Multivoxel magnetic resonance spectroscopy of the brain in HIV+ patients

UVOD: Neurokognitivni poremećaj udružen sa HIVinfekcijom (HIV associated neurocognitive disorder- HAND) javlja se u oko polovine pacijenata sa HIV-om. HAND obuhvata spektar neuroloških poremećaja koji variraju od asimptomatskog neurokognitivnog poremećaja(ANI), preko blagog kognitivnog oštećenja, koje se naziva blagi kognitivni poremećaj (MND) do demencije udružene sa HIV-om (HAD). U evaluaciji i dijagnostici ovog poremećaja koriste se razne laboratorijske, kliničke i metode imidžinga, među kojima magnetno-rezonantni imidžing (MRI) zauzima posebno mesto u pogledu dijagnostike strukturnih poremećaja. Međutim, za dijagnostiku suptilnih supcelularnih neurobiohemijskih poremećaja neophodno je korišćenje magnetno-rezonantne spektroskopije (MRS). Klasično shvatanje promena u neurobiohemijskom profilu kod pacijenata sa HIV infekcijom uključuje: sniženje NAA (neuronskog markera) kao odraz neurodegeneracije, povišenje Cho (markera razgradnje membrana) kao odraz inflamacije/ apoptoze, povišenje mI (markera proliferacije mikroglije) kao odraz inflamacije i povišenje Glx+Gln (markera glutaminergične ravnoteže) kao odraz ekscitotoksičnosti. Do sada u literaturi nije opisano korišćenje multivokselske MRS mozga na HIV+ pacijentima. CILJEVI: Ciljevi studije su bili: utvrditi da li postoje promene u odnosima koncentracija metabolita u mozgu dobijenih metodom multivokselske MRS u neurološki asimptomatskih HIV+ pacijenata u poređenju sa zdravim kontrolnim ispitanicima,zatim da li postoje razlike između pacijenata na kombinovanoj antiretroviralnoj terapiji (cART) i pacijenata bez cART; utvrditi da li postoji i kakva je povezanost odnosa metabolita dobijenih MRS sa imunološkim parametrima HIV+ pacijenata i nivoom nadir CD4+ T-limfocita; i konačno, utvrditi da li postoji i kakva je povezanost dobijenih odnosa metabolita sa parametrima penetracije antiretroviralne terapije u centralni nervni sistem. ISPITANICI I METODE: U studiju je uključeno ukupno 114 ispitanika (32 HIV+ pacijenta na cART, prosečne starosti 41.97 godina (25-61); 28 HIV+ pacijenata bez cART, prosečne starosti 35.21 godina (24-52); 50 kontrolnih, zdravih subjekata prosečne starosti 36.56.godina (19-53)). Svi ispitanici su potpisali informisani pristanak za učešće u studiji. Ispitivanje je odobreno od strane Etičke komisije Instituta za onkologiju Vojvodine, kao i Etičke komisije Medicinskog fakulteta Univerziteta u Novom Sadu. Kriterijum za uključenje pacijenata u studiju je bio prisustvo HIV infekcije. Kriterijumi za isključivanje pacijenata iz studije su bili: prisustvo aktivne oportunističke infekcije, prisustvo aktivnog neurološkog oboljenja, podatak o aktivnoj zloupotrebi narkotika, koinfekcija virusom hepatitisa B i C, prisustvo lezija bele ili sive mase i kontraindikacije za snimanje na aparatu za magnetnu rezonancu. 4 ispitanika su isključena iz ispitivanja. Svim ispitanicima urađeni su skrining neurokognitivni testovi, kao i rutinska laboratorijska ispitivanja (broj CD4+, CD3+ i CD8+ limfocita). Nakon toga, svim ispitanicima urađen je konvencionalni MRI praćen multivokselskom MRS supratentorijalne suprakalozalne bele mase. Ispitivano je ukupno 12 voksela (6 u sivoj i 6 u beloj masi), odnosno preko 7900 spektara. Određeni su pikovi karakterističnih metabolita. Na metodi dugog eha analizirana su tri glavna pika: NAA na 2.0ppm, Cho na 3.2ppm i tCr na 3.ppm, izražena preko odnosa koncentracija NAA/Cr i Cho/Cr. Na metodi kratkog eha analizirani su signali NAA, Cho, Cr te mI na 3.5ppm i Glx+Gln na 2.2-2.4ppm. Ovi signali su izraženi kroz odnose koncentracija NAA/Cr, Cho/Cr, mI/Cr i (Glx+Gln)/Cr. Za statističku obradu podataka korišćen je IBM SPSS ver. 21.0. Deskriptivna statistika je uključila određivanje srednje vrednosti, minimuma, maksimuma i standardne devijacije. Razlike između posmatranih grupa ispitanika za sve kontinuirane varijable ispitivane su jednofaktorskom analizom varijanse (ANOVA) sa naknadnim (post-hoc) testovima za koje je korišćena metoda po Tukey-ju. Rezultati su prikazani u vidu srednjih vrednosti, standardne devijacije, najviše i najniže izmerene vrednosti (maksimum i minimum), i za svaki ispitivani parametar pridružena je vrednost F i p. Veza između kontinuiranih varijabli je ispitivana pomoću koeficijenta Pirsonove linearne korelacije, uz prethodnu proveru zadovoljavanja uslova o homogenosti varijansi, normalnosti raspodele i linearnosti. Vrednosti p<0.05 su uzimane kao statistički značajne. REZULTATI: Pokazano je da su HIV+ pacijenti na cART značajno stariji od druge dve grupe ispitanika. Nije pokazana značajna razlika u stepenu obrazovanja među grupama. Pokazano je da godine života statistički značajno utiču samo na koncentracije NAA/Cr, dok na odnose drugih metabolita ne utiču značajno. Utvrđeno je statistički značajno sniženje (p<0.05) koncentracija NAA/Cr dobijenih metodom dugog eha između tri grupe ispitanika na svim posmatranim vokselima. Naknadnim analizama utvrđena je statistički značajna razlika na 10/12 voksela između HIV+ pacijenata sa cART i zdravih, kao i između HIV+ pacijenata bez terapije i zdravih, dok su se koncentracije NAA/Cr značajno razlikovale između HIV+ pacijenata sa i bez cART samo na jednom vokselu (duboka frontalna bela masa levo). Utvrđena je statistički značajna razlika u smislu sniženja Cho/Cr odnosa dobijenih metodom dugog eha u 5/12 voksela, sa pojedinačnim vokselima koji su prikazivali razlike između grupa. Na metodi kratkog eha utvrđeno je značajno sniženje odnosa koncentracija NAA/Cr kod HIV+ pacijenata samo na tri voksela, pri čemu nisu prikazane značajne razlike između dve grupe pacijenata sa HIV infekcijom (sa i bez cART). Rezultati odnosa koncentracija Cho/Cr između tri gurpe pacijenata dobijeni metodom kratkog eha slični su rezultatima dobijenim na metodi dugog eha (statistički značajna razlika dobijena je na 5/12 voksela). Što se tiče odnosa koncentracija mI/Cr, uočeno je značajno povišenje ovog odnosa kod HIV+ pacijenata u odnosu na zdrave na 6/12 voksela. Prikazano je statistički značajno povišenje ovog markera kod pacijenata bez cART u odnosu na pacijente sa cART samo u regiji levog dorzalnog anteriornog cinguluma. Statistički značajno povišenje (Glx+Gln)/Cr odnosa je prikazano u regiji zadnjeg cinguluma desno kod pacijenata na terapiji u odnosu na pacijente bez terapije, dok na drugim vokselima nije prikazana značajna razlika. Vokseli 4, 7 i 10 su dali najviše informacija(supkortikalna bela masa frontalno levo, dorzalni prednji cingulum levo te parijetalni supkorteks leve cerebralne hemisfere), sa prikazanim značajnim razlikama u bar 4 odnosa metabolita. Prikazana je značajna pozitivna korelacija nadir CD4 + broja limfocita sa koncentracijama NAA/Cr, a negativna sa odnosima Cho/Cr i mI/Cr, što čini nadir CD4+ najboljim serološkim prediktorom neurodegenerativnog oštećenja. Pokazana je pozitivna korelacija indeksa penetracije lekova u monocite (ME) sa odnosima NAA/Cr i negativna korelacija indeksa penetracije lekova u centralni nervni sistem (CPE) sa Cho/Cr i mI/Cr. Došli smo do zakljulka da je ME indeks bolji marker neurodegenerativnog odgovora a CPE indeks bolji u monitoringu kontrole inflamacije. ZAKLJUČAK: Smatra se da HIV virus uzrokuje prerano starenje mozgašto je prvenstveno posledica direktnog oštećenja nervnih ćelija samim virusom (preko viralnih proteina, indukovanih citokina i hemokina). Pokazali smo da su neuronski gubitak i neurodegeneracija proces koji zahvata celokupan volumen mozga, dok su procesi inflamacije i proliferacije mikroglije svedeni na tačno određene regione, pretežno sive mase. Visoka senzitivnost multivokselske 1H-MRS sa korišćenjem senzitivnih površinskih kalemova omogućava mapiranje metabolita sa prostornom rezolucijom. MRS može dati ključni uvid u promene koncentracija metabolita mozga tokom razvoja infekcije od akutne i primarne do hronične. Vrlo brzo nakon serokonverzije, dolazi do detektabilnih promena u metabolitima mozga u smislu neuronskog oštećenja i inflamacije. U našem istraživanju su po prvi put analizirani rezultati protonske multivokselske MRS bele i sive mase velikog mozga u suprakalozalnom regionu. Utvrđeno je da postoje difuzne, ali ipak visoko regionalno-zavisne promene u odnosima neurometabolita kod pacijenata koji dobijaju antiretroviralnu terapiju i kod pacijenata koji je ne dobijaju, u poređenju sa zdravim kontrolnim ispitanicima (odgovarajućim po polu i starosti). Dodatne studije sa posmatranjem apsolutnih koncentracija neurometabolita, kao i longitudinalne studije u koje su uključeni HIV+ pacijenti u različitim fazama bolesti, su neophodne za dalje i bolje razumevanje neuropatogeneze HAND-a. MRS se pokazala uspešnom u detekciji efikasnosti određenih terapijskih opcija. Dva postojeća indeksa za procenu efikasnosti antiretroviralne terapije (CPE, ME) odvojeno pogađaju dva puta neuropatogeneze kognitivnog poremećaja, sa različitim uspehom u sveobuhvatnoj proceni efekta i efikasnosti terapije. U budućnosti je potrebna njihova pojedinačna modulacija ili kreiranje jedinstvenog indeksa, koji bi obuhvatio i efikasnost prolaza leka kroz hematoencefalnu barijeru i dejstvo na latentni rezervoar HIV-a u ćelijama monocitno-makrofagne loze.INTRODUCTION: HIV associated neurocognitive disorder- HAND appears in about half of the HIV+ patients. HAND represents a spectrum of neurological disorders varying from asymptomatic neurocognitive impairment (ANI), over mild neurocognitive disorder (MND) to HIV associated dementia (HAD). For evaluation and diagnostics of this disorder, many laboratory, clinical and imaging methods are used, first of all magnetic resonance imaging (MRI). Nevertheless, for detecting subtle subcellullar neurobiochemical disorders, the use of magnetic resonance spectroscopy (MRS) is necessary. Classical pattern of neurobiochemical changes in HIV infection consist of: decrease in NAA (neuronal marker) depicting neurodegeneration, increase in Cho (metabolism on membrane marker) depicting inflammation/ apoptosis, increase in mI (marker of microglial proliferation) depicting inflammation and increase in Glx+Gln (glutaminergic balance marker) depicting the effect of excytotoxicity. To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV+ patients. AIMS: The aims of this study were: to show whether there are differences in metabolites' ratios on multivoxel MRS in neurologically asymptomatic HIV+ patients compared to control subjects; whether there are differences in metabolites' ratios between patients on combined antiretroviral therapy (cART) and therapy-naive ones; whether there are correlations between matebolites' ratios and immunological parameters in HIV+ patients as well as with nadir CD4+ count; whether there are correlations between metabolites' ratios with parameters of drugs' penetration in central nervous system (CNS). SUBJECTS AND METHODS: Overall of 114 subjects were enrolled in the study (32 HIV+ paients on cART, average age 41.97 years (25-61); 28 HIV+ patients off cART, average age 35.21 years (24-52); 50 control subjects, average age 36.56 years (19-53)). All the subjects signed the informed consent. The study was ethically approved by Ethical committee of Vojvodina Oncology Institute and Ethical committee of Faculty of Medicine, University of Novi Sad. Inclusion criteria for HIV+ subjects were: the presence of HIV infection. Exclusion criteria included: active opportunistic infection, active neurological illness, usage of drugs of abuse, hepatitis B or C coinfection, presence of both white or grey matter lesions, and contraindications that apply for magnetic resonance (MR) examination. 4 subjects were excluded from the study due to the presence of white matter lesions (3 HIV+ and one control subject). Each patient performed International HIV Dementia Scale (IHDS), a screening test for evaluation of global cognitive status in HIV-infected patients. Baseline study laboratory variables were assessed (CD4+ T-lymphocyte count and plasma HIV RNA, nadir CD4+ counts and CD4+ T-cell counts at the moment of MR scan. Conventional MRI scan was followed by multivoxel MRS with both long and short echo. We analyzed 12 voxels (6 in grey and 6 in white matter) with overall of over 7900 spectra. Finally, we analyzed following dominant signals: on the long echo tCr (creatine plus phosphocreatine) at 3.0 ppm, NAA (N-acetyl-aspartate) at 2.0 ppm and Cho (choline containing compounds) at 3.2ppm (ratios of NAA/Cr and Cho/Cr were assessed); on the short echo tCr, NAA, Cho, (Glx+Gln) at 2.2-2.4ppm and mI (myoinositol) at 3.5ppm (ratios of NAA/Cr, Cho/Cr, (Glx+Gln)/Cr and mI/Cr were assessed. All statistical calculations were performed using IBM SPSS software (version 21.0, Chicago, IL, USA). Descriptive statistics included determination of mean values, minimum, maximum and standard deviation. Among-group differences (HIV infected subjects versus healthy controls) in acquired metabolite ratios were evaluated using ANOVA with post hoc Tukey test to determine the differences between separate groups. Due to a known impact of age and education on the NAA concentrations, differences in NAA/Cr ratios among groups were tested using ANCOVA, with age as a covariate variable. Testing relationships between continuous variables was performed using Pearson linear correlation. Statistical significance was set at value p<0.05. RESULTS: We showed that HIV+ patients on therapy were significantly older than the other two groups of patients. There was no significant difference in the level of education. We confirmed that the age significantly affects the level of NAA/Cr only.There was significant decrease (p<0.05) in NAA/Cr level on long echo MRS among three groups on all the observed voxels. Post hoc analysis showed that there was significant difference in 10/12 voxels between HIV+ patients on cART and healthy controls and between HIV+ patients off cART and controls, while NAA/Cr differed significantly between HIV+ patients on and off cART in only one voxel (deep frontal white matter on the left). There was decrease in Cho/Cr levels on long echo MRS in 5/12 voxels among three groups. On short echo MRS, we showed decrease in NAA/Cr level in 3/12 voxels, while there were no differences between two groups of HIV+ patients. Results of short echo MRS in the means of Cho/Cr resembled long echo MRS. There was significant increase in mI/Cr level in HIV+ patients in 6/12 voxels compared to healthy controls, while there was difference in only one voxel between HIV+ patients on and off therapy (dorsal part of anterior cingulate on the left). Significant increase in (Glx+Gln)/Cr level was present between HIV+ patients on and off therapy in the region of right posterior cingulate. Voxels 4, 7 and 10 were the most informative ones (subcortical frontal white matter on the left, dorsal part of left anterior cingulate and right posterior cingulate), showing significant differences in 4 metabolites' ratios. We showed positive correlation between nadir CD4+ count and NAA/Cr and negative correlation between nadir CD4+ count and Cho/Cr, and nadir CD4+ count and mI/Cr, which made nadir CD4+ count the best serological predictor of neurodegeneration. Positive correlation was showed between monocyte efficacy (ME) index and NAA/Cr, while negative correlation was present between CNS penetration efficacy (CPE), Cho/Cr and mI/Cr. We concluded that ME better depicted neurodegenerative process while CPE was better in monitoring of inflammation. CONCLUSIONS: HIV causes premature ageing of the brain, in the means of cognition, attention, working memory and executive function. These effects are due to direct affection of neurons by virus per se (viral proteins, induced cytokines and chemokynes). We showed tha neuronal loss and neurodegeneration affect the whole volume of the brain while inflammation and glial proliferation affect restricted areas predominantly in grey matter. High sensitivity of multivoxel MRS with use of sensitive surface coils enables metabolite mapping with high spatial resolution. MRS can give essential data on metabolites' changes during the evolution of the infection from acute, over primary to chronic. Early after seroconversion, metabolites' changes can be detected (neuronal dysfunction and inflammation).To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV infection in human population, analyzing data from supracallosal grey and white matter. We showed the presence of diffuse but regionally highly specific changes in metabolites' ratios in patients on cART and off cART, compared to age and gender matched healthy controls. Additional studies with absolute concentrations of metabolites, as well as longitudinal studies with HIV+ patients in different stages of the disease, are necessary for better understanding of neuropathogenesis of HAND. We showed that MRS can be useful tool in evaluation of therapy regimens efficacy. Two available indices for evaluation of cART efficacy target two separate pathways of cognitive disorder pathogenesis, with different reliability in evaluation of effect and efficacy of applied therapy. In the future, their modulation or creation of new index is needed, in order to include drug delivery through the blood-brain barrier as well as the effect on latent reservoir of HIV in monocyte/macrophage cells

Multivoxel magnetic resonance spectroscopy of the brain in HIV+ patients

UVOD: Neurokognitivni poremećaj udružen sa HIVinfekcijom (HIV associated neurocognitive disorder- HAND) javlja se u oko polovine pacijenata sa HIV-om. HAND obuhvata spektar neuroloških poremećaja koji variraju od asimptomatskog neurokognitivnog poremećaja(ANI), preko blagog kognitivnog oštećenja, koje se naziva blagi kognitivni poremećaj (MND) do demencije udružene sa HIV-om (HAD). U evaluaciji i dijagnostici ovog poremećaja koriste se razne laboratorijske, kliničke i metode imidžinga, među kojima magnetno-rezonantni imidžing (MRI) zauzima posebno mesto u pogledu dijagnostike strukturnih poremećaja. Međutim, za dijagnostiku suptilnih supcelularnih neurobiohemijskih poremećaja neophodno je korišćenje magnetno-rezonantne spektroskopije (MRS). Klasično shvatanje promena u neurobiohemijskom profilu kod pacijenata sa HIV infekcijom uključuje: sniženje NAA (neuronskog markera) kao odraz neurodegeneracije, povišenje Cho (markera razgradnje membrana) kao odraz inflamacije/ apoptoze, povišenje mI (markera proliferacije mikroglije) kao odraz inflamacije i povišenje Glx+Gln (markera glutaminergične ravnoteže) kao odraz ekscitotoksičnosti. Do sada u literaturi nije opisano korišćenje multivokselske MRS mozga na HIV+ pacijentima. CILJEVI: Ciljevi studije su bili: utvrditi da li postoje promene u odnosima koncentracija metabolita u mozgu dobijenih metodom multivokselske MRS u neurološki asimptomatskih HIV+ pacijenata u poređenju sa zdravim kontrolnim ispitanicima,zatim da li postoje razlike između pacijenata na kombinovanoj antiretroviralnoj terapiji (cART) i pacijenata bez cART; utvrditi da li postoji i kakva je povezanost odnosa metabolita dobijenih MRS sa imunološkim parametrima HIV+ pacijenata i nivoom nadir CD4+ T-limfocita; i konačno, utvrditi da li postoji i kakva je povezanost dobijenih odnosa metabolita sa parametrima penetracije antiretroviralne terapije u centralni nervni sistem. ISPITANICI I METODE: U studiju je uključeno ukupno 114 ispitanika (32 HIV+ pacijenta na cART, prosečne starosti 41.97 godina (25-61); 28 HIV+ pacijenata bez cART, prosečne starosti 35.21 godina (24-52); 50 kontrolnih, zdravih subjekata prosečne starosti 36.56.godina (19-53)). Svi ispitanici su potpisali informisani pristanak za učešće u studiji. Ispitivanje je odobreno od strane Etičke komisije Instituta za onkologiju Vojvodine, kao i Etičke komisije Medicinskog fakulteta Univerziteta u Novom Sadu. Kriterijum za uključenje pacijenata u studiju je bio prisustvo HIV infekcije. Kriterijumi za isključivanje pacijenata iz studije su bili: prisustvo aktivne oportunističke infekcije, prisustvo aktivnog neurološkog oboljenja, podatak o aktivnoj zloupotrebi narkotika, koinfekcija virusom hepatitisa B i C, prisustvo lezija bele ili sive mase i kontraindikacije za snimanje na aparatu za magnetnu rezonancu. 4 ispitanika su isključena iz ispitivanja. Svim ispitanicima urađeni su skrining neurokognitivni testovi, kao i rutinska laboratorijska ispitivanja (broj CD4+, CD3+ i CD8+ limfocita). Nakon toga, svim ispitanicima urađen je konvencionalni MRI praćen multivokselskom MRS supratentorijalne suprakalozalne bele mase. Ispitivano je ukupno 12 voksela (6 u sivoj i 6 u beloj masi), odnosno preko 7900 spektara. Određeni su pikovi karakterističnih metabolita. Na metodi dugog eha analizirana su tri glavna pika: NAA na 2.0ppm, Cho na 3.2ppm i tCr na 3.ppm, izražena preko odnosa koncentracija NAA/Cr i Cho/Cr. Na metodi kratkog eha analizirani su signali NAA, Cho, Cr te mI na 3.5ppm i Glx+Gln na 2.2-2.4ppm. Ovi signali su izraženi kroz odnose koncentracija NAA/Cr, Cho/Cr, mI/Cr i (Glx+Gln)/Cr. Za statističku obradu podataka korišćen je IBM SPSS ver. 21.0. Deskriptivna statistika je uključila određivanje srednje vrednosti, minimuma, maksimuma i standardne devijacije. Razlike između posmatranih grupa ispitanika za sve kontinuirane varijable ispitivane su jednofaktorskom analizom varijanse (ANOVA) sa naknadnim (post-hoc) testovima za koje je korišćena metoda po Tukey-ju. Rezultati su prikazani u vidu srednjih vrednosti, standardne devijacije, najviše i najniže izmerene vrednosti (maksimum i minimum), i za svaki ispitivani parametar pridružena je vrednost F i p. Veza između kontinuiranih varijabli je ispitivana pomoću koeficijenta Pirsonove linearne korelacije, uz prethodnu proveru zadovoljavanja uslova o homogenosti varijansi, normalnosti raspodele i linearnosti. Vrednosti p<0.05 su uzimane kao statistički značajne. REZULTATI: Pokazano je da su HIV+ pacijenti na cART značajno stariji od druge dve grupe ispitanika. Nije pokazana značajna razlika u stepenu obrazovanja među grupama. Pokazano je da godine života statistički značajno utiču samo na koncentracije NAA/Cr, dok na odnose drugih metabolita ne utiču značajno. Utvrđeno je statistički značajno sniženje (p<0.05) koncentracija NAA/Cr dobijenih metodom dugog eha između tri grupe ispitanika na svim posmatranim vokselima. Naknadnim analizama utvrđena je statistički značajna razlika na 10/12 voksela između HIV+ pacijenata sa cART i zdravih, kao i između HIV+ pacijenata bez terapije i zdravih, dok su se koncentracije NAA/Cr značajno razlikovale između HIV+ pacijenata sa i bez cART samo na jednom vokselu (duboka frontalna bela masa levo). Utvrđena je statistički značajna razlika u smislu sniženja Cho/Cr odnosa dobijenih metodom dugog eha u 5/12 voksela, sa pojedinačnim vokselima koji su prikazivali razlike između grupa. Na metodi kratkog eha utvrđeno je značajno sniženje odnosa koncentracija NAA/Cr kod HIV+ pacijenata samo na tri voksela, pri čemu nisu prikazane značajne razlike između dve grupe pacijenata sa HIV infekcijom (sa i bez cART). Rezultati odnosa koncentracija Cho/Cr između tri gurpe pacijenata dobijeni metodom kratkog eha slični su rezultatima dobijenim na metodi dugog eha (statistički značajna razlika dobijena je na 5/12 voksela). Što se tiče odnosa koncentracija mI/Cr, uočeno je značajno povišenje ovog odnosa kod HIV+ pacijenata u odnosu na zdrave na 6/12 voksela. Prikazano je statistički značajno povišenje ovog markera kod pacijenata bez cART u odnosu na pacijente sa cART samo u regiji levog dorzalnog anteriornog cinguluma. Statistički značajno povišenje (Glx+Gln)/Cr odnosa je prikazano u regiji zadnjeg cinguluma desno kod pacijenata na terapiji u odnosu na pacijente bez terapije, dok na drugim vokselima nije prikazana značajna razlika. Vokseli 4, 7 i 10 su dali najviše informacija(supkortikalna bela masa frontalno levo, dorzalni prednji cingulum levo te parijetalni supkorteks leve cerebralne hemisfere), sa prikazanim značajnim razlikama u bar 4 odnosa metabolita. Prikazana je značajna pozitivna korelacija nadir CD4 + broja limfocita sa koncentracijama NAA/Cr, a negativna sa odnosima Cho/Cr i mI/Cr, što čini nadir CD4+ najboljim serološkim prediktorom neurodegenerativnog oštećenja. Pokazana je pozitivna korelacija indeksa penetracije lekova u monocite (ME) sa odnosima NAA/Cr i negativna korelacija indeksa penetracije lekova u centralni nervni sistem (CPE) sa Cho/Cr i mI/Cr. Došli smo do zakljulka da je ME indeks bolji marker neurodegenerativnog odgovora a CPE indeks bolji u monitoringu kontrole inflamacije. ZAKLJUČAK: Smatra se da HIV virus uzrokuje prerano starenje mozgašto je prvenstveno posledica direktnog oštećenja nervnih ćelija samim virusom (preko viralnih proteina, indukovanih citokina i hemokina). Pokazali smo da su neuronski gubitak i neurodegeneracija proces koji zahvata celokupan volumen mozga, dok su procesi inflamacije i proliferacije mikroglije svedeni na tačno određene regione, pretežno sive mase. Visoka senzitivnost multivokselske 1H-MRS sa korišćenjem senzitivnih površinskih kalemova omogućava mapiranje metabolita sa prostornom rezolucijom. MRS može dati ključni uvid u promene koncentracija metabolita mozga tokom razvoja infekcije od akutne i primarne do hronične. Vrlo brzo nakon serokonverzije, dolazi do detektabilnih promena u metabolitima mozga u smislu neuronskog oštećenja i inflamacije. U našem istraživanju su po prvi put analizirani rezultati protonske multivokselske MRS bele i sive mase velikog mozga u suprakalozalnom regionu. Utvrđeno je da postoje difuzne, ali ipak visoko regionalno-zavisne promene u odnosima neurometabolita kod pacijenata koji dobijaju antiretroviralnu terapiju i kod pacijenata koji je ne dobijaju, u poređenju sa zdravim kontrolnim ispitanicima (odgovarajućim po polu i starosti). Dodatne studije sa posmatranjem apsolutnih koncentracija neurometabolita, kao i longitudinalne studije u koje su uključeni HIV+ pacijenti u različitim fazama bolesti, su neophodne za dalje i bolje razumevanje neuropatogeneze HAND-a. MRS se pokazala uspešnom u detekciji efikasnosti određenih terapijskih opcija. Dva postojeća indeksa za procenu efikasnosti antiretroviralne terapije (CPE, ME) odvojeno pogađaju dva puta neuropatogeneze kognitivnog poremećaja, sa različitim uspehom u sveobuhvatnoj proceni efekta i efikasnosti terapije. U budućnosti je potrebna njihova pojedinačna modulacija ili kreiranje jedinstvenog indeksa, koji bi obuhvatio i efikasnost prolaza leka kroz hematoencefalnu barijeru i dejstvo na latentni rezervoar HIV-a u ćelijama monocitno-makrofagne loze.INTRODUCTION: HIV associated neurocognitive disorder- HAND appears in about half of the HIV+ patients. HAND represents a spectrum of neurological disorders varying from asymptomatic neurocognitive impairment (ANI), over mild neurocognitive disorder (MND) to HIV associated dementia (HAD). For evaluation and diagnostics of this disorder, many laboratory, clinical and imaging methods are used, first of all magnetic resonance imaging (MRI). Nevertheless, for detecting subtle subcellullar neurobiochemical disorders, the use of magnetic resonance spectroscopy (MRS) is necessary. Classical pattern of neurobiochemical changes in HIV infection consist of: decrease in NAA (neuronal marker) depicting neurodegeneration, increase in Cho (metabolism on membrane marker) depicting inflammation/ apoptosis, increase in mI (marker of microglial proliferation) depicting inflammation and increase in Glx+Gln (glutaminergic balance marker) depicting the effect of excytotoxicity. To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV+ patients. AIMS: The aims of this study were: to show whether there are differences in metabolites' ratios on multivoxel MRS in neurologically asymptomatic HIV+ patients compared to control subjects; whether there are differences in metabolites' ratios between patients on combined antiretroviral therapy (cART) and therapy-naive ones; whether there are correlations between matebolites' ratios and immunological parameters in HIV+ patients as well as with nadir CD4+ count; whether there are correlations between metabolites' ratios with parameters of drugs' penetration in central nervous system (CNS). SUBJECTS AND METHODS: Overall of 114 subjects were enrolled in the study (32 HIV+ paients on cART, average age 41.97 years (25-61); 28 HIV+ patients off cART, average age 35.21 years (24-52); 50 control subjects, average age 36.56 years (19-53)). All the subjects signed the informed consent. The study was ethically approved by Ethical committee of Vojvodina Oncology Institute and Ethical committee of Faculty of Medicine, University of Novi Sad. Inclusion criteria for HIV+ subjects were: the presence of HIV infection. Exclusion criteria included: active opportunistic infection, active neurological illness, usage of drugs of abuse, hepatitis B or C coinfection, presence of both white or grey matter lesions, and contraindications that apply for magnetic resonance (MR) examination. 4 subjects were excluded from the study due to the presence of white matter lesions (3 HIV+ and one control subject). Each patient performed International HIV Dementia Scale (IHDS), a screening test for evaluation of global cognitive status in HIV-infected patients. Baseline study laboratory variables were assessed (CD4+ T-lymphocyte count and plasma HIV RNA, nadir CD4+ counts and CD4+ T-cell counts at the moment of MR scan. Conventional MRI scan was followed by multivoxel MRS with both long and short echo. We analyzed 12 voxels (6 in grey and 6 in white matter) with overall of over 7900 spectra. Finally, we analyzed following dominant signals: on the long echo tCr (creatine plus phosphocreatine) at 3.0 ppm, NAA (N-acetyl-aspartate) at 2.0 ppm and Cho (choline containing compounds) at 3.2ppm (ratios of NAA/Cr and Cho/Cr were assessed); on the short echo tCr, NAA, Cho, (Glx+Gln) at 2.2-2.4ppm and mI (myoinositol) at 3.5ppm (ratios of NAA/Cr, Cho/Cr, (Glx+Gln)/Cr and mI/Cr were assessed. All statistical calculations were performed using IBM SPSS software (version 21.0, Chicago, IL, USA). Descriptive statistics included determination of mean values, minimum, maximum and standard deviation. Among-group differences (HIV infected subjects versus healthy controls) in acquired metabolite ratios were evaluated using ANOVA with post hoc Tukey test to determine the differences between separate groups. Due to a known impact of age and education on the NAA concentrations, differences in NAA/Cr ratios among groups were tested using ANCOVA, with age as a covariate variable. Testing relationships between continuous variables was performed using Pearson linear correlation. Statistical significance was set at value p<0.05. RESULTS: We showed that HIV+ patients on therapy were significantly older than the other two groups of patients. There was no significant difference in the level of education. We confirmed that the age significantly affects the level of NAA/Cr only.There was significant decrease (p<0.05) in NAA/Cr level on long echo MRS among three groups on all the observed voxels. Post hoc analysis showed that there was significant difference in 10/12 voxels between HIV+ patients on cART and healthy controls and between HIV+ patients off cART and controls, while NAA/Cr differed significantly between HIV+ patients on and off cART in only one voxel (deep frontal white matter on the left). There was decrease in Cho/Cr levels on long echo MRS in 5/12 voxels among three groups. On short echo MRS, we showed decrease in NAA/Cr level in 3/12 voxels, while there were no differences between two groups of HIV+ patients. Results of short echo MRS in the means of Cho/Cr resembled long echo MRS. There was significant increase in mI/Cr level in HIV+ patients in 6/12 voxels compared to healthy controls, while there was difference in only one voxel between HIV+ patients on and off therapy (dorsal part of anterior cingulate on the left). Significant increase in (Glx+Gln)/Cr level was present between HIV+ patients on and off therapy in the region of right posterior cingulate. Voxels 4, 7 and 10 were the most informative ones (subcortical frontal white matter on the left, dorsal part of left anterior cingulate and right posterior cingulate), showing significant differences in 4 metabolites' ratios. We showed positive correlation between nadir CD4+ count and NAA/Cr and negative correlation between nadir CD4+ count and Cho/Cr, and nadir CD4+ count and mI/Cr, which made nadir CD4+ count the best serological predictor of neurodegeneration. Positive correlation was showed between monocyte efficacy (ME) index and NAA/Cr, while negative correlation was present between CNS penetration efficacy (CPE), Cho/Cr and mI/Cr. We concluded that ME better depicted neurodegenerative process while CPE was better in monitoring of inflammation. CONCLUSIONS: HIV causes premature ageing of the brain, in the means of cognition, attention, working memory and executive function. These effects are due to direct affection of neurons by virus per se (viral proteins, induced cytokines and chemokynes). We showed tha neuronal loss and neurodegeneration affect the whole volume of the brain while inflammation and glial proliferation affect restricted areas predominantly in grey matter. High sensitivity of multivoxel MRS with use of sensitive surface coils enables metabolite mapping with high spatial resolution. MRS can give essential data on metabolites' changes during the evolution of the infection from acute, over primary to chronic. Early after seroconversion, metabolites' changes can be detected (neuronal dysfunction and inflammation).To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV infection in human population, analyzing data from supracallosal grey and white matter. We showed the presence of diffuse but regionally highly specific changes in metabolites' ratios in patients on cART and off cART, compared to age and gender matched healthy controls. Additional studies with absolute concentrations of metabolites, as well as longitudinal studies with HIV+ patients in different stages of the disease, are necessary for better understanding of neuropathogenesis of HAND. We showed that MRS can be useful tool in evaluation of therapy regimens efficacy. Two available indices for evaluation of cART efficacy target two separate pathways of cognitive disorder pathogenesis, with different reliability in evaluation of effect and efficacy of applied therapy. In the future, their modulation or creation of new index is needed, in order to include drug delivery through the blood-brain barrier as well as the effect on latent reservoir of HIV in monocyte/macrophage cells

Metod topografske inventarizacije i GPS tehnologija u modelovanju geoprostora

By the use of digital cartographic visualization, a wide range of relevant data can be systematically integrated, presented and analyzed. In particular, this refers to the connection between thematic and topographic maps and their application in the study of the environment. The specific purpose of the map implies that in certain situations there is no clear difference between the thematic and topographic maps, that is, there is no clear boundary between the thematic and the topographic content of the representation. The paper points to the importance of modeling geoenvironmental data (topographic and thematic) for different scientific and practical research needs. Bearing in mind the global size of orienteering, the heterogeneity of the geoenvironment in which competitions and ways of orientation are organized, it is necessary to standardize this topic at the international level. The conducted experiment of cartographic modeling in the orienteering function has examined the possibilities of application of topographic inventory methods of geoenvironment for this purpose, the importance of using GPS devices for collecting geoenvironmental data and CAD software for cartographic visualization of the issues in question.Digitalnom kartografskom vizuelizacijom širok spektar relevantnih podataka može se sistemski integrisati, predstaviti i analizirati. Posebno se to odnosi na vezu tematskih i topografskih karata i njihovu primenu pri izučavanju geoprostora. Specifična namena karte uslovljava da u određenim situacijama nema jasne razlike između tematske i topografske karte, odnosno, nema jasne granice između tematskog i topografskog sadržaja prikaza. U radu se ukazuje na značaj modelovanja geoprostornih podataka (topografskih i tematskih) za različite naučne i praktične potrebe istraživanja. Imajući u vidu globalne razmere orijentiringa, heterogenost geoprostora na kome se organizuju takmičenja i načine orijentisanja, neophodno je standardizovati ovu tematiku na međunarodnom nivou. Sprovedenim eksperimentom kartografskog modelovanja u funkciji orijentiringa, sagledane su mogućnosti primene metoda topografske inventarizacije geoprostora za ovu namenu, značaj korišćenja GPS uređaja za prikupljanje geoprostorih podataka i CAD softvera za kartografsku vizuelizaciju tretirane tematike

Significance of proper oral hygiene for health condition of mouth and teeth

Background/Aim. Proper mouth and teeth hygiene has influence on the prevention of a great number of diseases and very often some of them are not related only to oral cavity. Most frequent diseases of mouth and teeth such as caries and periodontal diseases are caused, among other factors, by poor oral hygiene. They are also more frequent in young population. The aim of this study was to estimate the influence of sociodemographic characteristics and hygienic habit on oral health in children aged 11-15 years. Methods. This crosssectional study was conducted by the dental teams in dentist surgeries in Tivat Health Center between May and September 2009. The sample consisted of patients 11 to 15 years of age. A questionnaire and dental examination of mouth and teeth were used as research instruments. The examinations were conducted in accordance with the World Health Organization methodology and criteria. The data obtained from the interviews were correlated with those obtained from the clinical examinations. Results. The results show that the majority of respondents brush their teeth twice a day and visit the dentist once in every six months. The research also shows that 57% respondents have caries of deciduous teeth and over 63% respondents of permanent teeth. Gingivitis was found in 14% and orthodontic anomalies in 44.7% respondents. A half of respondents who brush their teeth rarely have problems with gingivitis. There is a highly statistically significant difference between the occurrence of gingivitis and the frequency of teeth brushing. Conclusion. There is a significant difference between mouth and oral hygiene and sex as well as other sociodemographic characteristics of respondents. The study showed the correlation between occurrence of caries and the gingivitis and frequency of teeth brushing and dental visits

Zastupljenost pušenja i pušačke navike učenika dobi 13 do 15 godina u Crnoj Gori: rezultati globalnog istraživanja o potrošnji duhana kod mladih (GYTS) 2008.

The Global Youth Tobacco Survey (GYTS) is an international study that provides data on youth tobacco use for development of tobacco control programs. It is a school-based survey that uses a standardised methodology for sampling, core questionnaire items, training protocol, fi eld procedures, and data management. This article reports the fi ndings from a GYTS conducted in Montenegro in 2008, which included 5723 adolescents. More than 30 % of students aged 13 to 15 tried smoking, 5.1 % smoked cigarettes, and 3.6 % of students used tobacco products other than cigarettes. Four in 10 ever smokers started to smoke before the age of 10. More than half the students reported secondary smoke exposure at home. Almost all (96.5 %) current smokers bought cigarettes in a store. Two in 10 students owned an artifact with a cigarette or tobacco brand logo on it. The GYTS study has shown that there is an urgent need to introduce effective child-oriented smoking prevention programmes in early elementary school classes. These should be accompanied by public awareness campaigns on smoke-free homes.Globalno istraživanje o potrošnji duhana kod mladih (GYTS) međunarodna je studija koja pruža podatke o pušačkim navikama mladih i omogućava razvijanje programa čiji je cilj kontrola duhana. U ovom radu dati su rezultati istraživanja GYTS koje je provedeno 2008. godine u Crnoj Gori. Istraživanje GYTS provodi se u školama pri čemu je standardizirana metodologija za formiranje uzorka, upitnik, prikupljanje i obrada podataka. Ukupno su 5723 učenika sudjelovala u istraživanju. Više od 30 % učenika dobi 13 do 15 godina eksperimentiralo je cigaretama. U ovoj populaciji ima 5.1 % stalnih pušača cigareta, dok 3.6 % učenika koristi neke druge duhanske proizvode. Četiri od 10 pušača počela su pušiti prije desete godine života. Više od pola ispitanika odgovorilo je da su izloženi duhanskom dimu u svojoj kući. Gotovo svi pušači (96.5 %) najčešće kupuju cigarete u prodavaonicama. Dva od 10 ispitanika imaju neki predmet s logom proizvođača cigareta. Rezultati istraživanja pokazuju da je potrebno hitno pokrenuti preventivne programe kontrole duhana u nižim razredima osnovne škole. Također, potrebno je osmisliti i implementirati kampanje čiji je cilj podizanje svijesti građana o štetnosti pušenja u zatvorenom prostoru

Distillation products of Stanley plum obtaining

The sort of plum used for distillation is Stanley picked in August 2013 in Gračac, Vrnjačka Banja, Serbia. 200 kg of ripe fruit was fermented with 40 dm3 of potable water. Wine was removed from the obtained product, and the solid part was washed with water. The mixture of wine and water, 240 dm was distilled to a soft brandy, where 36 dm3 was obtained with 30 % of ethanol. After that, this soft brandy was redistilled after 44 dm3 of potable water was added. By this procedure, 20 dm3 with 50 % of ethanol was distillated, which means that the yield was 10 dm3 of brandy for 100 kg of plums

Distillation products of Stanley plum obtaining

The sort of plum used for distillation is Stanley picked in August 2013 in Gračac, Vrnjačka Banja, Serbia. 200 kg of ripe fruit was fermented with 40 dm3 of potable water. Wine was removed from the obtained product, and the solid part was washed with water. The mixture of wine and water, 240 dm was distilled to a soft brandy, where 36 dm3 was obtained with 30 % of ethanol. After that, this soft brandy was redistilled after 44 dm3 of potable water was added. By this procedure, 20 dm3 with 50 % of ethanol was distillated, which means that the yield was 10 dm3 of brandy for 100 kg of plums

Zadovoljstvo korisnika uslugama u reformiranom sustavu primarne zdravstvene zaštite u Crnoj Gori

Cilj: Istraživanje je provedeno s ciljem otkrivanja zadovoljstva korisnika pruženim uslugama u sustavu primarne zdravstvene zaštite.Metoda: Uzorak istraživanja činili su svi pacijenti koji tijekom istraživanja (u prva tri mjeseca 2009. godine) posjetili Dom zdravlja Tivat, radi zadovoljenja svojih zdravstvenih potreba. Instrument istraživanja bio je upitnik, sastavljen od 30 pitanja kombiniranog tipa, usmjerenih na otkrivanje subjektivnog zadovoljstva korisnika. Za obradu rezultata korištene su metode deskriptivne statistike.Rezultat: Istraživanjem je obuhvaćeno 340 korisnika, od čega je oko 62% bilo žena. Rezultati istraživanja pokazuju da su korisnici vrlo zadovoljni pruženim uslugama u smislu odnosa zdravstvenih radnika prema korisnicima, osjećaja povjerenja prema izabranome liječniku, obrazloženja o poduzetim aktivnostima, kao uključivanje u proces donošenja odluka o liječenju, čekanja na pregled kod izabranoga liječnika i drugih aspekata kroz koje se može sagledati kvaliteta rada izabranoga liječnika. Zadovoljstvo korisnika uslugama pruženim u zdravstvenim institucijama svakako je najznačajniji pokazatelj kvalitete rada izabranog liječnika i preporučeno je ispitivati zadovoljstvo korisnika na svim mjestima pružanja usluga, jer je vrijedna povratna informacija liječnika. Osim toga, zadovoljstvo korisnika može poslužiti kao važna smjernica nastavka aktivnosti reforme sustava zdravstva, jer precizno ukazuje na stupanj zadovoljstva korinika uslugama pruženim u reformiranome sustavu zdravstvene zaštite. Iskustva iz susjednih zemalja pokazuju da stalno praćenje zadovoljstva korisnika može imati značajne implikacije na rad izabranih liječnika. Istraživanje, provedeno 2001. godine, pokazalo da je 72% korisnika bilo zadovoljno uslugama pruženim u domu zdravlja u službi opće prakse.Zaključak: Osnovni zaključak istraživanja jest taj da su korisnici vrlo zadovoljni uslugama izabranoga liječnika na primarnoj razini zdravstvene zaštite

Early Introduction of cART Reverses Brain Aging Pattern in Well-Controlled HIV Infection: A Comparative MR Spectroscopy Study

Introduction: The aim of this study was to compare age-related changes in chronically infected, asymptomatic HIV-positive patients under combination antiretroviral therapy (cART), with age-, gender-, and educational-level-matched healthy subjects, using multi-voxel magnetic-resonance spectroscopy (MRS).Methods: There were 66 chronically infected HIV-positive subjects and 65 age-, gender-, and educational-level-matched control subjects, divided into four groups according to the age: group 1 (20–29 years old), group 2 (30–39), group 3 (40–49) and group 4 (50–59). MRS was performed and ratios of N-acetyl-aspartate (NAA)/creatine (Cr) were analyzed in ten locations of the supracallosal gray matter. For the comparison of NAA/Cr ratios in healthy and HIV-positive subjects, ANCOVA with age and education as covariates was performed. Correlations of NAA/Cr ratios with duration of cART were performed using Pearson’s correlation test. Statistical significance was set at p &lt; 0.05.Results: The NAA/Cr ratios were decreased in the 20–29-year-old HIV-positive subjects in 8/10 locations (p &lt; 0.005) compared to the healthy controls, while in the 50–59-year-old groups they were significiantly lower only in one location (p = 0.004). There were significant positive correlations of NAA/Cr levels with the duration of cART in the oldest group of HIV-positive subjects, while in the youngest group there were no significant correlations.Conclusion: The aging pattern in chronic HIV infection under cART is accentuated rather than accelerated. There is an initial HIV-related neuronal damage with a significant decline in NAA/Cr ratios; after the initiation of cART, however, NAA/Cr ratios increase continuously to become similar to healthy aging individuals, probably due to beneficial effect of long-standing cART.Summary: Brain aging in chronic HIV infection under cART is accentuated, with an initial HIV-related neuronal damage followed by a subtle NAA/Cr increase after the initiation of cART. Under cART, in advanced age, NAA/Cr ratios become similar to healthy aging individuals