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Introduction: Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory infection. In serological diagnosis of M. pneumoniae infections tests have been described based on the purified P1 protein, which is the most important virulence factor of this pathogen, as antigen. The aim of his study was to express and purify a recombinant protein P1 M. pneumoniae and next evaluate this protein as high specific antigen in serodiagnosis of mycoplasmosis., Methods: Protein P1 M pneumoniae was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Based on published literature, we decided to express C-terminal region [P1-C1] encompassing amino acid residues 1160 to 1521. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His-trap). Serum samples collected from 221 patients with mycoplasmosis, positive in complement fixation test (CFT), 87 patients with other then mycoplasmosis bacterial infections and 80 blood donors were screened for anti-P1 recombinant protein IgA, IgG and IgM antibodies by using the home-made ELISA., Results: SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P1 protein preparation with an expected molecular mass of 39,7 kDa. The specificity of the recombinant protein was confirmed by western blot analysis using serum samples from rabbits immunized by M pneumoniae. The results of ELISA revealed that more then 70.0% of patients with mycoplasmosis confirmed by CFT, had antibodies to recombinant P1 protein in diagnostically significant level (x + 2SD). The antibodies were found only sporadically in sera obtained from patients with other then mycoplasmosis bacterial infections and clinically healthy persons. A comparison of results obtained in home-made ELISA with results of commercial western blot (Virotech) showed similar, ranged from 84.2% to 97.4%, compatible of results. The IgM antibodies to recombinant P1 protein were found in 87.2% sera obtained in acute phase of disease, in 80.0% sera obtained 2-4 weeks after onset of clinical symptoms and only in 43.8% sera obtained in chronic mycoplasmosis., Conclusions: The present study confirmed the earlier observations of the high usefulness of recombinant P1 protein for reliable serologic diagnosis of M. pneumoniae infection.

Unlabelled: OBJECTIVES. The ST-17 clone of Streptococcus agalactiae (Group B Streptococcus, GBS) identified by Multi Locus Sequence Typing (MLST) is recognized as a hypervirulent international clone mainly associated with invasive neonatal infections. There is no data on the occurrence of the ST-17 clone of GBS in pregnant women and newborns in Poland., Methods: 169 strains of GBS, isolated from pregnant women's carriage, and 42 from newborns with invasive infection or asymptotic carriage, which were derived from the collection of the Chair of Microbiology Jagiellonian University Medical College, collected between 2006-2010, were genetically characterized. The PCR with specific primers ST17S and ST17AS was adopted to characterize the gbs2018 gene variant specific to ST-17 clone. All positive results in direction of ST-17 were confirmed by MLST. The genetic relatedness of the ST-17 clone was determined by PFGE., Results: The hypervirulent ST-17 clone was detected in 2.4% of women with GBS asymptotic carriage, whereas it was detected in 18.2% newborns with invasive infection. No ST-17 was isolated from healthy neonates. Statistically significant correlation between the presence of ST-17 and neonatal GBS infection was showed, in comparison to healthy neonates colonized by GBS (p = 0.0398) and to GBS-positive pregnant women (p <0.00001). The ST-17 strains belonged to 2 clearly distinguishable PFGE type, but were closely related to one another at a level of 60% similarity. CONCLUSIONS. The results emphasize the necessity of a regular study of the ST-17 GBS clone occurrence among pregnant women and newborns in Poland.

Introduction: Infections caused by Staphylococcus epidermidis form frequent complications after implantation of foreign bodies, including orthopedic implants. The ability of adhesion to synthetic surfaces is influenced by both the properties of the microorganisms under concern as well as physicochemical properties of the implant surface. In this study, biofilm formation abilities by S. epidermidis strains on orthopedic implants made of different materials have been evaluated., Methods: Researches have been performed on 60 S. epidermidis strains. The implants for osteosynthesis--cortical bone screws: steel, steel coated with titanium and steel coated with nanocrystalline diamond were used for the analysis. Slime production ability was assessed using Congo Red Agar method. Cell surface hydrophobicity was determined by salt aggregation test (SAT) and bacterial adhesion to hydrocarbon test (BATH). Presence of the icaA, icaD, icaC and icaB genes was detected by PCR. Biofilm formation of S. epidermidis strains was analysed according to Richards method., Results: Out of the 60 strains 43% were labeled as slime producing. 48% of S. epidermidis strains formed biofilm on steel screws, 43% on titanium implants and 45% of strains on implants coated with nanocristalline diamond. IcaA, icaD, icaC and icaB genes have been detected in 31%, 25%, 20% and 27% of the strains respectively., Conclusions: The degree of biofilm formation was the lowest on the steel screws coated with titanium.

Introduction: Varicella-zoster virus (HHV-3) is spread by the respiratory route and disseminates to lymph nodes and then via lymph back to the skin, resulting with the rash of chickenpox. Like other alpha-herpesviruses, HHV-3 infects the neurons of the dorsal root ganglia, where it causes lifelong latency. Virus reactivation causes episode of herpes zoster (shingles). During severe HHV-3 infections molecular methods, such as real-time PCR (qPCR) assay, are recognized as a method-of-choice for detecting viral DNA in clinical specimens. The aim of this study was to develop the qPCR assay for detection and quantification of varicella-zoster virus DNA in different clinical samples, using specific primers targeting a HHV-3 DNA ORF62 gene and a fluorescent TaqMan probe., Methods: The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 100 and 3 125 000 copies/ml. Limit of detection (LOD) was calculated using probit analysis, and was determined as 750 copies/ml. In further studies 18 clinical samples (sera, whole blood, skin swabs), taken from a small group of 5 children with symptoms of chickenpox were tested for the presence of varicella-zoster virus DNA, using LightCycler 2.0 system., Results: Described in-house qPCR method detected viral DNA in all examined specimens. Detected viral load was between 5750 copies/ml for sera samples and 27 300 000 copies/ ml for skin swabs, respectively., Conclusions: The results of this work showed that developed real-time PCR assay based on TaqMan probe was very reliable and valuable for detection and quantification of varicella-zoster virus DNA in different clinical samples. The high level of sensitivity, specificity, accuracy, and rapidity provided by the developed method are favorable for its use for the detection of HHV-3 DNA in laboratory practice.

Introduction: Adhesion of pathogenic bacteria to host cells is a crucial step during infection. The presence of specific adhesion factors on the bacterial cell surface determines the tropism of the pathogen to the tissues expressing certain surface receptors. The adhesion is mediated primarily by filamentous structures called pili. Three distinct pilus structures can be produced by Corynebacterium diphtheriae: SpaA-, SpaD- and SpaH-type pili. Pilus genes encode a total of nine pilus proteins, named SpaA through SpaI, and six sortases, named SrtA through SrtF. All the pilus genes are located on pathogenicity islands and can be acquired and lost by different strains. The aim of presented studies was assessment of occurence of pili genes among C. diphtheriae strains isolated from different infections, including invasive infections, in Poland., Methods: Thirty-one toxigenic and nontoxigenic C. diphtheriae strains isolated from wounds, blood, nose and pharynx were investigated for presence of 15 pili genes. The studies were conducted using PCR. Gene specific primers were designed on the basis of the complete genome sequence of C. diphtheriae NCTC 13129., Result: All the nontoxigenic C. diphtheriae strains isolated from invasive infections possess every tested genes in contrast to toxigenic strains that revealed highly mosaic structure of pili gene clusters. Differences in gene content were detected in SpaA- and SpaH-type pili gene clusters. Complete set of genes in SpaD-type pili gene cluster was detected in all but two strains. The two strains did not possess any of SpaD-type pili genes., Conclusions: Invasiveness of C. diphtheriae strains could be related to adhesive factors. Results of our studies suggest that ability to express all types of pili is indispensable for causing invasive infections by nontoxigenic C. diphtheriae. Whereas full set ofpili genes is not necessary for causing classical diphtheria.

Introduction: Among different laboratory techniques used in influenza surveillance, methods based on molecular biology, such as real-time PCR, play increasingly important role. They allow detection and identification of viruses currently circulating in human population and responsible for infections and diseases. The aim of the study was to develop duplex real-time PCR assay for detection and differentiation of influenza virus subtype A(H1N1) pdm09 and A(H3N2)., Methods: Specific primers targeting a highly conservative region ofhaemagglutinin gene and a dual-color TaqMan probes, labeled with fluorophore JOE (560 nm) and quencher BHQ-1 or CalFluor Red 610 (610 nm) and BHQ-2 were designed. The specificity of duplex reaction was evaluated using RNA isolated from reference strains of influenza virus A(H1N1)pdm09, A(H3N2), A(H1N1) and A(H5N1), B and other respiratory pathogens. Sensitivity of the assay was tested using serial dilutions ofplasmid constructs carrying fragment of specific viral HA gene, in range between 10 and 10(5) copies/sample. A number of 116 clinical samples were tested using developed duplex qPCR assay in comparison with the CDC monoplex assay,, Results: Positive duplex qPCR results were obtained only in samples containing RNA of influenza viruses of a specific subtype. The limit of detection (LOD) of developed method amounted up to 27 copies/sample for A(H1N1)pdm09 and up to 37 copies/sample for A(H3N2)., Conclusions: The results show that developed TaqMan-based duplex qPCR assay is a valuable diagnostic tool in influenza virus surveillance for using in direct study of clinical specimens as well as identification of isolated strains of influenza virus.

Aim: The aim of the study was the evaluation of clinical symptoms suggesting Epidermolysis bullosa dystrophica in a 5 years old boy and identification of an underlying molecular defect., Material and Methods: The patient was a 5 years old boy with the history of transient bullae and milla formation in the fi rst three years of life In the last two years such tendency has not been observed, with the exception of dystrophic toenails. The family history of bullous diseases is negative. Molecular analysis was performed using direct sequencing of COL7A1 gene encoding collagen type VII., Results: A novel mutation p.Gly2064&#95;Arg2069del was identified in the patient. The identified mutation does not appear in the patient's parents nor in the patient's brother. Molecular analysis in the family members revealed that the mutation occurred de novo. Biparental inheritance of genetic material for chosen genetic markers was confirmed., Conclusions: On the basis of clinical features observed, results of molecular studies and literature data, diagnosis of mild dominant Epidermolysis bullosa dystrophica can be established in the index case.

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.

Giardia is the most common intestinal protozoan parasite found in humans and animals worldwide. Although it has been known for three hundred years, the nomenclature, taxonomy, host specificity, and pathogenicity of Giardia still arouse numerous controversies and ambiguities. Giardia is classified into six species, that are characterised by various ranges of hosts. The most dubious species is G. intestinalis, which includes a dozen or so genotypes, and only two of them (genotype A and B) have wide ranges of hosts, including humans. Moreover, in some genotype assemblages of G. intestinalis certain subgenotypes were distinguished and it was proven that in the same host species various subgenotypes of this parasite may occur. Bearing in mind the significant genetic heterogeneity of G. intestinalis and the fact that various genotypes and subgenotypes of this parasite are characterised by the broad or narrow host specificity, the data concerning the frequency of giardiosis occurrence are insufficient. It is necessary to use molecular biology techniques in order to define the genotype and/or the subgenotype of G. intestinalis that are found in humans and in certain animal species. Furthermore, since more and more pieces of evidence connected with a possibility of the sexual recombination of Giardia are gathered, it is unknown if genotypes and subgenotypes of this parasite are stable in time. The aim of this thesis was to define the frequency of Giardia occurrence in humans and animals in Wielkopolska region, to identify species and genotypes of Giardia that occur in humans and animals, as well as to obtain an axenic culture of the chosen isolates of Giardia from animals and to compare the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites in order to check if the axenisation of G. intestinalis leads to the selection of genotypes or if Giardia genotypes are stable in time. Altogether, 2183 faecal samples were examined for the presence of Giardia cysts; 447 faecal samples were taken from 232 persons coming from 5 cities situated in Wielkopolska, and 1736 faecal samples were obtained from 123 animal species, including: 266 faecal samples from 113 species of animals kept in the Zoological Garden in Poznań, 1286 samples from 4 species of breeding animals, 118 samples from dogs, and 66 samples from 5 species of wild animals. Faecal samples were taken from animals coming from 25 places in Wielkopolska. Moreover, seven isolates of G. intestinalis were used in the studies, which were obtained from various species of hosts and kept in an axenic in vitro culture. Microscopic, molecular and bio-informative methods were used in the studies. From each faecal sample fresh smears were made in a 0.6% solution of physiological salt and in Lugol's solution, as well as a permanent smear stained with trichrome was made. Moreover, the following molecular techniques were implemented in the studies: DNA extraction and purification, the PCR technique (two molecular markers), electrophoresis and visualisation of PCR products, and sequencing. A fragment of the beta-giardin gene was used as a molecular marker in order to define the genotype and subgenotype of Giardia. Only in the case of genotyping of two Giardia isolates obtained from Peromyscus eremicus another molecular marker (SSU rRNA)was additionally used. Some widely available computer programmes (Chromas, CAP 3, BioEdit, BLASTn, MEGA version 4.0) were utilised in the analysis of the sequence of the beta-giardin gene fragment and in the phylogenetic analysis. The culture of Giardia trophozoites was established to compare the sequence of the partial beta-giardin gene from cysts and trophozoites. Concentration and purification of Giardia cysts in the saccharose gradient, and the excystation technique were applied in the studies to obtaining an axenic in vitro culture. In this study, Giardia cysts were found in 12 faecal samples obtained from 3 persons and 5 animal species. Giardia cysts were found only in faecal samples from humans living in Poznań and the samples obtained from animals coming from Poznań and around Puszczykowo. The highest frequency of infection was stated in domestic animals (2.5%) and in animals kept in the Zoological Garden (2.0%), whereas a slightly lower frequency was noticed in wild animals (1.5%) and in humans (1.3%). No Giardia cysts were found in the faecal samples collected from breeding animals. Two new species of Giardia hosts were identified, namely Rhinella marina and Peromyscus eremicus; however, due to a minimal amount of faecal samples supplied for the study it was impossible to define the species and genotype of this parasite. PCR products (the partial of beta-giardin gene) were obtained in seven faecal samples out of the ten studied, including three samples from people and four faecal samples derived from three animal species (i.e. dog, tamandua, red deer). Moreover, molecular characterization of seven Giardia isolates from three persons and four animal species (red-bellied monkey, silver marmoset, Thomson's gazelle, and sheep) kept in an axenic in vitro culture was performed. Based on the beta-giardin sequence fragment analysis, four assemblages of G. intestinalis genotypes were identified (A, B, C and D). In humans, A and B G. intestinalis genotypes and three subgenotypes, including a cosmopolitan subgenotype A2 and two new subgenotypes A and B were detected. Furthermore, four G. intestinalis genotypes were found in animals, including three genotypes which are non-infectious to humans, namely: genotypes C and D in dogs and a cervids-specific genotype A in red deer (Cervus elaphus), which indicate that these animals do not constitute the source of infection to humans. On the other hand, in a tamandua from the Zoological Garden in Poznań a new subgenotype B of G. intestinalis was identified, which due to a close relationship with Giardia isolates obtained from humans is potentially infectious to man. In none of the studied faecal samples a mixed infection of Giardia was found. To date, nine sequences of the partial beta-giardin gene have been deposited in the National Center for Biotechnology Information (NCBI), including two sequences of Giardia isolates obtained from humans (GenBank accession numbers FJ009207, FJ009208), three sequences of isolate obtained from red deer (GenBank accession numbers EU621373, EU626198, EU216429), two sequences of both Giardia isolates obtained from dogs (GenBank accession numbers FJ009205, FJ009206), and the single sequences obtained from tamandua (GenBank accession number FJ009209) and from Thomson's gazelle (GenBank accession number EU626199). According to the literature, an axenic in vitro culture of G. intestinalis was obtained from a red deer for the first time. Based on the analysis of the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites it was proven that the red deer was infected with a single population of Giardia and that during the axenisation of the culture no mutation in the DNA of the parasite's trophozoites took place. Probably the time distance that the DNA was isolated from the trophozoites kept in the culture was too short to cause the mutation. This suggestion is confirmed by the results of the genotyping of seven G. intestinalis isolates obtained from various host species and kept in an axenic in vitro culture for at least a number of years. Based on the molecular characteristics it was stated that all the studied isolates from the axenic culture were identical and belonged to the same assemblage B. The comparision with the sequences from GenBank database revealed that all mentioned isolates were 99% similar to the sequence of Giardia Nij5 isolate obtained from a person from the Netherlands and characterised as genotype B1. Due to the sameness of the molecular marker sequences it seems improbable that the identical G. intestinalis genotype occurred in various time periods (the largest difference was 14 years) in humans and in a number of animal species in diverse areas of Wielkopolska region. Quite opposite, the long-term keeping of these isolates in the homogenous conditions of an axenic in vitro culture leads to the selection of a genotype or proves the instability of genotypes of this parasite. Long-term studies need to be conducted in order to verify these hypothesis. Their results will have a key meaning in explaining the genetic structure of the Giardia population and in understanding the molecular epidemiology of giardiosis.

The aim of study was to evaluate the applicability of the PCR multiplex method for the identification of Streptococcus agalactiae (group B streptococci; GBS) serotypes and to indicate the participation of serotypes in the group of pregnant women. 1178 pregnant women at 35-37 weeks of pregnancy were studied between 2007-2009. Vaginal and rectal swabs collected from these women were tested for the presence of GBS. S. agalactiae identification was done basing on the culture method according to the CDC recommendations. For all isolated GBS strains the surface polysaccharides which conditioning serotypes were determined using commercially available serological latex kit and multiplex PCR method with two sets of specific primers. The frequency of GBS carriage among 1176 pregnant women was 30% (353 cases). Simultaneous colonization of the vagina and anus was demonstrated in 80% women, whereas GBS carriage only in the vagina or only in the anus was indicated in 9% and 11% cases, respectively. In pregnant women colonized by GBS serotype III (35%) dominated. The other serotypes were detected with the following frequency: Ia (20%), V (17%), II (15%), Ib (8%) and IV (5%). In the analyzed women population, GBS strains belonging to the VI, VII, VIII serotypes were not found. The results of our study show a great diagnostic usefulness of multiplex PCR method for identification of genes encoding GBS surface polysaccharides that are typical for individual serotypes.

Cutaneous tuberculosis is a specific form of tuberculosis, with various clinical pictures and resulting from either endo- or exogenous way of infection, immunological mechanisms and unfavourable conditions for mycobacterium development. The atypical course and symptoms of the disease may cause difficulties in obtaining proper diagnosis and, in consequence, result in delayed onset of appropriate treatment. When diagnosing cutaneous tuberculosis, a broad spectrum of differential diagnoses should be applied, taking into account other diseases, such as, among others, leishmaniasis, actinomycosis, leprosy or deep mycoses. In this report, a case of lymph node tuberculosis and of colliquative tuberculosis of the skin, at first erroneously diagnosed as actinomycosis, complicated by multiform erythema. In the reported case, no tuberculous bacilli were identified in bacteriological evaluations of bioptates, collected from the skin changes. The final diagnosis of the disease was determined by the presence of specific granulation tissue in the last of performed histopathological studies, as well as by hypersensitivity to tuberculin and the presence of mycobacterial DNA in PCR evaluation. According to the authors, in case of clinically suspected cutaneous tuberculosis, repeated (several) histopathological studies of samples from observed changes seem to be fairly justified. The results of histopathological studies should be completed by one of the methods of oligomycobacterial material evaluation, e.g. by identification of mycobacterial genetic material by means of nucleic acid amplification in the PCR method.

Incontinentia Pigmenti (IP, Bloch-Sulzberger syndrome, OMIM 308300) is a rare X-linked dominant genodermatosis, usually lethal in males in the prenatal period. Wide spectrum of clinical expression consists of skin hyperpigmented lines and swirling patterns, dysplastic teeth and nails, and in 30% central nervous system abnormalities including seizures, microcephaly and intellectual disability (10% of cases). In 80% of IP cases, the disease is caused by a large-scale deletion of exons 4 to 10 of the NEMO gene. Three cases of variable expression of Incontinentia Pigmenti are presented. In a one-year-old girl, her mother and grandmother molecular analysis revealed the same typical deletion of the NEMO gene. In the proband, characteristic skin lesions were detected located over the trunk and lower limbs. Characteristic evolution of the changes was observed. In the mother, expression of the disease was much milder, whereas in the grandmother lesions were restricted to the fingernails. Clinical characteristics and pedigree data are described.

The ABO human blood group system consists of A antigens, B antigens, and antibodies against these antigens. The antigenic determinants are synthesized in the Golgi apparatus by specific glycosyltransferases which transfer proper sugars to an oligosaccharide acceptor, called H antigen. N-acetylgalactosaminotransferase (transferase A) uses a UDP-GalNac donor to convert the H antigen to A antigen, whereas galactosyltransferase (transferase B) uses a UDP-galactose donor to convert the H antigen to B antigen. The amino-acid sequences of transferases A and B differ by four residues, of which only two cause a change in enzyme specificity. These residues are Leu/Met266 and Gly/Ala268 in transferases A and B, respectively. Structural studies revealed that the presence of amino acids with bulky side chains (methionine and alanine) in transferase B cause its inability to bind N-acetylgalactosamine. The recessive trait O, in which antigens A and B are not present, is caused by the expression of an incomplete enzyme as a result of a base deletion and a subsequent reading frame change. In addition to the basic ABO gene variants, several alleles are rarely found that may lead to the expression of enzymes with different specificities. In this article the mechanism of the synthesis of A and B antigens, the molecular background of ABO gene variablity, their allelic variants, and possible mechanisms by which they emerge are described.

The availability of sensitive cell line for isolation of measles virus from clinical samples and establishment RT-PCR and molecular sequencing metods have allowed for rapid genetic characterization of wild-type strains of measles virus. This sequence information makes it possible to identify the source of wild-type viruses and differentiate between native and reported cases. The aim of this study was evaluation of virus isolation in cell culture in National Laboratory in Poland (Department of Virology in NIH), presentation of RT-PCR and molecular genotyping results performed in Regional Reference Laboratory in Berlin (Robert Koch Institut).

Protozoa Babesia and Theileria are a group of parasites transmitted by ticks and invading mammals' blood cells. Infections by these protozoa cause heavy losses among farm animals. Since human babesiosis was described forty years ago, the number of reported cases has been increasing. Conventional methods of parasitological diagnostics are unreliable in these diseases, especially in stages with low parasitemia. Methods of nucleic acids detection have been developing fast in recent years and are more and more specific and sensitive. Polymerase Chain Reaction (PCR) enables detection of DNA of Babesia and Theileria, even in parasitemia as low as a few organisms per milliliter. Real-time PCR is useful especially in early stage of babesiosis when results of serological tests are negative and the blood smear does not reveal the pathogen or for distinction between intererythrocytic forms and Plasmodium. RLB is a method more sensitive than PCR and enabling detection of coinfections with different organisms. LAMB is a method of DNA amplification in isothermal conditions, offered as faster, easier, but still highly specific alternative to PCR. NASBA is a method where RNA of Babesia and Theileria is an initial template. The choice of a specific method highly depends on cost of laboratory equipment needed.

Aim: The aim of the study is to identify HLA-DQA1, HLA-DQB1 allele and to assess the risk of early pregnancy loss of women, couples with reproductive failure in the first trimester of pregnancy in comparison with fertile women, couples. The study group (B) enrolled 61 couples with reproductive failure and the control group (C) enrolled 20 fertile couples with at least 2 children., Method: HLA-DQA1 gene typing was performed using PCR-sequence-specific primer (SSP) on the high resolution level according to established procedure of labeling and using the detection kit (FASTYPE DQASSP Typing, FASTYPE DQA "High Resolution" Typing Sheet) purchased from Bio-Synthesis (USA)., Results: In female patient the highest risk quotient was associated with alleles HLA-DQA 01101/0105 OR 7.19 (95% CI 1.18-5.23; p=0.03) and HLA-DQB5 OR 3.67 (95% CI=1.11-12.0; p=0.037). The lowest but statistically significant risk of pregnancy failure in this group was related to allele HLA-DQB6 OR 0.48 (95% CI=0.22-1.04; p=0.087). In patient and control couples the significantly increased risk of pregnancy failure was related to the frequency of HLA-DQB5 allele OR 2.3 (95% CI 1.09-4.82; p=0,035). The lowest risk quotient in the patient couples was associated with HLA-DQ 0302/0303 allele OR 0.44 (95% CI 0.14-1.36; p=ns)., Summary: HLA-DQA and HLA-DQB allele might influence pregnancy outcome in the Polish population, but further studies are necessary in this regard.

Formation of a phenotype during development of the human being may result from a specific dialogue between genes and environmental factors. Expression of particular genes is controlled not only on the transcriptional level but also on the level of accessibility of genetic information through the influence for remodelling of chromatin. We characterized Rett syndrome and its molecular basis as an example of the relation between genes and environment and their influence on epigenetic processes determining the gene expression. Examples of importance of DNA methylation in psychiatric disorders were shown.

Introduction: The aim of this study was to analyse the distribution and frequency of mutations and their correlations with clinical phenotypes of patients with MTC, to reveal the differences between sporadic and familial type of MTC, and to describe the phenotypes of patients., Materials and Methods: 212 patients with medullary thyroid cancer (MTC) were treated in Cancer Centre in Warsaw between 1997 and 2005. In most patients, DNA isolated from peripheral blood leukocytes was tested for RET gene mutations by sequencing and accordingly MTC form was assessed. Genetic testing was performed in the relatives of patients with familial MTC in order to distinguish asymptomatic mutation carriers from noncarriers., Results: RET gene mutations were identified in 46 patients (22%). The others were found noncarriers and sporadic MTC was diagnosed. MEN 2A/FMTC syndrome (multiple endocrine neoplasia type 2A/ familial type of MTC) was diagnosed in 44 patients, MEN 2B syndrome (multiple endocrine neoplasia type 2B) in 2 patients. In patients with sporadic and familial MTC, age at diagnosis and multifocal occurrence was analysed, and the results were found to be in accordance with those of other research centres. However, the distribution and frequency of mutations, as well as some clinical data, such as the frequency of pheochromocytoma occurrence as the first manifestation of MEN syndrome, differed from the published data, and further studies are necessary to reveal the reasons of these differences., Conclusions: DNA testing for RET gene mutations is reliable as a diagnostic tool and therefore it should be performed for screening of all patients with MTC or other diseases of MEN syndrome.

Introduction: Leptotrichia bacteria belong to the group of Gram-negative anaerobes most frequently colonizing the oral cavity and reproductive organs. Leptotrichia sanquigenes constitute one of the bacterial factors in postpartum bacteremia of mothers and newborns, whilst Leptotrichia amnionii sp. nov., described for the first time in 2002, has turned out to be the etiopathogenetic factor in missed abortion in the second trimester of pregnancy., Aim: The purpose of this study was evaluation of the frequency of occurrence of Leptotrichia amnionii sp. nov. in patients in the 1st, 2nd and 3rd trimester of pregnancy., Materials and Methods: The study involved 69 patients. Amplification reactions detecting the presence of Leptotrichia amnionii so. RNA in the smears were performed using specific starters complementary to 16SrRNA Leptotrichia amnionii--primer 1 Lam upper and primer 2 Lsp lower--complementary to Leptotrichia 16SrRNA. After amplification, the specimens were placed on 1% agarose gel in the presence of MassRuler marker (Fermentas). Next, PCR products were ligated to pGEM-T Easy Victor and then competent E. coli DH5 alpha cells were transformed with pGEM vector with an insert. Plasmid DNA isolation was performed using Plasmid Mini kit from A&A Biotechnology. Sequencing of inserts from the purified plasmids was performer in Molecular Biology Techniques Laboratory in the Department of Biology at Adam Mickiewicz University in Poznań., Results: In the group of 69 pregnant patients Leptotrichia amnionii sp. nov. was identified in 8 women, which is 11.6%., Conclusion: Recently identified Leptorichia amnionii sp. nov. is a bacterie, which relatively frequently occurs in pregnant women.

The article presents results of valuation for B. anthracis-specificity and usefulness for its identification obtained for different chromosomal markers. In the first part of the study markers SG-749, SG-300 and SG-450 were analyzed. For the investigation RFLP-PCR and MSSCP techniques were used and different electrophoresis methods were tested. The results gave an information not only about specificity of tested markers but also about the possibility of shorten time necessary to obtain results of B. anthracis identification.

Unlabelled: Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis., Aim: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland., Material: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland., Methods: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains., Results: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.

Unlabelled: Mutations in the LAMB2 gene encoding laminin beta2, a component of the glomerular basement membrane and the neuro-muscular junction are responsible for the characteristic renal and eye abnormalities of Pierson syndrome. We report a girl with confirmed LAMB2 mutation who presented with early onset Congenital Nephrotic Syndrome (CNS) with renal failure and ocular findings of bilateral microcoria, persistent hyperplastic primary vitreous, right microphtalmia and left eye cataract. Automated peritoneal dialysis was started from the 3rd month of life. Severe muscle hypotonia with motor and mental delay were observed during the first year of life. She experienced numerous serious infections from birth and died at the age of 15 months due to a fulminant infection. Genetic studies revealed two novel mutations in LAMB2 gene (compound heterozygosity)., Conclusions: 1. Mutations in LAMB2 gene should be included in the work-up of patients with CNS in the presence of eye anomalies. 2. Severe phenotypes of Pierson syndrome are associated with marked handicaps and a poor outcome.

All living organisms possess adaptive responses to environmental stresses that are essential to ensuring cell survival. One of them is the stringent response, initially discovered forty years ago in the gram-negative model organism E. coli. Recently plant homologues to the bacterial relA/spoT genes were identified (RSH genes--RelA/SpoT Homologues). Also the products of rsh proteins activity--(p)ppGpp were identified in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to some biotic and abiotic stresses. Elevation of ppGpp levels was elicited also by treatment with plant hormones. What is more--in vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp. It is supposed that plant stringent response is a conserve stress-response pathway possibly operating via regulation of chloroplast gene expression and, thus, the regulation of plastid metabolism.

The classification of amino-acid substitutions into pathogenic mutations and harmless polymorphisms should be revised. In the recent years it was shown that some amino-acid substitutions considered as pathogenic mutations were polymorphisms. Similarly, some 'harmless' polymorphisms have been shown to be pathogenic mutations. Functional analysis considered as a good method to estimate the pathogenic nature of mutations is also limited. The selection of DNA samples for the control group is also difficult. Due to the molecular mechanism mediated by recently discovered exonic splicing enhancers and silencers (ESE and ESS) it is hard to predict a pathogenic effect of some mutations. In addition, the phenotype variability observed between unrelated patients harboring the same mutation may reflect the effects of modifying genes as well as the lack of association between mutation and "its" phenotype. The aim of this study is to describe the problem of the pathogenic effect of mutations.

The article presents results of valuation for B. anthracis-specificity and usefulness for its identification obtained for different chromosomal markers. In the second part of the study markers SSH241, SSH196, SSH163, SSH133 as well as a fragment of the house-keeping gene rpoB were analyzed. For the investigation MSSCP and multiplex-PCR assays were used. There were also tested different techniques of electrophoresis. The results gave an information about specificity of tested markers and their usefulness for B. anthracis identification.

At the 5' and 3' end of genomic HCV RNA there are two highly conserved, untranslated regions, 5'UTR and 3'UTR. These regions are organized into spatially ordered structures and they play key functions in regulation of processes of the viral life cycle. Most nucleotides of the region located at the 5' side of the coding sequence serve as an internal ribosomal entry site, IRES, which directs cap-independent translation. The RNA fragment present at the 3' end of the genome is required for virus replication and probably contributes to translation of viral proteins. During virus replication its genomic strand is transcribed into a strand of minus polarity, the replicative strand. Its 3' terminus is responsible for initiation of synthesis of descendant genomic strands. This article summarizes our current knowledge on the structure and function of the non-coding regions of hepatitis C genomic RNA, 5'UTR and 3'UTR, and the complementary sequences of the replicative viral strand.

The immunoglobulins of the Camelidae family belonging to subclasses IgG2 and IgG3 consist of heavy chains only. The lack of light chains is caused by a point mutation in the heavy-chain gene, resulting in the loss of the splice consensus signal and the removal of the entire CH1 domain together with introns. The heavy-chains antibodies also contain longer hinge regions and conservative amino-acid substitutions in the framework regions. Despite the lack of light chains, the heavy-chain antibodies reveal normal antigen binding ability and effector functions. The heavy-chain antibodies are relatively easy to clone and possess good stability, high specificity, low molecular weight, and the ability to recognize unique epitopes. Possible areas of application of heavy-chain antibodies include their use as in vivo imaging reagents and sources of peptide-based drugs.

The article presents the current knowledge on the proteines under question. The first analysed E-S products released by G. duodenalis was a polydisperse hydrophobic complex (16.5-225 kDa), protease VI sensitive, chloroform-methanol insoluble. Based on inhibition studies cysteine protease and metalloprotease were detected in the complex. The further analysis revealed that a 58 kDa heat stable as wall as protease sensitive glycoprotein secreted by G. duodenalis trophozoites is highly immunogenic for the hosts. Before encystation, G. duodenalis trophozoites secrete leucine-rich cyst wall proteins: CWP1, CWP2 and CWP3. Steady-state levels of CWPs gene transcripts are low in non-encysting trophozoites but increase more than 100-fold during encystation. Another protein, which expression also increases during encystation is gGSP (Giardia Granule-Specific Protein). The protein possesses typical strucutre of calcium-binding proteins. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this protein regulates Ca(2+)-dependent degranulation of encystation-specific secretory vesicles (ESVs) during cyst wall formation.

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.

Leukaemia inhibitory factor (LIF) is a multifunctional cytokine, which plays a role in growth-promotion and differentiation, regulates calcium and bone metabolism, induces acute phase proteins and causes cachexia in organisms with neoplastic disorders. Moreover, LIF participates in the induction of inflammation, and therefore represents an important pathogenic factor of many disorders. The multifunctional properties of LIF have become of special interest to investigators from different disciplines of medicine.

Peptide histidine-isoleucine (PHI) and its human analogue peptide histidine-methionine (PHM) are members of a superfamily of structurally related peptides embracing, among others, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), peptide histidine-valine (PHV), and helodermin. All the peptides display a pleiotropic biological activity. PHI, PHM, PHV and VIP are co-synthesized from the same precursor and share high levels of structural and functional similarity. These peptides may act through common receptors and are widely distributed throughout the body tissues (the central nervous system, gastrointestinal tract, respiratory system, and reproductive system); however, their role remains largely unknown. Changes in the levels of the peptides in the course of different diseases suggest their possible importance and usefulness in diagnostics. Moreover, the neurotrophic and neuroprotective properties of PHI suggest, by analogy to VIP or PACAP, its therapeutic potential in many neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

The problem of species recognition in relation to parasite organisms is discussed, based on literature data. Taking into account that over 20 species concepts have hitherto been formulated, the choice of characters, which should define a species differs from author to author. Lately the differences in some gene fragments are regarded as one of most important feature for species differentiation. On this basis some lineages, genotypes or strains have been differentiated within some species, but in most cases the respective authors did not decide to elevate these units to the species level. The present paper focuses on the studies on species representing three genera (Taenia, Echinococcus, Trichinella) as they show, especially the complex studies on the species within the genus Trichinella, how big material is necessary to formulate any proper conclusion and, on the other hand, that the differences in the DNA sequences cannot be the only characters defining species; some other features (morphological, biological, behavioral) should not be considered redundant.

There are many methods for detection of Cryptosporidium oocysts. Most of them (more than 20) enable the microscopic detection of Cryptosporidium oocysts in faecal smears. Such a great variability of diagnostic methods may lead to confusion as far as the choice of an appropriate technique by a given laboratory is concerned. This study evaluated the diagnostic usefulness of Cryptosporidium oocysts and coproantigen detection methods in the diagnosis of cryptosporidiosis in human (266 stool specimen) and animals (205 from cattle, 160 from sheep, 30 from horses, 80 from cats, 227 from dogs and 11 from wild animals). The total number of human and animal stool specimens processed was 266 and 713, respectively. In this study the usefulness of several diagnostic methods was compared. The following techniques were taken into account: wet mounts, hematoxylin staining, four different specific methods (modified Zeihl-Neelsen, Kinyoun's, safranin-methylene blue, as well as carbol-methyl violet and tartrazyne) and commercially available kit based on enzyme-linked immunoassay (ProspecT(r) Cryptosporidium Microplate Assay). The final number of positive specimens was 123. Out of them 77 were positive in all specific methods. The oocysts found in stool specimens were measured. Humans were infected with C. parvum and animals with C. parvum, C. andersoni or C. felis. The statistical analysis has shown that EIA test was a better than microscopy method for identification of Cryptosporidium in faecal samples in human and wild animal. Sensitivity and specificity are important factors for the choice of a proper diagnostic method for Cryptosporidium detection, however other factors such as cost, simplicity and ease of interpretation of results are also important considerations.

Evolution and systematics of nematodes based on molecular investigation. The use of molecular phylogenetics to examine the interrelationships between animal parasites, free-living nematodes, and plant parasites versus traditional classification based on morphological-ecological characters was discussed and reviewed. Distinct differences were observed between parasitic nematodes and free-living ones. Within the former group, animal parasites turned out to be distinctly different from plant parasites. Using small subunit of ribosomal RNA gene sequence from a wide range of nematodes, there is a possibility to compare animal-parasitic, plant-parasitic and free-living taxa. Nowadays the parasitic nematodes expressed sequence tag (EST) project is currently generating sequence information to provide a new source of data to examine the evolutionary history of this taxonomic group.

Many laboratories have been involved in characterization of the estrogen receptors (ER-alfa, ER-beta) with special emphasis on the number of isoforms of this receptors and their role in the pathogenesis of many gynecological disorders. ER isoforms, generated by alternative mRNA splicing have been defined in several tissues and are postulated to play a role in modulating the estrogen response. These splicing variants were shown to act differently from the wild-type receptors and their biological significance remain unknown but same studies have implicated a role of specific isoforms in the pathogenesis of estrogen dependent tissues. Expression of the estrogen receptors, as well as their splicing variants may impact on the biologic properties of same tissues and affect their ability to respond to estrogen and antiestrogen therapies. From the clinical point of view it is importance to know what molecular characteristic of ERs and their isoforms occures in estrogen dependent tissues before histopathological and clinical changes. All of these developments have had a great impact on our understanding of the estrogen action.

Recently, the single strand conformation polymorphism (SSCP) analyses were shown to be useful for identification a variety of bacterial genes. Although, SSCP was successfully applied for detection of single nucleotide polymorphism (SNP), it was also considered a time consuming and insufficiently reliable technique. Therefore, the modified Multitemperature-SSCP method was introduced. It was shown to be reliable and time effective technique due to a stringent control of the gel temperature and utilization of a high voltage up to 1 kV. In this study the usefulness of MSSCP for differentiation of gene variants and detection of the single point mutations was evaluated, using Yersinia enterocolitica O3 and O8 ail alleles or genes blaCTX-M-15 and blaCTX-M-3, which differs by a single point mutation. The 425 and 251 bp fragments of O3 and O8 ail alleles containing 15 and 11 point mutations respectively, as well as 277 and 208 bp fragments of both blaCTX-M genes differing in positions 243 and 62 were PCR amplified, denatured and loaded onto 7% and 9% polyacrylamide nondenaturing gel. Electrophoresis was carried out in the DNA-Pointer apparatus (Kuchrczyk, Poland) at voltage ranging from 500 to 750 V. The thermal profile consisted of 50 min. at 35 degrees C, 40 min at 20 degrees C and 40 min at 5 degrees C. Obtained results showed, MSSCP was capable to differentiate ail alleles independently of the length of analyzed fragment. However, the 425 bp fragment profile consisted of three bands, whereas 251 bp revealed two bands. The single point mutation in blaCTM, genes was also successfully distinguished by MSSCP in both tested fragments. Surprisingly, 277 bp fragment profile showed differences more apparently than 208 bp. Summarizing, MSSCP was found to be useful, sensitive and time efficient tool for detection of multiple and single point mutations in DNA fragments ranging from 208-425 bp.

The human beta 2-adrenoceptor is a member of the seven-transmembrane family of receptors. It is expressed in many cell types such as airway smooth muscle cells, neutrophils, eosinophiles, alveolar macrophages and airway epithelial cells. The beta 2-adrenoceptor agonists are the most important group of bronchodilator drugs used in the treatment of asthma. They are classified by their selectivity, affinity for the receptor, potency and pharmacological efficacy. The gene encoding the beta 2-adrenergic receptor is highly polymorphic in the human population. Polymorphism affecting amino acids 16, 27 and 164 are the most common and they have been shown to correlate with some clinical features of asthma, including airways reactivity. They can modulate the behaviour of the beta 2-receptor, altering ligand binding and the characteristics of down-regulation following agonist exposure. The homozygous glycine-16 (Arg-->Gly) variant of the beta 2-adrenoceptor is known to predispose to agonist-induced down-regulation and desensitization, and may play a role in the pathogenesis of asthma severity. The polymorphism at position 27 (Gln-->Glu) is associated with decreased airway responsiveness. The polymorphic variant 164 (Thr-->Ile) shows impaired agonist binding and decreased adenylyl cyclase activity. No convincing evidence has been presented demonstrating a linking of asthma per se with this receptor polymorphism.

Mental retardation (MR) is a symptom in a large group of clinical conditions and affects around 3% of the population. MR is divided into syndromic, if it is characterized by distinctive clinical features and nonspecific when mental retardation is the only defining manifestation. Although genetic causes of X-linked mental retardation (XLMR) are heterogenous and complex, recent findings have led to the identification of an increasing number of genes involved in these conditions. Eight genes involved in nonspecific X-linked mental retardation have been identified so far, including FMR2, GDI1, OPHN1, PAK3, ARHGEF6, IL1RAPL, TM4SF2, and FACL4. Four other MECP2, RSK2, ARX, ATR-X are involved in syndromic and nonspecific forms of MR. Recent research has shown that these genes encode for proteins involved in signaling pathways which regulate cytoskeleton organization, synaptic vesicle transport and establishment of connections between neuronal cells. These findings provide insight into the molecular mechanisms of crucial processes for the development of intellectual and cognitive functions.

Vaccinology became interdisciplinary field of knowledge. It takes advantage of epidemiological and immunological researches and achievements of molecular biology, biotechnology and genetic engineering. Nowadays vaccinology remains mainly immunoprevention, but immunization of organism, as a result of vaccine injection, opens new possibilities in medicine. Directions of development present vaccinology are subordinated to one task--to invent an ideal vaccine. According to Edelman's criterions this vaccine has to be safe, effective in immunization, efficient in disease prevention and accessible for people all over the world. Realization of this aim causes modification of antigens, changes of adjuvant and preservatives and applying new technologies in production of vaccines.

The purpose of the study was the analysis of regulatory (region 5 gen) and encoding sequences of RAR-alpha receptor in cervical dysplasia and invasive cancer associated with HPV infection. A PCR method with the use of specific primers was applied to amplify and detect DNA sequence of various HPV types. A PCR-SSCP method was used to analyse the sequence of RAR--a receptor. The study revealed changes in structure of regulatory and encoding sequences of the investigated RAR receptor. Utmost changes were found in DNA isolated from tissues with diagnosis of adenocarcinoma colli uteri.

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.

To establish a prenatal diagnosis of congenital toxoplasmosis the PCR test was done on amniotic fluids from 47 women suspected of primary Toxoplasma infection during pregnancy. Fragments of Toxoplasma B1 gene were found in 5 examined samples. Positive tests were confirmed by mouse inoculation or by serologic testing of newborns. It was concluded that the PCR performed on amniotic fluid should be recommended for the prenatal diagnosis of congenital toxoplasmosis, however, negative results of the test can not rule out congenital infection.

Prometalloproteinases are activated by serine proteases, MMP-3, leucocytic elastase, furin, furin-like proteases and by membrane-type metalloproteinases as well. They form complexes with some proMMPs and thus they modify their activation.

The studies were undertaken to evaluate the usefulness of a nested PCR assay in the diagnosis of Pneumocystis carinii pneumonia (PCP) in AIDS patients. To achieve the end, 51 excretions samples from the respiratory tract were collected from HIV-infected patients with respiratory symptoms, and examined for the presence of specific DNA. A portion of the mitochondrial large-subunit rRNA gene of Pneumocystis carinii was amplified with outer primers pair pAZ 102E, pAZ 102H and internal primers pAZ 102X, pAZ 102Y. Positive nested PCR results were obtained with 36 out of 51 examined samples. Some 52.8% of the positive results were obtained with samples collected from patients without clinical diagnosis of PCP. It was concluded, that the nested PCR method, being too sensitive, is not suitable for the routine diagnosis of PCP in AIDS patients.