Semi-nested PCR Method Research Articles

Prevalence of Torque Teno Virus, Hepatitis and Cytomegalovirus Infection in Iranian Patients With Different Types of leukemia

Background and Objectives: Torque teno virus (TTV) infection can be seen in different types of leukemia and may affect clinical outcomes. This study aims to investigate the prevalence of TTV, hepatitis B and C virus, and cytomegalovirus (CMV) in Iranian patients with different types of leukemia. Methods: In this descriptive cross-sectional study, 95 patients with different types of leukemia participated. The molecular prevalence of TTV infection was evaluated using the semi-nested PCR method. The prevalence of HBs-Ag and HCV-Ab viruses was measured by the ELISA method. Also, the prevalence of active CMV infection was investigated by the semi-quantitative antigenemia test. The data analysis was done in SPSS software version 12 using logistic regression analysis, chi-square test, and Fisher’s exact test. Results: The TTV infection was found in 40 out of 95 patients (42.1%). The presence of HBs-Ag was reported in 27 out of 95 patients (28.4%). HCV-Ab was found in 18 out of 69 patients (26.1%), and active CMV infection was found in 11 out of 69 (15.9%). Among 40 leukemia patients with TTV infection, 15 (37.5%) had hepatitis B, 14 (35%) had hepatitis C, and 7 (17.5%) had CMV infection. Conclusion: The high prevalence of single or comorbid TTV infection among Iranian patients with leukemia emphasizes the importance of this virus in causing or aggravating the clinical complications observed in these patients.

Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats.

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10-8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured.

Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera Aspergillus and Fusarium. Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Seminested assays specific to Mucorales (V4/V5 regions of 18S rDNA), Aspergillus (mitochondrial tRNA), and Fusarium (internal transcribed spacer [ITS]/28A regions of DNA) were compared with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology. Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus level for Aspergillus and Fusarium. When compared with histopathology, sensitivity and specificity of seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.

A semi-nested PCR method with increased sensitivity for the specific, direct detection of Salmonella enterica strains in poultry ectoparasites.

The darkling beetle, Alphitobius diaperinus, and the poultry red mite, Dermanysuss gallinae are among the most common pests of poultry farms. Both pests can be carriers and reservoirs of various pathogens including zoonotic ones like Salmonella. Salmonellosis is one of the most common foodborne diseases reported in the EU. We developed a semi-nested PCR method for the direct detection of Salmonella enterica. When testing the specificity of the novel PCR, we successfully detected various S. enterica strains, whereas Escherichia coli and Citrobacter strains gave negative results. The authenticity of the PCR products was confirmed by DNA sequencing. The sensitivity of the semi-nested PCR was tested on serial dilution of bacterial cultures and extracted DNA. We found our new method more sensitive than the previous PCRs. We also screened ectoparasite samples, collected from a poultry farm in Hungary, and three out of the eight samples were positive for S. Enteritidis. This novel PCR seems suitable for the detection of S. enterica strains in poultry ectoparasites without the need of sample pre-enrichment.

Phylogenetic analysis of Ehrlichia species isolated from horses and dogs in Iran

This study aimed to determine the prevalence and phylogenetic analysis of Ehrlichia spp. in horses and dogs in Iran. Blood samples were collected from 400 animals, including 200 horses and 200 dogs, from five different provinces in Iran. Polymerase chain reaction (PCR) was used to detect Ehrlichia spp. based on amplification of the 16S rRNA gene. The semi-nested PCR method was used to amplify the dsb, TRP36, and gltA genes. The results showed that 4.5 % of the samples (3 % horses and 6 % dogs) were positive for Ehrlichia sp. The highest prevalence was observed in Kerman and Khuzestan, while the lowest was found in West Azerbaijan, Golestan, and Mazandaran. The study suggests that the populations of dogs and horses in the country should be considered important factors in the epidemiology of ehrlichiosis. Phylogenetic analysis based on the dsb and TRP36 genes revealed that the prevalent species were E. canis and E. ruminantium.

Genotyping of G. duodenalis in the People Referred to Health Centers of Semnan City.

Giardia duodenalis (G. duodenalis), is one of the major causes of gastrointestinal disorders worldwide, infecting the small intestine of humans and animals. Based on the genetic characteristics of the parasite, eight genotypes (A to H) have been identified in clinical samples. The main purpose of the present study was to find the genetic diversity of Giardia in people referred to health centers in Semnan, Iran, using PCR. Totally, 300 stool samples were collected from people referred to health centers in Semnan. The stool samples were first examined using the microscopic method (direct method and Lugol staining), and the samples were checked with trichrome staining. After DNA extraction, the GDH gene of positive samples was amplified by the semi-nested PCR method. The genotype of positive samples was determined by the sequencing method. Out of 300 samples, only 20 (6.66%) samples were found to be positive in the microscopic examination of the stool. In the PCR test, only 13 (4.33%) of the samples were positive. According to the multiple alignment results, it was found that the isolates belonged to AII, BIII, and BIV genotypes. Most of which are related to people without clinical symptoms of diarrhea. Identification of AII, BIV, and BIII genotypes indicates the anthroponotic and anthropozoonotic transmission cycle of Giardia infection in Semnan.

Genotyping of G. duodenalis in the People Referred to Health Centers of Semnan City

Giardia duodenalis, is one of the main causes of gastrointestinal disorders worldwide, which infects the small intestine of humans and animals. Based on the genetic characteristics of the parasite, eight genotypes (A to H) have been identified in clinical samples. The main purpose of the present study was to find the genetic diversity of Giardia in people referred to health centers in Semnan city using PCR. Totally, 300 stool samples were collected from people who were referred to health centers in Semnan city. The stool samples were first examined using the microscopic method (direct method and Lugol staining) and the samples were checked with trichrome staining. After DNA extraction, the GDH gene of positive samples was amplified by semi-nested PCR method. The genotype of positive samples was determined by the sequencing method. Out of 300 samples, only 20 (6.66%) were positive in the microscopic examination of the stool. In the PCR test, only 13 (4.33%) of the samples were positive. According to the multiple alignment results, it was found that the isolates belong to AII, BIII, and BIV genotypes. Most of which are related to people without clinical symptoms of diarrhea. Identification of AII, BIV, and BIII genotypes indicates the anthroponotic and anthropozoonotic transmission cycle of Giardia infection in Semnan city.

Molecular identification of Leishmania RNA virus in cutaneous leishmaniasis patients and rodent reservoirs in Isfahan province, Iran.

Leishmania RNA virus (LRV) is a double-strand RNA virus that was first detected in members of the Leishmania viannia in the New World. The present study aimed to investigate the presence of LRV in the Leishmania species isolated from cutaneous leishmaniasis (CL) patients and rodents as reservoirs in Isfahan province an old zoonotic CL focus, center of Iran. Totally, 85 samples were collected from CL patients (n=80) and rodent reservoirs (n=5) from different regions of Isfahan province. Species identification was determined using the PCR-RFLP method. Viral dsRNA was extracted and for observation of 5.3kb dsRNA on an agarose gel. The presence of LRV was surveyed using the Semi-nested PCR method. For phylogenetic analyzes, 6 samples of 13 isolates were sequenced and a phylogenetic tree was drawn by MEGA7 version 7.0.26. Of 80 Leishmania isolates recovered from the patients with CL, 79 and only one were identified as L. major and L. tropica, respectively. Also, the PCR assays detected four L. major and one L. turanica in five assessed Rhombomys opimus as the rodent reservoirs. LRV was detected only in Leishmania species isolated from 13 species of 85 (15.3%) CL including (L. major, n=12) and (L. tropica, n=1). Phylogenetic analysis showed that they were belonged to LRV2 and had the highest similarity with Iranian reference LRV2 in GenBank. Our results showed that the LRV2 was present in cutaneous Leishmania species in Isfahan province is the most historical and touristic province of Iran. In the study LRV was not reported from rodent reservoirs, it may be due to the small sample size. Phylogenetic analysis of current sequences demonstrated that these isolates belong to the registered LRV2 of the Old World.

Gurltia paralysans: a neglected parasite of domestic cats

Gurltia paralysans (order Strongylida; family Angiostrongylidae) is a metastrongyloid parasite that causes chronic meningomyelitis in domestic cats in South America. The geographic distribution of G. paralysans includes rural and peri-urban areas of Chile and Argentina. However, feline gurltiosis has recently been reported in other South American countries, including Uruguay, Colombia, and Brazil, and was also recently reported in Tenerife, Canary Islands (Spain). Feline gurltiosis is increasingly detected in domestic cats in southern Chile and its apparent geographic range is also increasing, together with an awareness of the disease among veterinarians. The life cycle of the parasite is unknown, but is probably indirect, involving gastropods as the intermediate host, as in other metastrongyloid nematode species. The clinical signs of G. paralysans infection include progressive pelvic limb ataxia, paraparesis, paraplegia, faecal or urinary incontinence, and/or tail paralysis. A definitive diagnosis of feline gurltiosis is still challenging and only possible with necropsy, when adult G. paralysans nematodes are detected within the spinal cord vasculature, together with macroscopic lesions, and characteristic morphological features. A semi-nested PCR method was recently developed for the in vivo diagnosis of this neglected parasite. Current treatment options include macrocyclic lactones and mylbemicn oxime, but the prognosis is poor in severe cases. In this article, we review G. paralysans infection in cats, focusing on the diagnosis shortcomings and the future directions of research into its biology and the associated neurological disease. Comprehensive updates on the epidemiology and clinical features, diagnosis, treatment, and prevention of feline gurltiosis are provided.

Frequency and Molecular Identification of Cryptosporidium Species among Immunocompromised Patients Referred to Hos-pitals, Central Iran, 2015-16

The purpose of this study was to determine the prevalence and genotype of Cryptosporidium spp. in different groups of immunocompromised patients admitted to the referral hospitals in center of Iran during 2015-2016. This cross-sectional study was conducted on 346 immunocompromised patients (HIV+/AIDS, Lymphoma, Leukemia and organ transplants) in referred hospitals from central parts of Iran including Isfahan, Markazi, Yazd and Chaharmahale Bakhtiari provinces. Stool samples were analyzed for Cryptosporidium species, modified Ziehl-Neelsen staining techniques followed by the semi-nested PCR and DNA sequencing methods. The total rate of Cryptosporidium spp. was 3.46% (12/346) in the patients, however, the prevalence of the parasite, was 4.6% (4/87) in HIV+/AIDS patients, 3.6% (6/168) in patients with blood malignancy and 2.1% (2/91) in organ transplant recipients. The SSU rRNA gene of Cryptosporidium spp. in all microscopic-positive samples was effectively amplified by the semi-nested PCR and DNA sequences, exposed the existence of two Cryptosporidium species, including C. hominis 91.6% (11/12) and C. parvum 8.3% (1/12). The predominance of C. hominis in the present study may be certifies the importance of anthroponotic transmission of cryptosporidiosis in center of Iran.

A validated semi-nested PCR for rapid detection of scale drop disease virus (SDDV) in Asian sea bass (Lates calcarifer)

Scale drop diseases virus (SDDV), a newly characterized virus of farmed Asian sea bass (Lates calcarifer), has been reported in several countries in Southeast Asia. However, no fully validated detection method is publicly available for disease diagnosis and surveillance. Here, we described a newly developed semi-nested PCR (snPCR) method for detection of the virus from field samples. The designed primers targeting a gene encoding ATPase generated amplicons of 738 bp and 412 bp in the first and second step PCR, respectively. The established protocol could detect down to 100 viral copies/μL template and was 100-fold more sensitive than single step PCR. A Specificity test against extracted DNA from ten bacterial pathogens, tissues from viral infected specimens and fish host revealed no cross amplification. The SDDV snPCR method could detect the virus from all clinical samples showing symptoms of scale drop disease (n = 25) and all samples from outbreaks of an unknown disease (n = 6) whereas all clinically healthy fish sea bass (n = 161) and grouper (n = 45) collected from different provinces tested negative. The newly established protocol might be useful for Asian sea bass farming countries to initiate disease diagnosis and surveillance.

High frequency and prognostic value of MYD88 L265P mutation in diffuse large B-cell lymphoma with R-CHOP treatment.

The aim of this study was to analyze the prevalence and prognostic value of myeloid differentiation factor 88 (MYD88) L265P in diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). We assessed the MYD88 L265P mutation using an allele-specific semi-nested polymerase chain reaction method in 53 DLBCL patients treated with R-CHOP. The MYD88 L265P mutation was detected in 16 of 53 DLBCL (30.19%) samples from patients treated with R-CHOP. Age and location were statistically significantly associated with MYD88 L265P (P=0.025, 0.033, respectively), while treatment response and tumor recurrence were not. Univariate analysis showed that B symptoms (P=0.004) and Ki-67 (P=0.03) were significantly associated with progression-free survival (PFS), while MYD88 L265P showed no significant association with overall survival and PFS. Multivariate analysis showed that B symptoms were significantly associated with PFS. Our study suggests that the prognostic value of MYD88 L265P in DLBCL patients with R-CHOP requires further research.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

Using EGFP as a reporter to confirm the function of phytoene desaturase promoter in Duanliella bardawil

Phytoene desaturase (PDS) is an upstream key enzyme of carotenoid metabolic pathway in Dunaliella bardawil. This research attempts to clone the Pds gene and its promoter and terminator from D. bardawil and identify the promoter activity. Using LA-PCR based genomic walking approach and semi-nested PCR method, the DbPds gene was cloned and its structure was analyzed. 5′ RACE technique was used to determine its transcriptional start site. The bioinformatics analysis hypothesized that DbPds is one of the isoenzyme genes in D. bardawil. The full length of DbPds gene isolated included 8113bp coding region (from ATG to TAA), 3010bp upstream sequence and 1555 bp downstream sequence. By constructing the DbPds promoter-EGFP-DbPds terminator exogenous gene expression cassette to drive EGFP expression in D. bardawil, the activities of promoter and terminator of DbPds upstream and downstream sequence separated in this experiment were confirmed. Online promoter analysis tool PlantPAN was utilized to analyze the potential related osmotic stress-regulated elements in DbPds promoter. The result suggested that DbPds promoter does not contain any related elements about salt regulation. Furthermore, through in vivo detection of the responsive transcriptional level of DbPds genes under different salt concentrations, it was found that salt stress could not apparently affect DbPds expression, indicating that DbPds genes does not participate in the response to salt stress.

Adaptive immune response in symptomatic and asymptomatic enteric protozoal infection: evidence for a determining role of parasite genetic heterogeneity in host immunity to human giardiasis

The genetic basis of the ultimate clinical outcomes of human giardiasis has been the subject of numerous investigations. We previously demonstrated roles for both host and parasite factors in determining the outcome of enteric infection in a murine model of Giardia duodenalis infection. In the current study, fecal and serum specimens from healthy controls and human subjects infected with the intestinal parasite G. duodenalis were assessed. Using a semi-nested PCR method, clinical isolates were genetically characterized based on the gdh and tpi loci, and the phylogenetic trees were constructed. Using a sandwich ELISA method, the serum levels of representative TH1 and TH2 cytokines were measured in infected human subjects and healthy controls. Here we showed that symptomatic human giardiasis was characterized by significantly elevated serum levels of the TH1 cytokine IFN-γ compared to healthy controls, whereas asymptomatic human subjects and healthy controls had comparable levels of serum IFN-γ. Further analyses showed that human subjects infected with G. duodenalis genotype AI had significantly elevated levels of serum IFN-γ and IL-10, but not IL-5, whereas human subjects infected with AII had similar levels of those cytokines compared to healthy controls. These data demonstrate roles for both host and parasite factors in the determination of the outcome of enteric infections and may further broaden our understanding of host-parasite interaction during enteric protozoal infections.

Association Between TT Virus Infection and Cirrhosis in Liver Transplant Patients.

Background:Cirrhosis is one of the most severe liver complications, with multiple etiologies. The torque teno virus (TTV), also known as transfusion transmitted virus, which has a high incidence in the world population, is one of the possible increasing risk factors in patients with idiopathic fulminant hepatitis and cryptogenic cirrhosis.Objectives:The aim of this study was to evaluate solitary and co-infection with TTV, in patients with cryptogenic and determined cause of cirrhosis.Patients and Methods:In this cross-sectional study, 200 liver transplant patients were consecutively recruited between years 2007 and 2011. Patients were classified, based on recognition of the etiology of cirrhosis to determined (n = 81) and cryptogenic (n = 119) patient groups. The existence of TTV infection was analyzed, using a semi-nested polymerase chain reaction method. The presence of hepatitis B virus (HBV) infective markers, including HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis B core antibody (HBcAb), and hepatitis B e antibody (HBeAb), was evaluated using qualitative polymerase chain reaction and enzyme linked immunosorbent assay protocols, respectively.Results:The TTV infection was found in 37 of 200 (18.5%) and 53 of 200 (26.5%) plasma and tissue samples of studied liver transplanted patients, respectively. The TTV genomic DNA was found in 32 (26.9%) and 28 (23.5%) of 119 liver tissue and plasma samples of transplanted patients with cryptogenic cirrhosis, respectively. The genomic DNA of TTV was also diagnosed in 21 (25.9%) and nine (11.1%) of the 81 liver tissue and plasma samples of patients with determined cirrhosis, respectively. Significant associations were found between TTV infection with HBV molecular and immunologic infective markers, in liver transplanted patients, with determined and cryptogenic cirrhosis.Conclusions:The diagnosis of the high frequency of solitary TTV and co-infection with HBV, in both liver transplanted patients with cryptogenic and determined cirrhosis, emphasized on the importance of TTV infection in the development of cirrhosis, especially in the cases of cryptogenic ones, prompting for further studies the confirm this agent in the etiology of determined cirrhosis.

Study of the Associations Between TT Virus Single and Mixed Infections With Leukemia.

Background:The pathogenic association of Transfusion Transmitted Virus or Torque teno Virus (TT Virus) single and mixed infections with leukemia was under evaluation in these years but confront with controversies. This hypothesis is based on the higher prevalence of TT Virus infection in patients with leukemia compared with controls.Objectives:The aim of this study was to determine the frequency of TT Virus, Cytomegalovirus (CMV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) infections in patients with leukemia and healthy controls.Patients and Methods:In this cross sectional study, 95 patients with leukemia and 100 healthy controls who were admitted to the Namazi Hospital affiliated to the Shiraz University of Medical Sciences, Shiraz, Iran, were enrolled between years 2012 and 2013. Blood samples treated with EDTA were collected from each patient with leukemia and controls. The existence of TT Virus infection was analyzed using the semi-nested PCR method. The immunological prevalence of HBV and HCV infections were evaluated using HBs-Ag and HCV-Ab ELISA based protocols, respectively. Active CMV infection was also evaluated using an immunofluorescence method. Also risk factors of leukemia and viral infections were statistically analyzed in patients with leukemia.Results:The TT Virus infection was significantly found in 40 of 95 (42.1%) and 12 of 100 (12%) patients with leukemia and controls, respectively. The HBs-Ag and HCV-Ab were detected in 27 of 95 (28.4%) and 18 of 69 (26.1%) patients with leukemia but were not found in the controls. Active CMV infection was also found in 11 of 69 (16%) patients and none of the controls. Significant co-infection of TT Virus was found with HBV (15 of 40; 37.5%), HCV (14 of 40; 35%) and CMV (7 of 40; 17.5%) in patients with leukemia.Conclusions:Confirmation of the significantly higher frequency of TT Virus, HBV, HCV and CMV single infection and their co-infection in patients with leukemia compared with healthy controls, emphasizes the determinative role of TT Virus pathogenesis in clinical outcomes observed in patients with leukemia, which requires extensive evaluation by further studies.

Development of loop-mediated isothermal amplification methods for detecting Taylorella equigenitalis and Taylorella asinigenitalis.

ABSTRACTTaylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. Multiplex LAMP worked well without nonspecific reactions, and the analytical sensitivities of multiplex LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods.

Ocular Adnexal Marginal Zone Lymphomas (OAMZL): Lack of Evidence for Chlamydia Psittaci (C. Psittaci) Infection in a Clinicopathologic Study from a Single Institution

Ocular adnexal lymphomas (OAL) represent up to 55% of all orbital tumors, being extranodal marginal zone lymphoma (OAMZL) the most frequent histological subtype reported in approximately 50% of patients and its etiology and pathogenesis are still not well understood. There are conflicting reports regarding the association between C. Psittaci infection and ocular adnexal lymphoma (OAMZL), in part because this association may vary between different geographical regions. Herein, we analyzed the clinical features and the presence of C. Psittaci infection in a cohort of 28 patients with OAMZL diagnosed from a single Spanish institution. All tumor samples were centrally reviewed by two pathologists, and C. Psittaci infection was determined in tumor specimens by a Semi-nested PCR method. Between January 1984 and April 2009, 28 patients were diagnosed with primary OAMZL. The median age was 70 years (range: 31- 89) and 40% were male. The great majority of patients (79%) were diagnosed in stage I-extranodal (IE), and six patients (21%) had bilateral ocular involvement. IgH rearrangement was confirmed in 25 out of 28 cases with OAMZL. PCR for C. Psittaci infection was negative in all 28 tumor specimens analyzed. Most patients (82%) were treated with immunochemotherapy combinations, being chlorambucil based regimens the most frequently used. Overall, 18 out of 24 patients (75%) achieved a complete response after first-line treatment. Median PFS was of 79 months (IC 95% 36 - 121). Whereas C. Psittaci infection in OAMZLs has been proved in up to 90% of patients in Northern Italy, in this Spanish cohort OAMZLs were not associated with C. Psittaci infection. These findings confirm that the association between C. Psittacci infection and OAML is highly heterogeneous even between the same geographic regions. In these regions where C. Psittaci infection is not prevalent, the search for alternative antigenic stimuli driving the appearance of this lymphoma is warranted. DisclosuresNo relevant conflicts of interest to declare.

Detection of Toxoplasma gondii oocysts in soils in northwestern China using a new semi-nested PCR assay.

BackgroundToxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China.ResultsThe one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 102 T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0–1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%.ConclusionsThe present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.