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Background: Today, Methicillin-Resistant Staphylococcus Aureus (MRSA) has become one of the public health concerns due to its resistance to antimicrobial drugs, and this problem makes treating patients with infections caused by this bacterium difficult. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are pervasive in both community and hospital settings, primarily attributable to Staphylococcus aureus' capacity to colonize areas like the nose or skin. In this study, with the aim of comparing phenotypic (disc diffusion method) and genotypic (PCR) methods, to detect methicillin-resistant Staphylococcus aureus isolated from patients of hospitals under supervision of Tehran university of medical sciences, and also detection of nor A, that is the one of the most important genes in efflux pump cluster genes. Methods: The present research was a cross- sectional study that was conducted from February 2022 to September 2023. In this research, 43 isolated strains of Staphylococcus aureus from wound discharge and blood samples, were collected from different departments of Tehran hospitals and had submitted to the research laboratory of the school of allied medical sciences in Tehran university of medical sciences. After identifying the strains, the resistance of the isolates to 14 types of antibiotics was checked by disk diffusion method. Results: Staphylococcus aureus diagnostic tests including gram staining on colonies, catalase, coagulase, DNase tests were performed and it was found that all strains were Staphylococcus aureus. In the next step, all samples were resistant to Cloxacillin by disc diffusion method, and the presence of mec A gene in them was confirmed by PCR method, thus the presence of MRSA strains was confirmed from the genotypic point of view. Of the 43 Staphylococcus aureus strains, 26 samples were identified as having the nor A gene by PCR and electrophoresis. Conclusion: The results of the present research have shown that the prevalence of Staphylococcus aureus bacteria in hospital samples is significant and resistance to methicillin and ciprofloxacin has increased in the strains of this bacteria.

Aim. Ticks are the vectors of many pathogens, which cause diseases with fatal consequences. Polymerase chain reaction (PCR) was used to detect the presence of these different pathogens in ticks, but there is a need of isolated nucleic acid to conduct the molecular assays. In our previous research, we found that some ticks give huge yield of isolated nucleic acid during spectrometric measurements, therefore aim of this study is to find whether there is any relation between spectrometric values of DNA, RNA and presence of Borrelia burgdorferi as example pathogen in single tick samples. Methods. DNA and RNA were isolated with mini column method from single tick samples. They were run in real time as well as conventional PCR tests for the presence of Borrelia burgdorferi. The nucleic acid yields of isolated nucleic acid samples were measured with a spectrophotometer. Results. It was found that there were 47 ticks positive for Borrelia burgdorferi and 40 were negative. Average isolated DNA and RNA quantity was higher in pathogen positive ticks than those of negative ticks. There was no correlation between the yield of nucleic acid and presence of pathogen in a single tick, but there was tendency that pathogen positive tick gave higher yield of DNA and RNA during the isolation. Conclusions. This study shows some of Borrelia burgdorferi positive ticks give very high yield of DNA and RNA during the isolation. There is no correlation between presence of pathogen and nucleic acid in a single tick, but there is tendency that pathogen positive tick may have higher nucleic acid yield. Therefore, our recommendation is that laboratory should always measure the nucleic acid yield along with conducting the PCR tests.

Background and Aim: Infectious coryza (IC) or snot, is caused by Avibacterium paragallinarum and leads to upper respiratory disease in poultry. Various diagnostic methods are available, including isolation and identification through bacterial culture and biochemical tests. However, the isolation and subsequent identification of A. paragallinarum are challenging because the bacteria are fastidious and require specific growth factors. This study aimed to detect A. paragallinarum in clinical samples taken from the exudate of the infraorbital sinus of layer hens showing clinical signs of IC. Materials and Methods: Samples were collected from 10 layer hens with IC symptoms. Following DNA extraction, HPG-2 polymerase chain reaction (PCR) assays were performed. The PCR amplicons underwent electrophoresis to determine those of the correct target size (511 bp), and these were sequenced. The resultant sequences were analyzed using the National Center for Biotechnology Information (NCBI) basic local alignment search tool. MEGA X was used for bioinformatics analysis. Results: The presence of A. paragallinarum was confirmed by HPG-2 PCR in 4/10 samples. Bioinformatics analysis showed that the amino acid sequence of the samples and the A. paragallinarum reference sequences in the NCBI database were grouped within the same cluster. Furthermore, the nucleotide sequences showed 98.64%–100% of similarity with the reference sequences. The phylogenetic reconstruction of partial pyrG sequences from 55 A. paragallinarum strains/isolates deposited in GenBank confirmed that the four HPG-2 PCR-positive samples fell within the A. paragallinarum cluster, separate from the Pasteurella multocida, Avibacterium spp., and Rodentibacter pneumotropicus clusters. Conclusion: Avibacterium paragallinarum infection was molecularly confirmed in 4/10 (40%) samples by HPG-2 PCR amplicon detection. Clustering of the pyrG partial gene sequences revealed that the positive samples fell within the A. paragallinarum cluster.

Visceral leishmaniasis (VL) is a neglected disease and represents an important public health problem. The present study aimed to compare the conventional laboratory methods with the molecular method for VL diagnosis in human blood samples. Direct parasitological test of bone marrow aspirate (direct parasitological test), rapid immunochromatographic test (RIT), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used to evaluate 26 human samples with clinical suspicion of VL. The positivity rates obtained for direct parasitological test, RIT, IFAT and PCR were 80.8%, 80.8%, 73.1% and 84.6%, respectively. PCR had greater positivity and agreement with the other methodologies. Therefore, the use of PCR in the diagnostic routine should be encouraged to complement the serological results.

Background and Objective: Studies indicate that single nucleotide polymorphism of ICB-1 gene rs1467465 is associated with breast cancer and ovarian cancer. This study was conducted with the aim of investigating the association between ICB-1 gene polymorphism rs1467465 (A>G) and thyroid cancer susceptibility. Methods: In this case-control study, the peripheral blood of 92 thyroid cancer patients referred to Tabriz hospitals and 203 healthy individuals were prepared. After DNA extraction by saturated salt solution, single nucleotide polymorphism rs1467465 of ICB-1 gene was investigated by TETRA-ARMS-PCR method. Then, allelic and genotypic frequencies of patient and control groups were evaluated and compared. Findings: In this study, the genotypic frequency of AA, AG and GG in the patient group was 4.34%, 68.47% and 27.17%, respectively, and in the control group was 4.92%, 55.66% and 39.40% respectively. The frequencies of A and G alleles were calculated as 38.58% and 61.41% in the patient group and 32.75% and 67.24% in the control group, respectively. In this study, the relationship between this polymorphism and some clinical and pathological characteristics of thyroid cancer, including gender, age, type, grade and size of tumor, involvement of lymph nodes and involved side, was investigated. Conclusion: This study showed that AG and GG genotypes have a significant relationship with the risk of developing thyroid tumors in the studied population, and this polymorphism may contribute to determining the relative risk of developing thyroid tumors.

Faez Almohsen,1 Haithem A Al-Rubaie,1 Manal A Habib,1 Sherif A Nasr,2 Rajendra Perni,2 Lubab Al-Quraishi2 1College of Medicine, University of Baghdad, Baghdad, Iraq; 2siParadigm Diagnostic Informatics, New Jersey, NJ, USACorrespondence: Faez Almohsen, College of Medicine, University of Baghdad, Baghdad, Iraq, Tel +964 7902834062, Email dr_faez2005@yahoo.comBackground: Acute myeloid leukemia (AML) results from sequential genetic alterations in a normal hematopoietic stem cell or its progenitors giving rise to an autonomous clone that dominates the bone marrow leading to marrow failure. MicroRNAs are short non-coding nucleic acid sequences that regulate post-transcriptional gene expression by base-pairing with their target mRNAs. MiRNAs can be secreted into extracellular fluids and carried to target cells by vesicles or bound to proteins. Intracellular and circulating miRNAs are believed to be useful markers in the diagnosis, prognosis, and treatment of various cancers. Practically, circulating miRNAs are more stable at room temperatures and extreme conditions.Purpose: This study aimed to compare the expression of miR-126-3p and miR-423-5p in patients and normal subjects and correlate their expression with response to induction therapy and with their 2-year overall survival rate.Patients and Methods: Circulating miR-126-3p and miR-423-5p was measured in the plasma of 43 adult AML patients and 35 age- and sex-matched controls by quantitative reverse transcriptase PCR. The fold change in differential expression for each gene was calculated using the comparative cycle threshold method.Results: There was an increase in the expression of the studied miRNAs in patients compared to the control group. The average expression fold change of miR-126-3p was 3.02 (p= 0.010). The average expression fold change of miR-423-5p was 4.09 (p= 0.003). No significant correlation was found between the expression of miR-126-3p and miR-423-5p in the studied AML patients (r = 0.094, p = 0.22). Furthermore, no relationship was found between the expression of the studied miRNAs and response to induction therapy or the 2-year survival rate.Conclusion: Although further studies are needed, our findings highlight the studied circulating miRNAs as possible diagnostic markers for AML.Keywords: acute myeloid leukemia, miR-126-3p, miR-423-5p, response to induction therapy, overall survival, polymerase chain reaction

Acinetobacter baumannii is widely recognized in clinical environments due to its infectious capacity, antimicrobial adaptability, and lethality. Analyzing the prevalence of this agent in intra- and extra-hospital environments may reveal target indicators for appropriate management interventions. In this observational cross-sectional study, we evaluated the prevalence of A. baumannii within hospitals with intensive care units and in their external surroundings in a macro-health region of Brazil. Samples of Columba livia (pigeon) droppings from the external environment of four hospitals (n = 40), from floor surfaces (n = 20), and door handles (n = 20) of different hospital wards were collected based on random sampling, all of which were evaluated for the presence of A. baumannii using polymerase chain reactions (PCR). The sensitivity and specificity of the technique was verified after the collected samples were contaminated with clinical samples positive for A. baumannii. We detected a significantly higher A. baumannii prevalence (87.50%, CI = 71.29–100.00) in samples collected within the hospital environment compared with those obtained from the external environment (12.50%, CI = 0.00–28. 71) (p = 0.003). In addition, samples collected from floor surfaces contained bacterial densities (181.3 ± 11.58) that exceeded those in environmental (93.32 ± 1.56) and door handle (142.70 ± 17.14) samples by 94% and 78.71%, respectively. The findings of this study will enhance our understanding of the spatial distribution of A. baumannii and additionally, validate the efficiency of PCR for diagnosis of this infectious agent.

The application of quality control tests, such as the comet assay, are essential when adipose-derived stem cells are cultured for therapeutic purposes. However, the steps involved in the development of this assay should be investigated, in order to reduce their influence on genomic damage in cells. The study aimed to evaluate if the cell culture process causes DNA damage, and if variations in the lysis time and pH of the electrophoresis buffer interfere in the genotoxicity results. Four different comet assay protocols were evaluated, and the effects of lysis time and pH conditions of the electrophoresis buffer solution were stated as follows: 2 hours and pH 12; 24 hours and pH 12; 2 hours and pH ≥ 13 and 24 hours and pH ≥ 13. The tail moment was analyzed, and results indicated that at the time cells were detached from the flasks, there was little damage to the DNA in the adipose-derived stem cells, which was confirmed by evaluation of the expression of mRNA genes involved in damage and repair processes of genetic material. Also, the tail moment values ​​did not show significant differences among the four evaluated protocols (p < 0.05), with no indication of damage when compared to the positive control (p < 0.05). Thus, any of the tested protocols can be applied in genotoxicity tests with adipose-derived stem cells, without causing damage to them.

Today, Babesia is recognized as one of the most common blood parasites in the world, which in terms of the number of cases of invasion is second only to trypanosomes (the causative agent of African trypanosomiasis and Chagas’ disease). These microorganisms can cause parasitism in erythrocytes and hematopoietic organs. They cause an infectious process, the clinical course of which can vary from asymptomatic, subclinical, mild or moderate influenza-like forms – to severe progressive disease (fulminant form) with fatal outcome. Thus, the latter determines the significant burden of babesia for the leading branches of medicine, veterinary medicine and the economy as a whole. The presented work is devoted to the study of the prospects for verification of babesiosis causative agents by the polymerase chain reaction (PCR) method. Blood, erythrocyte suspension, homogenized tick-carriers of babesiosis, culture of Babesia spp. were used as research material (samples). In order to obtain an objective assessment, the PCR-diagnostics method was used in two formats – standard and multiplex (multi-primer). Multiple PCR testing of multiplex format using primers in model samples containing cells of different species of Babesia (B. microti, B. divergens, B. bovis, B. canis), allowed us to establish the level of reproducibility of the results of such studies, which ranged 94.6–96.4%, to determine the level of PCR sensitivity of the multiplex format for detection/identification of human pathogenic babesia (B. microti, B. divergens and B. venatorum). It is established that the advantages of the PCR-diagnostic method of babesiosis pathogens in the samples of the studied biomaterial were: speed of research (2–4 hours); high sensitivity, specificity, reproducibility of Babesia detection results, prospects of species identification, differentiation with apicomplex spores (Plasmodium falciparum, Toxoplasma). In view of the above, the PCR method is recommended for use in cases of persistent suspicion of babesiosis infection (in cases of negative results of microscopic/cytological studies, to identify asymptomatic, subclinical and chronic forms of babesiosis, verification of active invasion in seropositive individuals and for Babesia species and their differentiation).

Background: Herpes simplex virus (HSV) is a widely distributed human pathogen that is known for its ulcerative lesions at the infection site. HSV can cause persistent infection in the host that is often followed by a period of latency within the neurons. Considering the high rate of HIV infection in South Africa, it is important to assess the seroprevalence of HSV with a focus to determine the epidemiological association between HSV-DNA and HIV-1 in the population. Methods: A total of 44 sera samples were screened for HSV and HIV-1 using the highly sensitive enzyme-linked immunosorbent assay (ELISA). The ELISA positive samples were characterized using polymerase chain reaction (PCR) to confirm the positivity of both viruses and to further differentiate HSV into HSV-1 and -2. Thereafter, the samples were analysed for relatedness using phylogenetic analysis. Results: Of the 44 samples, 36 (81.8%) were positive for HIV-1, while 35 (79.5%) were positive for HSV when screened with ELISA kits. The PCR results, with the use of type specific primers, showed that 4/35 (11.4%) samples were specific for HSV-1 while 30/35 (85.7%) were specific for HSV-2. Statistical analysis performed using the chi-squared goodness-of-fit test showed that there is a significant relationship between HSV-2 and HIV-1 transmission. Conclusions: There is a significant positive association between HSV-2 and HIV-1 in the study population. Our study shows that some of the HSV-2 isolates are not related to the clinical isolate SD90e from South Africa, suggesting diversity in HSV-2 viral transmission.

Objective: To study the clinical features and the state of general immunological reactivity in non-gonococcal urethritis (NGU) in men. Methods: Examined 100 men between the ages of 20 and 48 years: 75 patients of NGU (main group) and 25 healthy (control group). The average age of the patients was 26.7±1.7 years, and the male of control group was 27.9±1.7 years. Clinical, microscopic, immunological research methods were used, including direct immunofluorescence (DIF), polymerase chain reaction (PCR). Results: Ureaplasma urealyticum was found in 37 (49.3%) patients, 33 (44%) had Chlamydia trachomatis, 23 (30.7%) had Mycoplasma genitalium, 16 (21.3%) had Trichomonas vaginalis. In 24 (32%) of NGU patients had a mixed infection: in 14 (18.7%) had a combination of two STIs and in 10 (13, 3%) had three infections. In 51 (68%) of patients the process passed in the form of monoinfection. Various complaints (dysuric disorders, pain, discomfort and agglutination of the labium urethra) were presented by 51 (68%) of sick patients. The excretions from the urethra were marked in 46 (61.3%) of patients, reproductive disorders are 3 times less common. Immunological disorders were manifested by a decrease in CD4 and CD8 lymphocytes, PHA, PN and IL-10, increase – IgM, IgG, CIC, TNFα, IL-1β. Conclusion: The most common cause of NGU is Ureaplasma urealyticum and Chlamydia trachomatis. In most cases NGU proceeds in the form of monoinfection. Subjective and objective symptoms occur in 64% and 59% of patients, respectively. Immunological disorders were detected in 71% of patients.

Background: Herpes simplex virus (HSV) is a widely distributed human pathogen that is known for its ulcerative lesions at the infection site. HSV can cause persistent infection in the host that is often followed by a period of latency within the neurons. Considering the high rate of HIV infection in South Africa, it is important to assess the seroprevalence of HSV with a focus to determine the epidemiological association between HSV-DNA and HIV-1 in the population. Methods: A total of 44 sera samples were screened for HSV and HIV-1 using the highly sensitive enzyme-linked immunosorbent assay (ELISA). The ELISA positive samples were characterized using polymerase chain reaction (PCR) to confirm the positivity of both viruses and to further differentiate HSV into HSV-1 and -2. Thereafter, the samples were analysed for relatedness using phylogenetic analysis. Results: Of the 44 samples, 36 (81.8%) were positive for HIV-1, while 35 (79.5%) were positive for HSV when screened with ELISA kits. The PCR results, with the use of type specific primers, showed that 4/35 (11.4%) samples were specific for HSV-1 while 30/35 (85.7%) were specific for HSV-2. Statistical analysis performed using the chi-squared goodness-of-fit test showed that there is a significant relationship between HSV-2 and HIV-1 transmission. Conclusions:There is a significant relationship between HSV-2 and HIV-1 in the study population. Our study shows that some of the HSV-2 isolates are not related to the clinical isolate SD90e from South Africa, suggesting diversity in HSV-2 viral transmission.

Background: Herpes simplex virus (HSV) is a widely distributed human pathogen that is known for its ulcerative lesions at the infection site. HSV can cause persistent infection in the host that is often followed by a period of latency within the neurons. Considering the high rate of HIV infection in South Africa, it is important to assess the seroprevalence of HSV with a focus to determine the epidemiological association between HSV-DNA and HIV-1 in the population. Methods: A total of 44 sera samples were screened for HSV and HIV-1 using the highly sensitive enzyme-linked immunosorbent assay (ELISA). The ELISA positive samples were characterized using polymerase chain reaction (PCR) to confirm the positivity of both viruses and to further differentiate HSV into HSV-1 and -2. Thereafter, the samples were analysed for relatedness using phylogenetic analysis. Results: Of the 44 samples, 36 (81.8%) were positive for HIV-1, while 35 (79.5%) were positive for HSV when screened with ELISA kits. The PCR results, with the use of type specific primers, showed that 4/35 (11.4%) samples were specific for HSV-1 while 30/35 (85.7%) were specific for HSV-2. Statistical analysis performed using the chi-squared goodness-of-fit test showed that there is a significant relationship between HSV-2 and HIV-1 transmission. Conclusions: The prevalence of HSV in the population is high with an increased HSV-2 infection in women. Our study shows that some of the HSV-2 isolates are not related to the clinical isolate SD90e from South Africa, suggesting diversity in HSV-2 viral transmission.

Background: A significant association between endometrial vascularity and pregnancy has been shown in previous research, while poor vascularization was attributed to repeated implantation failure (RIF). One possible approach to enhance angiogenesis for successful implantation is endometrial scratching (ES)., Objective: The purpose was to investigate endometrial responses to scratching by profiling angiogenesis-related gene expression in unexplained RIF participants., Materials and Methods: In this randomized controlled trial study, 20 infertile women with unexplained RIF were assigned to 2 groups by the balanced block randomization method (n = 10/each group): the intervention group (group A) (who received ES in the follicular phase) and the control group (group B). Endometrial biopsy was performed in the secretory phase. Gene expression profiling was performed using a polymerase chain reaction-array kit for human-angiogenic growth factors. The implantation and clinical pregnancy rates were also assessed., Results: Among the angiogenesis-promoting genes, FGF1 , FGF13 , FGF2 , TGFA , ANG , ANGPT1 , and VEGFA were significantly upregulated (p < 0.05). IL12A (an angiogenesis-inhibiting cytokine) was significantly upregulated (p < 0.01). In contrast, 15 genes with angiogenesis-related functions, including CXCL11 , CXCL13 , CXCL3 , CXCL5 , CXCL6 , EREG , FIGF , FST , IL10 , LEP , PPBP , PROK1 , RHOB , TNF , and TYMP, were downregulated after ES. No significant differences were observed between the intervention (group A) and control (group B) groups in terms of implantation (43.75% vs. 28.57%) or clinical pregnancy rates (75% vs. 57.1%)., Conclusion: ES induced significant alterations in the expression of angiogenesis-related genes, with notable up/downregulation of key angiogenic/antiangiogenic factors. These findings enhance our understanding of the molecular responses triggered by ES, underscoring the potential influence of ES on the complex processes of angiogenesis crucial for implantation., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2024 Aghajanpour et al.)

Background: Herpes simplex viruses (HSVs) are highly pervasive and show a strong synergistic interaction with human immunodeficiency virus (HIV). High prevalence of HSV type 1 (HSV-1) has been reported in Africa with a prevalence rate of 20-80% in women and 10-50% in men. Studies on the prevalence of HSV in South Africa are few considering the rate of HIV infection in the country. Our focus was to determine the molecular prevalence of HSV-DNA in HIV-1 sera. Methods: In total, 44 convenience samples were screened for HSV and HIV-1 using the highly sensitive enzyme-linked immunosorbent assay (ELISA). The ELISA positive samples were characterized using polymerase chain reaction (PCR) to confirm the positivity of both viruses and to further differentiate HSV into HSV-1 and -2. Thereafter, the samples were analysed for relatedness using phylogenetic analysis. Results: Of 44 samples, 36 (81.8%) were positive for HIV-1, while 35 (79.5%) were positive for HSV when screened with ELISA kits. The results of PCR with type specific primers showed that 4/35 (11.4%) samples were specific for HSV-1 while 30/35 (85.7%) were specific for HSV-2. Statistical analysis performed using chi-squared goodness-of-fit test showed that there is a significant relationship between HSV-2 and HIV-1 transmission. Conclusions: High prevalence of HSV-2 recorded in HIV-1 sera corroborate with similar studies conducted within different cohorts in the continent. SPSS Pearson’s chi-squared test established that there is a significant relationship between HSV-2 and HIV-1 transmission.

ABSTRACT Background and Objectives: Pterygium is a common ocular surface lesion that manifest as wing-shaped, benign conjunctival growth, which can extend onto the corneal surface. Presence of some oncogenic viruses in pterygium and the neoplastic nature of the lesion led us to the postulated involvement of the viruses in the etiology of pterygia. The aim of this study was to evaluate prevalence and possible role of human cytomegalovirus (HCMV) in the formation of pterygia. Methods: Fifty pterygium specimens and 10 normal conjunctival biopsy specimens (controls) were investigated by polymerase chain reaction using primers specific for the highly conserved regions of major capsid protein gene of HCMV. Data were analyzed using SPSS statistical software (IBM SPSS Statistics 18; IBM Corporation, USA) at significance level of 0.05. Results: The HCMV DNA was detected in seven (14%) patients with pterygium but in none of the control subjects. All subjects were β-globin positive. Conclusion: Given the results, direct involvement of HCMV in the development of pterygium seems less probable, thus suggesting that other agents might be involved in the multistep process of the disease. Keywords: Human Cytomegalovirus, Pterygium, Polymerase Chain Reaction.

ABSTRACT Background and objectives: Bacillus licheniformis is a potential cause of spoilage in pasteurized products. The aim of this study was to identify and isolate B. licheniformis from commercial pasteurized fruit juices distributed in the West Azarbaijan Province, Iran. Methods: Sixteen fruit juice samples including four apple juice and 12 orange juice samples were collected from five fruit juice manufacturing companies in Iran. The samples were tested for the presence of B. licheniformis by culture in specific media and biochemical testing. Suspected samples were also investigated for the presence of the bacterium by polymerase chain reaction using specific primer for the gyrB gene. Results: Three samples (18.75%) from the 16 tested fruit juice samples were found as positive. In other words, one apple juice sample (25%) and two orange juice samples (16.66%) were contaminated with B. licheniformis. Conclusion: Isolation of this bacterium indicates the unsuitable manufacturing conditions and ineffective bacterial decontamination, which might also be favorable for the growth of other fruit juice spoilage bacteria. KEYWORDS: Bacillus licheniformis, Fruit and Vegetable Juices, Polymerase Chain Reaction.

Tick-borne diseases have a significant worldwide impact in the veterinary and medical fields. The Serra da Tiririca State Park (PESET) is an Atlantic forest reserve that is home to a wide variety of wild animal species, but is suffering from unauthorised human activities, such as unregulated construction, such as private dwellings and the introduction of domestic pets. The objective of this study is to determine the prevalence of piroplasms and Hepatozoon spp. in dogs and ticks in the Atlantic Forest reserve. Blood samples were taken from 124 asymptomatic canines and 88 Rhipicephalus sanguineus ticks collected from animals inhabiting the PESET. Molecular techniques targeting fragments of the 18s rRNA gene from piroplasms and Hepatozoon spp. were applied to all samples obtained, and one blood sample (0.81%) positive for Babesia vogeli was discovered. All dogs tested negative to Hepatozoon spp. and no tick sample showed positive results for either PCR assay. Despite the low frequency of B. vogeli infection found in dogs that inhabit the PESET, parasitized dogs could be a source of transmission for new vectors, other dogs and even wild canines that are part of the native fauna of the park.

Background and Objectives: Mycoplasmas hominis is one of the important causes of bacterial infections of the reproductive system, which through changes in the volume, pH, motility, morphology, and sperm count in men, can lead to reduction in the level of fertility or infertility and sterility. In this research, the frequency of Mycoplasma hominis, was assessed by PCR method, and biological parameters, were investigated in the semen samples of infertile men referred to Qom jihad daneshgahi infertility treatment center. Methods: In this descriptive-analytic study, a total of 187 semen samples were collected from infertile men referred to Qom jihad daneshgahi infertility treatment center from April to September 2016. Semen analysis was performed according to the guideline of World Health Organization. After the extraction of genomic DNA, detection of Mycoplasma hominis, was performed by PCR method using 16S rRNA specific primers. Data were analyzed using dependent and independent t-test. Results: Among 187 semen samples studied by PCR, 71 samples (38%), were positive for Mycoplasma hominis. In the assessment of parameters, there was a significant association between bacterial infection and C class mean difference (in situ sperm motility( (p=0.008). Conclusion: The results of the present study showed that high percentage of infertile men are infected with Mycoplasma hominis, which may lead to pelvic inflammatory diseases, urethritis, and infertility. PCR method is a rapid and efficient technique for detection of mycoplasma, which can be useful in the control of sexual transmitted diseases.

Background and Objectives: Salmonella is one of the authentic bacteria found in food and raw materials. The existence of this bacterium in food, in addition to causing disease, can lead to drop in production quality and decrease in economic growth of the country. The aim of this research was to isolate and identify Salmonella spp. from local dairy products in Maragheh city. Methods: In this cross sectional study, a total of 100 samples, were randomly selected from local dairy products, including curd, cheese, butter, dough, and ice cream (20 samples of each). First, the samples were cultured and tested by standard method, then, biochemical and serotyping complementary tests, were performed to detect Salmonella bacterium, and polymerase chain reaction (PCR) using hilA gene primers, was performed for molecular confirmation of the isolates from local dairy products. Antibiogram test was carried out on the obtained isolates. Results: Overall, 3% of all samples, were contaminated with Salmonella. Two Salmonella cases (10%), including B and D serogroups were isolated from 20 local crud samples and 1 Salmonella case (5%) of C serogroup, was isolated from 20 local cheese. No Salmonella was isolated from local butter, dough, and ice cream samples. Among the obtained isolates, the highest susceptibility was related to trimethoprim-solfamethoxazole antibiotic, and the highest resistance was related to nitrofurantoin antibiotic. Conclusion: The results of this study showed that in order to prevent poisoning from local dairy products, pasteurization of milk and its products, personal hygiene compliance, and control of health monitoring of production and distribution centers, are necessary.

Background and Objectives: Pathogenic bacteria, such as Escherichia coli are dangerous for human population due to acquisition of resistance genes to beta-lactam antibiotics. This resistance is due to extended spectrum β lactamase (ESBL) genes, which are caused by plasmids, transposons, and/or mutation. The main cause of resistance to beta-lactam antibiotics is lactamase enzymes. In this study, the frequency of ESBL producing E. coli and molecular detection of CTX-M-1 gene, were investigated. Methods: In this descriptive cross-sectional study, 58 E. coli samples isolated from patients with urinary tract infection, were identified using standard biochemical tests. Then, drug susceptibility test was performed using standard disk diffusion method (Kirby-Bauer). In the following, combined disk test was performed to identify ESBL producing strains among the resistant isolates. Plasmid DNA was extracted from ESBL-producing strains and CTX-M-1 genes were detected using PCR. Results: Out of 58 E.coli strains, 28 strains (48.27%), were ESBLs producer, which in the molecular analysis, 20 strains (71.42%) had CTX-M-1 gene. Conclusion: The results of the preset study is indicative of a high percentage of beta-lactamase resistance among E. coli strains. Considering the high percentage of antibiotic resistance, precise antibiogram testing is necessary in infections caused by ESBL-producing organisms.

Aim: The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP) infected eggs, and histopathological changes of chorioallantoic membrane (CAM) in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR). Materials and Methods: After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell) embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus. Results: Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076) showed homology with the sequences data available in GenBank. Conclusion: The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.

Introducción. La infección crónica por el virus de la hepatitis C es un problema de salud pública y se estima que hay más de 180 millones de personas infectadas en el mundo. En Colombia no se conoce la epidemiología de la infección ni los genotipos virales más frecuentes. Objetivo. Describir los genotipos y subtipos del virus de la hepatitis C en pacientes colombianos infectados. Materiales y métodos. Se hizo un estudio descriptivo y retrospectivo de pruebas realizadas en dos laboratorios de referencia nacional, entre el 2003 y el 2015, mediante electroforesis en geles de poliacrilamida (Polyacrylamide Gel Electrophoresis, PAGE) o reacción en cadena de la polimerasa en tiempo real (quantitative PCR, qPCR). Resultados. Se estudiaron 1.538 aislamientos del virus de la hepatitis C de 1.527 pacientes con una edad promedio de 53 años, de los cuales 70 % tenía entre 40 y 70 años y 52 % eran mujeres. El 57 % de las pruebas se ordenaron en la ciudad de Bogotá, y el 80 % de los casos provenía de los departamentos de Cundinamarca, Valle y Atlántico. Se encontró el genotipo 1 en 88,6 % de los casos, distribuido así: subtipo 1b, 70 %, subtipo 1a, 13,5 %, y no determinado, 5,1 % de los casos; el genotipo 2 se encontró en el 5,4 % de los casos, el 3, en el 2 %, y el 4, en el 4 %. Se encontraron genotipos mixtos en 0,8 % de la muestra. Conclusión. El genotipo 1 del virus de la hepatitis C es el que circula con mayor frecuencia en el país, con predominio del subtipo 1b.

Background: The importance of common diseases between humans and animals (zoonosis), and whose health impacts on the community individuals are obvious to everyone. One of these diseases is human infection with Coxiella burnetii and Q fever. Due to the importance of this subject and lack of published documents regarding the presence and distribution of this bacterium in Lorestan province, we aimed to determine the prevalence of C. burnetii in this area. Methods: A cross-sectional study was conducted from July 2013 to March 2014. A total of 500 raw milk samples was collected from 130 cattle, 200 sheep, and 170 goats in both warm and cold seasons from 20 selected villages in Khorramabad and Nourabad regions, Lorestan province, Iran. The presence of C. burnetii in collected specimens was tested by polymerase chain reaction (PCR). Results: A total of 9 (1.8%) milk specimens were positive for the presence of C. burnetii (3 sheep milk samples and 6 goat milk samples). All positive milk samples were obtained from 2 villages (Daraei and Ghalebi) around of the Khorramabad region during the winter season. However, all collected samples from Nourabad were negative for presence of C. burnett. Conclusion: The presence of C. burnetii in raw milk of ruminants in the cold seasons could be related to climate changes. Therefore, further investigations via various methods seems to be needed.

Abstract Background and Objective: Cryptosporidium parvum is a zoonotic protozoan parasite causing diarrheal cryptosporidiosis. Numerous outbreaks of cryptosporidiosis have been reported worldwide. The transmission via milk, water and raw animal products is one of the important ways. The aim of this study was the identification of hsp70 gene in Cryptosporidium parvum in raw cow’s milk samples. Material and Methods: In this cross sectional study, 38 raw cow’s milk samples of bulk tank were randomly collected from traditional and semi industrial cattle farms in Isfahan. To identify the protozoa in milk samples, the extracted DNA was evaluated by Nested polymerase chain reaction (PCR). Results: Based on Nested polymerase chain reaction, 2 samples (5.26%) were infected to Cryptosporidium parvum. Conclusions: The contamination of milk with Cryptosporidium Parvum is less than that of the other foodstuffs. Thus, it is necessary to reduce food contamination and to have appropriate health education programs. Keywords: Cryptosporidium Parvum, Milk; Polymerase Chain Reaction.

Background and Objectives: Enterotoxins and toxic shock syndrome toxin–1 (TSST-1) are an important virulence factor of S. aureus. The purpose of this study was to analyze the presence of S. aureus enterotoxin (sea-see) and tst genes in the samples that collected from different sources with PCR method. Methods: During 5 months from 150 collected samples, 80 strains were identified as S. aureus. PCR reaction was used for investigation on the presence of genes for staphylococcal enterotoxins (A-E) and toxic shock syndrome toxin-1 (TSST-1). Results: Fifty three samples (66.25%) out of 80 samples were positive for one or more ETs and TSST-1 genes. of these positive strains, 17 (32.07%) were positive for sea 39 (73.58%) for seb, 30 (56.6%) for sec, 2 (3.7%) for sed, 21 (39.62%) for see, and 14 (26.41%) for tst. Enterotoxins and tst in 40 samples (75.47%) observed.Conclusion: In this study, high prevalence of S. aureus, its enterotoxin, and tst genes were observed in clinical samples, food samples, and healthy people. This fact emphasizes on the role of human as original source and carrier of S. aureus. Also, use of PCR reaction for detection of these genes in S. aureus that isolated from various sources is recommended.

Per contribuire all’individuazione di un possibile nesso epidemiologico tra consumo di mozzarella di bufala preparata con latte crudo (non trattato al calore) e casi di brucellosi umana, sono stati analizzati, per ricerca di Brucella spp., 80 campioni di latte bufalino crudo e 315 campioni di mozzarella. Gli alimenti esaminati sono stati prelevati in caseifici della provincia di Caserta dove è presente la più alta concentrazione di allevamenti bufalini sierologicamente positivi alla brucellosi in Campania, regione che, nel periodo 2000-2005, ha registrato il 96,02% dei casi di brucellosi umana notificati in Italia. Al fine de valutare possibili variazioni stagionali, i campioni sono stati acquistati in 72 rivendite associate a caseifici nel periodo febbraio 2006-marzo 2007. La ricerca di Brucella spp. è stata effettuata utilizzando polymerase chain reaction (PCR) ed eseguendo contemporaneamente l’isolamento microbiologico. I campioni esaminati sono risultati negativi alla ricerca di Brucella con entrambi i metodi utilizzati. Sono stati, inoltre, definiti i parametri di sensibilità, specificità, ripetibilità, riproducibilità e il limite di rilevazione del metodo molecolare, esaminando campioni artificialmente contaminati, sia con metodo PCR sia con isolamento microbiologico classico. Il limite di rilevazione è risultato inferiore a 1 UFC/g, ripetibiltà e riproducibilità sono stati pari a 100% (p=0,95), sensibilità a 96,7% (p=0,95) e specificità a 100% (p=0,95).

To help identify an epidemiological link between the consumption of buffalo mozzarella prepared with raw milk (not heat-treated) and cases of human brucellosis, 80 samples of raw buffalo milk and 315 samples of mozzarella were tested for the presence of Brucella spp. Samples originated from Caserta, the province with the highest number of Brucella-positive buffalo herds in Campania, the region in which 96.02% of all cases of human brucellosis reported in 2000-2005 were recorded. To take into account possible seasonal variations, between February 2006 and March 2007, samples were purchased directly from 72 dairy outlets that were representative of the province. Samples were tested for Brucella spp. using the polymerase chain reaction (PCR) and bacterial isolation. Samples tested negative for Brucella using both methods. Spiked samples were then prepared and tested by PCR and bacterial isolation and the sensitivity, specificity, repeatability, reproducibility and limit of detection were determined. The PCR limit of detection was below 1 colony-forming unit (cfu)/g. The repeatability and reproducibility of the method were 100% (p = 0.95), the sensitivity was 96.7% (p = 0.95) and the specificity was 100% (p = 0.95).

Objective: To study the association between hepatitis C virus viral load by real-time PCR and core antigen value of HCV and define an antiviral treatment monitoring cut-off value for HCV core antigen. Study Design: Cross-sectional validation study. Place and Duration of Study: Department of Infectious Diseases, Chughtai Lab Lahore Pakistan, from Jun 2017 to Mar 2018. Methodology: To establish the association between these two parameters, we took a hundred positive plasma samples for HCV RNA and subjected them to an HCV Core antigen test. Furthermore, the samples were divided into three categories based on their viral load; <2000 IU/ml, 2000-10,000 IU/ml and >10000 IU/ml. Results: Our results showed that the hepatitis C virus core antigen was concordant with hepatitis C virus RNA by PCR when the viral load was above 2000 IU/ml. Below the HCV RNA load of 2000 IU/ml, the HCV core antigen had a sensitivity of 94.95% and specificity of 88.89%. Conclusion: In patients having a viral load above 2,000 IU/ml, the hepatitis C virus core antigen value can be used as a marker for diagnosis and monitoring antiviral therapy. After the antiviral treatment, HCV RNA real-time PCR should be performed to validate viral clearance. [ABSTRACT FROM AUTHOR]

Background: Preimplantation genetic diagnosis (PGD) has been used as an option for couples with the possibility of having a baby with a genetic disorder. The common method for performing this test involves isolating 1 cell from day 3 or a few cells from day 5 embryos and performing genetic studies on the cell-extracted DNA. This method is invasive and can cause abortion after implantation in the uterus. Because of this, 2 noninvasive methods for performing a PGD have been studied: PGD using blastocyst fluid and PGD using embryo culture medium., Objective: The aim of this study is to determine the sensitivity of the polymerase chain reaction (PCR) technique to detect the Y chromosome using cell-free DNA within a culture medium for gender prediction of blastocysts., Materials and Methods: In this study, the gender of 30 embryos on day 5 was determined using embryonic DNA extraction from the culture medium and the PCR technique to evaluate the sex-determining region Y and fragile X mental retardation genes. Then, the accuracy was assessed using ultrasound., Results: The results of the PCR technique showed that 7 embryos were male, but an ultrasound revealed that 13 were male., Conclusion: The given results indicated that, because of the low amount of DNA extracted from the culture medium, the diagnosis of the existence of the Y chromosome by this method is still not accurate enough for detecting the gender of the embryo., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2022 Barzanouni et al.)

Diagnosis of HIV in children younger than 18 months can be challenging in developing countries because requires the use of HIV virological tests. In this study we describe the experience with three commercial assays, HIV- 1 DNA polymerase chain reaction (PCR) (Roche Amplicor 1.5) with dried blood spot, HIV-1 RNA PCR (Roche COBAS TaqMan) with plasma and reverse transcriptase activity assay (Cavidi Exavir Load 3) with plasma in a rural setting of India. Sensitivity and specificity were 98.1% (95% confidence interval [CI] 90- 100) and 99.3% (95% CI 97.9-99.9) for the HIV- 1 RNA PCR assay, 66.7% (95% CI 29.9-92.5) and 100% (95% CI 96.8-100) for the HIV-1 DNA PCR assay, and 100% (95% CI 48-100) and 98.7% (95% CI 92.8-100) for the reverse transcriptase activity assay respectively. The low sensitivity of the HIV-1 DNA PCR assay in this setting is worrisome and warrants further investigations.

The surveillance of foodborne pathogens in food industries has shown the urgent need for rapid and dependable methods to detect and characterize the organisms in food and environments of clinical and epidemiologic importance. Recent studies on rapid methods in microbiology have been focused on biochemical characterization, immunoassays, and molecular methods. Many molecular methods have been developed and applied to the detection and characterization of foodborne pathogens in laboratories and food industries. They can be mainly divided into DNA banding pattern-based tests and DNA sequence-based tests. The former includes nucleic acid hybridization, polymerase chain reaction (PCR), amplified restriction length polymorphism, and randomly amplified polymorphic DNA, etc. Most of these methods in commercial applications are based on PCR or hybridization techniques. The principle, characteristics, and application of molecular methods for the detection and characterization of foodborne pathogens were reviewed in this article. [ABSTRACT FROM AUTHOR]

The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. The yield of clinical examination in the diagnosis of keratomycosis is 63–83% and KOH mount is 91%. This still highlights the limitation of routine clinical examination and smear examination, which is not performing 100% efficiently. It is for these 37%, 17% and 9% of cases, every day advanced technologies are called for. Those who deal with patient care are aware of certainties and uncertainties of results of clinical examination. The best reported figures at specialized centres might not translate into clinical practice. Another factor to be kept in mind is that many patients who come after secondary and tertiary referrals are already treated with antibiotics, antivirals, steroids and sometimes even antifungals that distort the clinical picture completely. Further, one has to consider as well the cases caused by yeast-like fungi, which resemble bacterial keratitis. Confirmation of diagnosis, not only in case of mycotic keratitis but also for other diseases, to initiate prompt and accurate therapy would avoid unnecessary and indiscriminate use of steroids/antibacterials/antivirals and antifungals. [ABSTRACT FROM AUTHOR]

Abstract: Eight polymorphic nuclear microsatellite loci were identified from the invasive Indo-Pacific Mediterranean strain of Asparagopsis taxiformis. Microsatellite markers were tested against a panel of specimens collected along the Italian (Elba, Naples) and Californian (Catalina Island) coasts, all belonging to the same mitochondrial lineage. In addition, we used Hawaiian specimens, belonging to a closely related mitochondrial lineage. The markers amplified in all of the specimens but failed consistently in thalli of two more distantly related mitochondrial lineages of A. taxiformis as well as in specimens belonging to the sister species Asparagopsis armata. Since haploid female individuals among the Mediterranean specimens contained cystocarps, genotyping was performed on supposedly haploid female specimens and supposedly diploid cystocarps separately. As expected, external allelic contribution was detected in the cystocarps. However, even after removal of these reproductive structures, gametophyte thalli exhibited patterns consisting of up to three alleles in all of the tested populations indicating polyploidy. An elevated number of distinct genotypes (up to 85%) were found per population, suggesting high intra-population variation. Results showed high genetic similarity between the two Mediterranean populations screened and lower similarity between these two and the Californian one within the same mitochondrial lineage. Lowest similarity was found between these three and the Hawaiian population belonging to the other related mitochondrial lineage 1. [Copyright &y& Elsevier]

Abstract: p21-activated kinases (PAKs) are a family of serine/threonine kinases whose activity is regulated by the binding of the small Rho family GTPases as well as by RhoGTPase independent mechanisms. PAKs have wide-ranging functions which include cytoskeletal organisation, cell motility, cell proliferation and survival. We have identified a PAK from Entamoeba histolytica — EhPAK3 that is distributed in the cytoplasm of unstimulated cells and localizes to the caps after induction of capping with Concanavalin A. EhPAK3 contains a GTPase interacting (CRIB) domain, an N-terminal pleckstrin homology (PH) domain and a C-terminal kinase domain. Among the PAKs of E. histolytica studied so far, EhPAK3 bears the maximum similarity to Dictyostelium discoideum PAKC (DdPAKC). Phylogenetic analysis showed that EhPAK3 was closely related to DdPAKC and forms a group with DdPAKA, Dd Myosin I heavy chain kinase (DdMIHCK), and a PAK reported earlier from E. histolytica EhPAK2. Recombinant full-length EhPAK3 undergoes auotophosphorylation and phosphorylates histone H1 in vitro in the absence of any small GTPase. This is the first comprehensive characterization of a PAK protein from E. histolytica, which has constitutive activity and has demonstrated a strong involvement in receptor capping. [Copyright &y& Elsevier]

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized have translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSXl or SSX2 gene, thus creating SYT-SSXI or SYT-SSX2 rearrangement. The 2 types of gene fusion have been courted with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSXi and SYT-SSX2 rearrangements by using a LightCycler real-time one step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX! sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of renouncement in the same reaction but distinguishes them hashed on differences in melting temperature detected hay melting cun'e analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 non synovial sarcomas) were .studied and showed SYT-SSXI rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In con- elusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX real Tangement by using LightCycler RT-PCR and melting cui-ve analysis. [ABSTRACT FROM AUTHOR]

Cairo F, Gaeta C, Dorigo W, Oggioni MR, Pratesi C, Pini Prato GP, Pozzi G. Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial. J Periodont Res 2004; doi: 10.1111/j.1600-0765.2004.00761.x.© Blackwell Munksgaard2004A possible relationship between periodontitis and cardiovascular disease has been suggested. The aims of this controlled clinical study were: (i) to ascertain the presence of periodontal bacteria DNA[ Actinobacillus actinomycetemcomitans,Fusobacterium nucleatum,Porphyromonas gingivalis,Prevotella intermedia,Tannerella forsythensis(formerlyBacteroides forsythus)] in carotid atheromatous plaques and (ii) to assess the concomitant presence of the same periodontal bacteria DNA, if any, in periodontal pockets and in carotid atheroma in the same patient.A total of 52 patients scheduled for carotid endarderectomy were enrolled in this study. The test group consisted of 26 dentate patients; the control group included 26 edentulous patients. A complete periodontal examination, including radiographic orthopanoramic and subgingival plaque sample, was performed in the test population. Oral and X-ray examinations were performed in the control group. Atheromatous plaques were harvested during surgical procedure for each dentate and edentulous patient and then sent to the microbiological laboratory. Subgingival plaque samples and carotid specimens were examined using the polymerase chain reaction (PCR) technique by means of specific primers for periodontal bacteria. Amplification of extracted DNA was tested using human beta-globin specific-primers.Out of 52 endarterectomy samples, 12 (seven dentate, five edentulous patients) were excluded as negative to DNA amplification. In subgingival plaque samples of 19 test patients,T. forsythensis(79%),F. nucleatum(63%),P. intermedia(53%),P. gingivalis(37%) andA. actinomycetemcomitans(5%) were found. No periodontal bacteria DNA was detected by PCR in any of the carotid samples in either patient group.The presence of periodontal bacteria DNA in atheromatous plaques could not be confirmed by this study and thus no correlation could be established between species associated with periodontal disease and putative bacteria contributing to atheromatous plaques. [ABSTRACT FROM AUTHOR]

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 × 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 °C or above for one or more days. In contrast, in the compost microcosms incubated at 23 °C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 °C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes. [ABSTRACT FROM AUTHOR]

We consider a single-type supercritical or near-critical size-dependent branching process {Nn}n such that the offspring mean converges to a limit m ≥ 1 with a rate of convergence of order Nα n as the population size Nn grows to : ∞ and the variance may vary at the rate Nβ n, where -1 ≤ β < 1. The offspring mean m(N) = m + μN-α + 0(N-α) depends on an unknown parameter θ0 belonging either to the asymptotic model (θ0 = m) or to the transient model (θ0 = p. We estimate θ0 on the nonextinction set from the observations {Nh, ..., Nn,} by using the conditional least-squares method weighted by {N-y k-1}k (where γ &epsiv; &Rscript;) in the approximate model mθ &vcirc;n (·), where &vcirc;n is any estimation of the parameter of the nuisance part (O (N-α) if θ0 = μ). We study the strong consistency of the estimator of θ0 as γ varies, with either h or n - h remaining constant as n → ∞. We use either a minimum-contrast method or a Taylor approximation of the first derivative of the contrast. The main condition for obtaining strong consistency concerns the asymptotic behavior of the process. We also give the asymptotic distribution of the estimator by using a central-limit theorem for random sums and we show that the best rate of convergence is attained when γ = 1 + β. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: A common G to A polymorphism (UCSNP-43) in the Calpain 10 gene was recently found to be associated with Type II (non-insulin-dependent) diabetes mellitus and variations in post-absorptive and insulin stimulated glucose metabolism in vivo. We aimed to study the influence of Calpain 10 polymorphism on insulin action in fat cells. Methods:Calpain 10 polymorphism (UCSNP-19, -43 or -63) were set in relation to lipolysis and lipogenesis in isolated subcutaneous adipocytes of 46 apparently healthy non-obese subjects. Results: For UCSNP-43 the G/G genotype had twofold higher basal and insulin stimulated rates as compared with AA/AG genotypes. However, there was no genotype effect on basal or insulin inhibited lipolysis rates in fat cells. The protein amount of GLUT 4 in adipocytes was not influenced by the polymorphism. Fat cells expressed mRNA for the Calpain 10 gene at a relatively high concentration, about 4 amol/μg RNA, which is similar to that of uncoupling protein-2. Neither a UCSNP-19 nor a UCSNP-63 polymorphism in the Calpain 10 gene was found to be associated with basal or insulin-induced adipocyte lipolysis and lipogenesis. None of the polymorphisms influenced body mass index or fasting plasma concentrations of insulin and glucose in 693 non-obese healthy subjects. Conclusions/interpretation: The Calpain 10 gene could be involved in the regulation of glucose metabolism but not lipolysis in human fat cells, although it does not involve adipocyte GLUT-4 protein content. It is possible that the Calpain 10 gene predisposes to diabetes by influencing the glucose metabolism. [Diabetologia (2002) 45: 276–282] [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes and T lymphocytes, and could thus contribute to mononuclear cell infiltration in Type I (insulin-dependent) diabetes mellitus. Cytokines induce MCP-1 mRNA expression in pancreatic rat beta cells. To investigate this issue, we analysed the signal transduction for IL-1β-induced MCP-1 expression in rat beta cells and in vitro MCP-1 mRNA expression and protein release by human islets as well as in vivo islet MCP-1 mRNA expression in prediabetic non-obese diabetic mice. Methods. Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1β (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6–72 h with the cytokines IL-1β, IFN-γ or the inducible nitric oxide synthase (iNOS) inhibitor NG-methyl-l-arginine or both. We measured MCP-1 mRNA by RT-PCR and protein by ELISA. The MCP-1 mRNA expression in islets from male and female non-obese diabetic mice (2–12 weeks of age) was measured by real time reverse transcription-polymerase chain reaction (RT-PCR). Results. Interleukin-1β induced MCP-1 mRNA expression in rat beta cells, with a maximum induction after 6 h. A combination of p38 and ERK1/2 inhibitors decreased MCP-1 expression by 70 %. IL-1β induced both MCP-1 mRNA expression and a threefold increase in medium MCP-1 protein accumulation in human islet cells. This effect was not prevented by iNOS blockers. In vivo there was an age-related increase in MCP-1 mRNA expression in islets from male and female non-obese diabetic mice, reaching a peak at 8 weeks. Conclusion/interpretation. In rat and human islet cells MCP-1 mRNA is induced by IL-1β. Both ERK1/2 and p38 MAPK, but not nitric oxide, contribute to MCP-1 expression. In non-obese diabetic mice MCP-1 mRNA expression increases with age, peaking at the early phases of insulitis. The production of MCP-1 by pancreatic beta cells could contribute to the recruitment of mononuclear cells into pancreatic islets in early Type I diabetes. [Diabetologia (2001) 44: 325–332] [ABSTRACT FROM AUTHOR]

Aims/ hypothesis. The regulation of uncoupling protein 2 and uncoupling protein 3 gene expression in skeletal muscle has recently been the focus of intense interest. Our aim was to determine expression of uncoupling protein 2 and 3 in skeletal muscle from tetraplegic subjects, a condition representing profound muscle inactivity. Thereafter we determined whether exercise training would modify expression of these genes in skeletal muscle. Methods. mRNA expression of uncoupling protein 2 and 3 was determined using quantitative reverse transcription-polymerase chain-reaction. Results. Expression of uncoupling protein 2 and 3 mRNA was increased in skeletal muscle from tetraplegic compared with able-bodied subjects (3.7-fold p < 0.01 and 4.1-fold, p < 0.05, respectively). A subgroup of four tetraplegic subjects underwent an 8-week exercise programme consisting of electrically-stimulated leg cycling (ESLC, 7 ESLC sessions/week). This training protocol leads to increases in whole body insulin-stimulated glucose uptake and expression of genes involved in glucose metabolism in skeletal muscle from tetraplegic subjects. After ESLC training, uncoupling protein 2 expression was reduced by 62 % and was similar to that in able-bodied people. Similarly, ESLC training was associated with a reduction of uncoupling protein 3 expression in skeletal muscle from three of four tetraplegic subjects, however, post-exercise levels remained increased compared with able-bodied subjects. Conclusion/ interpretation. Tetraplegia is associated with increased mRNA expression of uncoupling protein 2 and 3 in skeletal muscle. Exercise training leads to normalisation of uncoupling protein 2 expression in tetraplegic subjects. Muscle disuse and physical activity appear to be powerful regulators of uncoupling protein 2 and 3 expression in human skeletal muscle. [Diabetologia (1999) 42: 826–830] [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) has been implicated in the genesis of oral hairy leukoplakia (OHL). Initially, OHL was also associated with human papillomavirus (HPV) as evidenced by staining with antiserum to papillomavirus common structural antigens and reports of two HPV-positive OHL as detected by in situ DNA hybridization. The aims of this study were to determine the prevalence of EBV and HPV DNA in OHL and normal oral mucosa and to explain the basis for the staining of 01- IL tissues with antibodies to papillomavirus common structural antigens. EBV DNA was detected by in situ hybridization in 47 of 47 cases of OHL from human immunodeficiency virus (HIV)-seropositive individuals and in I of 10 biopsies of clinically normal buccal mucosa from the same group of individuals. Twenty-five of 35 OHL specimens stained with antibody to papillomavirus common structural antigens. There was no staining of two EBV-containing lymphoblastoid lines, indicating that the staining with anti-papillomavirus anti- body was not due to antigenic cross-reactivity with EBV antigens. HPV DNA was detected by polymerase chain reaction amplification in 10 of 18 OHL specimens and in 6 of 10 normal buccal mucosa specimens. Our results indicate that EBV and HPV are present frequently in OHL and that HPV can be found regularly in histologically normal mucosa. [ABSTRACT FROM AUTHOR]

The purpose of the present investigation was to identify 2 putative periodontal pathogens: Eikenella corrodens and Actinobacillus actinomycetemcomitans by polymerase chain reaction (PCR) in vitro and in subgingival plaque. On the basis of published sequences coding for 16S rRNA two primer pairs were designed which amplify a 410 bp sequence from E. corrodens DNA and a 547 bp fragment from A. actinomycetemcomitans DNA, respectively. As few as 50 cells could be detected from pure bacterial cultures. Each of the two primer pairs was found to be specific in that it did not give any amplification product neither with cell lysates from the respective alternative bacterium nor with lysates obtained from other putative periodontal pathogens and other bacteria. The PCR method developed turned out to be a simple, rapid and reliable diagnostic tool for the detection of the target microorganisms in clinical samples. [ABSTRACT FROM AUTHOR]

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6-kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6-kb plasmid from T. denticola e' was shown to be similar to pTD1, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e' share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2-kb plasmid in 4 of these strains. [ABSTRACT FROM AUTHOR]

Archival oral tissues comprising 51 squamous cell carcinomas, 18 non-malignant lesions and 7 normal mucosa samples were investigated for human herpesvirus-6 (HHV-6)-encoded antigens and HHV-6 DNA. The virus-specific antigens were detected by an immunohistochemical method using monoclonal antibodies. Two further techniques used for HHV-6 DNA detection included the polymerase chain reaction (PCR) with virus-specific primers and in situ hybridization using digoxigenin-labelled oligonucleotides specific for HHV-6A and HHV-6B genotypes. A high proportion (79-80%) of the squamous cell carcinomas were positive for HHV-6 with the various detection methods. In cases of lichen planus and leukoplakia a high prevalence rate (67-100%) was noted with in situ hybridization and immunohistochemical techniques but a lower proportion (22-33%) was detected with the PCR method. All 7 normal tissues tested were negative for HHV-6. The HHV-6 variant B was found in 60% of the oral carcinoma tissues analysed. The study demonstrates the frequent presence of HHV-6 in neoplastic and non-malignant lesions of the oral cavity. While the role of HHV-6 in oral mucosal tissues remains to be determined, the in vitro tumorigenic potential of the virus suggests a possible role in the etiopathogenesis of oral lesions. [ABSTRACT FROM AUTHOR]

Exfoliated cervical cells from 321 Japanese women were examined for human papillomavirus (HPV) DNA types 6, 11, 16, 18, 31, 33 and 35 using polymerase chain reaction (PCR) and dot-blot hybridization methods. HPV DNA was present in 9.3% of patients with normal cervixes, 72.7% of patients with cervical intraepithelial neoplasia (CIN) and 77.8% of patients with invasive carcinoma. Younger patients (29 years) with normal cervixes had a 18.5% incidence of HPV DNA, whilst similar older patients (50 years) had a 1.9% incidence, a significant difference (χ2= 6.478, P < 0.01). In the CIN I and II groups, an incidence of 11.1% of types 16 and 18 was found, while in the CIN III or invasive carcinoma group the incidence was 58.1%, again a significant difference (χ2 = 12.075, P < 0.01). Furthermore, persistence or progression of CIN showed a significant correlation with infections by types 16 and 33 (χ2= 4.904, P < 0.01). However, no significantly different incidence of HPV infection was found between the CIN and the invasive carcinoma groups. It is suggested that (a) younger patients with normal cervixes have a higher incidence of HPV infection than do older patients; (b) HPV types 16, 18 and 33 are important etiologic agents of CIN III and invasive carcinoma, as well as in the persistence and progression of CIN; (c) progression of CIN to invasive carcinoma may depend on factors other than HPV infection in the cervix. [ABSTRACT FROM AUTHOR]

Chetsanga C, Pettersson B, Pettersson U, Gyllensten U. Detection of human papillomavirus DNA in cervical smears by the polymerase chain reaction. Int J Gynecol Cancer 1991; 1: 209-213.A PCR-based method has been developed which allows human papillomavirus DNA to be detected in routinely obtained cervical smear specimens. The results showed that 76% of the smears that were collected from women who were carrying HPV-positive lesions by cervical swab analysis were positive by the PCR-based method. The results suggest that it is possible to carry out population-based retrospective studies of archival cervical smears. [ABSTRACT FROM AUTHOR]