Comparative Evaluation of Nested Polymerase Chain Reaction for Rapid Diagnosis of Human Brucellosis.

Brucellosis is recognized as a zoonotic disease with high morbidity in the absence of treatment. The primary diagnosis of brucellosis can be effective in the achievement of satisfying treatment results and prevention of chronic infections. The present study aimed to compare the efficiency of conventional microbiological and serological approaches with nested Polymerase chain reaction (nested PCR) for rapid diagnosis of human brucellosis. A total of 120 subjects with symptoms of brucellosis were included in the study. The sensitivity and specificity of nested PCR for the detection of Brucella bacteria were compared with serological and blood culture methods. Out of 120 patients enrolled, brucellosis was detected in 73 (60.83%) cases based on serological tests with a blood culture confirmation in 8.33% of participants. Based on the obtained results, 55% of cases were positive in serum agglutination test (SAT≥1:160), and Coombs (C-SAT≥1:160) tests. Furthermore, seven negative SAT cases were positive in C-SAT as evidence of chronic brucellosis. The results of the 2-mercaptoethanol (2-ME) ≥ 1:80 test were negative in six SAT-positive cases. Based on nested PCR results, 68.18% and 56.06% SAT positive samples were also detected by blood nested PCR and serum nested PCR, respectively. The sensitivity of blood nested PCR was significantly more than serum nested PCR, SAT≥1:160, and blood culture (p <0.001). Moreover, the specificity of blood and serum nested PCR was obtained at 100%, compared to blood culture and SAT≥ 1:160. In the present study, the nested PCR was able to identify chronic brucellosis in SAT negative patients. As evidenced by the obtained results, the nested PCR showed higher efficiency for rapid diagnosis of human brucellosis, as compared to the blood culture method. Furthermore, the findings pointed to the high performance of nested PCR for rapid diagnosis of both chronic and acute brucellosis.

Does the BCG vaccine have different effects on strains of tuberculosis?

Several explanations have been suggested concerning the variety in bacille Calmette-Guerin (BCG) vaccine efficacy on strains of Mycobacterium tuberculosis (Mtb). This study aimed to compare the effect of BCG vaccination history in the prevention of the occurrence of Mtb-Beijing and non-Beijing strains. In this cross-sectional study, 64 patients with pulmonary tuberculosis (TB) were recruited from the Iranian border provinces (North West and West). Isolates were subjected to restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 as a probe (IS6110 RFLP) and drug susceptibility testing using the proportion method. Samples were analyzed with Gel Compare II 6.6 and spss version 18. The mean age [standard deviation (SD)] of the patients was 54·4 (SD = 17·0). Overall, 49 cases (76·56%) had no BCG vaccination scar. The prevalence of Beijing strains was 9·38% and drug resistance proportion among the isolates was 14·1% (nine cases). There was a significant relationship between Beijing strains and tuberculosis (TB)-drug resistance in isolates (χ2  = 26·29, P < 0·001). There was also a strong association between vaccination history and Beijing strains (χ2  = 13·23, P = 0·002). Also, a statistical relationship was observed between Beijing strains and drug-resistant TB among patients with a history of vaccination (χ2  = 7·47, P = 0·002). This association was not maintained in the unvaccinated group (P = 0·102). These findings confirm the claim that the vaccine has different effects on different subspecies of tuberculosis. The cause of the high probability of drug resistance in patients with Beijing-TB and vaccination history requires further investigation with a higher sample size.

A Non-pathogenic Recombinant Leishmania Expressing Lipophosphoglycan 3 Against Experimental Infection with Leishmania infantum.

Visceral leishmaniasis (VL) is caused by Leishmania infantum in the Mediterranean basin and affects primarily children and immunosuppressed individuals. Various strategies of vaccination have so far been examined by either protein or DNA without achievable complete protection against the disease. The live non-pathogenic lizard parasite, Leishmania tarentolae, expressing elected Leishmania antigens has recently provided a promising new approach as a safe and effective live vaccine candidate to prevent leishmaniasis. Here, we evaluated the immunoprotective potential of a live recombinant L. tarentolae expressing Lipophosphoglycan 3 (LPG3) antigen against L. infantum infection in BALB/c mice. Results indicated that the administration of live recombinant Leishmania produced a significant high level of IFN-γ accompanied by reduced levels of IL-10 as compared to wild-type parasites as live vaccine control, thus suggesting the induction of a Th1-type immune response in a mouse model of visceral leishmaniasis. Analysis of the IgG antibody response also showed high levels of IgG2a relative to IgG1 in sera of mice immunized with recombinant Leishmania parasites. However, immune responses elicited by this live vaccine conferred partial protection against infectious challenge. Therefore, further studies are required to increase its protective efficacy.

Evaluation of Cytotoxic Effect and Antioxidant Activity of Grape Seed Extract, Crocin, and Phenytoin.

Antioxidant compounds inhibit formation of free radicals, chelate catalytic metals, and scavenge free radicals in biological systems. In addition, antioxidants play a decisive role in prevention of numerous physiological dysfunctions, cancers, and metabolic disorders. This study sought to evaluate the antioxidant capacity and cytotoxic effect of grape seed extract (GSE), crocin (CRO), and phenytoin (PHEN) on a human breast cancer cell line (MCF-7). Methanol extracts of the three mentioned agents were prepared and their antioxidant activity was evaluated by diphenyl-1-picrylhydrazyl method, using Quercetine (QUER) as positive control. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effect of the extracts on Michigan Cancer Foundation-7MCF-7 cell line, using doxorubicin hydrochloride (DOX) as the positive control. Given the results, greater scavenging activity was achieved by using GSE in comparison to CRO and PHEN. Further, a significant correlation was found between the antioxidant activity and cytotoxic effects of these agents, and GSE had the highest antioxidant capacity and cytotoxic effect in comparison to CRO and PHEN.

Epidemiology and patterns of drug resistance among tuberculosis patients in Northwestern Iran.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has emerged as an important global health concern and is on the rise throughout the world. OBJECTIVE: The aim of this study was to examine the epidemiology and pattern of TB drug resistance. METHODS: In this cross-sectional study, 180 pulmonary TB patients from two Northwestern provinces of Iran were selected. The first and second line drug susceptibility testing was carried out using the 1% proportion method on the Lφwenstein-Jensen medium. Full demographic, environmental and clinical history was evaluated. RESULTS: Prevalence of resistance to any TB drug was 13.8%. Eight (4.4%) patients had MDR-TB (2.4% in the province of East Azerbaijan and 9.3% in the province of Ardabil) and one patient had extensively drug-resistant TB. Patient resistance to both isoniazid and streptomycin was the most prevalent at a rate of 8.3%. Patients showed the least resistance to ethambutol (2.8%). There was a significant relationship between the previous history of TB drug treatment and TB drug resistance. Migrants from rural to urban areas were in high-risk groups for the occurrence of TB drug resistance. CONCLUSION: In our study, prevalence of MDR was less than the global average. It is essential to monitor the patients with previous history of TB treatment and migrants by rapid and accurate techniques in terms of drug-resistance odds.

New insights into the mechanisms of multidrug resistance in cancers.

Drug resistance is one of the major obstacles in the treatment of various cancers. Since chemotherapy serves as a most beneficial method for the repression of tumor progression and due to its desirable cell death potency in tumors which reducing metastasis, failure of such a pivotal treatment lead to tumor recurrence and consequent mortality. Multidrug resistance, the principal mechanism by which many cancers develop resistance to chemotherapy drugs, is a major factor in the failure of many forms of chemotherapy. MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. In this review, we briefly look inside the recent mechanisms of chemotherapeutic resistance, the MDR1 gene expression in tumors and some novel inhibition-based approaches.

Molecular Epidemiology of Mycobacterium Tuberculosis Strains in the North-West and West of Iran.

BACKGROUND: Identifying Mycobacterium tuberculosis (MTB) transmission type is a key step in the control of this disease. AIM: This study aimed to determine the path and transmission type of MTB and the insertion sequence IS6110 band number and verify their relationship to demographic and clinical risk factors. SUBJECTS AND METHODS: In this cross-sectional study, 64 MTB patients from three border provinces of Iran were selected after full clinical history and physical evaluation design. The drug susceptibility testing was carried out using the standard proportion technique on sputum samples. Isolates tested with restriction fragment length polymorphism technique used IS6110. RESULTS: Recent transmission of disease was 33/50 (66%) based on clustering rate. The IS6110 band number had a significant relationship with drug resistance detected in proportion method tested by univariate linear regression (P < 0.01). Furthermore, the IS6110 band number had association with Bacillus Calmette-Guérin vaccination history (P = 0.02), sex (P < 0.01), and purified protein derivative (PPD) reaction size (P < 0.01) tested by multiple analysis. The risk of recent transmission inferred from the clustering rate was significantly higher in patients from Western provinces compared to those from the North-West province (P = 0.048). However, age (P = 0.39), gender (P = 0.16), vaccination history (P = 0.57), drug susceptibility, and PPD (P < 0.6) were independent of clustering. The largest cluster of up to six subjects was found in the Western provinces. CONCLUSION: Recent MTB transmission was much more common in the West compared to the North-West of Iran. Large MTB clusters with strong epidemiological links may be reflective of a disease outbreak. Correlation noted between the IS6110 band number and vaccination history; PPD size and female gender necessitates further studies.

Effect of Chronic Administration of Buspirone and Fluoxetine on Inflammatory Cytokines in 6-Hydroxydopamine-lesioned Rats.

Neuro-inflammation in Parkinson's disease (PD) is associated with glial cell activation and production of different inflammatory cytokines. In this study we investigated the effect of chronic administration of buspirone and fluoxetine on cerebrospinal fluid (CSF) levels of inflammatory cytokines, TNF-α, IL-1ß and IL-6 in 6-hydroxydopamine (6-OHDA)-lesioned rats.6-OHDA (8 µg/2 µl/rat) was injected unilaterally into the central region of the substantia nigra pars copmacta (SNc) and after 21 days lesioned rats were treated with buspirone and fluoxetine intraperitonealy (i.p.) for 10 days. CSF samples were collected at tenth day of drugs administration and were analysed by ELISA method to measure TNF-α, IL-1ß and IL-6 levels.The results showed that the CSF levels of TNF-α was increased 3 weeks after 6-OHDA injection while there was a significant decrease in TNF-α levels of parkinsonian animals treated with buspirone (1 mg/kg) and fluoxetine (1 mg/kg). IL-1ß and IL-6 both were decreased in parkinsonian rats, while their level was increased in buspirone (1 mg/kg) and fluoxetine (1 mg/kg) treated parkinsonian rats.Our study indicates that chronic administration of buspirone and fluoxetine in 6-OHDA-lesioned rats restores central concentration of inflammatory cytokines to the basal levels. We suggest that serotonergic agents can be used as adjuvant therapy along with commonly used anti-parkinsonian drugs by modulation of cerebral inflammatory cytokines. We suggest that the further clinical investigations may be carried out to prove this hypothesis.

Development and Evaluation of a Cetuximab-based Humanized Single Chain Antibody Against EGFR-overexpressing Tumors.

Production of humanized single chain antibodies (hscFv) can potentially be a powerful solution to limitations imposed by large size and murine nature of cetuximab. The present study describes generation of a cetuximab-based hscFv using CDR-grafting method. Cetuximab CDRs were grafted on frameworks selected from human germline antibody sequence repertoire. The strategy employed in selecting human sequences was the highest sequence similarity of variable domains between human and parental antibodies as well as similarity in the CDRs canonical structures. To maintain the binding affinity, the parental vernier zone residues were retained murine in hscFv. Recombinant hscFv was expressed in E. coli and affinity purified by Ni-NTA column. The potency of hscFv in targeting EGFR was evaluated using A431, a cell line over-expressing EGFR. Dot blot and ELISA tests were used to assess the reactivity and MTT assay to evaluate the growth inhibition of hscFv on A431 cell line. The humanization of cetuximab variable regions resulted in 22.2% increase in humanness of hscFv. Reactivity analyses of hscFv on A431 cells showed better binding affinity and higher growth inhibition effect (2.6 times) comparing to murine counterpart. In conclusion, hscFv produced in this study displayed reduced potential immunogenicity as well as enhanced cytotoxic effect on EGFR- overexpressing tumor cells.

Detection of Dientamoeba fragilis among diarrheal patients referred to Tabriz health care centers by nested PCR.

Dientamoeba fragilis is a protozoan parasite of the human large intestine which is implicated as a cause of gastrointestinal diseases. The diagnosis of this parasite in direct smear preparations is difficult due to the lack of a cyst stage. The permanent staining method is generally used for diagnosis of D. fragilis, but the technique is laborious and time consuming. The purpose of this study was to evaluate the performance of PCR for detection of D. fragilis in clinical specimen of health care center in Tabriz, northwest of Iran. Stool samples of 1000 patients were collected from different laboratories and were immediately examined via wet mount and permanent staining methods. All positive samples and 55 randomly selected negative samples were studied by PCR technique. Using direct smear examination, no positive sample was found among 1000 stool samples, whereas 21 (2.1%) positive and 26 suspicious cases were reported in stained smears. PCR screening indicated that from 21 positive cases, 17 were positive by primary PCR, whereas nested PCR detected all 21 positive cases as well as 3 new positive samples from the suspicious cases (overall 24 (2.4%) positive samples), yet all negative cases remained negative through both stages of PCR amplifications. In comparison with nested PCR (if considered as gold standard), primary PCR showed 81% sensitivity and 100% specificity and those of microscopy was determined to be 87.5% and 100%, respectively. Considering the favorable sensitivity and specificity of nested PCR and its other advantages such as relative simplicity and speed this technique is proposed for rapid diagnosis of D. fragilis in clinical samples.

A short and simple improved-primer extension preamplification (I-PEP) procedure for whole genome amplification (WGA) of bovine cells.

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS/GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (~3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.

Class 1 integron and Imipenem Resistance in Clinical Isolates of Pseudomonas aeruginosa: Prevalence and Antibiotic Susceptibility.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa is one of the most important causative agents of nosocomial infections especially in ICU and burn units. P. aeruginosa infections are normally difficult to eradicate due to acquired resistance to many antibiotics. Recent appearance of carbapenem resistant P. aeruginosa isolates is considered a major healthcare problem. The present study was conducted to detect class 1 integron and antibiotic susceptibility profiles of imipenem-sensitive and resistant clinical isolates of P. aeruginosa. MATERIALS AND METHODS: Antibiotic susceptibility profiles and minimum inhibitory concentration against imipenem was studied in 160 clinical isolates of P. aeruginosa by disk agar diffusion method and Etest, respectively. Detection of class 1 integron was performed by the PCR method. Demographic and microbiological data were compared between imipenem susceptible and non-susceptible isolates by the SPSS software. RESULTS: PCR results showed that 90 (56.3%) of P. aeruginosa isolates carried class 1 integron. Antibiotic susceptibility results revealed that 93 (58.1%) were susceptible and 67 (41.9%) were non-susceptible to imipenem. Comparison of antibiotic susceptibility patterns showed high level of drug resistance among imipenem non-susceptible isolates. We found that MDR phenotype, presence of class 1 integron and hospitalization in ICU and burn units were significantly associated with imipenem non-susceptible isolates. CONCLUSION: The high frequency of imipenem resistance was seen among our P. aeruginosa isolates. Since carbapenems are considered as the last drugs used for treatment of P. aeruginosa infections, it is crucial to screen imipenem non-susceptible isolates in infection control and optimal therapy.

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran.

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.

Evaluation of selective and nonselective media for isolation of Helicobacter pylori from gastric biopsy specimens.

The aim of the present study was to compare six media, three selective and three nonselective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 8 months, mucosal antral biopsy specimens were obtained from 97 dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all six media. Egg yolk emulsion agar (EYE), Skirrow's medium and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase Soy Agar (TSA) and brain heart infusion agar were used as nonselective media. Overall, by using these six media, H. pylori were recovered from biopsy specimens from 48 of 97 patients, yielding an isolation rate of 49%. Comparison of all possible combinations of the six media showed that the highest rate of isolation of H. pylori was 100% (48 of 48) with EYE-MCHOC, followed by 97% (47 of 48) when EYE-SK was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 92%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs.

Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response.

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Molecular characterization of a novel amastigote stage specific Class I nuclease from Leishmania major.

Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.