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DOI: http://dx.doi.org/10.20534/ELBLS-17-1-27-35
Morozov Igor Aleksandrovich, Chumakov Institute of Poliomyelitis and Viral Encephalitides Deputy Head of the Institute, Head of the Department of Pathomorphology
E-mail: moroz38@gmail.com Kyuregyan Karen Karenovich, Mechnikov Research Institute of Vaccines and Sera, Research Center at Russian Academy of Postgraduate Education
Head of the Department of Viral Hepatitis E-mail: karen-kyuregyan@yandex.ru Karlsen Anastasia Andreevna, Mechnikov Research Institute of Vaccines and Sera, Research Center at Russian Academy of Postgraduate Education
researcher
E-mail: karlsen12@gmail.com Ilchenko Liudmila Yur'evna, Pirogov Russian National Research Medical University Professor of the Department of Hospital Therapy E-mail: Ilchenko-med@yandex.ru Fedorov Il'ya Germanovich, City Clinical Hospital named after V. M. Buyanova Head of Gastroenterology Department E-mail: _fig-med@yandex.ru
Viruses of the Anelloviridae family in cases of chronic liver pathology and in primary blood donors
Abstract: Prevalence of anelloviruses (TTV, TTMDV and TTMV) in blood serum and liver biopsy specimens from 203 patients with chronic liver pathology of viral and non-viral etiology was studied using molecular, histopathological, immunohistochemical and electron microscopic methods. Serum samples from 115 primary blood donors were used as controls.
Keywords: anelloviruses, prevalence, chronic liver diseases, hepatotropism, morphometry, electron microscopy.
In 1997 during the search of yet unknown hepatitis viruses T. Nishizawa et al. [1] found a new DNA virus in a patient with transfusion-associated hepatitis of unknown etiology. The new virus with non-enveloped 30-32 nm virions and single-stranded circular DNA 3,853 nt genome was preliminary named as "TT virus" (TTV) after the patient's initials. It was later renamed as "Torque teno virus" (Latin for "thin necklace") without change in abbreviation. Multiple genetic variants of the virus were detected in patients with liver diseases and healthy persons by polymerase chain reaction (PCR) with primers to conservative 5'-non-translated region of its genome [2].
TTV is currently assigned to genus Alphatorquevirus within family Anelloviridae [2] which includes 9 genera, 3 of which contain human viruses. The other two human anelloviruses are Torque teno mini virus (TTMV, genus Betatorquevirus) with 2,8-2,9 kb genome [3] and Torque teno midi virus (TTMDV, genus Gammatorque-virus) with 3,2 kb genome [4]. The taxonomic classification is based on >56% sequence divergence between genera and >35% between species.
Anelloviruses are among the most abundant infectious agents with their prevalence approaching 100% not only in humans but also in some mammals [5]. Global dispersion of these viruses and their detection in various
biological samples suggest both parenteral and enteral transmission. Different genera of Anelloviridae can simultaneously infect the host, being detectable by polymerase chain reaction (PCR) in blood serum, while tissue tropism of some of them is not yet determined. Extremely high infection rates in human populations throughout the World make the studies of association between anelloviral infection and pathology really challenging. Cases of monoinfection with one genus of Anelloviridae are extremely rare. Currently the role of TTMDV in pathology is not determined, its size is unknown, and morphology is not characterized.
The aim ofour study was to assess the prevalence ofthree anelloviruses (TTV, TTMDV and TTMV) in blood serum of healthy persons (voluntary blood donors) as well as in blood serum and liver biopsy samples ofpatients with chronic hepatitis of viral and nonviral origin as well as EM verification of anellovirus size in cytoplasm ofhepatocytes.
Material and methods
Total of203 patients with various viral and non-viral liver diseases and clinical indications for liver biopsy were selected for this study from a large database of people admiTTed to Gastroenterology Department of State Budgetary Unitary Institution City Clinical Hospital named after V. M. Buyanov of Moscow Healthcare Department (SBUI CCH12 of MHD) from 2009 to 2014. In all cases the material for morphological and molecular testing (serum and liver biopsy) was present (Table 1). The study cohort included 110 men (mean age 45,9 ± 6,5 years) and 93 women (mean age 46,1 ± 7,3 years). Serum samples from 115 healthy voluntary donors matched for gender and age were used as controls. Serologic and molecular markers of hepatitis C virus (HCV) and hepatitis B virus (HBV) were tested in all cases and controls.
Serum and liver biopsy samples were appropriately processed and then used for histopathological, immu-nohistochemical and EM studies and tested by ELISA and PCR. Serum and portions ofbiopsy samples (2 mm) were frozen at -20 °C within 3 minutes after obtaining and stored for up to 1 week before transportation (maintaining the "cold chain") to the laboratory for analysis. Another portion of biopsy samples was placed into fixing solution of 2% paraformaldehyde and 2,5% glu-taraldehyde in Hanks buffer, fixed, dehydrated and embedded into Epon-Araldite epoxy resin mixture. Subsequently ultrathin sections stained with uranyl acetate and lead citrate were studied using JEM-100C (Jeol, Japan) electron microscope. Morphometric analysis of the virions was performed using program BioVision (Austria) software with 30k magnification.
The part of ultrathin sections of the liver was mounted on nickel meshes (200 meshes), covered with formvar and dusted with carbon (Agar Scientific Ltd), and after osmium deplating with Na-metaperiodate was used for conduct of immunocytochemical verification of TTM-DV-virions. The procedure of drug preparation for im-munoelectron microscopy with the use of "Protein A — Colloidal Gold" (10 nm, produced by EMS) was carried out according to the method, which was described in detail by Bendayan M. [6]. Due to the lack of commercial antibodies against the examined virus we used the whole blood serum (dilution 1:50) of the donor, who had monoinfection with only one TTMDV-anellovirus, but the biopsy material was absent, what excluded morphological study.
As negative control we used the gating of incubation with antibodies, or incubated with commercial anti-IgG antibodies or with 1:50 diluted serum of the healthy donor, who had no viruses of the family Anelloviridae.
All blood serum samples were also tested for GBV-C RNA (virus, formerly known as hepatitis G virus) by RT-PCR using protocol described by J. HaTTori and coauthors [7]. Serologic markers of HBV and HCV (HBsAg, anti-HBc, anti-HCV) were determined by ELIZA (Diagnostic systems, Russia) according to the manufacturer's protocol. All samples positive for HBsAg and/or anti-HBc, were tested for HBV DNA by PCR following the protocol described by A. Basuni and W. Carman [8]. To confirm the presence of latent form of chronic hepatitis B (CHB) in patients with the clinical diagnosis "chronic hepatitis of alcoholic etiology" positive for anti-HBc in the blood serum, we carried out the immunohistochemi-cal detection of HBsAg in paraffin sections from archival blocks from the Pathoanatomical Department of State Budgetary Health Care Facility of City Clinical Hospital named after V. M. Buyanov with the help of monoclonal antibodies against HBsAg (anti-HBs, Cell Marque S1-210 USA), according to the standard manufacturer's procedure.
Extraction of nucleic acids from serum samples was performed using "Kit for isolation of DNA/RNA from serum or plasma on magnetic particles MP@SiO2" (Sileks, Russia) according to the manufacturer's protocol.
Detection of Anelloviridae DNA in serum samples was performed using PCR with nested primers proposed by M. Ninomiya et al. [9], which allow differentiation of TTV, TTMDV and TTMV based on the size of the amplified fragment. The reaction conditions for the first and second round of PCR were identical: 94 °C for 5 minutes, then 35 cycles of94 °C — 30 seconds, 55 °C —
30 seconds, 72 °C — 30 seconds, and final elongation at 72 °C — 7 minutes. Sizes of amplification products were: 112-117 nt for TTV, 88 nt for TTMDV and 70-72 nt for TTMV.
To confirm the specificity of identification of TTV, TTMDV and TTMV by PCR, we performed direct sequencing of the resulting fragments of viral genomes. PCR products of corresponding sizes were excised from agarose gel and purified using QIAquick Gel Extraction kit (QIAGEN). Sequencing of the fragments was performed on SEQ8800 automatic sequencer (Beckman Coulter) using GenomeLab Methods Development Kit (Beckman Coulter) according to manufacturer's protocol. Sequencing of each PCR fragment was carried out in two directions using the forward and reverse primers. The resulting chromatograms were assembled into con-
tigs using Seqman 4.03 (LASERGENE, DNASTAR, USA) software. Specificity of PCR was assessed using BLAST search in NCBI (National Center for Biotechnology Information) database.
The differences in detection rates of the indicators in the two groups were evaluated using Fisher's exact test, the differences were considered significant at 95% confidence interval (p <0,05).
Results
Screening of serum samples for HBV and HCV showed that isolated anti-HBc, in the absence of HBsAg and HBV DNA in serum, was found in 69/151 patients with presumably non-viral chronic liver disease (alcoholic liver disease, non-alcoholic steatohepatitis, and chronic hepatitis of unknown etiology) and in 15/30 patients with chronic hepatitis C (CHC) (Table 1).
Table 1. - The detection rate of anelloviruses (TTV, TTMDV, and TTMV) in cases of chronic liver disease and in primary blood donors
Primary clinical diagnosis Quantity The viruses, detected in PCR, ELISA and morphological study
The number of positive individuals (%)
HBV anti-HB core + HCV TTV TTMDV TTMV
Alcoholic liver disease 80 0 35 (43.8) 0 77 (96.3) 47 (57.5) 62 (72.5)
Chronic hepatitis of unknown etiology 57 0 29 (50.9) 0 54 (94.7) 33 (57.9) 45 (78.9)
Nonalcoholic steatohepatitis 14 0 5 (35.7) 0 13 (92.9) 9 (56.3) 11 (78.6)
Chronic hepatitis C 30 1 15 (50.0) 30 29 (96.7) 25 (83.3) 26 (86.7)
Chronic hepatitis B 22 22 - 1 20 (90.9) 15 (68.2) 19 (86.4)
"Isolated" anti-HBc 84 - 84 (100.0) 0 78 (92.9) 52 (61.9) 70 (83.3)
Primary blood donors 115 0 0 0 111 (96.5) 84 (73.0) 103 (89.6)
The presence of isolated anti-HBc even in the absence of HBV DNA in serum can indicate the presence of occult HBV infection ofhepatocytes; this is clinically relevant and affects understanding of mechanisms for the pathogenesis ofmultiple viral infection. Therefore, for the purpose ofas-sessing prevalence of anellovirus infection among patients with chronic liver diseases of different etiology, we assigned patients with isolated anti-HBc to a separate group.
Prevalence of anelloviruses in the healthy population (first-time volunteer blood donors in the Moscow region) was extremely high. Among 115 donors, TTV DNA was found in 111 (96.5%), TTMDV in 84 (73%), and T TMV in 103 (89.6%). TTV monoinfection was found only in 4 individuals, TTMDV in one person, and TTMV DNA was found only in combination with other Anelloviridae. Only three persons did not have anellovirus infection, while triple infection with all three viruses was found in 60 (52.2%) healthy individuals (Table 2).
In patients with chronic liver disease (CLD), T TV DNA in serum was found in all study groups with prevalence similar to the healthy population, ranging from 90.9% in patients with CHB to 96.7% in patients with HCV infection (Table 1).
The detection rate for TTMV DNA in patients with HBV and HCV infection as well as in persons with isolated anti-HBc (86.7%, 86.4%, and 83.3%, respectively) was practically identical to the rate observed in healthy population (89.6%). In most cases the differences between the groups were not statistically significant, except for patients with alcohol hepatitis, in whom TTMV DNA was found less frequently compared to first-time blood donors (72.5% vs. 89.6%; P=0.006). A similar trend was observed with TTMDV: prevalence of this virus was 20% lower in all groups of patients with non-viral liver disease as compared to healthy individuals (56.3-57.9%; P<0.05).
Table 2. - The detection rate of mono - and multi-viral anelloviral infection in cases of chronic liver pathology and in primary blood donors
Monoinfection Triple infec- Without infection TTV, TTMDV and TTMV
Quantity tion TTV,
Group TTV TTMDV TTMV TTMDV and TTMV
The number ofpositive individuals (%)
Patients with chronic
hepatitis B and chronic 52 4 (7.7) 0 1 (1.9) 36 (69.2) 1 (1.9)
hepatitis C
Patients with hepatitis of non-viral origin 151 16 (10.6) 2 (1.3) 0 75 (49.7) 1 (0.66)
Primary blood donors 115 4 (3.5) 1 (0.9) 0 60 (52.2) 3 (2.6)
The most interesting findings, in our view, relate to patients without serologic markers of HBV or HCV who nonetheless presented with clinical and morphological signs of chronic immunoinflammatory reaction. Among these patients (excluding anti-HBc-positive ones), 41 (51.9%) were infected by all three anelloviruses, comprising 20.2% of all examined patients. 16 cases of T TV monoinfection and two cases of T TMDV monoinfection were detected.
Our interest in this virus dates to 2007 and was spurred in part by the paucity of information other than the genomic DNA sequence on the basis of which TTMDV was included in Anelloviridae. Morphology of the virus had not been visualized; there were no suggestions on its potential hepatotropism and pathogenicity.
Morphological examination of biopsy samples taken from patients with T TMDV monoinfection showed signs of chronic hepatitis very similar to those observed in TTV monoinfection [10]. These signs of chronic hepatitis included: mild lymphocytic infiltration of portal tracts extending out of the limiting plate; presence of small intralobular accumulations of lymphocytes; hydropic and protein dystrophy (cytopathy) of hepatocytes; intracellular cholestasis; dystrophy and necrobiosis of bile duct epithelium; stage 1-2 fibrosis ofportal tracts, and presence of collagen fibers in the walls of central veins and in the perisinusoidal space (Fig. 1a, b). Enzyme activity (alanine and aspartate aminotransferase [ALT, AST]) did not exceed 2-4 times the upper limit of normal (ULN) in these two patients as well as in most cases of TTV infection in ongoing follow-up. Finally, diagnosis was made of chronic hepatitis of minimal activity with minimal portal cholangi-tis, and stage 2 TTMDV-associated moderate portal and perisinusoidal fibrosis.
An EM image of virions present in liver biopsy samples taken from patients with TTMDV monoinfec-
tion (Fig. 2 a, b) shows morphological similarity between TTMDV and TTV virions. The difference between these two genera of anelloviruses is the virion size as determined morphometrically: TTV virions measured 42.25 ± 3.48 nm, compared to TTMDV virions of 35.4 ± 1.36 nm.
EM examination allowed us to characterize liver damage in TTMDV monoinfection as manifested by virus accumulation in hepatocytes, significant cytopathy with cytoplasm matrix clarification and reduction of the intracellular organelles involved in protein synthesis (polyribosome and rough endoplasmic reticulum), and portal (Fig 1b), perivascular, and perisinusoidal fibrosis (Fig. 3b).
Direct evidence, that the detected virions, namely TT midi, are representatives of Anelloviridae family, is the results of their verification with the use of immunocyto-chemical electron microscopy with "Protein A — Colloidal Gold" (Fig. 3a). It is clearly seen that the reaction product is observed only in location area of virions and is absent on the profiles of mitochondria. The absence of nonspecific reaction in negative tests confirmed that the cause of hepatitis in patients is TTMDV mono-infection, that we identified by molecular-biological methods in blood serum and liver tissue.
Regarding TTMV, we should note its constant presence in white blood cells (neutrophils, lymphocytes) and platelets found in liver sinusoids (in 70% of cases), as has been reported previously [11]. For morphological verification, we were guided by PCR data and a single image of this virus in the nuclei of Hodgkin's lymphoma L1236 cell-line cells published in a brief report by H. Hausen and E-M. de Villiers [12]. Morphological analysis indicated that TTMV is the smallest anello-virus, with a diameter (20-22 nm) that is half the size of TTV virions. EM examination of the localization of TTMV virions supports the hypothesis of lymphotro-pism of this virus (Fig. 4 a, b).
a b
Figure 1. Pathological liver changes in the patient with TTMDV-infection.
a) Pronounced cytopathy, intracellular cholestasis, perisinusoidal fibrosis.
b) Fibrosis of portal tract, necrobiosis of the biliary tract epithelium x 800
a b
Figure 2. Multiple virions in the cytoplasm of hepatocytes: a) TTV. b) TTMDV x 30 000
However, during the analysis of liver biopsy speci- (Figure 5 a, b), thereby confirming virus hepatotropism. mens from 4 individuals with "isolated" anti-HBc us- Moreover, the cytoplasm of most hepatocytes coning immunocytochemical method, we identified large tained virions that were similar in size to viral particles amounts of TTMV in karyoplasm of some nuclei of HBV, located in hepatocytes (49.3±2.6 nm).
a b
Figure 3. Electron microscopy of liver biopsy with monoinfection TTMDV. a) Immunocytochemical verification of TTMDV virions with the use of "Protein A - Colloidal Gold" b) Collagen fibers (arrow) in perisinusoidal space x 10 000
The structure and shape of TTMV virions located in liver tissue is similar to all anelloviruses: all the viruses are non-enveloped, featuring moderate electron density and irregular roundish shape with ruffled borders by surface, which is consistent with images obtained by Y. Itoh
et al. [13] by negatively contrasting TTV isolated from a patient's serum and feces. The size of TTV virions was smaller in their study (30-32 nm) due to overlap of the marginal surface by phosphotungstic acid contrast.
a b
Figure 4. TTMV in lymphoid blood elements of the patient with a latent form of chronic hepatitis B. a) TTMV in lymphocyte x 6 thousand. (insert х 30 000). b) TTMD in neutrophil (arrow) x 15 000
Figure 5. TTMV virions in hepatocyte of the a) TTMV in karyoplasm of the nucleus, b) TTMV virions in the nucleus
Discussion
Study of TTV prevalence worldwide began subsequent to discovery of the virus in 1997. In 1999, a collaborative study of researchers from several countries was published [14], which showed a very high infection rate across diverse human populations: from 70-75% in Japan and the USA to 98-100% in Myanmar and Cambodia, with low influence of age, geographic location, and socioeconomic factors. Almost simultaneously, reports were published on lower TTV prevalence among blood donors in Brazil (46-62%) [15], Turkey (51.6%) [16], and China (53.3%) [17]. Data to indicate much higher virus prevalence was reported following discovery of two other viruses of the Anelloviridae family. In the same year, several authors of the current work also performed prevalence studies in Russia [18]. These studies suggested that previous reports indicating relatively low prevalence of TTV in the general population might be due to use of PCR primers with low sensitivity for TTV DNA detection.
Using the most modern PCR techniques, the Japanese team led by H. Okamoto that discovered all three anelloviruses published an article6 with data on the prevalence of these viruses in all age groups of the healthy population (from newborns to 81-year-old individuals). Sets of universal and species-specific primers for highly
patient with a latent from of chronic hepatitis B HBV virions in cytoplasm (arrow). x 8 000 (fragment of Figure 5 a). x 50 000
conserved regions of the TTV, TTMDV, and TTMV genomes were used; we used the same primer sets in our study. M. Ninomiya et al. [9] report high prevalence of TTMDV viremia in adults (75%), consistent with the prevalence rates of TTV viremia (100%) and TTMV viremia (82%). Although no anelloviral DNA was found in 10 samples of umbilical blood, anelloviral prevalence increases after birth and reaches 100% in infants by the first year of life. Total prevalence of double and/or triple TTV, TTMDV, and TTMV infection in all age groups (0 to 81 years old) was 99.2% for TTV 82.4% for TTMDV, and 89.7% for TTMV.
These values are comparable to our results obtained among first-time blood donors (TTV: 96.5%, TTMDV: 73.0%, TTMV: 89.6%). However, contrary to the Japanese study, we found 3 cases without infection of any anellovirus, 4 cases with TTV monoinfection, and 1 case with TTMDV monoinfection.
Literature search did not uncover any publications on prevalence of all three anelloviruses in patients with chronic liver disease. Our data demonstrate that TTV prevalence is very high and independent of the etiology of liver disease. Similarly, TTMV prevalence in chronic hepatitis B and C and in isolated anti-HBc was identical to prevalence among healthy individuals, but significantly lower in patients with alcoholic liver disease (73.7%
b
a
vs. 89.6%; P=0.006). The same correlation was observed for TTMDV prevalence: this virus was significantly less prevalent in patients with non-viral liver disease compared to healthy donors and patients with hepatitis B and C (P<0.05). The reason for these differences is unclear.
Although some anelloviruses are known to be lym-photropic, our results show that at least some variants of these viruses are able to infect hepatocytes and might be pathogenic to the liver. We consider this fact to be proven at least in relation to TTV and TTMDV.
In 1999 shortly after discovery of T TV and evidence suggesting its high prevalence, P. Simmonds et al. [19] hypothesized that TTV may be the part of human normal microflora. Presence of TTV, TTMDV, and TTMV DNA in the blood of the majority of the population indicates that viremia (and thus active virus replication) is not always related to disease. However, active replication of some widely prevalent viruses (such as herpes viruses) with chronic potential is known to be associated with pathology. This indicates that such commensalism between humans and viruses (in this case, microflora) is contingent on a fragile balance between virus replication, high genetic variability, and the human immune system.
Therefore the high prevalence of TTV, T TMDV, and TTMV infection reported here should not be interpreted to rule out a role by these viruses in pathogenesis in humans. To the contrary, the high genetic heterogeneity of anelloviruses, as well as their potential for intragenom-ic rearrangement and changes in protein synthesis components as described by in vitro and in vivo data [20] may lead to changes in the pathogenicity of the virus and cause development of disease.
We believe that the presence of characteristic signs of chronic hepatitis in patients with TTMDV monoinfection is evidence that the virus has both an affinity and pathogenic effects with regard to the liver.
Our work in this area continues. Directions for continuing research include the search for monoinfection cases in healthy individuals, deep genome-wide sequence analysis, and genotyping of TTMDV strains that cause liver disease. Intensive studies are needed to investigate the hepatopathogenic potential of TTMV. While hepatotropism of TTMV has been established in some cases, identification of monoinfection in patients with chronic liver disease promises to offer valuable insight in this regard.
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DOI: http://dx.doi.org/10.20534/ELBLS-17-1-35-39
Naska Jonida, Lecturer of Gender Sociology, Social Sciences Faculty, University of Tirana, Albania
Puca Edmond, Service of Infectious Diseases, University Hospital Center, Tirane, Albania
Zekja Ilirjana,
Service of Neurology, University Hospital Center, Tirane, Albania
Kruja Jera,
Service of Neurology, University Hospital Center, Tirane Department of Neurology, University Hospital Center, Tirane, Albania
E-mail: Edmond-puca@yahoo.com
Indicators of social support in patients with multiple sclerosis in Albania
Abstract:
Multiple sclerosis is a chronic disease that attacks the central nervous system.
Objective: The aim of this one year study was to assess the impact of neurological disability in the overall social support domains. Indicators of quality of life are assessed too and social functions and roles, economic constrains, marital status, family relationships and education of patients are included in the study as complementary evidences.
Methods: We randomly selected 67 patients diagnosed with multiple sclerosis. Female were 51 (76.1%) and male were 16 (23.9%), mean disease duration was 6 years, (m)education 12 years. In this study social support constrains were measured by MSSS (Modified Social Support Survey) questionnaire (MSQLI scale) which has 4 subscales (I) — Tangible Support Subscale (TAN) II- Emotional/Informational Support
