Hepatitis B surface antigen seroconversion is associated with favourable long-term clinical outcomes during lamivudine treatment in HBeAg-negative chronic hepatitis B patients.

The aims of this study were to assess hepatitis B surface antigen (HBsAg) seroconversion and to determine its impact on the natural course of the disease in patients with HBeAg-negative chronic hepatitis B (CHB) during lamivudine (LMV) treatment. A total of 183 consecutive patients with HBeAg-negative CHB who were treated with LMV were included in the study. Data were retrospectively collected from outpatient visit charts. The primary endpoint was HBsAg seroconversion to anti-HBs. The secondary endpoint was to determine the development of cirrhosis. Loss of HBsAg was confirmed in 10 patients and seroconversion to anti-HBs in nine patients during LMV treatment or after its discontinuation. HBsAg seroconversion was achieved on-treatment in four patients after a median treatment duration of 30 months and off-treatment in the remaining five patients in a median 61 months after LMV discontinuation. The cumulative probability of HBsAg seroconversion increased from 0.6% at 1 year and 1.9% at 5 years to 21.5% at 10 years of LMV during and after LMV treatment. HBsAg clearance was preceded by undetectable serum hepatitis B virus (HBV) DNA. The majority of the patients responding to treatment had undetectable HBV DNA levels at 24 weeks of treatment. The cumulative probability of LMV resistance increased from 2.2% at 1 year to 37.3% at 5 years. No baseline parameter predicting either HBsAg seroconversion or the emergence of LMV resistance was identified. None of the patients with HBsAg seroconversion experienced virological breakthrough or disease progression during the follow-up period. These results indicate that HBsAg seroclearance can occur in patients with HBeAg-negative CHB under LMV therapy and predicts better clinical outcome.

The effects of montelukast against amikacin-induced acute renal damage.

BACKGROUND AND OBJECTIVES: The therapeutic and protective effects of montelukast against amikacin-induced acute renal damage were investigated. MATERIAL AND METHODS: 35 Wistar albino female rats were divided into 5 groups as follows: Group I: Control; Group II: Control+montelukast; Group III: Amikacin; Group IV: Amikacin+montelukast; Group V: Montelukast+amikacin. At the end of the experiment, the kidney tissues and the blood of rats were collected. Malondialdehyde (MDA), myeloperoxidase (MPO), and reduced glutathione (GSH) levels were determined from kidney tissues. Blood urea nitrogen (BUN), creatinine (Cr), TNF-alpha, and IL-1beta levels were assessed in the serum. In addition the kidney tissues were examined histologically. RESULTS AND DISCUSSION: The MDA, MPO, BUN, and Cr levels of group III significantly increased when compared to groups I and II. These parameters of group IV decreased when compared to group III. In addition, GSH levels significantly increased when compared to the first three groups. MDA, BUN and Cr levels of group V did not reach significant level in comparison with the control group. The most significant histological damage was observed in the group III followed by the groups IV and V. Immunohistochemically, group III showed a significantly increased apoptotic staining. In group IV, it was observed that montelukast treatment reduced the expression of apoptotic cells. CONCLUSIONS: Montelukast treatment after amikacin injection could reduce the amikacin-induced kidney damage.

A short course of add-on adefovir dipivoxil treatment in lamivudine-resistant chronic hepatitis B patients.

The aims of the study were to investigate the efficacy of rescue therapy with lamivudine (LAM) and adefovir (ADV) combination for 6 months followed by ADV monotherapy in lamivudine-resistant chronic hepatitis B (LAM-R CHB) patients, and to analyze the frequency of ADV resistance mutant development in such patients. A total of 170 consecutive LAM-R CHB patients (male/female: 130/40, mean age: 42.9+/-13.4 years) with viral breakthrough under LAM therapy were analyzed. A total of 68 had HBeAg-positive. Patients received rescue therapy with LAM [100 mg (qd)]+ADV [10 mg (qd)] for 6 months after which LAM was discontinued. HBV-DNA was assessed with the HBV-DNA 3.0 bDNA assay. ADV-resistant mutations were identified by sequencing the reverse transcriptase region. The median duration of rescue therapy was 24 months. Cumulative probability of becoming HBV-DNA undetectable was 33.8%, 59.6% and 68.2% after 24, 48 and 96 weeks of treatment, respectively. These figures were 43.2%, 58.0% and 73.1% for ALT normalization. Among 68 HBeAg-positive CHB patients, 10 patients had an e-antigen seroconversion. Low baseline HBV-DNA level (<10(7) copies/mL) was a significant predictor of response to ADV treatment (P<0.01). Cumulative probability of ADV resistance was 1.2%, 15.1% and 37.3% at 12, 24 and 36 months of therapy, respectively. By multivariate analysis, baseline high viral load and primary nonresponse to treatment at week 24 predicted ADV resistance. The data indicate that a time limited add-on strategy does not provide benefit over the switch strategy with respect emergence of ADV resistant mutants in LAM-R CHB patients.

Distribution and relative frequency of immunohistochemically detected endocrine cells in the stomach of New Zealand White rabbit (Oryctolagus cuniculus).

BACKGROUND: Gastrointestinal (GI) endocrine cells produce many GI hormones that perform various physiological functions of the digestive system. AIMS: We aimed to investigate the presence and distribution of immunoreactive (IR) endocrine cells to glucagon, somatostatin, cholecystokinin-8 (CCK-8), serotonin, secretin and histamine in the stomach of adult male New Zealand White rabbit (Oryctolagus cuniculus). METHODS: For immunohistochemical staining, peroxidase anti-peroxidase (PAP) method was applied to stomach samples. RESULTS: Glucagon-IR cells of closed- and open type were found throughout all the stomach parts examined. Somatostatin-IR cells of closed- and open type in the cardiac and oxyntic glands were localized to deep portions of foveola gastrica. CCK-8 IR cells that were not observed in the cardia and fundus were mostly localized to the glands and lamina epithelialis in the pyloric part near the duodenum. Oval-shaped open and closed type serotonin-IR cells were mostly dispersed throughout the fundic and pyloric glands. Secretin-IR cells were rare in the pyloric and cardiac region although they were not observed in the fundic glands. Histamine-IR cells were rarely found in the cardia, fundus and pylorus. CONCLUSION: Our findings show that glucagon, histamine, somatostatin, secretin and serotonin might be produced by all the stomach regions while pyloric region had only CCK-8 IR. These distribution patterns also provide further evidence of species-specific differences, which might be important from the evolutionary aspect of the digestive tract in relation to evolutional niches and nutrient resources.

Immunolocalization of natriuretic peptides and their receptors in goat (Capra hircus) heart.

Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors.

Regional and mucosal distributions of some intestinal immunoreactive endocrine cells in New Zealand white rabbit (Oryctolagus cuniculus L.).

The aim of this study was to detect the regional and mucosal distribution of endocrine cells that secrete gulcagon, somatostatin, Chyholecystokinin-8 (CCK-8), serotonin, secretin, substance P (SP) and histamine in the small and large intestine of New Zealand white rabbit (Oryctolagus cuniculus L.) using immunohistochemical peroxidase-antiperoxidase (PAP) method. It was found that most of the immunoreactive (IR) endocrine cells, which are oval- or spindle-shaped, are spotted in the basal parts of the relevant glands. It was noticed that cells in the lamina epithelialis of small and large intestine is linked to the lumen and that the cells in their glands cannot reach the lumen. Immunoreactive cells for glucagon, somatostatin, serotonin, secretin and SP were identified in lamina epithelialis of the small and large intestine. It was seen that secretin, SP and histamine-IR cells are rarely deployed throughout the intestinal tract. It was determined that somatostatin-IR cells were identified throughout the intestinal tract. In conclusion, the immunohistochemical study shows that gastrointestinal tract of this species contained different types of endocrine cells similar to those found in other vertebrate species. However, some species-dependent unique distributions and frequencies of endocrine cells were also observed in the present study.

Some lectin binding properties of the tongue of the mole rat, Nannospalax xanthodon.

We investigated carbohydrate residues on the epithelial surface, in the epithelial cells and in gland cells of the tongue of the mole rat using histochemical methods. We used horseradish peroxidase-conjugated lectins from Helix pomatia (HPA), Arachishypogaea (PNA), Ulexeuropaeus (UEA I), Canavaliaensiformis (Con A). The most intense reactivity was observed in the keratin layer with HPA, UEA I and Con A, and in the epithelial cells with UEA I and Con A. In the glands, we found strong reactivity in serous cells with HPA and Con A, and in mucous cells with HPA and UEA I. PNA did not bind to epithelial or gland cells. Consequently, GlcNAc, fucose and α-D-mannose terminal glycoconjugates are distributed widely; GalNAc terminal glycoconjugates appeared in small amounts.

Penetrating shrapnel injuries of the posterior fossa.

BACKGROUND: Gunshot injuries of the posterior fossa are rare and may follow a fatal course. In posterior fossa gunshot injuries, cerebellar hematoma, contusion, obstruction of cerebrospinal fluid (CSF) circulation by the shrapnel, and intracranial hypertension caused by autoregulation loss lead to mortality in the early stage. METHODS: In this study, four cases of patients who underwent surgical intervention after penetrating shrapnel injuries of the pure posterior fossa were evaluated. RESULTS: All of the patients were male; their mean age was 26.5 ± 5 years. The lowest and highest Glasgow Coma Scale scores were 4 and 12, respectively. Neural injury was detected by computed tomography performed after systemic and neurological examination following admission to the emergency service. The shrapnel was found in the cerebellar tissue in three cases and in the fourth ventricle in one case. Following preoperative procedures, surgery was performed with the patient in the prone position. Postoperative monitoring revealed no CSF fistula, meningitis, or hydrocephalus. None of the patients required revision surgery. There were no postoperative mortalities. CONCLUSION: Due to the small volume of the posterior fossa, acute pathologies may lead to rapid neurological deterioration and death. Early surgical intervention and close postoperative follow-up after penetrating shrapnel injuries of the posterior fossa play a significant role in reducing mortality and morbidity.

Circulating IL-2, IL-10 and TNF-alpha in chronic hepatitis B: their relations to HBeAg status and the activity of liver disease.

BACKGROUND/AIMS: Preferential production of immunoregulatory cytokines may play an important role in the pathogenesis of chronic hepatitis B. We aimed to determine the serum levels of IL-2, IL-10 and TNF-alpha in patients with chronic hepatitis B and to correlate these findings with the activity of liver disease, HBeAg/anti-HBe status and replication level of the virus. METHODOLOGY: Seventy-two chronic hepatitis B patients were categorized into 4 groups according to activity of liver disease and HBeAg status. Group 1 (n = 13): HBeAg and HBV DNA-positive with persistently normal ALT. Group 2 (n = 20): HBeAg and HBV DNA-positive patients with persistently elevated ALT. Group 3 (n = 19): HBeAg and HBV DNA-negative patients with persistently normal ALT. Group 4 (n = 20): HBeAg-negative patients with persistently elevated ALT and variable serum HBV DNA. IL-2, IL-10 and TNFa levels were determined in stored patient sera. RESULTS: Apart from group 1 patients, all patients groups had higher IL-2 levels compared to controls suggesting that IL-2 production is increased when liver disease becomes active in HBeAg-positive phase of HBV infection. Only group 2 patients had elevated IL-10 levels compared to controls. None of the HBeAg-negative patients had detectable TNF-alpha levels while 64% HBeAg-positive patients had elevated levels of TNF-alpha irrespective of the activity of liver disease. Except TNF-alpha, no association was found between HBV DNA status and the presence or absence of detectable cytokines in circulation. CONCLUSIONS: Our results suggest that circulating cytokine profile in chronic hepatitis B is related with the HBeAg status, replication level of the virus and the activity of liver disease.

Serum cytotoxin and oxidant stress markers in N-acetylcysteine treated thioacetamide hepatotoxicity of rats.

N-acetylcysteine (NAC) is a glutathione precursor used to treat several clinical conditions where intracellular oxidant-antioxidant balance is disturbed, among which, acetaminophen induced hepatotoxicity may be counted. In this study, administering thioacetamide (TAA) as a hepatotoxic agent, a rat model of hepatotoxicity has been established, to investigate some of the immune mediated basic oxidant-antioxidant homeostatic mechanisms involved, and potential serum markers for follow-up of disease and treatment. To do this, four experimental groups receiving saline/saline, saline/NAC, saline/TAA and NAC/TAA as intraperitoneal injections, have been formed. Rat serum tumor necrosis factor-alpha (TNF-alpha), Interleukin1-beta (IL1-beta), malondialdehyde (MDA) as a measure of final oxidant damage and the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) have been assayed. Hepatocellular damage has been measured via the biochemical estimates ALT, AST and LDH as well as histopathological grading. It was found that both TNF-alpha and IL1-beta were significantly elevated in saline/TAA receivers (P<0.01) when compared to NAC/TAA receivers. Serum MDA was also increased in TAA receivers in addition to SOD (P<0.05) and GSH-Px (P<0.05). Serum nitrite levels have also been assayed to give an estimate of nitric oxide that is suggested as a counter-balancer of oxidant stress. NAC/saline receivers had the highest levels of nitrites in the serum (P<0.05). Our results indicate that part of the hepatocellular injury to rat liver, induced by TAA is mediated by oxidative stress caused by the action of cytokines imparted by the enzymatic SOD and GSH-Px and non-enzymatic gaseous nitric oxide mechanisms causing an alleviation on administration of NAC. In addition, TNF-alpha, IL1-beta, MDA, SOD, GSH-Px and nitrites are potential candidates of serum indicators for monitorization of pathophysiological stage of liver disease.

The distribution of taste buds in Garra rufa.

The distribution and relative frequency of external taste buds (TB) of Garra rufa were studied. TB of Garra rufa were observed on different body location (lips, lateral and ventral areas, forehead, operculum and dorsal-pelvic-pectoral-anal fins). TB are at the highest frequency in lips, forehead, pectoral and anal fins. These structures are moderate as a number in operculum, La2 (between pelvic and anal fins) areas, and then decreased a few in other areas.

Histological and histochemical characterization of the mucosa of the digestive tract in flower fish (Pseudophoxinus antalyae).

The wall of the digestive tract is composed of the tunica mucosa, tunica muscularis and tunica serosa. Lamina-submucosa separation or any glands were not observed in tunica mucosa. Goblet cells were determined to constitute a much larger reserve at digestive tract mucosa. Histochemical analysis of the intestine of flower fish (Pseudophoxinus antalyae) showed that gastrointestinal mucous content included sulphate-esters and/or carboxylic [Alcian blue (AB) 0.06+], glycogene and/or oxidable dioles [periodic acid/Schiff+ (PAS+)], neutral or acid-rich (PAS/AB pH 2.5+), sialic acid residues (KOH/PAS) and strong acid sulphated [Aldehyde fuchsin+ (AF+)] glycoproteins (GPs). Except these mucosubstances to lower densities, densely sulphate (AB pH 2.5+), O-sulphate esters (AB pH 1+) strong and weak sulphated (AB 0.3 M+), GPs were also determined.

The diagnostic value of on-site cytopathological evaluation and cell block preparation in fine-needle aspiration cytology of liver masses.

OBJECTIVE: The aims of this study were to evaluate the typing accuracy of conventional smear (CS), cell block (CB) preparations and combined use of both procedures (CS + CB) for the diagnosis of hepatic malignancies and to determine whether immediate on-site cytopathological evaluation improves the diagnostic yield of liver fine-needle aspiration cytology (FNAC). METHODS: Ultrasound-guided FNABs were performed on 323 consecutive cases with liver masses between December 2002 and December 2004. Histologically and/or clinically correlated 167 cases were included in the study. Preliminary FNAB results, results of CS, CB, and combined use of CS and CB were compared regarding diagnostic sensitivity, specificity, and accuracy for the diagnosis of malignancy. Subtyping accuracies of different methods were also compared. RESULTS: The sensitivity of on-site cytopathological examination and CS were both 92.8%. The sensitivity of CS + CB was slightly better than that of CB (93.5% versus 84.8%). Specificity of all procedures was achieved 100%. Diagnostic accuracy of on-site cytopathological evaluation, CS, CB, and CS + CB were 93.9%, 93.9%, 87.2%, and 94.5%, respectively. A specific subtype diagnosis of malignant tumours could be rendered accurately on the basis of preliminary diagnosis in 71%, CS in 75.4%, CB in 78.3% and combined approach in 92% of cases. In terms of typing accuracy, 87.5% of HCCs, 93.2% of adenocarcinomas, 92.3% of neuroendocrine carcinomas, 100% of lymphomas and 100% of other malignant tumours were correctly subclassified in the final cytopathological diagnosis. The agreement between preliminary diagnosis and final cytopathological diagnosis was 77.2%. CONCLUSION: With use of on-site cytopathological evaluation and combined use of CS and CB, the diagnostic accuracy of liver tumours approaches 100% and also significantly improve diagnostic and subtyping accuracy of liver malignancies.

The distribution of external taste buds in flower fish (Pseudophoxinus antalyae).

Purpose of this study was to determine the distribution of external taste buds of flower fish (Pseudophoxinus antalyae) on different body locations (lip, lateral, ventral and dorsal areas, dorsal-pelvic-pectoral-anal fins). In this species, it was found that the density of taste buds was greater on lip epithelium than that of lateral (between pectoral-anal, anal-caudal fins), ventral (between pelvic and anal fins) and dorsal (close to the head) locations. It was observed that the shape of taste bud changed depending upon the epithelium thickness and it was found that some of these structures protruded towards to the exterior of epithelium.

Multimodality demonstration of primary splenic angiosarcoma.

Angiosarcomas are rare, accounting for only 1-2% of all soft tissue sarcomas. Primary abdominal angiosarcomas usually arise in the liver or spleen. We report the first color Doppler findings of a rare, low-grade splenic angiosarcoma in a 52-year-old woman.

Combination treatment of hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure in the rat with benzodiazepine and opioid receptor antagonists.

BACKGROUND/AIMS: Treatment of hepatic encephalopathy with drugs acting on the target organ of this syndrome, the brain, is unsatisfactory. Combination treatment with different neurotransmitter receptor antagonists may be a rational option to optimize treatment. METHODS: The effects of various doses of the benzodiazepine receptor antagonist Ro 15-3505 and the opioid receptor antagonist naloxone, alone or in combination, were tested on hepatic encephalopathy in rats with thioacetamide-induced hepatic failure in an open-field activity meter. Comparison of single and combination treatment was also done using a neurological test battery. In addition, we compared survival of treatment-responder rats with treatment non-responders. RESULTS: Naloxone dose dependently increased ambulatory activity and improved neurological score. Ro 15-3505 also improved ambulatory activity and neurological score; however, the improvement was less evident at higher doses. Combination treatment was not superior to single treatment. Survival was increased in treatment-responder rats. CONCLUSIONS: The failure of combination treatment with Ro 15-3505 and naloxone to further improve hepatic encephalopathy may suggest that the two neurotransmitter systems are interrelated or that hepatic encephalopathy may not be further improved by drugs acting on the brain.

Ligand-induced expression of peroxisome proliferator-activated receptor alpha and activation of fatty acid oxidation enzymes in fatty liver.

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates lipid metabolism upon activation by ligands. Peroxisome proliferator-activated receptor alpha may play a role in the pathogenesis of fatty liver disease. The aim of this study was to assess the PPARalpha expression pattern and mitochondrial/peroxisomal enzyme activities in response to high fat diet (HFD) and clofibrate, a well known PPARalpha ligand. MATERIALS AND METHODS: Four groups of Wistar-Albino rats were included: (1) rats fed a control diet (CD) for 6 weeks, (2) rats fed CD (6 weeks) plus clofibrate (last 2 weeks), (3) rats fed HFD for 6 weeks, and (4) rats fed HFD (6 weeks) plus clofibrate (last 2 weeks). Peroxisome proliferator-activated receptor alpha expression was evaluated by immunohistochemistry. Fatty acid beta-oxidation (peroxisomal-acyl-CoA-oxidase and mitochondrial-acyl-CoA-dehydrogenase) and catalase enzyme activities, and malondialdehyde and glutathion levels were measured spectrophotometrically in liver tissues. RESULTS: All animals were fed HFD but only 2/12 animals were fed HFD plus clofibrate-developed fatty liver. Both HFD and clofibrate induced PPARalpha expression, clofibrate induction being more prominent than HFD. Clofibrate plus HFD did not further increase PPARalpha expression. Activities of peroxisomal-acyl-CoA-oxidase and mitochondrial-acyl-CoA-dehydrogenase enzymes were not induced by HFD alone. Clofibrate increased the activity of these enzymes in both CD- and HFD-fed animals. However, an increase of acyl-CoA-oxidase activity was blunted in rats fed HFD. Catalase activity and malondialdehyde levels were increased but glutathion levels were unchanged in rats fed HFD plus clofibrate. CONCLUSIONS: Clofibrate was a more potent inducer of PPARalpha expression than HFD in our rat fatty liver model. The finding of blunted peroxisomal enzyme response to clofibrate in fatty livers suggests that alterations in postreceptor events may exist and further contribute to liver steatosis. Clofibrate seems to stabilize glutathion content and this might contribute to the prevention of liver steatosis.

Circulating IL-2 and IL-10 in chronic active hepatitis C with respect to the response to IFN treatment.

BACKGROUND: The importance of circulating immunoregulatory cytokines in response to IFN treatment and the change of in vivo production of these cytokines during interferon (IFN) treatment are not well known. We aimed to determine whether pretreatment serum levels of IL-2 and IL-10 are predictive of the response to IFN treatment and to investigate if treatment response or nonresponse has any effect on the circulating levels of these cytokines. PATIENTS AND METHODS: 37 patients (18 responders and 19 non-responders) with chronic hepatitis C virus (HCV) infection who received IFN-alpha2b for 6 months were studied. Responders were defined by complete alanine aminotransferase (ALT) normalization and loss of HCV RNA as detected by bDNA assay while patients who had elevated ALT levels and positive HCV RNA after 6 months were considered as nonresponders. RESULTS: Genotype distribution, ALT and HCV RNA levels were similar in responders and nonresponders. A significant number of patients with chronic hepatitis C (20/37 = 54%) had elevated IL-2 levels while IL-10 levels were not different from controls. No difference in baseline cytokine levels was observed between responders and non-responders. In the posttreatment serum samples some patients lost their detectable IL-2 or IL-10; some patients developed detectable cytokine levels after treatment irrespective of the treatment response. CONCLUSION: These results suggest that active liver injury in chronic hepatitis C is associated with increased circulating Th1 cytokine IL-2 but not with Th2 cytokine IL-10 and that circulating levels of these cytokines do not predict the response to IFN treatment. There is no constant and regular change in circulating levels of these cytokines under IFN treatment with respect to treatment response.

YSDD: a novel mutation in HBV DNA polymerase confers clinical resistance to lamivudine.

The emergence of drug-resistant virus in hepatitis B virus (HBV) patients treated with lamivudine is well documented. In this study, we determined the mutations occurring in the tyrosine-methionine-aspartate-aspartate (YMDD) amino acid motif of the HBV DNA polymerase gene, as well as upstream and downstream of this region, in patients with breakthrough virus during lamivudine therapy. Thirty-one Turkish patients (20 patients HBeAg positive, 11 patients HBeAg negative and anti-HBe positive) with chronic HBV infection who completed at least 104 weeks of lamivudine treatment were investigated. All patients received lamivudine, (150 mg/day), for 104 weeks, with or without 4 months of interferon (IFN) combination. HBV-specific sequences were amplified by polymerase chain reaction (PCR) from sera of patients with breakthrough virus, and the PCR products were directly analysed by sequencing. Breakthrough virus was detected in seven of the 31 patients (22.6%) between 9 and 18 months of therapy. Of the seven patients, six were HBeAg positive at baseline, and four had a double mutation consisting of rtM204V and rtL180M, while two had an rtM204I change. In one patient, two base substitutions at rt204 (ATG --> AGT; T to G and G to T) lead to a methionine to serine change (YMDD --> YSDD). This novel DNA pol mutation was detected at month 18 of lamivudine treatment. In addition, this new variant had the rtL180M mutation and a 12 base pair deletion in the pre-S1 region between nucleotides 43-54. The YSDD mutation was still present 6 months after lamivudine discontinuation. In vitro transfection studies also confirmed that the YSDD strain is resistant to lamivudine. In conclusion, the results indicate that, in addition to a Met --> Val and Met --> Ile change in YMDD, a Met --> Ser change at rt204 (YMDD --> YSDD) associated with the rtL180M change can also emerge during lamivudine treatment, which confers lamivudine resistance in vivo and in vitro, leading to virological breakthrough and ALT increases.