecal material allowed detection of Cryptosporidium sp. in human fecal samples collected from children with diahorrea, and detection of Trichinella spiralis in stool samples obtained, on days 8 to 12 post infection, from mice experimentally infected with 500 muscle larvae per mouse. Thorough mechanical disruption with simultaneous chemical lysis allows efficient isolation ofDNA of the investigated intestinal parasites present in stool and the subsequent PCR detection.

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair.

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.

Detection of Trichinella spiralis DNA in mouse faeces during the early stage of infection.

Trichinella spiralis is an etiological agent of trichinellosis, a parasitic disease present worldwide, affecting both animals and humans. The infection starts from the intestinal stage which usually lasts a few weeks and develops into the muscle phase when larvae enter the muscle cells and create a nurse cell-larva-complex. In animals, the infection is asymptomatic even if a high number of larvae is ingested. The aim of this work was to develop a sensitive test to monitor the early phase of trichinellosis in laboratory animals. T. spiralis DNA presence was assayed in faeces of mice during the first 21 days of experimental infection. The target gene fragment of the mitochondrial ATP6 synthase F0 subunit was amplified by PCR from faecal samples of mice infected with 50, 250 or 500 larvae per mouse (lpm). Trichinella DNA was detected in 45% (116) of the examined faecal samples from days 1 to 21 post infection (p.i.). The time of appearance of the parasite DNA in the faeces and the percentage of PCR positive results were dose dependent. In collective samples consisting of faeces from 3 mice average 80%, 83% and 90% of PCR positive results were obtained for 50, 250 and 500 lpm doses, respectively, throughout 21 days of the observation period. In individual mice faeces DNA was detected on days 2-19 and 9-18 p.i. for 250 lpm and 50 lpm doses, respectively. For both infective doses the highest probability of DNA detection was on day 11 p.i. Since the developed test is simple and non-invasive, it can be useful for monitoring the intestinal phase of Trichinella sp. infection in laboratory animals.