Vitamin D3 application and factors of its activity in the adrenal cortex of andropausal rats: A functionally-histological study.

INTRODUCTION AND AIM: Vitamin D supplementation in aging subjects manifests a positive effect on various health-related parameters. We performed a functionally-histological analysis of the adrenal cortex regarding the factors of vitamin D activity and corticosterone output after vitamin D3 application in a rat model of the andropause. MATERIAL AND METHODS: Middle-aged Wistar rats were divided into sham operated (SO; n=8), orchidectomized (Orx; n=8) and vitamin D3-treated orchidectomized (Orx+vit. D; n=8) groups. Vitamin D3 (5 µg/kg b.m.) was administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using histochemistry/immunohistochemistry, stereology, ultrastructural and biochemical analyses. RESULTS: Orchidectomy (Orx) decreased the adrenal cortex-related volume densities of vascular (p<0,0001), vitamin D receptor (VDR; p<0,0166), cytochrome P450 oxidase 2R1 (CYP 2R1; p<0,0001) and cytochrome P450 oxidase 24 (CYP 24; p<0,0001) depots, but increased the volume density of cytochrome P450 27B1 (CYP 27B1; p<0,0001) depots. In Orx+vit. D rats, increase of the adrenal cortex-related volume densities of collagen (p<0,0001), VDR (p<0,0001) and CYP 2R1 (p<0,0001) depots as well as the lipid-droplet diameter (p<0,0001) in adrenocortical outer zona fasciculata cells was observed, while a decrease of volume densities of the vascular (p<0,0001), CYP 27B1 (p<0,0001) and CYP 24 (p<0,0001) depots was registered, all versus Orx group. Plasma level of ACTH was decreased (p=0,0155) and serum concentrations of 25-hydroxyvitamin D3 and corticosterone were increased (p<0,0001 and p=0,0187, respectively), all after the same treatment. CONCLUSIONS: Increased corticosterone output after vitamin D3 application to andropausal rats appears not to be related to increased availability of 25-hydroxyvitamin D3 and decreased degradation of 1,25-dihydroxyvitamin D3 in adrenal tissue, but rather involves the central regulatory mechanisms.

All-cause and AIDS-related mortality among people with HIV across Europe from 2001 to 2020: impact of antiretroviral therapy, tuberculosis and regional differences in a multicentre cohort study.

Background: All-cause and AIDS-mortality in Europe has been decreasing between 1996 and 2020. However, regional differences as well as their drivers remain unclear. This study investigates mortality differences and their drivers, including usage of and response to antiretroviral therapy (ART) and active tuberculosis (TB), among people with HIV across Europe. Methods: People with HIV enrolled in EuroSIDA were followed from 2001 through 2020. Immunologic-virologic status (IVS) was categorized as poor (CD4-cell count ≤350 cells/mm3 and viral load (VL) > 200 copies/ml), good (CD4 ≥ 500 and VL < 200), or intermediate (remaining combinations). Participants missing either CD4-cell count or VL were categorized as unknown. Regional differences in mortality were analyzed using multivariable Poisson regression with interaction analyses between regions of Europe and IVS, ART, or TB status. Findings: 20,364 people with HIV were included: 13,715/20,346 (67.3%) from Western, 3020/20,364 (14.8%) from Central Eastern, and 3629/20,364 (17.8%) from Eastern Europe. At enrolment, median age was 40 years (inter-quartile range (IQR): 33-48), median CD4-cell count 449 cells/mm3 (IQR: 291-638), and most were male 14,993/20,346 (73.3%). A total of 2639 died during 192,591 person-years of follow-up (crude mortality rate 13.7/1000 person-years, 95% CI: 13.2-14.2), 519/2639 (19.7%) from AIDS (2.7/1000 person-years, 2.5-2.9). All-cause and AIDS-mortality rates decreased over time but remained higher in Eastern Europe after adjusting for confounders. Being off ART (aIRR 2.42; 95% CI 2.14-2.74), poor IVS (aIRR 4.2; 95% CI 3.39-5.20) and prior TB (aIRR 3.33; 95% CI 2.75-4.03) were associated with higher all-cause mortality. For all-cause mortality the effect of ART (test for interaction: p < 0.001) and IVS (p = 0.02), but not TB (p = 0.5) varied across regions. Interpretation: Overall mortality and AIDS-mortality rates decreased over time, but remained higher in Eastern Europe. A poor IVS, being off ART and prior active TB were related to higher mortality. Eastern Europe had the highest proportion of people with poor or unknown IVS, emphasizing the continued need to improve HIV care with a focus on early diagnosis, ART initiation, and adherence. Funding: EuroSIDA has received funding from ViiV Healthcare LLC, Janssen Scientific Affairs, Janssen R&D, Bristol-Myers Squibb Company, Merck Sharp & Dohme Corp, Gilead Sciences and the European Union's Seventh Framework Programme for research, technological development and demonstration under EuroCoord grant agreement n˚ 260694. The study is also supported by a grant from the Danish National Research Foundation and by the International Cohort Consortium of Infectious Disease (RESPOND).

Mpox across countries from Central and Eastern Europe - 2022 outbreak.

BACKGROUND: The aim of the study was to assess socio-demographical characteristics, clinical presentation, and outcomes in patients diagnosed with mpox. METHODS: A survey on patients diagnosed with mpox was performed in 14 countries from Central and Eastern Europe. Data was compared according to HIV status and country of origin (EU vs. non-EU). Mpox diagnosis was confirmed by RT-PCR from oropharyngeal swabs, skin lesions, and other body fluids. RESULTS: Out of 154 patients confirmed with mpox in 2022, 99.3% were males, with a median age (years) of 35 (IQR 30-39), 90.2% MSM and 48.7% PLWH. Compared to HIV-negative subjects, PLWH had more frequent high-risk behaviours:chemsex (p = 0.015), group sex (p = 0.027), and a history of sexually transmitted infections (STIs) (p = 0.004). Persons from EU were more often PLWH (p = 0.042), MSM (p < 0.0001), had multiple sexual partners (p = 0.025), practiced chemsex (p = 0.008) or group-sex (p = 0.005) and had more often history of STIs (p < 0.0001). The median CD4 cell count/mL at mpox diagnosis was 713 (IQR 486-996) and 73.5% had undetectable HIV VL. The commonest clinical features were fever (108 cases), lymphadenopathy (78), and vesiculo-pustular rash: penile (76), perianal (48), limbs (67). Fifty-one (31%) persons were hospitalized due to complications or epidemiological reasons. Three patients received tecovirimat or cidofovir. The outcome was favorable for all patients, including 4 with severe forms. CONCLUSIONS: Mpox was diagnosed predominantly in young MSM, with high-risk behaviors and history of STIs. Effective contact tracing and vaccination are important strategic pillars to control mpox outbreaks.

GPX3 Variant Genotype Affects the Risk of Developing Severe Forms of COVID-19.

In SARS-CoV-2 infection, excessive activation of the immune system intensively increases reactive oxygen species levels, causing harmful hyperinflammatory and oxidative state cumulative effects which may contribute to COVID-19 severity. Therefore, we assumed that antioxidant genetic profile, independently and complemented with laboratory markers, modulates COVID-19 severity. The study included 265 COVID-19 patients. Polymorphism of GSTM1, GSTT1, Nrf2 rs6721961, GSTM3 rs1332018, GPX3 rs8177412, GSTP1 rs1695, GSTO1 rs4925, GSTO2 rs156697, SOD2 rs4880 and GPX1 rs1050450 genes was determined with appropriate PCR-based methods. Inflammation (interleukin-6, CRP, fibrinogen, ferritin) and organ damage (urea, creatinine, transaminases and LDH) markers, complete blood count and coagulation status (d-dimer, fibrinogen) were measured. We found significant association for COVID-19 progression for patients with lymphocytes below 1.0 × 109/L (OR = 2.97, p = 0.002). Increased IL-6 and CRP were also associated with disease progression (OR = 8.52, p = 0.001, and OR = 10.97, p < 0.001, respectively), as well as elevated plasma AST and LDH (OR = 2.25, p = 0.021, and OR = 4.76, p < 0.001, respectively). Of all the examined polymorphisms, we found significant association with the risk of developing severe forms of COVID-19 for GPX3 rs8177412 variant genotype (OR = 2.42, p = 0.032). This finding could be of particular importance in the future, complementing other diagnostic tools for prediction of COVID-19 disease course.

Expression of CYP2B6 Enzyme in Human Liver Tissue of HIV and HCV Patients.

Background and Objectives: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections present significant public health challenges worldwide. The management of these infections is complicated by the need for antiviral and antiretroviral therapies, which are influenced by drug metabolism mediated by metabolic enzymes and transporters. This study focuses on the gene expression of CYP2B6, CYP3A4, and ABCB1 transporters in patients with HIV, HCV, and HIV/HCV co-infection, aiming to assess their potential association with the choice of therapy, patohistological and clinical parameters of liver damage such as the stage of liver fibrosis, serum levels of ALT and AST, as well as the grade of liver inflammation and other available biochemical parameters. Materials and Methods: The study included 54 patients who underwent liver biopsy, divided into HIV-infected, HCV-infected, and co-infected groups. The mRNA levels of CYP2B6, CYP3A4, and ABCB1 was quantified and compared between the groups, along with the analysis of liver fibrosis and inflammation levels. Results: The results indicated a significant increase in CYP2B6 mRNA levels in co-infected patients, a significant association with the presence of HIV infection with an increase in CYP3A4 mRNA levels. A trend towards downregulation of ABCB1 expression was observed in patients using lamivudine. Conclusions: This study provides insight into gene expression of CYP2B6 CYP3A4, and ABCB1 in HIV, HCV, and HIV/HCV co-infected patients. The absence of correlation with liver damage, inflammation, and specific treatment interventions emphasises the need for additional research to elucidate the complex interplay between gene expression, viral co-infection, liver pathology, and therapeutic responses in these particular patients population.

Antioxidant Genetic Variants Modify Echocardiography Indices in Long COVID.

Although disturbance of redox homeostasis might be responsible for COVID-19 cardiac complications, this molecular mechanism has not been addressed yet. We have proposed modifying the effects of antioxidant proteins polymorphisms (superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPX1), glutathione peroxidase 3 (GPX3) and nuclear factor erythroid 2-related factor 2, (Nrf2)) in individual susceptibility towards the development of cardiac manifestations of long COVID-19. The presence of subclinical cardiac dysfunction was assessed via echocardiography and cardiac magnetic resonance imaging in 174 convalescent COVID-19 patients. SOD2, GPX1, GPX3 and Nrf2 polymorphisms were determined via the appropriate PCR methods. No significant association of the investigated polymorphisms with the risk of arrhythmia development was found. However, the carriers of variant GPX1*T, GPX3*C or Nrf2*A alleles were more than twice less prone for dyspnea development in comparison with the carriers of the referent ones. These findings were even more potentiated in the carriers of any two variant alleles of these genes (OR = 0.273, and p = 0.016). The variant GPX alleles were significantly associated with left atrial and right ventricular echocardiographic parameters, specifically LAVI, RFAC and RV-EF (p = 0.025, p = 0.009, and p = 0.007, respectively). Based on the relation between the variant SOD2*T allele and higher levels of LV echocardiographic parameters, EDD, LVMI and GLS, as well as troponin T (p = 0.038), it can be proposed that recovered COVID-19 patients, who are the carriers of this genetic variant, might have subtle left ventricular systolic dysfunction. No significant association between the investigated polymorphisms and cardiac disfunction was observed when cardiac magnetic resonance imaging was performed. Our results on the association between antioxidant genetic variants and long COVID cardiological manifestations highlight the involvement of genetic propensity in both acute and long COVID clinical manifestations.

Biologia Futura: does the aging process contribute to the relativity of time?

In his Theory of relativity, Einstein determined that the time is relative to the reference frame of the observer. Under specific conditions, there is a difference in the elapsed time between two clocks, known as time dilation. A similar relativistic effect could be attributed to the brain operating at different frequencies, e.g., while it is slow and during the thought process. Time flow and the aging process are causally linked. Herein, we introduce physical relativity into the mind/thought context and elaborate changed perception of the time flow (the impression of the time acceleration) with aging. The phenomenology of time is observed in the context of physical and biological clock, as well as by introducing the category of 'mind time.' Mental processing impairment is crucial for the "aging-caused relativity of time," while adjusting of its' perception seems to be a matter of body/mind rest, mental hygiene and physical activity of the aging subject. We also provide a brief overview of the perception of time flow in some disease states that coincide with aging. Our main idea has a perspective for future development in the interdisciplinary synergy of philosophy, physical-mathematical elaboration, experimental biology and clinical investigations.

Incidence and risk factors for suicide, death due to substance use, and violent/accidental death in people with HIV.

OBJECTIVE: Deaths due to suicide, substance use and violence/accident may reflect similar risk factors and overlap in their classification. This study aimed to investigate incidence and risk factors of mortality among people with HIV (PWH) due to these three related causes. DESIGN: Prospectively collected data from PWH at least 18 years old and under active follow-up in the EuroSIDA study from 2007 to 2019 were analysed. METHODS: Cause-specific Cox regression analysis was used to assess risk factors. RESULTS: A total of 17 881 participants were included, comprising 149 327 person-years of follow-up (PYFU). Forty participants died by suicide {incidence rate [IR] [95% confidence interval (CI)]: 0.3/1000 PYFU (0.2, 0.4)} 93 from substance use [IR (95% CI): 0.6/1000 PYFU (0.5, 0.8)], and 57 by violence/accident [IR (95% CI): 0.4/1000 PYFU (0.3, 0.5)]. An AIDS diagnosis within the last 12 months was associated with nine-fold increased risk of suicide vs. no history of AIDS [adjusted hazard ratio (aHR): 9.06; 95% CI: 2.07, 39.7]. Male gender was associated with double the risk of violent/accidental death (aHR: 2.28; 95% CI: 1.09, 4.78). PWH in Eastern Europe and those who acquired HIV by injection drug use (IDU) demonstrated a greater risk of death due to substance use or violence/accident. CONCLUSIONS: The association between a recent diagnosis of AIDS and suicide highlights a critical period for intervention. HIV infection acquired through IDU demonstrated an expected relationship with death due to substance use and violent/accidental deaths. Increased risk of death due to substance use and violence/accident in Eastern Europe demands investigation into specific differences that may drive that association.

Antioxidant Genetic Profile Modifies Probability of Developing Neurological Sequelae in Long-COVID.

Understanding the sequelae of COVID-19 is of utmost importance. Neuroinflammation and disturbed redox homeostasis are suggested as prevailing underlying mechanisms in neurological sequelae propagation in long-COVID. We aimed to investigate whether variations in antioxidant genetic profile might be associated with neurological sequelae in long-COVID. Neurological examination and antioxidant genetic profile (SOD2, GPXs and GSTs) determination, as well as, genotype analysis of Nrf2 and ACE2, were conducted on 167 COVID-19 patients. Polymorphisms were determined by the appropriate PCR methods. Only polymorphisms in GSTP1AB and GSTO1 were independently associated with long-COVID manifestations. Indeed, individuals carrying GSTP1 Val or GSTO1 Asp allele exhibited lower odds of long-COVID myalgia development, both independently and in combination. Furthermore, the combined presence of GSTP1 Ile and GSTO1 Ala alleles exhibited cumulative risk regarding long-COVID myalgia in carriers of the combined GPX1 LeuLeu/GPX3 CC genotype. Moreover, individuals carrying combined GSTM1-null/GPX1LeuLeu genotype were more prone to developing long-COVID "brain fog", while this probability further enlarged if the Nrf2 A allele was also present. The fact that certain genetic variants of antioxidant enzymes, independently or in combination, affect the probability of long-COVID manifestations, further emphasizes the involvement of genetic susceptibility when SARS-CoV-2 infection is initiated in the host cells, and also months after.

SOD2 rs4880 and GPX1 rs1050450 polymorphisms do not confer risk of COVID-19, but influence inflammation or coagulation parameters in Serbian cohort.

Objectives: Due to the role of oxidative stress in the pathophysiology of COVID-19, it is biologically plausible that inter-individual differences in patients' clinical manifestations might be affected by antioxidant genetic profile. The aim of our study was to assess the distribution of antioxidant genetic polymorphisms Nrf2 rs6721961, SOD2 rs4880, GPX1 rs1050450, GPX3 rs8177412, and GSTP1 (rs1695 and rs1138272) haplotype in COVID-19 patients and controls, with special emphasis on their association with laboratory biochemical parameters.Methods: The antioxidant genetic polymorphisms were assessed by appropriate PCR methods in 229 COVID-19 patients and 229 matched healthy individuals.Results: Among examined polymorphisms, only GSTP1 haplotype was associated with COVID-19 risk (p = 0.009). Polymorphisms of SOD2 and GPX1 influenced COVID-19 patients' laboratory biochemical profile: SOD2*Val allele was associated with increased levels of fibrinogen (p = 0.040) and ferritin (p = 0.033), whereas GPX1*Leu allele was associated with D-dimmer (p = 0.009).Discussion: Our findings regarding the influence of SOD2 and GPX1 polymorphisms on inflammation and coagulation parameters might be of clinical importance. If confirmed in larger cohorts, these developments could provide a more personalized approach for better recognition of patients prone to thrombosis and those for the need of targeted antiox-idant therapy.

GSTO1, GSTO2 and ACE2 Polymorphisms Modify Susceptibility to Developing COVID-19.

Based on the close relationship between dysregulation of redox homeostasis and immune response in SARS-CoV-2 infection, we proposed a possible modifying role of ACE2 and glutathione transferase omega (GSTO) polymorphisms in the individual propensity towards the development of clinical manifestations in COVID-19. The distribution of polymorphisms in ACE2 (rs4646116), GSTO1 (rs4925) and GSTO2 (rs156697) were assessed in 255 COVID-19 patients and 236 matched healthy individuals, emphasizing their individual and haplotype effects on disease development and severity. Polymorphisms were determined by the appropriate qPCR method. The data obtained showed that individuals carrying variant GSTO1*AA and variant GSTO2*GG genotypes exhibit higher odds of COVID-19 development, contrary to ones carrying referent alleles (p = 0.044, p = 0.002, respectively). These findings are confirmed by haplotype analysis. Carriers of H2 haplotype, comprising GSTO1*A and GSTO2*G variant alleles were at 2-fold increased risk of COVID-19 development (p = 0.002). Although ACE2 (rs4646116) polymorphism did not exhibit a statistically significant effect on COVID-19 risk (p = 0.100), the risk of COVID-19 development gradually increased with the presence of each additional risk-associated genotype. Further studies are needed to clarify the specific roles of glutathione transferases omega in innate immune response and vitamin C homeostasis once the SARS-CoV-2 infection is initiated in the host cell.

Helicobacter pylori infection rates in dyspeptic Serbian HIV-infected patients compared to HIV-negative controls.

Helicobacter pylori infection does not belong to the spectrum of opportunistic infections in people living with HIV (PLHIV). To evaluate the Helicobacter pylori infection prevalence rate trends in HIV co-infected individuals in comparison to the HIV-negative population, we compared histopathological findings of H. pylori positive gastritis (gastritis topography and histopathology) between 303 PLHIV and 2642 HIV-negative patients who underwent esophagogastroduodenoscopy (EGD) between 1993 and 2014 due to dyspeptic symptoms. The prevalence of H. pylori infection was significantly higher in HIV-negative controls than in PLHIV (50.2% vs. 28.1%). A significantly positive linear trend of H. pylori co-infection in PLHIV was revealed in the observed period (b = 0.030, SE = 0.011, p = 0.013), while this trend was significantly negative in HIV-negative patients (b = - 0.027, SE = 0.003, p < 0.001). Patients with HIV/H. pylori co-infection had significantly higher CD4+ T cell counts and more often had undetectable HIV viremia, due to successful anti-retroviral therapy (ART). Stomach histopathological findings differed between HIV co-infected and H. pylori mono-infected patients. Our findings confirm that the ART has changed the progression of HIV infection, leading to a significant increase in the prevalence of H. pylori infection in dyspeptic PLHIV over time. Our data also suggests that a functional immune system may be needed for H. pylori-induced human gastric mucosa inflammation.

GSTP1 and GSTM3 Variant Alleles Affect Susceptibility and Severity of COVID-19.

Based on the premise that oxidative stress plays an important role in severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection, we speculated that variations in the antioxidant activities of different members of the glutathione S-transferase family of enzymes might modulate individual susceptibility towards development of clinical manifestations in COVID-19. The distribution of polymorphisms in cytosolic glutathione S-transferases GSTA1, GSTM1, GSTM3, GSTP1 (rs1695 and rs1138272), and GSTT1 were assessed in 207 COVID-19 patients and 252 matched healthy individuals, emphasizing their individual and cumulative effect in disease development and severity. GST polymorphisms were determined by appropriate PCR methods. Among six GST polymorphisms analyzed in this study, GSTP1 rs1695 and GSTM3 were found to be associated with COVID-19. Indeed, the data obtained showed that individuals carrying variant GSTP1-Val allele exhibit lower odds of COVID-19 development (p = 0.002), contrary to carriers of variant GSTM3-CC genotype which have higher odds for COVID-19 (p = 0.024). Moreover, combined GSTP1 (rs1138272 and rs1695) and GSTM3 genotype exhibited cumulative risk regarding both COVID-19 occurrence and COVID-19 severity (p = 0.001 and p = 0.025, respectively). Further studies are needed to clarify the exact roles of specific glutathione S-transferases once the SARS-CoV-2 infection is initiated in the host cell.

Exploring Evolutionary and Transmission Dynamics of HIV Epidemic in Serbia: Bridging Socio-Demographic With Phylogenetic Approach.

Previous molecular studies of Serbian HIV epidemic identified the dominance of subtype B and presence of clusters related HIV-1 transmission, in particular among men who have sex with men (MSM). In order to get a deeper understanding of the complexities of HIV sub-epidemics in Serbia, epidemic trends, temporal origin and phylodynamic characteristics in general population and subpopulations were analyzed by means of mathematical modeling, phylogenetic analysis and latent class analysis (LCA). Fitting of the logistic curve of trends for a cumulative annual number of new HIV cases in 1984-2016, in general population and MSM transmission group, was performed. Both datasets fitted the logistic growth model, showing the early exponential phase of the growth curve. According to the suggested model, in the year 2030, the number of newly diagnosed HIV cases in Serbia will continue to grow, in particular in the MSM transmission group. Further, a detailed phylogenetic analysis was performed on 385 sequences from the period 1997-2015. Identification of transmission clusters, estimation of population growth (Ne), of the effective reproductive number (Re) and time of the most recent common ancestor (tMRCA) were estimated employing Bayesian and maximum likelihood methods. A substantial proportion of 53% of subtype B sequences was found within transmission clusters/network. Phylodynamic analysis revealed Re over one during the whole period investigated, with the steepest slopes and a recent tMRCA for MSM transmission group subtype B clades, in line with a growing trend in the number of transmissions in years approaching the end of the study period. Contrary, heterosexual clades in both studied subtypes - B and C - showed modest growth and stagnation. LCA analysis identified five latent classes, with transmission clusters dominantly present in 2/5 classes, linked to MSM transmission living in the capital city and with the high prevalence of co-infection with HBV and/or other STIs.Presented findings imply that HIV epidemic in Serbia is still in the exponential growth phase, in particular, related to the MSM transmission, with estimated steep growth curve until 2030. The obtained results imply that an average new HIV patient in Serbia is a young man with concomitant sexually transmitted infection.

Severe strongyloidiasis and systemic vasculitis: comorbidity, association or both? Case-based review.

A possible association between strongyloidiasis and systemic vasculitis is rarely reported in the literature. We report the case of a patient with severe strongyloidiasis and an angiographic finding consistent with polyarteritis nodosa. Diagnosis of strongyloidiasis was made by finding of larvae and adult parasites in samples of the upper gastrointestinal tract mucosa and stool. The patient was treated with albendazole, ivermectin and corticosteroid withdrawal. This therapy led to the resolution of symptoms, with repeated stool samples negative for S. stercoralis. However, the clinical course was complicated with pulmonary tuberculosis. Despite tuberculostatic therapy and supportive measures, a lethal outcome occurred. The report is followed by a focused review of the available literature on the association of strongyloidiasis and systemic vasculitis.

High Frequency of Human Leukocyte Antigen-B*57:01 Allele Carriers among HIV-Infected Patients in Serbia.

Abacavir is an effective antiretroviral drug and one of the most commonly used nucleoside reverse transcriptase inhibitors in Serbia. А percentage of the treated patients experience a potentially life-threatening hypersensitivity reaction, which was shown to be associated with the presence of the class I MHC allele, HLA-B*57:01; hence genotyping for HLA-B*57:01 prior to starting abacavir is nowadays recommended in international HIV treatment guidelines. In Serbia, this testing became available in 2013. This study was designed to estimate the prevalence of the HLA-B*57:01 allele in Serbian HIV-1-infected patients. The presence of the HLA-B*57:01 allele was analyzed in 273 HIV-1-infected patients aged 18 years or more, who were abacavir naïve. Buccal swab samples were obtained from all participants and assayed for the presence of HLA-B*57:01 using a commercially available HLA-B*57:01 real-time PCR kit. The presence of the HLA-B*57:01 allele was found in 22 of 273 tested individuals (8%; 95% CI 5.4-11.9%). This is the first study that estimated the HLA-B*57:01 prevalence among HIV-infected patients in Serbia. The very high prevalence of HLA-B*57:01 found in our study strongly supports HLA-B*57:01 genotyping, which should be implemented prior to the initiation of an abacavir-containing therapy to reduce the risk of potentially life-threatening hypersensitivity reactions.

Intravenous drug use - an independent predictor for HCV genotypes 3 and 4 infection among HIV/HCV co-infected patients.

INTRODUCTION: About one quarter of human immunodeficiency virus (HIV) infected persons in Serbia have also been found to be hepatitis C virus (HCV) co-infected. In the general population, HCV genotype 1 has been shown to be the most prevalent one. Here, we present the first study on the distribution of HCV genotypes among HIV/HCV co-infected patients in Serbia, in relation to epidemiological and clinical features. MATERIAL AND METHODS: The study included HIV/HCV co-infected and a group of HCV mono-infected patients in the period 1998-2012, with collection of epidemiological, clinical, and behavioral data using a standardized questionnaire. The HCV genotyping to the level of pure genotype was performed by reverse hybridization. RESULTS: Intravenous drug use (IDU) was found to be significantly more prevalent among the co-infected patients (p < 0.01). HCV genotype 1 was detected in 87% of patients with mono-infection, compared to 46.3% of patients with co-infection (p < 0.01); genotypes 3 and 4 were significantly more common among co-infected patients (6% and 5%, vs. 27% and 25%, respectively). Multivariate logistic regression confirmed IDU, infection with non-1 HCV genotype and HCV viral load over 5 log to be predictors of HIV co-infection. CONCLUSIONS: The HCV genotypes 3 and 4 were found to be significantly more prevalent among HIV/HCV co-infected patients in Serbia, compared to HCV mono-infected patients, but also more prevalent compared to the European HIV/HCV co-infected cohort. History of IDU represents an independent predictor of HCV genotypes 3 and 4 infection, with important implications for treatment.

Forensic application of phylogenetic analyses - Exploration of suspected HIV-1 transmission case.

Transmission of human immunodeficiency virus (HIV) between individuals may have important legal implications and therefore may come to require forensic investigation based upon phylogenetic analysis. In criminal trials results of phylogenetic analyses have been used as evidence of responsibility for HIV transmission. In Serbia, as in many countries worldwide, exposure and deliberate transmission of HIV are criminalized. We present the results of applying state of the art phylogenetic analyses, based on pol and env genetic sequences, in exploration of suspected HIV transmission among three subjects: a man and two women, with presumed assumption of transmission direction from one woman to a man. Phylogenetic methods included relevant neighbor-joining (NJ), maximum likelihood (ML) and Bayesian methods of phylogenetic trees reconstruction and hypothesis testing, that has been shown to be the most sensitive for the reconstruction of epidemiological links mostly from sexually infected individuals. End-point limiting-dilution PCR (EPLD-PCR) assay, generating the minimum of 10 sequences per genetic region per subject, was performed to assess HIV quasispecies distribution and to explore the direction of HIV transmission between three subjects. Phylogenetic analysis revealed that the viral sequences from the three subjects were more genetically related to each other than to other strains circulating in the same area with the similar epidemiological profile, forming strongly supported transmission chain, which could be in favour of a priori hypothesis of one of the women infecting the man. However, in the EPLD based phylogenetic trees for both pol and env genetic region, viral sequences of one subject (man) were paraphyletic to those of two other subjects (women), implying the direction of transmission opposite to the a priori assumption. The dated tree in our analysis confirmed the clustering pattern of query sequences. Still, in the context of unsampled sequences and inherent limitations of the applied methods, we cannot unambiguously prove that HIV-1 transmission occurred directly between two individuals. Further exploration of the known and suspected transmission cases is needed in order to define methodologies and establish their reliability.

HIV/AIDS mortality in a south east European country versus a west European country.

INTRODUCTION: Antiretroviral (ARV) treatment available in low-middle income countries differs as suggested in international HIV-treatment guidelines. Thus, we compared ARV regimens introduced as a first-line therapy, time of initiation, frequency of making combination antiretroviral therapy (cART) switches, frequency of viral and immunological monitoring and treatment outcome in south east European (SEE) country (i.e. HIV Centre in Belgrade, Serbia, (HCB)) and west European country (i.e. Royal Free Centre for HIV Medicine at the Royal Free Hospital London, UK (RFH)). MATERIALS AND METHODS: ARV naïve patients starting cART from 2003 to 2012 were included. Comparisons of the two cohorts were made using a chi-square test or Fisher's exact test for categorical variables and a Mann-Witney U test for continuous variables. Kaplan Meier survival curves were compared using the log rank test. RESULTS: Of 597 patients from HCB, 361 (61%) initiated cART with prior AIDS diagnosed, while 337 (19%) of 1763 patients from RFH. Average baseline CD4+ T cell counts were significantly lower in Serbia than in UK (177 cells/mm(3) vs 238 cells/mm(3)). The total (mediana, IQR) CD4+ T cell count measurements in the first year of cART was 2 (1, 2) at the HCB, while it was statistically significant higher at the RFH 5 (3, 7), respectively (p<0.0001). At the RFH, it appeared that the cART switching is due to patient's preference or toxicity (46%), while the lack of supply and toxicity (37%) were the most important reasons for treatment change in HCB, within the same period of time (p<0.05). Mortality rates were higher at the HCB versus RFH (p<0.0001). After 12, 24 and 36 months of cART, 3%, 5% and 8% of patients died in HCB, whereas 2%, 3% and 4% of patients died in RFH, respectively (Figure 1). CONCLUSIONS: In south European countries, as a consequence of low testing rate, ARV treatment is introduced at an advanced stage of disease, having a high mortality rate as a consequence. Switching within ARV drugs appears often due to lack of drug supplies and frequently drug-related toxicity in south east Europe, while in the east European country due to patient's preferences and rarely due to drug-related toxicity.

Ten years survey of primary HIV-1 resistance in Serbia: the occurrence of multiclass resistance.

In Serbia, the first cases of HIV infection were reported in 1985, whereas antiretroviral (ARV) therapy has been in use since 1987. With this study we aimed to assess the occurrence and pattern of HIV resistance mutations among newly diagnosed patients in the period 2002-2011. The study prospectively included 181 adult patients. Genotypic HIV-1 drug resistance testing was performed and drug resistance was scored according to the 2009 WHO list for surveillance of drug resistance mutations (SDRMs). A bioinformatic approach was used to estimate the duration of infection by calculating the percentage of ambiguous basecalls per sequence, with a cutoff of 0.47% as the delimiter for recent infection. The overall prevalence of transmitted drug resistance (TDR) found in the study was 8.8% (16/181, 95% CI=5.5-13.8). Thirty-one percent of resistant samples contained multiple SDRMs. In particular, 5/16 patients with resistance carried viral strains with SDRMs to multiple ARV classes, hence one-third of resistant strains were multiclass resistant, including non-B strains. A total of 51.9% of samples (94/181) were classified as recent infection, with a significant increase in the second part of the study period. However, the prevalence of TDR in recent infection was 6.4% (6/94, 95% CI=2.9-13.2), not statistically different from that found in nonrecent infection. We showed a changing pattern of TDR mutations over the study period, with a substantial occurrence of multiclass resistance, across different HIV subtypes. Our results highlight the need for continued surveillance of primary resistance.