Long-term analysis of the resistance development in HIV-1 positive patients treated with protease and reverse transcriptase inhibitors: correlation of the genotype and disease progression.

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.

Genetic subtypes of HIV type 1 viruses circulating in the Czech Republic.

We analyzed the genetic diversity of HIV-1 strains circulating in the Czech Republic. Phylogenetic analysis of the env and gag gene sequence fragments from 39 isolates revealed that the majority of these strains (32 of 39, 82%) were of subtype B; other genetic subtypes identified were A, C, F, and recombinant circulating form CRF01_AE. The isolates that did not cluster with subtype B originated almost exclusively from a heterosexual route of transmission. The molecular epidemiological data are suggestive of multiple entry of HIV-1 infection into the Czech Republic and show that the genetic pattern of the HIV-1 strains circulating in this country corresponds to that found in other European countries.

Seroprevalence of HHV 8 antibodies among the general population and HIV positive persons in the Czech Republic.

BACKGROUND: The seroprevalence rates of herpesvirus 8 (HHV 8) antibodies were determined for the general Czech population and HIV-positive individuals. OBJECTIVES: Six hundred and sixty six serum samples from the general Czech population and 129 serum samples from HIV-positive persons were tested for the presence of antibodies to the HHV 8 lytic and latent antigens. STUDY DESIGN: HHV 8 antibodies were detected by the indirect immunofluorescence test. RESULTS: In the general Czech population, only 2.4 and 0.3% of the serum samples tested positive for antibodies against the lytic and latent HHV 8 antigens, respectively. As many as 34.9 and 10.9% HIV positive individuals had antibodies to the HHV 8 antigens, respectively. Only three of them have developed Kaposi's sarcoma (KS) to date. At the time of KS diagnosis, the three patients had antibodies to both HHV 8 antigens. HIV-positive homo/bisexuals were at significantly higher risk of acquiring HHV 8 infection compared with HIV-positive heterosexuals. The increase in HHV 8 seroprevalence was associated with progression of the HIV infection from stage A to stage B. No correlation was found between the HHV 8 seroprevalence and CD 4+T-lymphocytes counts or the HIV viral load. CONCLUSIONS: Among the general Czech population, the HHV 8 seroprevalence is as low as in the West European countries. The mean HHV 8 seroprevalence rate in HIV-positive individuals was 34.9% and was comparable with those reported in other low seroprevalence countries.

Detection of respiratory syncytial virus serum antibodies by an ELISA system.

An ELISA test for respiratory syncytial (RS) virus assay was adapted and standardized; it gave 10-15 times higher antibody titres than complement fixation (CF) but was not a more sensitive test for detecting recent RS virus infection in persons above 1 year of age. In testing normal-population sers, ELISA revealed twice more positive sera than the CF test. Owing to its high sensitivity and apparent ability to detect long-persisting antibodies, ELISA is the test of choice for sero-epidemiological surveys on RS virus infections.

Biophysical properties of coronavirus strain OC 43.

Coronavirus OC 43 from suckling mouse brains was subjected to sucrose density gradient centrifugation. Maxima of complement fixing (CF) activities occurred in the ribosomal (density 1.14) and mitochondrial (density 1.19) fraction which contained incomplete, damaged virus particles. The haemagglutinating (HA) activity was associated with the microsomal soluble protein fraction displayed no antigenic activity. The isolated purified virus proved to be very unstable.

An outbreak of respiratory disease due to a type 5 adenovirus identified as genome type 5a.

A cluster of adenovirus type 5-caused acute respiratory diseases was observed in Prague in the autumn-winter season of 1978/79. An analysis of viral DNA by sequence-specific restriction enzymes in two out of 39 isolates revealed that these isolates, identical to each other, were clearly distinct from adenovirus type 5 prototype strain. The designation "adenovirus genome type 5a" is suggested for these isolates.

Human immunodeficiency virus 1 strains resistant to nucleoside inhibitors of reverse transcriptase in isolates from the Czech Republic as monitored by line probe assay and nucleotide sequencing.

The genetic resistance to nucleoside inhibitors of the reverse transcriptase (RT) of human immunodeficiency virus I (HIV-1) isolates in the Czech Republic was examined by a line probe assay (LiPA) and nucleotide sequencing. The results of LiPA analysis of 294 blood specimens obtained from 156 patients revealed a high incidence of mutations in the RT gene related to resistance to various drugs (67.3%) in various combinations. Mutations in RT gene (M41L, K70R and T215Y/F) conferring the resistance to zidovudine (ZDV) were most frequent (62.6%), that (M184V) responsible for the resistance to lamivudine (3TC) was less frequent (33.7%), while those linked to the resistance to dideoxyinosine (ddl) and dideoxyinosine together with dideoxycytidine (ddl/ddC) were rather rare (6.5% and 5.1%, respectively). LiPA gave a high rate of uninterpretable results due to codon hybridization failure, especially in HIV-1 isolates of non-B subtype. Thirty-two specimens were analyzed also by direct sequencing of a part of RT gene. The results obtained by LiPA and the sequencing were highly concordant for codons successfully analyzed by both methods, but the sequencing provided information also about the codons that could not be analyzed by LiPA. A high prevalence of resistant strains in the Czech Republic and their heterogeneity justifies a regular HIV-1 resistance testing. LiPA turned out as a fast, powerful and most reliable tool for such a purpose. However, due to an increasing diversity of HIV-1 strains circulating in the Czech Republic, LiPA cannot replace the nucleotide sequence analysis.

Occurrence and aetiology of acute respiratory diseases: results of a longterm surveillance programme.

Totals of 58,661,000 acute respiratory disease (ARD) cases, 1,376,651 bronchitis and pneumonia complications, and 93,042 deaths from influenza, bronchitis, pneumonia or chronic pulmonary affection were notified during 11 years of ARD surveillance from 1975 to 1986. All ARD seasons started with the first phase in September-December; this increase in morbidity was caused chiefly by adenoviruses, parainfluenza viruses, rhinoviruses and M. pneumoniae. Second wave of ARD morbidity occurring in January-April used to be explosive and was associated with an influenza epidemic in 9 of the 11 seasons; only in 1978/79 and 1984/85 the ARD epidemics were caused by adenoviruses and especially RSV, the share of influenza being minimal. Pneumonia and bronchitis excesses occured during epidemics caused by M. pneumoniae in 1975/76, 1980/81 and 1985/86. Particularly high mortality excesses occurred in 1976, 1977 and 1983 during epidemics elicited by a new drift variants of influenza A(H3N2). Identification of viral agent of M. pneumoniae attempted in 5474 ARD cases was successful at 37.4%. The respective contributions of parainfluenza viruses, adenoviruses, influenza A virus and RSV to overall aetiologically identified morbidity were 14.2, 13.9, 13.8, and 12.0%. Mixed infections (2-3 agents identified simultaneously) accounted for 14.6% of cases. Type B influenza virus, rhinoviruses, enteroviruses and herpes simplex virus contributed only by 5.6-7.8%. In ordinary seasons the share of M. pneumoniae in aetiologically identified ARD morbidity was 0.6-3.8%; this agent displayed predominance at 5-year cycles, when accounting for 20.5-38.9% of cases. The most frequently detected agents in individual age groups were as follows: in preschool children parainfluenza (18.6%), RSV (16.6%), and adenoviruses (17.4%); in school children M. pneumoniae (26%), influenza A and B (10.2 and 14.7% respectively), and adenoviruses (10.7%); in adolescents and young adults influenza type A (20.2%), M. pneumoniae (15.0%), and rhinoviruses (13.3%); in adults above 25 years age influenza A virus (38%), and other respiratory viruses at a frequency lower than 10% each.

Bacterially expressed core and envelope proteins of human immunodeficiency virus type-1 (HIV-1): comparative evaluation in detection of type-specific antibodies.

Recombinant proteins derived from immunodominant conserved domains of HIV-1 env and gag genes were synthesized in E. coli. An immunoblot system using total cell lysates was employed for the analysis of recombinant bacterial clones. Together 427 serum samples obtained from asymptomatic anti-HIV seropositive individuals, AIDS patients, healthy donors and persons suffering from various conditions were comparatively evaluated for the presence of HIV-1 antibodies using recombinant peptides and commercially available western blot (WB) and ELISA assays. The recombinant antigen product of plasmid pEX41 was found to be superior, with respect to sensitivity and specificity, to the viral gp41 which represents a diagnostically important constituent of the WB.

Virus neutralizing antibodies at different stages of the HIV disease: increased levels after azidothymidine treatment.

Specific HIV-1 neutralizing activity was measured in single serum samples obtained from 52 individuals suffering from different stage of HIV disease, as well as in serum samples collected during a four years follow up of other 13 HIV-1 seropositive persons, from whose seven developed AIDS. Three of these persons were treated with azidothymidine. In the former group of single serum specimens, the specific neutralizing antibody positivity rate was 81 per cent in symptomless persons, 92 per cent in patients with ARC and 43 per cent in patients with AIDS. From 13 HIV-1 infected individuals, prospectively investigated from 1986 to 1990, six remained asymptomatic and no significant fluctuation of specific virus neutralizing antibody levels was noted. During this time period, remaining seven patients developed AIDS. In the sera of AIDS patients, specific neutralizing activity was either not detected or its titres were rather low before the appearance of clinical disease. Three AIDS patients were administered azidothymidine. Specific neutralizing antibody titres increased significantly one month after the beginning of azidothymidine administration and persisted at relatively high levels over several months of follow up.

Levels of immunoglobulins and antibodies to haemaglutinin and neuraminidase of influenza virus in nasal secretions after natural infection.

Nasal washings (NW) from 16 influenza patients in the course of an epidemic in November and December, 1974 were examined for the presence of influenza virus, immunoglobulins (Ig) and titres of haemagglutination inhibiting (HI) and neuraminidase inhibiting (NI) antibodies. Influenza virus identical with A/Port Chalmers/1/73 (H3N2), increased levels of IgA and occasionally IgG, and specific antibodies were detected in the NW. The dynamics of HI and NI antibody formation did not differ substantially, but there were individual differences in titres and persistence of antibodies. Convalescent sera always contained increased levels of HI and NI antibodies. In some cases, the titres of antibody to viral ribonucleoprotein did not increase.

[Detection of HIV virus antibodies in saliva]. / Detekce protilátek proti viru HIV ve slinách.

The authors evaluate the use of saliva as alternative biological material for detection of HIV antibodies. If collected properly and when using the appropriate ELISA methods, HIV antibodies can be assessed in saliva with considerable sensitivity and specificity. Testing in saliva eliminates many disadvantages found when assessing HIV antibodies in serum. It does not require trained staff for blood sampling, collection and processing of saliva reduces the risk of professional infection to a minimum and can be carried out also under field conditions. A great advantage is the easy and unpretentious transport of collected samples to the laboratory, even long distance transport at extreme temperatures (transport medium in the testing kit Omni-SAL ensures great stability of the sample as well as proper collection of the sample). Alternative testing in saliva is useful in particular for epidemiological surveillance and for screening of HIV antibodies in population groups with a high risk of infections such as drug users and commercial works where it is often very difficult to obtain blood samples for examination.

[Indicators of progression of HIV infection and their application to therapy]. / Ukazatele progrese infekce HIV a jejich vyuzití v terapii.

The authors evaluated in a group of 217 HIV positive patients the mutual relationship of the number of CD4+T lymphocytes and the level of the viral load of HIV RNA. Using correlation analysis evidence was provided of a not very marked negative correlation of the two indicators. As it was assumed that the relationship of the two parameters is influenced by the applied therapeutic procedures which reduce in particular the viral load, the two parameters were evaluated in relation to treatment. The closest relationship of the two investigated parameters was found in the group treated by monotherapy with zidovudine, followed by the group treated with a combination of two preparations (two nucleoside inhibitors of reverse transcriptase). In the group treated by three preparations (two nucleoside inhibitors of reverse transcriptase and a protease inhibitor) the relationship of the two parameters was least close. Investigation of the level of the viral load of HIV RNA and number of CD4+T lymphocytes is of major importance for the introduction of antiretrovirus treatment and selection of a suitable combination of antiretrovirus preparations. It makes it also possible to follow up the effectiveness of this treatment.

10th anniversary of the National Reference Laboratory on AIDS in the Czech Republic.

National Reference Laboratory on AIDS (NRL AIDS) was established in the National Institute of Public Health by the Ministry of Health in the late 1985. At that time the existence of HIV/AIDS and the potencial spread of this infection/disease even in a socialistic country has been officially recognized. The main tasks of NRL AIDS were and still are the laboratory diagnostic, epidemiology and research of HIV/AIDS. As one of its first steps NRL AIDS introduced HIV antibody testing methods and strongly recommended obligatory screening of all blood donations to assure the safety of blood and blood products. In 1986 NRL AIDS performed the first serological pilot study to gain basic data on HIV infection incidence and prevalence in the Czech population. National HIV/AIDS reporting system has been elaborated at NRL AIDS and connected with the WHO-EC European AIDS Monitoring System. Several national and international collaborative HIV surveillance studies in different population groups are performed and evaluated yearly to monitor the trends in the spread of HIV/AIDS in the Czech Republic. From 1986 through May 1995 a total of 230 cases of HIV infection were reported in the Czech Republic. Of these 62 cases met the criteria for AIDS case definition. Homo/bisexual men are still the most affected population group accounting for 59% of all HIV reported cases. Nevertheless the percentage of heterosexually transmitted infections is constantly increasing, presently accounting for 19% of all infected persons. The activities of NRL AIDS are very broad, including the field of applied research. Several research grants were obtained from the Grant Agency of the Ministry of Health. Results of some of these are included in the National HIV/AIDS Programme of the Czech Republic.

Characteristic features of HIV/AIDS in the Czech Republic.

First laboratory proved HIV infections in the Czech Republic were diagnosed in a homosexual couple in autumn of 1985. Total number of detected HIV+ cases reached 143 persons by the end of 1992 (132 men and 11 women), of that 31 cases were classified as fully blown AIDS (21 already died). Out of these 143 HIV+ cases, 93 (65.0%) are represented by homo/bisexuals, 10 (7.0%) by heterosexuals, 16 (11.2%) by haemophiliacs, 14 (9.8%) by blood recipients, 1 (0.7%) by IVDU and 9 (6.3%) by unknown transmission category. Approx. 2.1 mil. of tests have been done as routine screening of donated blood. Only 5 cases (0.0002%) of HIV positivity have been found in this group. All HIV positive blood recipients (30 cases) were infected before the mandatory HIV testing of blood supply has been introduced in 1987 (14 out of 16 HIV+ haemophiliacs were infected by imported clotting products). The twins born in 1990 to mother infected by contaminated blood in 1984, were declared at the age of 30 months as HIV free, with all laboratory tests (serology, virus cultivation, PCR) negative. The cummulative infection rate of HIV antibody in 1986-1992 reached 13.8 per million inhabitants. It may be concluded that slow steady increase in the number of reported HIV/AIDS cases has been registered during the whole follow-up period. The authors are aware that relatively low prevalence of HIV infection in the Czech Republic may change dramatically in consequences of recent deep social, political and economical changes in the country.

Detection of serum antibodies in tuberculosis patients.

Sera of 14 bacteriological confirmed pulmonary tuberculosis and 16 non-tuberculotic or healthy controls were sampled in Ignace Deen University Hospital in Conakry, Quinea. Samples were examined for IgG and IgM antibodies by means of enzyme-linked immunosorbent assay (ELISA) using sonicated M. bovis BCG and M. avium antigens and were tested for antibodies to HIV-1/HIV-2 as well. Median of IgG antibody titres to M. bovis BCG antigen was 1:445 and differed significantly from that of the control group (1:149). The median of IgM antibody titres was 1:79.1 and did not differ statistically from that of control group (1:69.3) as well as the antibody titres against M. avium antigen in the IgG and IgM classes for both analyzed serum groups. Seven of tested TB patients sera were positive for antibodies against HIV-1. The median of IgG antibody titres against M. bovis BCG antigen was 1:442 not differing significantly from values of remaining TB patients as well as the IgM antibody titre (1:109).

Mycobacterial infections in patients with AIDS in a low HIV prevalence area.

The Czech Republic is characterized here as both a low HIV prevalence area (1.64 registered positivity per 10(5) population and 45 AIDS patients notified in the 1986-93 period) and a low tuberculosis prevalence area (mortality rate 0.8, incidence of bacillary pulmonary cases 11.3 per 10(5) population and risk of infection 0.04 in 1990). From 1986 to 1993 a total of 10 cases of mycobacterial infections complicating AIDS or ARC disease were registered in the Czech Republic in 9 male patients aged from 29 to 55, 8 homosexuals, and one woman aged 48. Classical pulmonary tuberculosis caused by M. tuberculosis was diagnosed in one male patient and was controlled by antituberculosis chemotherapy. The remaining mycobacterial infections were caused by the following opportunistic mycobacterial pathogens. (a) M. avium in five individuals, four of them died of a disseminated disease; (b) M. kansasii--in two patients, one died of a disseminated infection; (c) M. xenopi--involved in a fatal generalized CMV, and cryptococcal and mycobacterial infection; (d) M. fortuitum caused a generalized disease in a case of exhaustive multifocal encephalopathy. Authors conclude that under conditions of low prevalence of both HIV and tuberculosis infection the risk to AIDS patients of contracting classical tuberculosis is low. A relatively frequent occurrence of non-tuberculosis mycobacterial disease in AIDS patients afflicted with the impaired immunity is explained by transmission of opportunistic mycobacteria from the environment.

High prevalence of HIV risk-behavior and the identification of predictors for sharing injecting materials among young drug users in Prague, Czech Republic.

This study is one of the first studies in the Czech Republic evaluating the extent of the HIV epidemic among drug users. Interview data on demographics, drug use, sexual practices, and HIV knowledge and risk perception were obtained from study participants and a saliva sample was obtained for an HIV antibody test. Although the HIV infection rate is low (0.2%), the prevalence of high risk behaviors, such as sharing injecting equipment and unsafe sexual practices, indicate a potential for rapid HIV spread in this high risk population. Preventive measures need to be targeted toward drug users, especially those of younger age and women.

[Childhood AIDS]. / Detský AIDS.

The authors emphasize the present importance of AIDS in children and explain the causes of the rising trend of this syndrome in the child population. They summarize the most important epidemiological characteristics of AIDS in children, its pathogenetic differences and the ensuing clinical symptoms. Attention is drawn to the pitfalls of microbiological diagnosis of child infections caused by HIV, and the authors discuss principles, possibilities and perspectives of causal treatment of this syndrome in children. Finally they summarize contemporary experience and knowledge regarding immunization of children infected with the human immunodeficiency virus.

[The expert diagnosis of HIV infection by radioimmunoprecipitation]. / Ekspertnaia diagnostika VICh-infektsii metodom radioimmunopretsipitatsii.

Using a panel of sera from HIV-infected persons and donors, the authors showed that radioimmunoprecipitation assays compare favourably with immunoblotting assays. With radioimmunoprecipitation, cross reactions were observed between HIV-2 antigens and HIV-2 antibodies, and that the nature of cross reactivity differs from that observed with immunoblotting. Potentials of radioimmunoprecipitation assays as a confirmatory test for use with sera that have given indeterminate results in immunoblotting assays and contradictory results in enzyme immunoassays are examined.