Pediatric visceral leishmaniasis: a retrospective study to propose the diagnostic tests algorithm in southern Iran.

Leishmania infantum is the most common cause of visceral leishmaniasis (VL) in Iran, where mainly the patients are children under the age of 5 years. Timely, less invasive, and accurate diagnosis and proper treatment of the disease are necessary. This retrospective study aimed to search for a less invasive but robust algorithm on VL diagnostic tests in children. Four hundred and fifteen patients with clinical suspicion of VL, 50 healthy children from VL endemic areas, 46 healthy individuals from non-endemic VL areas, and 47 non-VL diseases were tested using three diagnostic tests: indirect immunofluorescent antibody test (IFAT), rK39-rapid diagnostic test (rK39-RDT), and quantitative PCR (qPCR). One hundred and two suspected VL cases were positive in at least one test and were cured after receiving appropriate treatment. Of these 102 VL patients, 94 were positive in qPCR, 84 in IFAT, and 79 in rK39-RDT. None of the tests detected all the patients, but overall, qPCR is capable of detecting more VL patients than serological tests, i.e., 92.2%, compared to IFAT, 82.4%, and rK39, 77.5%. There was only a significant difference between the sensitivity of qPCR and rK39-RDT (p = 0.024). The specificity was 100% for qPCR and IFAT (≥128) and 98.6% for rK39-RDT. qPCR alone is capable of detecting most of the VL-suspected children. Serological tests like IFAT and rk39-RDT are recommended to increase the overall sensitivity of detection in patients with a negative molecular test. Combining qPCR with a serological test (IFAT or rK39-RDT) can help diagnose 98% of VL. In laboratories without molecular facilities, we recommend testing with the combination of rK39-RDT and IFAT yielding a combined sensitivity of 93.1% equivalent to that of qPCR in our study.

Interleukin-17A genetic variants can confer resistance to brucellosis in Iranian population.

On the subject of brucellosis, it seems that Th1/Th2 cytokines balance may be involved in the resistance or susceptibility to Brucella infection. In this respect, Th1 cytokines confer resistance, while Th2 cytokines predispose brucellosis. It is also clarified that IL-17 is required for the induction of IFN-γ and IL-12 in macrophages and dendritic cells. Then, it seems that IL-17 can affect the induction of Th1 immunity which is necessary for controlling Brucella. In the present study, we tried to investigate probable relationship between IL-17A genetic variants and susceptibility to the human brucellosis. One hundred and seventy six patients with brucellosis and 84 healthy animal husbandmen, who consumed contaminated raw milk and dairy products from animals with brucellosis, were included in this study. All individuals were genotyped for 9 single nucleotide polymorphisms (SNPs) (rs4711998AG, rs8193036CT, rs3819024AG, rs2275913AG, rs3819025AG, rs8193038AG, rs3804513AT, rs1974226AG and rs3748067AG) being selected by using NCBI SNP database and literature using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The distribution of IL-17 rs4711998, rs8193038, rs3748067 AA genotypes and AAGAA haplotype were significantly more frequent in the patients than in the controls (P=0.008, 0.0019, 0.003 and 0.002, respectively) while IL-17 genotypes rs3819024GG and rs3819025AA were more frequent in the controls than the patients (P=0.001 and 0.0035, respectively). Based on the results, IL-17 rs4711998, rs8193038, rs3748067 AA genotypes and AAGAA haplotype could be considered as susceptibility factors for brucellosis while the inheritance of IL-17 rs3819024GG and rs3819025AA genotypes might be resistance factors against the disease.

Association of interleukin-18 gene variants with susceptibility to visceral leishmaniasis in Iranian population.

Host resistance to Leishmania infection is mediated by cellular immune responses leading to macrophage activation and parasite killing. Interleukin-18 (IL-18) known as interferon-γ (IFN-γ) inducing factor, stimulates IFN-γ production by T cells. Taking into account the important role of IL-18 in the defense against visceral leishmaniasis (VL) and the known effect of IL-18 gene polymorphisms on its production, the aim of this study was to investigate the probable relationship between IL-18 gene polymorphisms and the susceptibility to VL. The study groups included 118 pediatric patients who suffered from VL and 156 non-relative healthy people as the controls from the same endemic area. IL-18 gene polymorphisms at the positions -656 G/T, -137 G/C and +105A/C (codon 35/3) were analyzed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). The results showed that the frequency of T allele at the position -656 was significantly higher in the controls, compared with that in the patients (P = 0.047), but it couldn't tolerate Bonferroni correction. Regarding the IL-18 genotypes, there was no significant difference between the patients and controls. Although the frequencies of ATG single haplotype and AGG/ATG double haplotype were significantly higher in the controls (P = 0.043) and the patients (P = 0.044), respectively, the two P values couldn't tolerate Bonferroni correction. Furthermore, a strong linkage disequilibrium was observed among the -656, -137 and +105 single nucleotide polymorphisms of IL-18 gene (all Ps < 0.001). In conclusion, this study suggests that the inheritance of T allele at the position -656 may be considered as a genetic factor for resistance to VL.

IL-1ß (-511T/C) gene polymorphism not IL-1ß (+3953T/C) and LT-α (+252A/G) gene variants confers susceptibility to visceral leishmaniasis.

Lymphotoxin-α (LT-α) and interleukin-1beta (IL-1ß) are proinflammatory cytokines playing important roles in immunity against Leishmania infection and the outcome of the disease. As cytokine productions are under the genetic control, this study tried to find any probable relationship between these cytokine gene polymorphisms and the susceptibility to visceral leishmaniasis in Iranian pediatric patients. Ninety-five pediatric patients involved with visceral leishmaniasis and 128 non-relative healthy people, from the same area as the patients, were genotyped for LT-α (+252A/G) and IL-1ß (+3953T/C and -511T/C) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was not found any significant differences in allele and genotype frequencies of LT-α (+252A/G) and IL-1ß (+3953) among the study groups. However, the frequency of IL-1ß -511TT genotype was higher in the controls (P = 0.0004) while the frequency of IL-1ß -511CC genotype and C allele were higher in the patients (P = 0.008 and P = 0.00006, respectively). Furthermore, IL-1ß CC (-511/+3953) haplotype was more frequent in VL patients compared with the controls (P = 0.0002) and the distribution of TT haplotype was higher in the controls compared with the patients (P = 0.003). In conclusion, based on the results, IL-1ß -511C allele, CC genotype and CC (-511/+3953) haplotype could be considered as the susceptibility factors for visceral leishmaniasis while IL-1ß -511TT genotype, T allele and TT haplotype (-511/+3953) might be counted as the influential factors for resistance to the disease.

Livestock infected with Leishmania spp. in southern Iran.

BACKGROUND: Query ID="Q1" Text="Graphical abstract: As per journal requirements, graphical abstract is necessary. Kindly check and provide the same."The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods. METHODS: Blood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing. RESULTS: Of the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5-46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9-58.9) livestock. The identified Leishmania spp. were L. infantum and L. major. CONCLUSIONS: The findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection.

Construction of AAV-rat-IL4 and Evaluation of its Modulating Effect on Aß (1-42)-Induced Proinflammatory Cytokines in Primary Microglia and the B92 Cell Line by Quantitative PCR Assay.

BACKGROUND: Interleukin-4 (IL-4), as the most prominent anti-inflammatory cytokine, plays an important role in modulating microglial activation and inflammatory responses in Alzheimer's disease (AD), a chronic inflammatory disorder. OBJECTIVES: The current study aimed to develop a new recombinant Adeno-associated viral (rAAV) vector that delivers IL-4 and then assess the counterbalancing effect of the new construct along with recombinant IL-4 (rIL-4) protein in in-vitro models of AD. MATERIALS AND METHODS: The rAAV-IL4 was originally prepared and then employed along with rIL-4 protein to counter Amyloid ß (1-42)-induced proinflammatory cytokines in a primary microglia cell culture and the B92 rat microglia continuous cell line, using relative Real-Time PCR assay. RESULTS: Aß (1-42) stimulated the production of the proinflammatory cytokines IL6, IL1ß, TNFα, and IL18 in both the primary microglia cell culture and the B92 cell line. Both the rAAV-IL4 construct and the rIL-4 protein were found to inhibit production of the most important Aß (1-42)-induced proinflammatory cytokine mRNAs in the two types of cells with different patterns. CONCLUSIONS: It seems that the new construct can serve as an appropriate option in the modulation of Aß-induced proinflammatory cytokine gene expression and microglia activation in patients affected by AD.

Seroprevalence of Hepatitis A and E Virus Infections Among Healthy Population in Shiraz, Southern Iran.

BACKGROUND: Enterically-transmitted acute viral hepatitis is caused predominantly by hepatitis A virus (HAV) and hepatitis E virus (HEV). The prevalence of HEV and HAV infections varies in different geographical regions. OBJECTIVES: This study was conducted to determine the prevalence of HEV and HAV infections among Iranian healthy individuals in southern Iran. PATIENTS AND METHODS: Totally, 1030 samples were collected from healthy subjects in schools, those referred to tertiary outpatient clinics and health centers in Shiraz between November 2011 and May 2012. Their ages ranged between six months and 95 years. The presence of total anti-HAV and anti-HEV immunoglobulin M (IgM) in plasma was assessed by ELISA. RESULTS: The results showed that 66.2% and 0.6% of the general population in this area were positive for total anti-HAV and IgM antibodies by ELISA, respectively. As seen, 13.4% and 0.9% were positive for total anti-HEV and IgM antibodies, respectively. The difference in total anti-HAV and anti-HEV antibodies was significant among the age groups (P < 0.001). CONCLUSIONS: This study showed that the prevalence rates of HAV and HEV antibodies were positively correlated with age. The results demonstrated that the infection with these two viruses in the region was high and some high-risk individuals including females at child-bearing age were more susceptible. HAV vaccination could be recommended for antibody-negative adults.

The incidence of epstein-barr virus primary infection among suspected patients referred to namazi hospital of shiraz, iran.

BACKGROUND: Many children become infected with Epstein-Barr virus (EBV) during their childhood. Since the clinical profile of EBV primary infection is challenging, it is important to use the best diagnostic clinical means. Detection of IgM against viral capsid antigen (VCA) by ELISA has been shown to be a reliable method. OBJECTIVES: This study was conducted to demonstrate the incidence of EBV primary infection, among suspected patients referred to Namazi hospital, Shiraz, Iran. PATIENTS AND METHODS: The sample included 346 patients with an age range of 0 to 20 years (6.31 ± 4.66: 10.97 years). A volume of 5 mL of blood was collected from each case. The patients were divided to four age groups. The sera were tested for the presence of VCA-IgM by commercially available Anti-EBV-VCA ELISA kit. RESULTS: The results indicated that 104 (30.0%) of the patients were EBV VCA IgM positive, with no significant difference in the incidence of EBV primary infection between males and females. However, the incidence of infection was significantly different between age group I (0 - 5 years) and III (11 - 15 years), and also between age group I (0 - 5 years) and IV (16 - 20 years) (P < 0.05). CONCLUSIONS: Considering the results, accurate and on time diagnosis of EBV primary infection in both children and adolescents will help prevent unnecessary hospitalization, medication and incorrect medical decisions. In addition, this will decrease further treatment costs and related medical procedures.

Diagnosing of herpes simplex virus infections in suspected patients using real-time PCR.

BACKGROUND: Herpes simplex virus infections are very common worldwide. The virus can cause infection in various body parts, especially eyes and the nervous system. Therefore, an early diagnosis and highly sensitive method is very helpful. OBJECTIVES: The present study sought to investigate the efficiency of Real-time TaqMan probe PCR in the diagnosis of HSV infection in suspected patients. PATIENTS AND METHODS: In this study, 1566 patients with suspected HSV infections were enrolled. They aged 17 days to 96 years. The collected specimens were classified into four groups; cerebrospinal fluid (CSF) from HSE suspected individuals, samples from eye epithelial scraping, tear fluid or aqueous humor from herpes simplex keratitis (HSK) suspected patients, plasma of immune compromised patients and mucocutaneous collected samples from different body parts. The samples were analyzed by Real-time PCR assays. RESULTS: In total, 44 (5.6%), 118 (26.8%), 23 (11.7%), 13 (44.8%) and 65 (45.5%) of 791 HSE, 407 HSK, 29 skin HSV, 143 oropharyngeal suspected patients and 196 patients with systemic HSV infection HSV had positive results by Real-time PCR assays, respectively. CONCLUSIONS: Real-time PCR assay, due to its high sensitivity and specificity, can help in early diagnosis and more effective treatment for patients. Also, it requires shorter hospital stay and promotes patients' survival.

Hepatitis B virus DNA level Among the Seropositive Afghan Immigrants, Southern Iran.

BACKGROUND: Diagnosis and control programs for infectious diseases among immigrants are the most important aspects of epidemiological studies for both origin and destination countries. Data about hepatitis B virus (HBV) infection among the Afghan immigrants in Iran is limited. OBJECTIVES: To the best of HBV treatment and prevention in Afghan immigrants in Iran, the present study was conducted to determine the virus DNA level, and the frequency of respective hepatitis B risk factors among the respective seropositive patients in Fars province, southern Iran. PATIENTS AND METHODS: A total of 64 HBsAg positive Afghan immigrants including 47 (73.4%) men and 17 (26.6%) women, with ages ranging between 15 and 74 years (mean ± standard deviation: 37.69 ± 15.02 years) participated in this study. From those, whole blood sample were collected and DNAs were extracted from the sera and analyzed by TaqMan real-time PCR assay with a set of primers and probe amplified core protein region of HBV genome. RESULTS: HBV DNA was detected in a total of 51/64 (79.7 %) serum samples; 37 (72.5%) male and 14 (27.5%) female. The copy number of HBV DNA ranged from 5 × 10(2) to 8.49 × 10(8) copies/mL in the serum samples; median 3.8 × 10(4) copies/mL. Demographic data and risk factors were also evaluated. The comparison of viral loads between the age groups and sex indicated no significant correlation (P > 0.05). However, the serum HBV DNA level significantly decreased in the treated patient group (P = 0.03). There was no significant difference in medicine usage between the two sexes in the study population (P > 0.05). CONCLUSIONS: Considering the results, determining the HBV DNA load and evaluation of treatment response can help to reduce the costs of diagnosis and treatment procedures in such patients, as well as, decreasing the risk of HBV transmission in immigrant Afghan population. Moreover, HBV screening strategies in country border entrances among immigrant should be performed. Moreover, free vaccination and treatment programs, and improving the level of HBV knowledge among Afghan immigrants in Iran is highly recommended.

Determining hepatitis C virus genotype distribution among high-risk groups in Iran using real-time PCR.

AIM: To assess hepatitis C virus (HCV) genotype patterns among high-risk Iranian groups, using real-time RT-PCR. METHODS: In this study, we evaluated the distribution of different HCV genotypes among injection drug users and other high-risk groups over a 4-year period (from 2009 to 2012) using real-time polymerase chain reaction (PCR). Sera from 888 HCV-infected patients residing in southern and southwest Iran were genotyped using real-time PCR with common primers and specific probes. These patients were grouped into distinct exposure categories. Illicit drug users constituted the primary group and were further evaluated for HCV genotype distribution and parameters such as age range. RESULTS: Of the examined HCV-infected patients, 62% were substance abusers, although the route of transmission could not be determined in approximately 30% of these patients. HCV genotyping revealed that Gt1 was the most prevalent genotype among the drug users as well as among patients with thalassemia, hemophilia, solid organ recipients and those on hemodialysis. Mixed infections were only seen in addict groups, where Gt2 genotype was also found. The highest frequencies in HCV-positive addict patients were observed in the 31-40 age group. Our research also showed that the addiction age has increased, whereas the addiction rate has dropped in this region. Most illicit drug users had more than one risk factor such as tattoo and/or a history of imprisonment. CONCLUSION: This study revealed that the most common HCV-infection route and HCV-genotype in southern and southwest Iran was illicit drug abuse and Gt1, respectively.

Interleukin-8 but not interleukin-6 variant may affect susceptibility to brucellosis.

BACKGROUND: Increased levels of interleukin-8 (IL-8) and interleukin-6 (IL-6) in acute human brucellosis have been reported. Previous studies have shown that the production and level of IL-6 and IL-8 cytokines are associated with the polymorphism of the encoding genes. OBJECTIVE: To investigate the probable association between IL-6 (-174 C/G) and IL-8 (-251 A/T) gene polymorphisms and susceptibility/resistance to brucellosis. METHODS: The patient group included 196 patients suffering from Brucella infection and the control group consisted of 82 healthy animal husbandmen from the same geographical area. IL-8 (-251 A/C) and IL-6 (-174 C/G) gene polymorphisms were analyzed by PCR-RFLP and Allele Specific PCR (AS-PCR) respectively. RESULTS: The frequency of -251 IL-8 AA genotype was significantly lower in the controls compared with that of the patients (p=0.0051), while the frequencies of other genotypes (AT and TT) and alleles (A and T) were not significantly different among the participants. No association was found between IL-6 (-174 C/G) polymorphism and brucellosis. CONCLUSION: This study indicates that the IL-8 -251 AA genotype may be considered as a genetic susceptibility factor for brucellosis.