[The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

[The identification of viruses of human papilloma of high carcinogenic risk and evaluation of physical status of viral DNA using technique of polymerase-chain reaction under affection of cervical epithelium].

The DNA of virus of human papilloma of high carcinogenic risk was detected in 116 cervical samples. At that, the morphological symptoms of background processes are detected in 19 samples, CIN 1 in 9, CIN 2 in 23, CIN 3 in 54 (and out of them carcinoma in situ in 13), epidermoid cancer (squamous cell carcinoma) in 11 cases. The viral load of human papilloma of high carcinogenic risk in all samples of DNA exceeded threshold of clinical value (3 lg copies of DNA of human papilloma/105 cells). The genetic typing of human papilloma of high carcinogenic risk revealed the dominance of human papilloma of type 16 in 49.7%, type 33 in 15.3%, type 31 in 12.3% and type 45 in 5.5%. In women with background processes in cervix of the uterus DNA of human papilloma type 16 was detected more often in episome form. In case of dysplastic alterations of epithelium and cervical cancer DNA of human papilloma type 16 is detected in mixt form with different degree of integration into cell genome.

[The development and testing of reagents kit for detection and qualitative evaluation of DNA of methicillin sensitive and methicillin resistant Staphylococcus aureus and also methicillin resistant coagulase negative Staphylococcus spp. applying technique of polymerase chain reaction in "real time" mode].

The reagents kit is developed to identify and quantitatively detect DNA of methicillin sensitive and methicillin resistant Staphylococcus aureus, methicillin resistant coagulase negative Staphylococcus spp. in biological material using technique of polymerase chain reaction with hybridizational fluorescent detection and having higher analytical and diagnostic characteristics. The application of the given reagents kit makes it possible to optimize the epidemiologic monitoring of propagation of methicillin resistant strains of Staphylococcus spp. Significantly decreasing duration and laboriousness of study.

[Use of immunological and molecular biological methods to diagnose cerebral toxoplasmosis in HIV infection].

Cerebral toxoplasmosis is one of the leading causes of neurologic diseases with high mortality rates in patients with HIV infection. Invasion was difficult to diagnose for a number of objective reasons. The objective of the investigation was to determine the clinical sensitivity of different laboratory techniques as both a single study and their various combinations to verify the diagnosis of cerebral toxoplasmosis in HIV-infected patients. Blood and cerebrospinal fluid were tested in 51 patients with Stage 4B HIV infection (AIDS) with the verified diagnosis of cerebral toxoplasmosis. Separate determination of specific antibodies of IgG, IgM, IgA and toxoplasma DNA in the blood and cerebrospinal fluid was shown to have an insufficient clinical sensitivity (37.3-68.6%). The benefits of various combinations of immunological and molecular biological assays enhancing the diagnostic efficiency up to 76.5-96.1% are demonstrated.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

AIM: Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. MATERIALS AND METHODS: During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. RESULTS: A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. CONCLUSION: Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

[Cytomegalovirus infection in infants with perinatal damage of the central nervous system].

Ninety children, aged from 7 days to 12 months, with perinatal damage of the central nervous system (CNS) were virologically tested. The persistence of cytomegalovirus (CMV) DNA in the blood, urine and oropharyngeal swabs was identified in 18.9% of children. In 14.4% of children, CMV DNA was found only in oropharyngeal swabs and urine samples. In some cases, the severity of neurologic symptoms and absence of positive changes in the restoration treatment could be caused by the activity of CMV infection, blood CMV DNA and the increase in blood-brain barrier permeability. The authors recommend performance of a comprehensive immunological and molecular-biological survey in patients with suspected CMV infection. In case of positive reactions, a specific antiviral treatment should be started. Tree clinical cases are reported.

[Are human papillomaviruses responsible for the occurrence of bladder cancer].

A female patient with recurrent bladder cancer underwent complex examination. The primary tumor removed in 2004 showed human papillomavirus (HPV) 16 DNA, mRNA corresponding to HPV16 oncogene E7, as well as HPV16 protein E7. The patient is a smoker who has been working at a chemical factory for over 20 years. During tumor recurrence in 2009, there was no DNA of high-risk HPV types in the cancer cells. HPV16 E7protein and cellular p 16(INK4alpha), an indicator of HPV-induced carcinogenesis, were not found. Colposcopy revealed no precancerous changes in the epithelium of the cervix uteri. The cervical epitheliocytes contained no high-risk HPV DNA, E7 and p16(INK4alpha) proteins. It seems expedient to continue in vitro studies of the possible role of HPV in urothelial carcinogenesis on an experimental model.

[Infection with high-risk human papillomavirus and prognosis of cervical carcinoma].

Real-time polymerase chain reaction procedure was used to evaluate bioptic tumor samples from patients suffering cervical carcinoma (CC) stages I-IV. Out of 110 patients, high-risk human papillomavirus (HPV) infection was identified in 98 (89.1%), HPV type 16--63, HPV type 18--10 and HPV type 45--5. One of genotypes 31.33, 35, 39, 52, 58, 59 was established in 8 and a combination of several genotypes of the virus--12 patients. Frequency of remission in CC patients associated with HPV type 16 who had survived 3 years was significantly higher than in the same category associated with HPV type 18 (p=0.03). Relapse frequency and mortality rates in patients with tumors associated with one of viruses 31.33, 35, 39, 52, 58 or 59 were higher as compared with HPV type 16--associated cases 2 years (p=0.03) or 3 years on (p=0.11), respectively. A similar trend was established for squamous-cell tumors stages 1 and 2 (p=0.07) (p=0.12), respectively. No difference was observed in efficacy of therapy for infection with one or a combination of several genotypes of high-risk HPV. Hence, the genotype of virus is believed to be a factor of prognosis in CC early cancers. However, a definitive conclusion cannot be reached until results of a larger body of evidence and longer follow-up are available.

[The pattern of central nervous system lesions in HIV-infected patients of the specialized unit in infectious disease hospital].

AIM: To define the incidence and features of brain lesion (BL) in HIV-infected inpatients. SUBJECTS AND METHODS: Four hundred and fifty-eight patients with Stage 4B HIV infection (AIDS) and central nervous system (CNS) lesion admitted to Infectious Diseases Hospital Two, Moscow, were followed up in 2003-2009. The authors used cerebrospinal fluid (CSF) microscopic and bacteriological assays for DNA of T. gondii, M. tuberculosis, herpes simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV), HSV type 6, and varicella-zoster virus, Cr. neoformans, C. albicans, C. glabrata, and C. krusei. Blood and CSF were tested for IgM and IgG T. gondii antibodies; brain magnetic resonance imaging was carried out. RESULTS: In patients with late-stage HIV infection, the principal cause of neurological diseases was cerebral toxoplasmosis (34.7% of BL cases) and a generalized process involving the brain, lung, heart, liver, and eyes in 11.5%. There was commonly cerebral toxoplasmosis concurrent with CMV infection with clinical manifestations. 16-32% of the inpatients developed tuberculosis meningoencephalitis that was a manifestation of hematogenous disseminated tuberculosis involving the lung. There was a rise in the incidence of cancers (brain lymphomas, astrocytomas) running with CNS lesion. Mental disorders progressing to dementia were a distinctive property of CMV ventriculoencephalitis, one of the leading factors in the development of AIDS dementia complex. Molecular diagnostic techniques are needed to ascertain the etiology of BL in HIV infection. CONCLUSION: The CSF test for DNA of causative agents is a specific and most sensitive method for diagnosing a relevant CNS lesion.

[Detection of oncoprotein E7 HPV16 in the cancer and normal urinary bladder urothelium].

Oncoprotein E7 HPV16 was detected by immunohistochemical staining with specific polyclonal antiserum [Fiedler et al., 2004] in 7 out of the 24 (29.2%) studied bladder cancer specimens. The result is in good agreement with the hypothesis that HPVs take part in the carcinogenesis of the urothelium. However, some of the observations made seem rather hard to be interpreted at present. The latter include the detection of E7 HPV16 in a small number of cancer cells in a few bladder cancer specimens being examined; the presence of this protein in the cytoplasm, rather in the cancer cell nuclei, and its detection in some morphologically normal bladder urothelial specimens from non-cancer patients. Thus, the hypothesis that HPVs are implicated in the carcinogenesis of the bladder urothelium deserves further verification.

[Cerebral toxoplasmosis in HIV-infected patients].

AIM: To detect clinical characteristics of cerebral toxoplasmosis in HIV-infected patients, to clarify diagnostic role of detection of DNA and antibodies to Toxoplasma gondii in the cerebrospinal fluid (CSF) and blood. MATERIAL AND METHODS: Diagnostic procedures were performed in 156 patients with HIV infection at the stage IVB (AIDS) in 2003-2006. All the patients suffered from diseases of the central nervous system (CNS). Toxoplasmosis was diagnosed in 57 (36%) cases. Lumbar puncture, MR imaging of the brain, reaction of indirect immunofluorescence, polymerase chain reaction and enzyme immunoassay were made to identify IgM and IgG to T. gondii. RESULTS: Typical for HIV-infected patients with cerebral toxoplasmosis were focal symptoms of CNS affection, hemipareses, adynamia, mental disorders, intoxication symptoms. CONCLUSION: MR imaging data are very important. Toxoplastosis is characterized by multiple destructive foci in the hemispheres and cerebellum with great amount of the parasites along the periphery of brain tissue necrosis. Detection of the infective agent DNA and specific IgG antibodies in cerebrospinal fluid confirms the presence of toxoplasmosis but sensitivity of the markers is low. IgG antibodies to T. gondii have diagnostic implications if they occur in high and moderate titers.

[Development and evaluation of the kit for detection of SARS-associated Coronavirus RNA]. / Razrabotka i aprobtsiia test-sistemy dlia vyiavleniia RNK virusa vizyvaiushchego tiagezhelyi ostryi resperativnyi sindrom.

AIM: To develop a diagnostic kit for detection of SARS (severe acute respiratory syndrome)-related coronavirus RNA based on reverse transcription and polymerase chain reaction and to estimate its specificity and sensitivity. MATERIAL AND METHODS: 68 virus and bacterial cultures, 240 clinical samples from people without SARS symptoms and also 22 RNA samples from patients with SARS symptoms received during the epidemic in Beijing were used. RESULTS: The specificity of the kit was determined using animal coronaviruses and other bacterial and viral strains, causing acute respiratory and intestinal infections, and was shown to be 100%. The sensitivity of the kit in different clinical samples was 2.2 x 10(3) genome equivalents of recombinant SARS RNA in 1 ml of the specimen. The kit was evaluated in the Institute of Microbiology and Epidemiology of Beijing (China) using SARS-cov viral suspension and clinical samples from patients with suspected SARS. It was shown that kit was able to detect 10 TCID/50 ml of SARS-Cov virus. Testing of clinical samples from patients with suspected SARS showed that diagnostic sensitivity of the kit was 95%. Detection of the SARS-Cov RNA was more effective in feces compared to sputum 990 and 40%, respectively). CONCLUSION: The kit "AmpliSens SARS" for qualitative detection of SARS-related coronavirus RNA by reverse transcription and polymerase chain reaction (PCR) in nasopharyngeal wash/aspirates, naso/oropharyngeal swabs, plasma, and extract from feces has been developed in the Central Research Institute for Epidemiology of the RF Ministry of Health. The kit contains reagents for RNA isolation and purification, cDNA synthesis by reverse transcription of RNA, for PCR and for electrophoretic analysis of amplified products. The kit also contains recombinant positive and internal control samples allowing to control efficiency of analysis and showed good analytical and diagnostic characteristics.

[The rate of HCV-RNA detection in the serum, saliva, and tissue of the minor salivary glands in chronic hepatitis C with Sjogren's syndrome]. / Izuchenie chastoty vyiavleniia HCV-RNK v syvorotke, sliune i tkani malykh sliunnykh zhelez pri khronicheskom gepatite s sindromom Sjogrena.

The trial enrolled 38 patients with chronic HCV-infection and Sjogren's syndrome (mean age 44.3 +/- 13.7 years). Biopsy of the minor salivary glands (MSG) was made in 20 patients. Polymerase chain reaction was used to study 20 MSG biopsies, 38 samples of native saliva for HCV-RNA. Saliva samples were also studied for Herpes virus DNA (EBV, CMV, HHV-VI type). All the patients with VHC appeared to have signs of xerostomia, 24 (63.2%) patients had xerophthalmia. MSG pathohistological changes were found in 19 (95%) patients. In the majority of cases (86.9%) they were characterized by mild infiltration and advanced fibrosis. HCV-RNA was found in the saliva of 23 (57.5%) patients, in MSG tissue--in 9 (39.1%) patients. HCV-RNA detection in the saliva did not depend on the degree of viremia, viral RNA in MSG correlated with viral load. EBV and HHV-VI, HHV-VI only and EBV were detected only in 7 (18.4%), 10 (26.3%) and 6 (15.8%) patients, respectively. Xerostomia occurred with the same rate (26.1 and 31.3%) in patients with and without herpes viruses in the saliva. Detection rate for HCV-RNA in the saliva was not related with viremia degree. Sjogren's disease symptoms in CHC patients did not depend on the presence or absence of DNA of herpes viruses in the saliva.

[Sjogren's syndrome in chronic hepatitis C: clinical features and diagnosis]. / Sindroma Shegrena pri khronicheskom gepatite C: kliknicheskie osobennosti i diagnostika.

AIM: To examine clinical features of Sjogren's syndrome (SS) and morphological picture of the lesser salivary glands (LSG) in chronic hepatitis C (CHC). MATERIAL AND METHODS: The examination of 42 patients with SS and chronic HCV infection (mean age 44.3 +/- 13.7 years) has detected signs of chronic hepatitis and hepatic cirrhosis, respectively, in 31 (71.4%) and 11 (26.2%) patients. "Dry syndrome" was diagnosed by criteria of European SS Study Group. LSG biopsy of the lower lip was conducted in 23 (54.7%) of 42 patients. RESULTS: The "dry" syndrome in CHC ran subclinically in 73.8% patients. Apparent symptoms of SS were seen primarily in middle-aged and aged women with CHC history over 10 years. The first signs of SS occurred in 25 (59.5%) patients 2.9 +/- 3.1 years prior to diagnosis of hepatic disease. All the patients had xerostomy. Xerophthalmia was recorded 1.5 times less frequently. In 16 (47.1%) patients with CHC "dry eye" and in 6 (17.6%) patients dry keratoconjunctivitis were detected. Pathohistological changes of LSG were diagnosed in 21 (91.3%) of 23 patients with CHC. In the majority of cases (86.9%) the glands exhibited insignificant inflammatory infiltration and advanced fibrosis. LSG in CHC is characterized by fibrosis prevalence over cell infiltration. 83.3% CHC patients had SS and other extrahepatic lesions. SS was most evident in 28.6% CHC patients with cryoglobulinemia. CONCLUSION: Registration of SS symptoms in CHC patients depends on targeted examination of patients with chronic HCV infection. The severity of the symptoms correlates directly with the infection duration and age of the patient. LSG lesions in CHC patients with SS are characterized by fibrosis pre-domination over cell infiltration.

[Evaluation of the diagnostic efficacy of a test system for detecting hepatitis virus C RNA by polymerase chain reaction "AmpliSens(TM) HCV-240/BKO-440)"]. / Otsenka diagnosticheskoi éffektivnosti tests-sistemy dlia vyiavleniia RNK virusa gepatita C metodom polimeraznoi tsepnoi reaktsii "AmpliSens(TM) HCV-240/VKO-440".

The test system developed at the Central Research Institute of Epidemiology, Ministry of Health of the Russian Federation for identification of hepatitis C virus RNA was studied. The sensitivity of the test system which the rate of similar results was 100% with its 5-fold reproduction was evaluated. That was 5 x 103 genomic equivalents (or international units) per ml of a sample. A scheme for evaluation of the reproductibility of test systems based on the polymerase chain reaction (PCR) by using model samples is proposed. Whether it can be used for intra- and extra-laboratory assessment of the quality of PCR analyses is discussed.

[Stability of hepatitis C virus RNA in human blood plasma in a panel of control samples]. / Stabil'nost' RNK virusa gepatita C v plazme krovi cheloveka v paneli kontrol'nykh obraztsov.

The stability of hepatitis C virus (HPC) RNA concentration in 5 human plasma samples after storage at +22 degrees C for two months, at -20 degrees C, and +4 degrees C for six months after 10 freezing-unfreezing cycles was evaluated. In this study, the concentration of HCV RNA in the samples was stable after six months of storage at -20 degrees C. The concentration of HCV RNA decreased on the average of 92% after 2-month storage at +22 degrees C. After six months of storage at +4 degrees C and after 10 freezing-unfreezing cycles, that decreased by 28 and 42%, respectively. Based on their own findings, the authors developed a HCV-RNA panel containing 5 positive human plasma samples with RNA levels of 103-105 IU/ml. The panel may be recommended both for the standardization of PCR kits and for the intra- and interlaboratory quality control of PCR laboratories.

[The prospects for the diagnosis of bacterial meningitis]. / Perspektivy diagnostiki bakterial'nykh meningitov.

The samples of spinal fluid arriving to the Clinical Infectious Hospital in 1994-1996 with the clinical diagnosis "generalized form of meningococcal infection" or "purulent meningitis of unclear etiology" were studied. The etiological agent was bacteriologically identified in 35% of 487 patients (in 25% of cases Neisseria meningitidis, in 7% of cases Streptococcus pneumoniae and in 2% of cases Haemophilus influenzae, type b, were detected). The method of latex agglutination, used in this study, was highly specific (100%) and moderately sensitive (67%); this method made it possible to diagnose 25% of cases additionally (N. meningitidis in 15% of cases, S. pneumoniae in 5% of cases and H. influenzae in 3% of cases). Diagnostics with the use of PCR was characterized by high specificity (> 97%) and sensitivity (> 85%) relatively to the "golden standard" of microbiological diagnostics. There were few false positive results (3 samples), caused probably by contamination at the moment of taking the samples. For this reason the results obtained by PCR could be used for diagnostic purposes even in cases of negative results given by other methods. Tests with the use of PCR made it possible to diagnose 29% more cases additionally (in 26% of cases N. meningitidis DNA and in 3% of cases S. pneumoniae DNA were detected. Thus the complex of methods used in this study permitted the detection of the etiological agent altogether in 87% of cases.

[Sensitivity and specificity of the PCR-test-system for diagnosing HIV infections]. / Chuvstvitel'nost' i spetsifichnost' PTsR-test-sistemy dlia diagnostiki VICh-infektsii.

Sensitivity and specificity of the Russian PCR test system (experimental lot) were assessed with materials from 107 Russian patients with HIV infection and 100 donors without specific antibodies to HIV-1. The electrophoretic and hybridization-enzymatic methods for amplicon detection were compared. The sensitivity of electrophoresis was 89.9%, specificity 91%; for hybridization-enzymatic detection these values were 96.1 and 100%, respectively. Samples which yielded false-negative results were repeatedly tested by two-staged amplification (nested PCR) with external and internal primers. The main cause of false-negative results was a low concentration of proviral HIV-1 DNA in some blood samples.

[Polymerase chain reaction in diagnosing cytomegalovirus infection in HIV-infected patients]. / Polimeraznaia tsepnaia reaktsiia v diagnostike tsitomegalovirusnoi infektsii u VICh-infitsirovannykh patsientov.

The authors propose a simple and standard approach to semi-quantitative detection of cytomegalovirus (CMV) DNA in blood leukocytes by the polymerase chain reaction. The method was used in early diagnosis and treatment of manifest CMV infection in HIV-infected patients. High titers of CMV DNA correlated with the manifestation of CMV disease and progressive loss of host immune function. Clinical significance of difference in DNA titers was revealed. The proposed method can be used to assess the efficacy of specific anti-CMV therapy.

[The diagnosis and clinical characteristics of cytomegalovirus infection in HIV-infected subjects]. / Diagnostika i klinicheskaia kharakteristika tsitomegalovirusnoi infektsii u VICh-infitsirovannykh lits.

Out of 180 HIV carriers active cytomegalovirus (CMV) infection was found in 30 patients, in 16 cases the infection manifested clinically. Most of the latter were patients with HIV infection IIIb or IIIc stage against persistent lowering of CD4-lymphocyte count under 100/mm3. Active CMV infection may be determined most significantly by the following criteria: high or moderate concentrations of CMV DNA in the blood, low concentrations of blood CMV DNA in the presence of long-term (at least 3 months) persistence of anti-CMV IgM and isolation of urinary CMV. CMV infection manifested usually as a generalized disease with typical signs of retinitis, myelitis, erosive-ulcerative colitis. Most patients had thrombocytopenia, functionally defective platelets. CNS involvement predicts poor prognosis in CMV-infected HIV carriers.