Sonja Zerjav

HIV-1 subtypes in Yugoslavia.

To gain insight concerning the genetic diversity of HIV-1 viruses associated with the HIV-1 epidemic in Yugoslavia, 45 specimens from HIV-1-infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty-one of 45 specimens (91.2%) were identified as pol subtype B, 2 of 45 as subtype C (4.4%), 1 of 45 as CRF01_AE (2.2%), and 1 as CRF02_AG recombinant (2.2%). Nucleotide divergence among subtype B sequences was 4.8%. Results of this study show that among HIV-1-infected patients in Yugoslavia subtype B predominates (91.5%), whereas non-B subtypes are present at a low percentage, mostly related to travel abroad.

Herpes zoster as an immune restoration disease in AIDS patients during therapy including protease inhibitors.

A prospective study to evaluate the incidence of herpes zoster (HZ) as an immune restoration disease in patients with AIDS during highly active antiretroviral therapy (HAART) was conducted in a series of 115 patients diagnosed with AIDS initiated on HAART between 1 January 2000 and 31 July 2001. Of these, a single dermatomal HZ episode occurred in 14 (12%) patients within one and 15 months of HAART (median eight months). The HZ patients were similar to the non-HZ patients in age, sex, and HIV transmission risk factor, but had a more advanced disease. Compared with the baseline values, the viral loads significantly (P<0.01) decreased, while the mean CD4+ T-cell counts increased by almost four-fold (P<0.01) in both groups at the time of the HZ episode (or equivalent in non-HZ), but remained below 400/mL in the HZ patients. HZ during HAART is an immunopathological consequence of the improvement of the host immune response, correlating with the beginning of immune restoration.

HBsAg as the antigen component of circulating immune complexes in HIV-infected patients

Seeing the same transmission pattern of HIV and HBV coinfection by these two agents is not an uncommon feature. Immunity impairment due to HIV infection can be the cause of a higher rate of HBV replication with less intensive liver damage and less effective immune response to HBV, while the pathological course in both infections involves elevated levels of circulating immune complexes (CIC). These were the reasons for us to examine the frequency of HBsAg involvement as the antigen component of circulating immune complexes formed in sera of HIV-infected patients in different stages of HIV disease. We tested 67 sera of HIV-positive patients in different stages of HIV disease for the presence of HBsAg and HIV antigen p24 (with and without acid dissociation of immune complexes), for the presence of anti-Hbc antibodies and circulating immune complexes. HBsAg was positive in 13.8% sera prior to and 33.8% after acid pretreatment. Anti-HBc antibodies were present in 76.9% serum samples tested. Fifty percent of sera were positive for both HBsAg and p24 antigen after dissociation of immune complexes. The level of CIC was elevated in 65.9% of sera. Our results suggest that HBsAg is commonly associated in immune complexes formed in the sera of HIV-infected patients and that they may simultaneously contain HIV and HBsAg in patients coinfected with both agents. This may contribute to their mutual interaction and influence the diagnosis and follow-up of patients.

ANALYSIS OF HUMAN TROPHOBLASTIC TISSUE IN DIAGNOSIS OF EMBRYONIC VIRAL INFECTIONS

Summary Thirty four pregnant women with serologically and by isolation (from cervix and urine) proven viral infection (20 with CMV, 7 with HSV tip II, 6 with VZV and one with Parvovirus B19 infection) were studied. Chorionic villus sampling and amniocentesis were performed between 9th and 13th gestational week. Isolation of viruses on the culture of human fibroblasts for CMV, and on monkeys kidney for HSV was performed. Testing for indirect immunofluorescence with anti VZV specific serum and FITC marked antihuman globulin was used for detection of VZV antigens. The presence of HSV antigens was investigated by direct immunofluorescence with monoclonal antibodies on HSV type I and II, with FITC marked antibodies. CMV antigens was detected by indirect immunofluorescence. ELISA testing was used for Parvovirus B19. Anti HSV antibodies were detected by indirect immunofluorescence. ELISA testing also was used for detection of specific anti CMV IgG and IgM antibodies. Embryonic infection was detected in one case of VZV and Parvovirus B19 infection, two cases of HSV infection and six pregnancies with CMV infection (17%, 100%, 29%, 30%, respectively). In two cases of proven embryonic infection (VZV and CMV), the pregnancy was terminated, and in one (Parvovirus) spontaneous abortion occurred. Other pregnancies were continued. Cordocentesis were performed after the 22nd gestational week. The presence of specific and nonspecific signs of fetal infection was evaluated. Fetal infection was found in two (8%-CMV and HSV) in which embryonic infection could not have been proven (false negative results). One false positive (CMV) finding was found. It may be concluded that trophoblast analysis is important for studying of infections produced by teratogenic agents during pregnancy. It contributes to the early diagnosis of such infections (sensitivity 81%). Excluding the presence of infection (specificity 96%) is of even greater importance. The predictive value of the positive finding (90%) suggests that the positive findings should greatly influence decisions involving the subsequent therapeutical protocol. Negative findings (predictive value 92%) mandate application of additional diagnostic procedures in order to rule out any fetal-related risk.

Molecular typing of the local HIV-1 epidemic in Serbia.

Worldwide HIV-1 pandemic is becoming increasingly complex, with growing heterogeneity of subtypes and recombinant viruses. Previous studies have documented HIV-1 subtype B as the predominant one in Serbia, with limited presence and genetic diversity of non B subtypes. In recent years, MSM transmission has become the most frequently reported risk for HIV infection among newly diagnosed patients in Serbia, but very little is known of the network structure and dynamics of viral transmission in this and other risk groups. To gain insight about the HIV-1 subtypes distribution pattern as well as characteristics of HIV-1 transmission clusters in Serbia, we analyzed the genetic diversity of the pol gene segment in 221 HIV-1-infected patients sampled during 2002-2011. Subtype B was found to still be the most prevalent one in Serbia, accounting for over 90% of samples, while greater diversity of other subtypes was found than previously reported, including subtypes G, C, A, F, CRF01 and CRF02. In total, 41.3% of analyzed subtype B sequences were found associated in transmission clusters/network, that are highly related with MSM transmission route.

Antigen/antibody content of circulating immune complexes in HIV-infected patients

Dual infection with HIV and hepatitis B virus (HBV) is not an uncommon feature. Immunity impairment due to HIV infection can be the cause of a higher rate of HBV replication with less intensive liver damage and less effective immune response to HBV. Many HIV-infected patients have an elevated level of circulating immune complexes (CIC) in serum, throughout all stages of illness evolution. The aim of our study was to estimate p24 and HBsAg content of CIC in dually infected patients, and the prevalence of major classes of complexed antibodies (IgM and IgG). We examined 146 samples of sera from 105 HIV positive patients of the Institute for Infectious and Tropical Diseases during 1992 and 1993. On those sera we performed p24Ag and HbsAg detection, with and without prior dissociation of CIC, we determined serum level of CIC and immunoglobulin classes IgM and IgG level in sera and in polyethilenglycol (PEG) precipitates of sera. Acid dissociation of immune complexes revealed a high proportion of HIV antigen positive sera in all stages of HIV disease progression. HbsAg in serum of HIV positive patients was also found coupled in immune complexes much more frequently than in the HIV negative control group. In many instances both antigens were simultaneously found coupled in CIC. Immune complexes detected have been shown to contain both IgM and IgG immunoglobulins, while IgM antibodies were associated to immune complexes in higher proportion than IgG, compared to total serum immunoglobulins.

CMV DNA in blood and CSF of HIV infected patients

Cytomegalovirus (CMV) infection frequently affects the central nervous system in HIV infected patients. Varied clinical manifestations of CMV disease make virological detection of CMV essential for proper diagnosis and treatment. Thus, in patients in different stages of HIV-induced disease, we attempted to detect cell associated and free, non cell associated CMV DNA in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells (PBMCs). Twenty-six blood samples were collected from 22 patients included in the study. Nine of these blood samples were tested in pair with the concomitant CSF sample for the presence of CMV DNA by a commercial hybridization test. CMV serostatus and avidity of IgG antibodies were detected by a commercial ELISA test. CMV DNA was present in the cells found in CSF in all but one of the AIDS patients, independently of the presence of neurological symptoms, suggesting that it represented a marker of advanced immunodeficiency, rather than of the specific CMV-related disease. Cell-associated CMV DNA in CSF tested positive even in the samples negative for cell-free CMV DNA in the CSF, and with no detectable CMV DNA in the PBMCs of concomitant blood sample. We believe that searching for CMV DNA in different compartments of CSF merits further attention.

Non-specific inflammatory parameters in patients with pandemic H1N1 influenza.

The measurement of non-specific inflammation parameters, such as erythrocyte sedimentation rate (ESR), fibrinogen, C-reactive protein (CRP) and procalctinon (PCT) are very important tools for diagnosis of infections, as well as for monitoring of treatment response. The aim of this study was to determine the significance of non-specific inflammatory parameters in patients with influenza H1N1 infection. ESR, fibrinogen, CRP and PCT were analyzed in patients with influenza H1N1 infection. The diagnosis of influenza H1N1 was established from the nasopharyngeal swabs using Real Time Polymerase Chain Reaction - (RT PCR) method. Chest X-ray was performed to diagnose pneumonia Sixty-three out of 340 hospitalized patients with influenza had pandemic influenza. Their mean age was 34.60±13.82 years. They were referred to hospital 1 to 7 (4.06±2.0) days after onset of symptoms. Of these, 46 had pneumonia, while the majority (41 patients) had interstitial pneumonia, and only five had lobar or segmental pneumonia. Patients with pneumonia had significantly higher levels of CRP and PCT in comparison with those without pneumonia. Patients with lobar pneumonia had significantly higher CRP than those with interstitial pneumonia. However, mean values of PCT between interstitial and lobar pneumonia cases did not differ significantly. Interstitial pneumonia was the most common complication of H1N1 infection among our patients. Non-specific parameters of inflammation, especially CRP and PCT were increased in all pneumonia cases, regardless of the etiology. Monitoring of non-specific inflammatory parameters in patients with H1N1 infection allows recognition of patients with complications, their prompt hospitalization and early initiation of antimicrobial therapy.

Forensic application of phylogenetic analysis – exploration of suspected epidemiological linkage

Phylogenetic analysis may serve as a valuable tool in assessing the epidemiological relation between viral DNA sequences. In order to increase the likelihood of observing phylogenetic separation of sequences as well as to give strong forensic evidence regarding transmission, phylogenetic analysis needs to be performed on appropriate local control sequences and by sequencing of at least two genetic regions of reasonable length, depending on the gene under investigation. The aim of this study was to explore the suspected epidemiological linkage between DNA sequences isolated from three HIV-1 infected patients by means of phylogenetic analyses. Phylogenetic analysis was performed on investigated sequences together with a number of local controls, that are viral sequences from infected individuals in the same location and diagnosed in a similar time period. Two genetic regions, of 1,6 kb for pol and 800 bp for env, were amplified and sequenced from viral RNA extracted from plasma. Transmission clusters were assigned as those phylogenetic clades consisting of three or more sequences, fulfilling the conditions of genetic distance of 1.5% or less with posterior probability higher than 0.9 in the Bayesian analyses, for both genetic region. Maximum likelihood (ML) phylogenetic analysis was performed as implemented in Phylogenetic Analysis Using Parsimony (PAUP), using the evolutionary model selected by jModeltest software. Phylogenetic analysis revealed the presence of a single transmission cluster that accomplished all predefined sets of criteria. This cluster was composed of three viral sequences isolated from patients whose epidemiological linkage was under investigation. The mean pairwise nucleotide divergence among all observed pol sequences was 7.1% (range 0.2-12.8%) while among sequences under investigation it was 0.8% (0.3-1.1%). Regarding env sequences, nucleotide divergence among all was 16.1% (range 0.3-26.3%), while among sequences under investigation it was 1.3% (1.1-1.5%). Our results strongly support the epidemiological linkage between sequences under investigation. Viral phylogenies can help to evaluate proposed epidemiological linkage and to infer the ancestral relationships of infections. However phylogenetic analysis cannot unambiguously prove that HIV-1 transmission occurred directly between two individuals, since any transmission chain may contain additional sequences not included in the analysis.

The prevalence of RT 245 codon polymorphisms and its association with duration of infection among HIV-1 patients in Serbia

Amino acid (aa) substitutions at position 245 of HIV-1 reverse transcriptase (RT) have been described to be associated to the presence of human leukocyte antigen (HLA)–B*5701 allele in the host, in particular in subtype B infection. Preliminary data show HLA–B*5701 prevalence of around 6.5% in Serbian population. In this study, we investigated the prevalence of RT codon 245 substitutions among HIV infected patients in Serbia. Furthermore, we analyzed the association between RT 245 aa substitutions with duration of infection, estimated by proportion of ambiguous basecalls per sequence. The study included 184 consenting, subtype B HIV infected patients aged 18 or more. The majority of patients were newly diagnosed, 150/184, while 34/184 patients were on treatment. Pol region sequences, covering protease and minimally 250 RT codons, were obtained within routine drug resistance testing. The fraction of ambiguous nucleotides in each sequence was calculated for samples drawn from naive patients. We used ambiguity percentage of 0.47% as a cut-off value delimiting recent (less than 1 year) vs. chronic infection (longer than 1 year). In total, predominant aa at RT codon 245 was the wild type valin (V) found in 118/184 (64.1%), hence 35.9% (66/184) contained mutation at this position (among naive patients this percentage was 38% (57/150). The most common substitution at RT codon 245 was methionine (M) 29/184 (15.7%), followed by glutamic acid (E) 20/184 (10.9%) and others. Based on the percentage of ambiguous basecalls, a total of 55.3% of naive samples (83/150) were classified as recent infection, while among these, 57.8% (48/83) had V at position 245. A total of 45.3% (68/150) were classified as chronic infection, with the presence of V at RT codon 245 found in 69.1% (47/68). We did not find statistically significant association between polymorphisms at codon 245 and duration of infection (p=0.2080). The frequency of RT 245 substitution found in our study exceeds the estimated prevalence HLA–B*5701 in Serbian population. This may be related to the presence of other, similar HLA alleles, limiting specificity of the correlation between HLA-B*5701 and RT codon 245 variation. Furthermore, no statistically significant difference was found in the prevalence of RT 245 substitutions between recent and chronic infection. This may suggest early fixation of an HLA induced selective imprint, during viral evolution in an infected patient.