HPV16 E6 polymorphism and physical state of viral genome in relation to the risk of cervical cancer in women from the south of Poland.

The aim of this study was to analyse the correlation between HPV16 E6 variants and the physical status of viral genome (integrated, mixed, episomal) among patients with cervical cancer (n=40) and low-grade squamous intraepithelial lesions - LSIL (n=40). The study was performed on 80 HPV16 positive samples. HPV16 E6 variants were identified using PCR and DNA sequencing. Nucleotide sequences of E6 were compared with the prototype sequence (EUR-350T). The physical state of HPV DNA was determined as the ratio of E2/E6 copy number per cell. Twelve different intratypic variants were identified as belonging to European (in 77 samples) and North-American 1 (in 3 samples) sublineages. The most prevalent non-synonymous variant was EUR-350G, which occurred with similar frequency in cervical cancer and LSIL. The frequencies of additional mutations in variants with EUR-350T or EUR-350G sequences differed significantly. For the first time, missense mutations G122A, C153T and G188A were discovered in EUR-350G variant. The integrated viral genome was predominant in women with cervical cancer. The EUR-350T prototype and EUR-350G without additional mutations variants were prevalent in cervical cancer samples with the HPV16 characterized by integrated DNA. In summary, European variants of HPV16 E6 dominated in both cancer and LSIL group. The presence of EUR-350G favoured the occurrence of additional nucleotide changes. We showed that nucleotide changes occur significantly more often in the mixed form of viral DNA and in LSIL group and that the variants without additional mutations may promote integration of HPV16 genome.

Multiplex real-time PCR to identify a possible reinfection with different strains of human cytomegalovirus in allogeneic hematopoietic stem cell transplant recipients.

Human cytomegalovirus (HCMV) infection remains the leading cause of serious contagious complications after allogeneic hematopoietic stem cell transplantation. These infections in HCMV-seropositive recipients can be due to reactivation or reinfection. Different HCMV strains were identified by determining the genotypes isolated from repeatedly tested patients. The UL55 sequences encoding viral glycoprotein B (gB) have been chosen as the target gene. The region, in which the gB precursor protein is cleaved into two fragments by a cellular endoprotease, is characterized by genetic variability, and based on that HCMV is classified into four major genotypes: gB1, gB2, gB3 and gB4. Multiplex real-time PCR assay enabled both, HCMV gB genotyping, as well as simultaneous quantitative assessment of the detected genotypes. This study was carried out in 30 transplant recipients, from whom 105 isolates of HCMV DNA were genotyped. In 40% of recipients, a mixed infection with two or three genotypes was detected. Genotype gB1 dominated in general, and characteristically for mixed infections, the genotype gB3 or gB4 was always present. Although there were no significant differences in the load for each genotype, in case of multiple infections, the number of copies of gB1 genotype was significantly higher when compared to a single gB1 infection. In patients with mixed genotypes, chronic HCMV infections and graft versus host disease were observed more often, as well as antiviral treatment was less effective. It was assumed that these adverse effects can be related to the presence of gB3 and gB4 genotypes.

Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods.

The aim of this study was to analyse the correlation between a new multiplex qPCR assay and a reference qPCR assay for assessment of the human papillomavirus (HPV16) load and the viral genome status. The study was performed on 100 HPV16 positive samples containing premalignant lesions and carcinomas. HPV16 E2 and E6 gene loads were assessed by two PCR methods. The load of E2 and E6 was normalized to the cell number by qPCR targeting the RNase P open reading frame. The physical state of the viral genome was determined as a ratio of E2/E6 copies number per cell. Among 100 samples analysed, there were no statistically significant differences in the E2 and E6 viral load evaluated by multiplex qPCR and qPCR, the correlation coefficients were 0.98 and 0.97, respectively. There were 19% of samples with the integrated, 73% with mixed and 8% with episomal state of viral genome detected by multiplex qPCR and 17%, 79%, 4%, respectively, found by qPCR. Prevalence of integrated and episomal forms estimated by multiplex qPCR was higher than the one obtained by qPCR (Chi2, p < 0.0001), but in samples with premalignant and malignant diagnoses no significant differences were demonstrated regardless of the methods used. Sensitivity and specificity of multiplex qPCR were 93.7% and 100% as compared with qPCR, the positive predictive value was 100%. In summary, the multiplex qPCR assay in respect of HPV16 load and the frequency of viral genome status was shown to be a sensitive and specific reference method. Simultaneous estimation of E2 and E6 genes in one reaction tube reduces the cost of testing.

Differences in the expression of human papillomavirus type 16 (HPV-16) E6 oncogene mRNA in SiHa cell line inoculated with CMV, HSV or ureaplasmas.

One of the factors associated with an increased risk of HPV-related malignant transformation may be bacterial and/or viral infections. The aim of our study was to examine whether the presence of infectious agents commonly detected in the genitourinary tract such as herpesviruses (HSV, CMV), and ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum) may lead to alterations in the expression of the HPV-16 E6 oncogene. Quantitative RT-PCR analysis was used to assess the level of HPV-16 E6 mRNA expression in SiHa cells. The presence of HSV-1 or HSV-2 in SiHa cells caused a 1.5-fold increase in HPV-16 E6 mRNA expression as compared with non-inoculated SiHa cells. Ureaplasma urealyticum presence but not Ureaplasma parvum stimulated the expression of HPV-16 E6 resulting in a nearly five-fold (4.8) up-regulated E6 mRNA level in SiHa cells. Our study is the first to suggest that infection of Ureaplasma urealyticum in an urogenital tract could increase the risk of cervical cancer by overexpression of the HPV E6 oncogene.

Cytomegalovirus glycoprotein H genotype distribution and the relationship with hearing loss in children.

Cytomegalovirus (CMV) is a leading cause of congenital infection and a leading infectious cause of hearing loss in children. The ORF UL75 gene encodes envelope glycoprotein H (gH), which is essential for CMV entry into host cells and the target of the immune response in humans. However, the distribution of gH variants and the relationship between the viral genotype, viral load, and sequelae in children infected with CMV is debated. The UL75 genetic variation of CMV isolates from 42 newborns infected congenitally with CMV and 93 infants with postnatal or unproven congenital CMV infection was analyzed. Genotyping was performed by analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. There were no differences in the distribution of gH genotypes in the children infected congenitally and postnatally. Mixed-genotype infections with both gH1 and gH2 variants were detected in approximately 25% of the examined patients. No relationship between UL75 gene polymorphisms and the symptoms at birth was observed. The results suggest that the infection with gH2 genotype diminishes the risk of hearing loss in children (P = 0.010). In addition, sensorineural hearing loss was associated with CMV gH1 genotype infection in infants (P = 0.032) and a high viral load in urine (P = 0.005). In conclusion, it was found that the gH genotype does not predict clinical sequelae in newborn infants following congenital CMV infection. However, these results suggest that the gH genotype might be associated with hearing loss in children.

[Ebola virus disease]. / Ebola virus disease.

Ebola is one of the most virulent zoonotic RNA viruses causing in humans haemorrhagic fever with fatality ratio reaching 90%. During the outbreak of 2014 the number of deaths exceeded 8.000. The "imported" cases reported in Western Europe and USA highlighted the extreme risk of Ebola virus spreading outside the African countries. Thus, haemorrhagic fever outbreak is an international epidemiological problem, also due to the lack of approved prevention and therapeutic strategies. The editorial review article briefly summarizes current knowledge on Ebola virus disease epidemiology, etiology, pathogenesis, clinical presentation, diagnosis as well as possible prevention and treatment.

General introduction into the Ebola virus biology and disease.

Epidemic of Ebola hemorrhagic fever which appeared in the countries of West Africa in 2014, is the largest outbreak which occurred so far. The virus causing this epidemic, Zaire Ebolavirus (ZEBOV), along with four other species of Ebolaviruses is classified to the genus Ebolavirus in the family Filoviridae. ZEBOV is one of the most virulent pathogens among the viral haemorrhagic fevers, and case fatality rates up to 90% have been reported. Mortality is the result of multi-organ failure and severe bleeding complications. The aim of this review is to present the general characteristics of the virus and its biological properties, pathogenicity and epidemiology, with a focus on laboratory methods used in the diagnosis of these infections.

Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants.

BACKGROUND: Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. OBJECTIVES: To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. STUDY DESIGN: The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. RESULTS: Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. CONCLUSION: Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns.

[UL55 genotype diversity of cytomegalovirus strains isolated from newborns and infants hospitalized in southern Poland]. / Róznorodnosc genotypowa UL55 szczepów wirusa cytomegalii izolowanych od noworodków i niemowlat hospitalizowanych w Polsce poludniowej.

Studies on cytomegalovirus (HCMV) infections more often draw attention to the differences in tropism, pathogenicity and virulence of the virus depending on its genotype. The aim of this study was to assess the individual gB genotypes which are encoded in UL55 region of HCMV genome in a population of newborns and infants from Southern Poland. Genotypic analysis was carried out on 53 children (16 newborns and 37 neonates) with confirmed HCMV infection. The children were tested several times. A total of 101 samples, mainly urine, less blood, swabs from the upper respiratory tract, in justified cases, the cerebrospinal fluid were used in our study. Both genotyping and quantitative assessment of HCMV were performed using real time-PCR (rt-PCR). For identification of four major gB genotypes in one reaction, a modification of multiplex rt-PCR was used. Studies confirmed the presence of all major genotypes: gB1, gB2, gB3 and gB4 in the examined groups of children. Only in one case, the genotype could not be determined, perhaps it belonged to subtypes outside the detectable majority ofgB genotypes. Genotype gB1 (63.5%) which was slightly more frequent in infants than in neonates, dominated in our studies. The other genotypes occurred at a rate: gB2 - 15.4%, gB3 - 21.2%, gB4 - 28.8%, respectively. Mixed infections, caused by two genotypes were found in 16 (31%) children, mainly in older infants. There were no statistically significant differences in viral load when comparing a group of newborns with infants and single vs. mixed infection, as well as individual genotypes. The observed differences in the proportional occurrence of different gB genotypes in the two study groups of children may suggest various preferences of particular HCMV genotypes in congenital and acquired infections. Moreover, by monitoring of HCMV infection and determination the genotypes in consecutive samples, it could be identified infection acquired during hospitalization in three children.

Lymphotropic herpesvirus DNA detection in patients with active CMV infection - a possible role in the course of CMV infection after hematopoietic stem cell transplantation.

BACKGROUND: The natural history of cytomegalovirus (CMV) infection and disease in transplant recipients prompts researchers to look for other factors contributing to this infection. The ubiquity of lymphotropic herpesviruses (EBV, HHV-6, and HHV-7) and the possibility of their activation during immunosuppression may suggest their participation in progression of CMV infection in patients after hematopoietic stem cell transplantation (HSCT). MATERIAL/METHODS: The presence of CMV, EBV, HHV-6 and HHV-7 was confirmed through detection of viral DNA isolated from leukocytes. Allo-HSCT recipients (n=55) were examined repeatedly within the average period of 14±7.3 months post-transplant. RESULTS: CMV DNA was detected in 24% of samples, while EBV, HHV-6 and HHV-7 were detected in 20%, 15% and 14% of samples, respectively. Based on the presence of CMV infection at particular time-points (months) after transplantation, the recipients were divided into 3 groups: Group I (N=15) with persistent infection, Group II (N=20) with transient infection, and Group III (N=20) without CMV infection. In Group I, the mean CMV load was significantly higher than in Group II, and the clinical condition of Group I patients was poorer. All these patients manifested clinical symptoms, and all had episodes of GvHD. All Group I patients developed multiple infections; EBV in 80%, HHV-6 in 47% and HHV-7 in 87% of patients. In the remaining groups, with the exception of HHV-6 in group II, the frequency of infected patients was lower. In addition, CMV presence was often preceded by another herpesvirus. CONCLUSIONS: The results suggest that other herpesviruses, mainly HHV-7, could predispose CMV to cause chronic infection.

[Differentiation of an integrated and episomal HPV-16 DNA using real-time PCR in cervical specimens of women diagnosed with intraepithelial lesions and invasive cervical cancer]. / Róznicowanie postaci zintegrowanej i episomalnej DNA HPV-16 metoda real-time PCR w wydzielinie szyjki macicy kobiet z rozpoznan sródnablonkowa neoplazja i rakiem szyjki macicy.

UNLABELLED: Persistent high-risk HPV infection, especially HPV-16, is considered to be an important step in the process of cervical carcinogenesis. Integration of viral DNA into the host genome through the destruction of HPV E2 sequences, increases the expression of viral proteins E6 and E7 and their participation in the transformation of cervical cancer. OBJECTIVE: The aim of this study was to apply real-time PCR (RT-PCR) to assess the prevalence of integrated and episomal HPV-16 DNA and determine viral DNA load in women with cervical intraepithelial lesions and invasive cervical cancer MATERIAL AND METHODS: A total of 84 women infected with HPV-16, including 44 with LSIL, 7 with HSIL and 33 with invasive cervical cancer participated in the study Cervical specimens were collected using the cytobrush. The presence of a sequence of E2 and E6 HPV-16 and human gene RNasy P was detected by quantitative RT-PCR. The viral load presented as the form of the virus genome copy numbers per 1,000 cells. RESULTS: The integrated form of HPV-16 genome was found in 97% of women with cervical cancer. In women with LSIL and HSIL mixed form (simultaneous occurrence of an integrated and episomal form) of the viral genome (84% and 57%, respectively) prevailed. The frequency of the integrated HPV-16 DNA increased with progression of dysplastic lesions of the cervix (p<0.001). Statistically significant differences in average number of copies of the virus in women with LSIL and HSIL compared to patients with cancer (p<0.001) were observed. The highest viral load was detected in women demonstrating an integrated HPV-16 DNA. CONCLUSIONS: Quantitative analysis of the sequence of E2 and E6 HPV-16 tested by RT-PCR can be used to determine the degree of integration of the viral genome and quantitative evaluation of viral load in clinical material. It can also serve as an additional parameter defining risk of progression of transformation in the cervix.

[Evaluation of vertical transmission of two species of ureaplasmas in term newborns without respiratory disorders--a preliminary study]. / Ocena wertykalnej transmisji dwóch gatunków ureaplazm u donoszonych noworodków bez zaburzen oddechowych--badania wstepne.

UNLABELLED: Pregnancy promotes ureaplasma vaginal colonization. This creates the possibility of vertical transmission of these organisms to the child. These microorganisms can cause complications during pregnancy and poor condition of newborn. OBJECTIVES: Objectives of this study were to analyze the vertical transmission of different species of ureaplasmas in term newborns without respiratory distress. MATERIALS AND METHODS: The study included 50 mothers and 50 of their newborn children. Swabs were obtained from swabs of the cervix in women and tracheal aspirates from neonates. The presence of ureaplasmas was confirmed by culture and PCR. Ureaplasmas species identification was performed using PCR. RESULTS: infection of ureaplasmas was found in 21 women (42%). Predominant species was U. parvum, which was found in 18 women. In 3 patients only the presence of U. urealyticum was confirmed. Ureaplasma infection in mother and her newborn baby was confirmed in 8 (17.4%) mother-child pairs, including 6 of these cases showing the presence of U. parvum and 2 U. urealyticum. The incidence of vertical transmission of ureaplasma infection was assessed at 33% for U. parvum and 67% for U. urealyticum, and the total for both species at 38%. It should be noted that in the group of 18 women infected with U.parvum, in 12 cases there was no transmission of infection to the child. However in 3 women infected with U. urealyticum 2 cases of transmission from mother to child were observed (67%). Although the group infected with U. urealyticum accounted for only 3 women, our preliminary observations may suggest that this species is probably more likely to be transferred from mother to child. CONCLUSIONS: Infection with U. urealyticum may be more frequently transferred from the genital tract of mother to child.

Herpesviruses as possible cofactors in HPV-16-related oncogenesis.

Cervical carcinogenesis is a complex problem with papillomavirus widely accepted as a causative agent. Integration of a human papillomavirus (HPV) of the high-risk type into the host cell genome is one of the major contributing factors to cervical malignant transformation. In this study, the correlation of CMV, EBV, HSV-1, HSV-2, HHV-6 and HHV-7 infections with the physical status of the HPV genome in cervical cancer and precancerous cervical lesions was investigated in sixty HPV-16-positive women. Cervical secretion samples were submitted to DNA extraction and analyzed by PCR. HPV-16 DNA was confirmed in genotyping with the reverse hybridization line probe assay. Multiplex PCR with specific primers for the E2/E6 genes was used to assess the viral integration status of HPV-16. Our results show that CMV DNA was more frequently present in samples with mixed forms of HPV-16 than in the episomal form (P < 0.025). Such a correlation was also observed in the case of EBV (P < 0.005). The presence of CMV resulted in a six-fold (OR 6.069; 95% CI 1.91-19.22; P = 0.002), while EBV caused a seven-fold (OR 7.11; 95% CI 1.70-29.67; P = 0.007) increase in the risk of the integrated or mixed HPV-16 genome occurrence. Our data suggest that coinfection with herpesviruses, especially CMV and EBV, may be involved in the integration of the HPV-16 genome and may contribute to the development of cervical cancer.

[Is the presence of herpesviruses in cervical secretions a prognostic factor for cervical pathology in HPV-positive women?]. / Czy obecnosc herpeswirusów w wydzielinie szyjki macicy moze byc czynnikiem rokowniczym patologii szyjki macicy kobiet zakazonych HPV?

Cervical carcinogenesis is a complex problem where papillomavirus is widely accepted as a causative agent. The correlation of CMV, EBV, HSV-1, HSV-2 with precancerous and cancer cervical lesions was investigated in 125 women with different diagnosis: LSIL- 44, HSIL- 12, cervical carcinoma-27 vs. 42 women without abnormality in cytology (control group). Cervical secretion samples were submitted for DNA extraction and determined by PCR and nPCR. HPV DNA genotyping was performed with the reverse hybridisation line probe assay. Among HPV-positive specimen,CMV was detected in 32% of samples, EBV in 14% and HSV-1 in 3%. The presence of CMV and EBV DNA was more frequent in cervical cancer specimen than in other study groups (p<0.001). The prevalence of EBV infection was increasing with the severity of cervical smear abnormality and was associated with HPV-16 (p=0.009). The risk for HPV-16 infection was 6.4 fold higher for CMV positive women (OR=6.44;95% CI 2.68-15.48; p=0.001) and 4.5 fold higher for EBV positive women (OR=4.58; 95% CI 1.45-14.46;p=0.009). HSV-1 and/or HSV-2 infections were detected rarely and only in the women with LSIL and in the control group. Our data suggest that EBV and/or CMV may be associated with HPV in cervical carcinogenesis.

The essential role of single Ig IL-1 receptor-related molecule/Toll IL-1R8 in regulation of Th2 immune response.

A novel cytokine IL-33, an IL-1 family member, signals via ST2 receptor and promotes Th2 responses, through the activation of NF-kappaB and MAP kinases. Previous studies reported that single Ig IL-1R-related molecule (SIGIRR)/Toll IL-1R8 acts as negative regulator for TLR-IL-1R-mediated signaling. We now found that SIGIRR formed a complex with ST2 upon IL-33 stimulation and specifically inhibited IL-33/ST2-mediated signaling in cell culture model. Furthermore, IL-33-induced Th2 response was enhanced in SIGIRR-deficient mice compared with that in wild-type control mice, suggesting a negative regulatory role of SIGIRR in IL-33/ST2 signaling in vivo. Similar to ST2, SIGIRR was highly expressed in in vitro polarized Th2 cells, but not Th1 cells. SIGIRR-deficient Th2 cells produce higher levels of Th2 cytokines, including IL-5, IL-4, and IL-13, than that in wild-type cells. Moreover, SIGIRR-deficient mice developed stronger Th2 immune response in OVA-challenged asthma model. Taken together, our results suggest that SIGIRR plays an important role in the regulation of Th2 response in vivo, possibly through its impact on IL-33-ST2-mediated signaling.

Physical state of human papillomavirus type 16 in cervical cell DNA.

Multiplex PCR with specific primers for E2/E6 genes was used to assess the viral integration status of HPV-16 in women with low and high grade squamous intraepithelial lesions (LSIL and HSIL, respectively) in comparison to cervical cancer patients. Women with confirmed HPV-16 infection were examined: 30 with LSIL, 12 with HSIL and 23 with cervical cancer. The PCR products were separated electrophoretically in agarose gels and densitometric analysis was performed using Bio-Rad Quantity One software. E2 and E6 sequences of HPV-16 were detected in 91% of the women. The free episomal viral genome was not detected in the cervical carcinoma group. Twenty six percent of the samples obtained from this group harboured the integrated form, whereas the remaining samples possessed a mixture, i.e. episomal and integrated forms of viral DNA. The free episomal form dominated in women with LSIL and HSIL. In 6 cases the episomal and integrated forms were detected simultaneously. HPV-16 integration occurred in a subset of LSILs and HSILs, not only in the cervical cancer patients and correlated with progression of cytological changes. The assessment of the status of HPV-16 may be the molecular factor preceding the morphological features leading to malignancy.

Detection of specific lytic and latent transcripts can help to predict the status of Epstein-Barr virus infection in transplant recipients with high virus load.

Epstein-Barr virus (EBV), a member of the family Herpesviridae, is widely spread in the human population and has the ability to establish lifelong latent infection. In immunocompetent individuals the virus reactivation is usually harmless and unnoticeable. In immunocompromised patients productive infection or type III latency may lead to EBV-associated post-transplant lymphoproliferative disorder (PTLD). The aim of our research was to investigate the utility of PCR-based methods in the diagnosis and monitoring of EBV infections in bone marrow transplant recipients. Thirty-eight peripheral blood leukocyte samples obtained from 16 patients were analysed, in which EBV DNA was confirmed by PCR. We used semi-quantitative PCR to estimate the viral load and reverse-transcription PCR (RT-PCR) to differentiate between latent and productive EBV infection. In 14 patients we confirmed productive viral infection. We observed a correlation between higher number of EBV genome copies and the presence of transcripts specific for type III latency as well as clinical symptoms.

Genotype-specific human papillomavirus detection in cervical smears.

Human papillomavirus (HPV) is widely accepted as a causative agent of cervical cancer. The distribution and prevalence of HPV types depend on geographic region and demographic factors. The aim of this study was to investigate the relationship between the presence of various HPV types and the outcome of cytological examination. Cervical smears were obtained from 125 women from southern Poland: low grade squamous intraepithelial lesions (LSIL) - 44, high grade squamous intraepithelial lesions (HSIL) - 12, cervical carcinoma - 27 and 42 women without abnormality in cytology as a control group. DNA was extracted from the smears and broad-spectrum HPV DNA amplification and genotyping was performed with the SPF 10 primer set and reverse hybridisation line probe assay (INNO-LiPA HPV Genotyping, Innogenetics). HPV DNA was detected in approximately 72% cases, more frequently in women with squamous intraepithelial lesions and cervical carcinoma than in the control group (P < 0.0005). The most frequent type found was HPV 16 (37%), followed by HPV 51 (28%) and HPV 52 (17%). A single HPV type was detected in 51% positive cases, more frequently in cervical cancer specimens. Multiple HPV infection was dominant in women with LSIL and normal cytology. Prevalence of HPV 16 increased with the severity of cervical smear abnormality. For women HPV 16 positive, the relative risk (odds ratio) of the occurrence of HSIL and cervical cancer versus LSIL was 14.4 (95% CI, 3.0-69.2; P=0.001) and 49.4 (95% CI, 6.5-372.8; P < 0.001), respectively. Genotyping of HPV will allow better classification of women with cervical abnormalities into different risk groups and could be useful in therapy.

[Herpesviruses mixed infections in allogeneic steam cell recipients (allo-HSCT)]. / Mieszane zakazenia herpeswirusowe u biorców alloprzeszczepów komórek hemopoetycznych (allo-HSCT).

AIM: Assessment of frequency and clinical course of infections with herpesviruses: CMV, EBV, HHV-6 and HHV-7 in patients that underwent non-manipulated allo-HSCT from matched-related donors. METHODS: 35 recipients of age 31 +/- 8 years. Serological status of donor and recipient against CMV and EBV assessed before transplantation. After transplantation, herpesviruses infection was confirmed based on the presence of viral DNA isolated from peripheral leukocytes, using nested PCR method. Patients were examined repeatedly, during 11.7 +/- 7 months of observation. RESULTS AND CONCLUSIONS: mixed infections appeared in 80% of allo-HSCT recipients. Infections with two or three viruses dominated, especially CMV and EBV (65%). We observed specific tendency of appearance of particular herpesviruses in each episode--in the first and the second episodes CMV dominated, EBV or HHV-6 infections were rare, whereas in the successive episodes EBV and HHV-7 were the leading viruses. In correlation with clinical symptoms mixed CMV and EBV infections were characterised by the most severe course. Superinfections with HHV-6 or HHV-7 had no significant influence on the progression of illness. Our observations may suggest that the serious CMV infections in allo-HSCT recipients are the result of not only the pathogenic properties of CMV but of the additive effect of replication of other herpesviruses.

In situ detection of DNA and mRNA of human cytomegalovirus to distinguish different forms of viral infection in leukocytes.

In situ PCR and in situ reverse transcription PCR (RT-PCR) were applied to discriminate between latent and productive infection of human cytomegalovirus (HCMV) in leukocytes. We investigated 28 samples, in which viral pp65 antigen was detected only in the cytoplasm of leukocytes. Additionally we assayed 12 specimens lacking pp65 antigen. Using nested PCR (nPCR), viral DNA was detected in 27 samples. In six samples the results of nPCR were unreadable due to the presence of polymerase inhibitors. By application of in situ PCR, we were able to confirm the presence of viral DNA in the nucleus and/or cytoplasm. Productive infection was recognized in 20 samples in which transcripts for late viral genes were detected. Among the 20 samples negative by in situ RT-PCR, we recognized phagocytosis of viral particles in eight and the latent form of HCMV infection in five.