Detection of Parvovirus 4 in Iranian patients with HBV, HCV, HIV mono-infection, HIV and HCV co-infection

Aim: In this study, we investigated the prevalence of PARV4 virus among the healthy population and four other groups of HBV infected, HCV infected, HIV infected and HIV/HCV co-infected individuals in Iran

Background: Parvovirus 4 [PARV4] was first discovered in 2005, in a hepatitis B virus-infected injecting drug user [IDU]. To date, the best evidence about PARV4 transmission is parenteral roots which comes from IDU individuals. It seems that the prevalence of the virus in the normal population is very low

Methods: A total of 613 patients, including chronic HCV [n=103], HBV [n=193], HIV [n=180] infected individuals, HIV/HCV [n=34] co-infected patients and 103 healthy controls, were studied by using nested-PCR and also real-time PCR techniques

Results: Of those 180 samples were positive for HIV RNA, co-infection of PARV4 was detected in 3 cases [1.66%]. All these three patients were male with the age of 28, 32 and 36 years [mean: 32]. No statistical differences were found between HIV positive group and the healthy individuals. [P>0.05] The result of PARV4 PCR was negative in all other samples and healthy controls as well

Conclusion: This study is the first to investigate the occurrence of PARV4 among these groups in Iran. The results show, that the virus is not significant in Iranian population, even in patients with blood born infections such as HCV, HBV or even HIV patients. Further studies in other areas and various groups are required

Protein-protein interaction analysis of Alzheimer`s disease and NAFLD based on systems biology methods unhide common ancestor pathways

Aim: Analysis reconstruction networks from two diseases, NAFLD and Alzheimer`s diseases and their relationship based on systems biology methods

Background: NAFLD and Alzheimer`s diseases are two complex diseases, with progressive prevalence and high cost for countries. There are some reports on relation and same spreading pathways of these two diseases. In addition, they have some similar risk factors, exclusively lifestyle such as feeding, exercises and so on. Therefore, systems biology approach can help to discover their relationship

Methods: DisGeNET and STRING databases were sources of disease genes and constructing networks. Three plugins of Cytoscape software, including ClusterONE, ClueGO and CluePedia, were used to analyze and cluster networks and enrichment of pathways. An R package used to define best centrality method. Finally, based on degree and Betweenness, hubs and bottleneck nodes were defined

Results: Common genes between NAFLD and Alzheimer`s disease were 190 genes that used construct a network with STRING database. The resulting network contained 182 nodes and 2591 edges and comprises from four clusters. Enrichment of these clusters separately lead to carbohydrate metabolism, long chain fatty acid and regulation of JAK-STAT and IL-17 signaling pathways, respectively. Also seven genes selected as hub-bottleneck include: IL6, AKT1, TP53, TNF, JUN, VEGFA and PPARG. Enrichment of these proteins and their first neighbors in network by OMIM database lead to diabetes and obesity as ancestors of NAFLD and AD

Conclusion: Systems biology methods, specifically PPI networks, can be useful for analyzing complicated related diseases. Finding Hub and bottleneck proteins should be the goal of drug designing and introducing disease markers

Prevalence of celiac disease in Iranian patients with rheumatologic disorders

Aim: Patients with Systemic Lupus Erythematosus [SLE], Rheumatoid Arthritis [RA], and Fibromyalgia [FM] may have underlying non-diagnosed celiac disease [CD]

Background: The aim of this study was to determine the prevalence of CD in patients with these underlying diseases in Iran

Methods: This cross-sectional study was performed among 300 consecutive patients with SLE, RA, and FM [each group 100 patients] since 2015 to 2017. The blood samples were collected and serum IgA anti-tissue trans-glutaminase [Anti-tTG] level was assessed for all patients. The seropositive patients underwent endoscopy and duodenal/jejunal biopsy according to the Marsh classification

Results: Out of 300 investigated patients with mean age of 41.2 years old, 92% of patients with SLE, RA and fibromyalgia were women. Among 100 patients with SLE, only 1 subject [1%], out of 100 patients with RA 3 subjects [3%], and none of the patients with fibromyalgia were seropositive for CD [with overall prevalence 1.4]. All four patients were female and categorized as Marsh III

Conclusion: The results of the study indicated that patients with lupus have the same prevalence, but subjects with RA had three times higher prevalence rate than normal population for CD. Therefore, CD investigation in these individuals can improve their quality of life

rapid and sensitive assay to identify HLA-DQ2/8 risk alleles for celiac disease using real-time PCR method

Aim: To perform a simple, rapid and sensitive Real-time PCR based SYBR Green method to determine the human leukocyte antigen [HLA]-DQ 2/8 alleles in celiac disease [CD] patients

Background: Many molecular techniques are available to determine the HLA-DQ2 and DQ8 alleles, but they are too expensive and have many steps that make them difficult to use

Methods: To determine the HLA-DQ 2/8 alleles we have developed a new real-time PCR assay, using SYBR Green technique with melting curve analysis on genomic DNA isolated from 75 CD patients and 94 healthy controls. The specific primers to examine HLADQA1* 05, HLA-DQB1*02 and HLA-DQB1*0302 alleles were used and results were compared with commercially available kits

Results: Using this method, the presence of HLA-DQ2 and HLA-DQ8 alleles were determined with sensitivity and specificity 80% and 100% respectively and compared to low resolution commercially available kits, the results of this method were more efficient. The frequency of DQ2 and DQ8 in patients was 76% and 29%, respectively and overall 96% of patients were carries DQ2 and/or DQ8 alleles

Conclusion: The result of this study showed that Real-time PCR using SYBR Green method with melting curve analysis has good efficiency to identify the HLA-DQ2/8 risk alleles

Small-scale risk assessment of transmission of parasites from wastewater treatment plant to downstream vegetable farms

Aim: The aim of the present study was to simultaneously investigate parasitic contamination of treated wastewater and downstream vegetable farms that are irrigated with treated sewage, during a year

Background: [Oo] Cysts and eggs of parasites are resistant to most of routine wastewater treatment process. Irrigation of vegetables farms with either treated wastewater or illegally use of raw wastewaters enhances the risk of contamination with enteric pathogens

Methods: The treated wastewater samples were taken after chlorination from a wastewater treatment plant located at the south of Tehran. In addition, 60 vegetable samples [5 samples from each farm] were collected from the selected downstream farms that routinely used treated wastewater for irrigation of crops. Parasitological tests were performed using Ziehl-Neelsen, conventional lugol's iodine staining and direct microscopical examination

Results: Parasites including free living larvae, eggs of Toxoascaris leonina, egg of Toxocara sp. Trichuris sp, Trichostrongylus sp and amoeboid trophozoite were seen in 5/12 [41.7%] of vegetable samples gathered during a year. There was no statistically significant correlation between the season and parasitic contamination of the vegetables [P= 1]. Furthermore, parasitic contamination was observed in 7/12 [53.8%] of treated wastewater samples. The correlation between season and parasitic contamination of treated wastewater was evaluated that the results showed a higher contamination of treated wastewater in spring and autumn [P<0.05].Fisher's exact test also showed that there was no significant correlation between parasitic contaminations of vegetable samples and treated wastewater according to seasonal change

Conclusion: The results showed parasites in both treated wastewater plant and downstream crops farms that suggests the public health importance of the quality of water resources that routinely used for irrigation of vegetable farms

rare presentation of simple renal cyst: gastrointestinal obstruction

Simple renal cysts are one of the most common lesions in elderly. These cysts are usually asymptomatic but when the size of these cysts increase, we would see symptoms such as hypertension, hematuria, flank pain or urinary obstruction. In this study, we explore a case of small bowel obstruction that presented with nausea, repeated vomiting that causes hematemesis, and a submucosal obstructive lesion that was seen in Esophagogastroduodenoscopy [EGD]. After endoscopic ultrasound [EUS] evaluation, we detected a large simple renal cyst and approved our diagnosis with CT scan. We planned a medical treatment for this patient that consist consuming small size meals, 5 to 6 times a day, and high calorie liquids in small volumes. We conclude that simple renal cyst can be one of the cause of extrinsic intestinal obstruction and EUS is affective for differentiation of intrinsic submucosal lesion from extrinsic compression

Up-regulation of miR-21, miR-25, miR-93, and miR-106b in gastric cancer

Background: Differential expression profile of microRNAs [miRNAs] could be a diagnosis signature for monitoring gastric cancer [GC] progression. In this study, we focus on the comparison of expression levels of miR-21, miR-25, miR-93, miR-106b, and miR-375 during the sequential pattern of GC development, including normal gastric, gastric dysplasia, and GC sample

Methods: We used SYBR Green-based quantitative-PCR to quantify miRNAs expression

Results: Our analysis revealed the increased expression levels of miR-21 [p = 0.034], miR-25 [p = 0.0003], miR-93 [p = 0.0406], and miR-106b [p = 0.023] in GC samples. In addition, GC patients with positive lymph node metastasis showed the up-regulation of miR-25, miR-93, and miR-106b [p < 0.05]

Conclusion: Our findings suggested that the expression of miR-21, miR-25, miR-93, and miR-106b altered in GC, and some of them may be further investigated as biomarkers for GC early detection and prognosis prediction

IgG4 associated cholangiopathy: diagnosis, treatment, and outcome

IgG4-associated cholangitis [IAC] is a subgroup of IgG4-related disease, which is more common in elderly men. IAC is frequently coincident with autoimmune pancreatitis [AIP]. However, some IAC cases do not have other organs involvement. The diagnosis of IAC is based on biochemical, radiological, and histological features. Among these, elevated serum levels of IgG4, extra- and intrahepatic biliary strictures [as visualized by cholangiography], lymphoplasmacytic infiltrations in the liver and bile duct tissue, and association with AIP are of key importance. IAC may mimic primary sclerosing cholangitis or cholangiocarcinoma [CC]. It is classically a corticosteroid-responsive condition and corticosteroid is regarded as the initial treatment of choice in this disease. However, relapse following corticosteroid withdrawal is a frequent event

Systematic review of pancreatic cancer epidemiology in Asia-Pacific Region: major patterns in GLOBACON 2012

Pancreatic cancer is one of the deadliest cancers with short-term survival rates. Trends for pancreatic cancer incidence and mortality varied considerably in the world. To date, the causes of pancreatic cancer are not known sufficiently, although certain risk factors have been identified such as, smoking, obesity, life style, diabetes mellitus, alcohol, dietary factors and chronic pancreatitis. Since there are no current screening recommendations for pancreatic cancer, primary prevention is very important. Therefore, up-to-date statistics on pancreatic cancer occurrence and outcome are essential for the primary prevention of this disease. Due to the lack of information on epidemiology of pancreatic cancer in most Asian countries, and limited of statistics and registration system in this area, we conducted a systematic review study to evaluate the most recent data concerning epidemiology of pancreatic cancer in Asia-Pacific region. In this review we focused on collected recent data on incidence, mortality, survival and risk factors of pancreatic cancer in this region. In addition, we reviewed and used the data of GLOBOCAN 2012 in this paper to complete the information as a source of compiling pancreatic cancer incidence and mortality rate

Economic burden made celiac disease an expensive and challenging condition for Iranian patients

Aim: The aim of this study was to estimate the economic burden of celiac disease [CD] in Iran

Background: The assessment of burden of CD has become an important primary or secondary outcome measure in clinical and epidemiologic studies

Methods: Information regarding medical costs and gluten free diet [GFD] costs were gathered using questionnaire and checklists offered to the selected patients with CD. The data included the direct medical cost [including Doctor Visit, hospitalization, clinical test examinations, endoscopies, etc.], GFD cost and loss productivity cost [as the indirect cost] for CD patient were estimated. The factors used for cost estimation included frequency of health resource utilization and gluten free diet basket. Purchasing Power Parity Dollar [PPP dollars] was used in order to make inter-country comparisons

Results: Total of 213 celiac patients entered to this study. The mean [standard deviation] of total cost per patient per year was 3377 [1853] PPP dollars. This total cost including direct medical cost, GFD costs and loss productivity cost per patients per year. Also the mean and standard deviation of medical cost and GFD cost were 195 [128] PPP dollars and 932 [734] PPP dollars respectively. The total costs of CD were significantly higher for male. Also GFD cost and total cost were higher for unmarried patients

Conclusion: In conclusion, our estimation of CD economic burden is indicating that CD patients face substantial expense that might not be affordable for a good number of these patients. The estimated economic burden may put these patients at high risk for dietary neglect resulting in increasing the risk of long term complications

[Evaluation of the survival time of the patients with colorectal cancer using exponential model]

Background: colorectal cancer is one of the important causes of death due to cancer worldwide. Survival time in this cancer should be controlled to decrease the risk of mortality. In this study survival time and factors that could affect it are evaluated

Materials and Methods: in the present study, the files of 446 patients afflicted with colorectal cancer who had referred to Taleghani Hospital, Tehran from 1985 to 2013 were chosen as the study group. The exponential model was used here, for the purpose of investigating the survival of patients with colorectal cancer and finding proper variables affecting this survival and longevity

Results: in this study, the subjects had the mean [standard deviation] survival time 4.52 [0.182] year. The patients' age at the time of diagnosis [P=0.002] and tumor size [P=0.032] were the only significant variables affecting the survival of patients in the exponential model. Sex, the family history of colorectal cancer, the tumor site, and body mass index had no significant effect on the survival time of the patients with colorectal cancer

Conclusions: the results of the study show that to improve the survival chance of the patients with colorectal cancer or decrease the mortality rate, due attention should be paid to the age of the patient at the time of diagnosis

Biomarkers in inflammatory bowel diseases: insight into diagnosis, prognosis and treatment

Inflammatory bowel disease [IBD] is a chronic disease of unknown etiology which mostly involves the intestine and requires a personalized approach for treatment. IBD represents a heterogeneous group of patients with inherently variable disease courses. Hence, the heterogeneity of patient populations may delay the diagnosis, clinical practice and initiation of appropriate treatment. Use of biomarkers for diagnosis and management of IBD is still necessary. Descriptions of the immunological pathway abnormalities in IBD improve assessment to identify the patient's disease status, and relative risk of progression to complicated disease behaviors, and this information may ultimately influence therapeutic decisions. In this study, we try to explain the role of biomarkers in early diagnosis, estimating prognosis, and target agents for correct managements of IBD's patients. This information might be important to provide insight into emerging panels of multiple IBD biomarkers and highlighting the essential role of personalizes panel for each patient

Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow

Aim: We describe the minimum requirements and a simplified method for isolation and characterization of mesenchymal stem cells [MSCs] from human bone marrow

Background: MSCs are well known adult stem cells present in many tissues such as adipocytes, chondrocytes, osteoblasts, and neurons. Many isolations and characterization methods have emerged to apply MSCs in the clinical applications, which many of them are expensive and time-consuming

Methods: MSC isolation was carried out from human bone marrow, and cultured in defined medium. Cultures were maintained at 370C in a humidified atmosphere containing 5% CO2 for 48h. The medium was exchanged every 3-4 days. Adherent cells were characterized according to main criteria defined by ISCT, such as differentiation capability to adipocyte and osteoblast using specific differentiation mediums; also, flow cytometry verified MSC specific markers

Results: Isolated MSCs had a fibroblastic-like appearance with adherent property to the culture plate. Differentiation function was proved with the formation of lipid drops and calcium oxalates on the differentiated MSCs and finally, purified MSCs from bone marrow were positive for cell surface markers, CD73, CD90, and CD105 while being negative for CD34 and CD45

Conclusion: These findings confirm that the represented method is capable of isolating MSCs from bone marrow with proven results according to all minimum criteria defined by the International Society for Cellular Therapy [ISCT]

Association between TNF-alpha rs1799964 and RAF1 rs1051208 MicroRNA binding site SNP and gastric cancer susceptibility in an Iranian population

Aim: The aim of this study was to find the relationship between rs1799964 in TNF-alpha gene as well as rs1051208 of RAF1 gene SNPs on GC in an Iranian population

Background: Gastric cancer [GC] is the second leading cause of cancer-related death worldwide after lung cancer. Tumor necrosis factor [TNF] is one of the most important factors in the pathogenesis of this cancer. Single nucleotide polymorphisms have a principle role in gene expression of TNF-alpha and miRNAs which may lead to gastric cancer

Methods: In a case-control study, we investigated the risk of GC in 198 Iranians. For this purpose, 5 mL of peripheral blood was collected in EDTA -containing tube and genomic DNA was isolated. Genotyping of SNPs was also performed by PCR-RFLP; to approve the outcome, 10% of genotyping results with RFLP were sequenced

Results: The comparison between case and control groups revealed a significant association between the rs1051208 C allele of RAF1 gene and GC [P = 0.04]. We did not observe any remarkable association between TNF-alpha -1031 in gastric cancer patients and the healthy control group

Conclusion: The results indicated that C allele in RAF1 gene plays a role in susceptibility to gastric cancer. Therefore, SNPs are among notable biomarkers for predicting susceptibility to dreadful diseases, especially cancers

molecular investigation of celiac disease

Celiac disease [CD] is an immune-mediated disorder resulting in nutrient malabsorption now thought to have a prevalence of 1:100 in the Iranian population.Symptoms of CD are included diarrhea, abdominal pain, steatorrhea, bloating, cramps, flatulence, weight loss, weakness and short stature. In addition to presenting symptoms, patients are also at increased risk of metabolic bone disease, lymphoma [enteropathy-associated with T-cell] and other malignancies in different parts of the body such as gastric, esophageal, bladder, breast and brain. There appears to be a strong genetic component to this disease. In this short review we provided the historical, clinical and genetic aspects of this disease and highlight numerous findings from recent molecular immunology studies

Fecal carriage of Escherichia coli and Klebsiella spp. as major reservoirs of clinically important resistance markers

Intestinal normal flora can become reservoirs of antibiotic resistance genes present among the strains responsible for nosocomial infections. It is suggested that gram negative intestinal bacterial flora have increased capacities to obtain antibiotic resistance genes and therefore can act as main reservoirs for transfer of resistance genes to other pathogenic bacteria. This study aimed to compare fecal carriage of clinically important resistance markers for more frequent members of enterobacteriacae between nondiarrheal and community associated diarrheal patients [control group] versus their counterparts from the patients with nosocomial infections [case group]. 261 stool and 190 clinical samples were collected from outpatient and hospitalized patients from 6 hospitals in Tehran, Iran. The samples were cultured on MacConkey agar plates and colonies were identified by standard biochemical methods. Antibiotic sensitivity testing of the isolates against 13 antibiotics was performed according to the CLSI guideline using the disk diffusion method. Among stool and clinical samples, more frequent identified enterobacteriaceae bacteria were included E. coli [58.99/ 3.15%], Klebsiella spp. [22.61/7.36%], and other members of enterobacteriaceae [8.86/1.06%], respectively. Overall, resistance against four of the main antibiotics [3[th] and 4[th] generation cephalosporins, gentamicin, imipenem, and ciprofloxacin] was significantly higher among the case group [50-75% versus 10-14%]. Analysis of these results showed similar dissemination of resistance phenotypes among the isolates from the control group in ranges of 1.5-7.6% and 4.4% for E. coli and Klebsiella spp., respectively. Our results suggested that the fecal carriage of resistant phenotypes related to the beta-lactam antibiotics in E. coli and Klebsiella spp. in compare to the clinical isolates is rapidly increasing. This may be caused by dissemination of beta-lactamase producing E. coli in the community from the hospitals. There were no significant correlations between the two groups of the samples, as the clinical samples had shown 3 to 7 folds excess resistance phenotypes. Surveillance studies of the resistance patterns among the samples from different regions will provide awareness about dissemination of these bacteria within the community as reservoirs of main resistance markers

Hydatic cyst prevalence in slaughtered animals, a neglected health problem

Lorestan and other part of Iran are one of the important endemic focuses for the major zoonotic parasitic diseases like cystic hydatid disease where several species of intermediate host are commonly infected with Echinococcus granulosus. Meat inspection records in a slaughtered house were used to determine the prevalence of Hydatidosis in sheep, cattle, and goats in Lorestan province [Aleshtar] located in South-West of Iran. The data were collected during a 5 year period from 2002 to 2006. A total of 40,431 animals [cattle 6993; sheep 14084; goats 19354] slaughtered in the 5-year period and overall 2885 [7.13%] lungs and 2885 [7.13%] livers and 1598 [3.95%] peritoneal cavity were contaminated by hydatid cyst. One thousand and eight hundred sixty eight [26.71%] out of 6993 of cattle, 2989/14084 [21.22%] of sheep and 2511/19354 [12.97%] of goats were infected by hydatid cyst in liver, lung and peritoneal cavity respectively. This study indicates that the highest prevalence of Hydatidosis was found in Aleshtar, Lorestan as compared to the rest of Iran [p<0.05]. The annual prevalence of lung condemnations due to hydatidosis was increased from 8.3, 5 and 1.3% in Sep 2002 to Sep 2003 to 20.5, 8 and 5.5% in Sep2005 to Sep2006 for cattle, sheep, and goats, respectively. An urgent attention is required to work on lack of awareness among farmers, lack of a policy to destroy the infected organs, prevention of access of dogs to raw offal's and other responsible factors

Imputation in missing not at random SNPs data using EM algorithm

The relation between single nucleotide polymorphisms [SNPs] and some diseases has been concerned by many researchers. Also the missing SNPs are quite common in genetic association studies. Hence, this article investigates the relation between existing SNPs in DNMT1 of human chromosome 19 with colorectal cancer. This article aims is to presents an imputation method for missing SNPs not at random. In this case-control study, 100 patients suffering from colorectal cancer consulting with the Research Institute for Gastroenterology and Liver Disease of Shahid Beheshti University of Medical Sciences were considered as the case group and 100 other patients consulting with the same research institute were considered as the control group and the genetic test was applied in order to identify the genotype of the 6 SNPs of the DNMT1 of chromosom 19 for all the patients under investigation. The obtained data were analyzed using logistic regression, then a fraction of the data was eliminated both at random and not at random and the imputation was done through the EM algorithm and the logistic regression coefficients variation before and after the imputation was compared. The results of this study implied that in both methods, at random and not at random missing SNPs, the estimation of the logistic regression coefficients after the imputation through EM algorithm has a greater correspondence to the results obtained from the complete data in comparison with the method of eliminating the missing values

efficacy and safety of peginterferon alpha-2a [pegasys] monotherapy in the treatment of chronic hepatitis C infected subjects with transfusion dependent thalassemia

Background and aims: interferon monotherapy is currently the only approved treatment for chronic hepatitis C [CHC] infection in transfusion dependent thalassemic patients, in whom ribavirin has limited use because of its hematologic complications. Our aim was to evaluate the efficacy and safety of pegylated Interferon monotherapy for the treatment of HCV infection in transfusion dependent thalassemic patients

Methods: the trial was a multicenteric, open label, single treatment prospective study of Peginterferon alfa-2 a [PEGASYS, 180 micg per week] for a period of 48 weeks. 32 subjects, 18 to 42 years old [mean +/- SD: 24.1 9.44 years], whose serum HCV RNA was positive and mean ALT remained greater than 1.5 times upper limit of normal were enrolled. A percutaneous liver biopsy was performed before treatment and all patients underwent monthly assessment of any adverse events and were monitored for serum ALT. Efficacy was assessed by measuring serum HCV RNA following 24 week treatment-free period . One patient missed follow up and another died due to a drug unrelated cause and 30 patients were evaluated

Results: liver biopsy showed mild fibrosis in 3 1.2%, moderate fibrosis in 53.1 % and cirrhosis in 15.6% of patients. Siderosis was severe in 16 patients [50%]. In 26 out of 30 patients [86.6%] HCV RNA was negative at the end of treatment [ETR response]. Data about 24 weeks post treatment was available in 23 patients, which showed a sustained virological response [SVR] of about 14/23 [60.8%]. Two patients had an elevated end of treatment serum ALT instead of negative HCV RNA but their ALT returned to normal assoon as the treatment stopped. These 2 patients were considered to have INF toxicity

Conclusion: our experience indicates that the cure of HCV-related liver disease in thalassemic patients is not an unrealistic aim and may be reached with Peginterferon alfa-2a monotherapy in a sizable portion of cases

Peginterferon alfa-2a [pegasys] and ribavirin in the treatment of chronic Hepatitis C

Background and aims: pegylation of interferon alfa-2a is a new modality for treatment of chronic hepatitis C. This clinical trial was conducted to evaluate the efficacy and safety of PEG IFN in combination with ribavirin in CHC patients

Methods: fifty-seven patients with HGV RNA in serum, persistently elevated ALT and chronic C hepatitis on liver biopsy enrolled to this study. The patients received PEG IFN 180 micg per week plus ribavirin 10- 15 mg/kg per day

Results: HCV RNA was negative in 37 patients [74%] after three months of beginning of study [EVR] and SVR occurred in 50% of all patients

Conclusion: peginterferon alfa-2a plus ribavirin is safe and effective in treatment of naive patients and relapses