The frequency of efflux pump genes expression in Acinetobacter baumannii isolates from pulmonary secretions.

Acinetobacter baumannii is an important opportunistic pathogen, and the cause of nosocomial infections worldwide in recent decades. Efflux pumps are considered as the important causes of multidrug resistance of A. baumannii. The aim of this study was to determine the frequency of efflux pump genes, and evaluate the antibiotic effect of Tigecycline on the expression of adeB gene in isolates of multidrug-resistant. A. baumannii. 70 isolates of A. baumannii were collected and confirmed by biochemical and molecular tests. Antibiotic resistance (Ciprofloxacin, Trimethoprim-sulfamethoxazole, and Tigecycline) was performed based on the minimum inhibitory concentration (MIC) method. Then, the effect of Carbonyl cyanide m-chlorophenyl hydrazone inhibitor (CCCP) on isolates was investigated and the frequency of adeB, adeG, adeJ and abeM genes were examined by PCR for isolates with reduced in MIC titer. Also, the antibiotic effect of Tigecycline on adeB gene expression in A. baumannii isolates was analyzed by Real-Time PCR. The antibiotic resistance for Ciprofloxacin, Trimethoprim-sulfamethoxazole, and Tigecycline was 97.1%, 95.8% and 37.2%, respectively. Following CCCP inhibitor use, the MIC titer had a decrease in MIC titer containing CCCP inhibitor was 64.3% for Ciprofloxacin, 51.5% for Trimethoprim-sulfamethoxazole and 50% for Tigecycline. The frequencies of genes associated with adeB, adeG, adeJ and abeM efflux pump were 100%, 92.8%, 86% and 98.5%, respectively. Real-Time PCR results showed a correlation between the antibiotic effects of Tigecycline on adeB gene expression. The antibiotic resistance of the isolates was relatively high. The isolates were resistant to Ciprofloxacin and Trimethoprim-sulfamethoxazole antibiotics, while more sensitive to Tigecycline. Also, efflux pump genes, which are the antibiotic resistance factors of A. baumannii, are frequently high in the isolates but it seems that isolates use other effluxe pumps than RND family to exit tigecycline.

Chitosan nanofiber biocomposites for potential wound healing applications: Antioxidant activity with synergic antibacterial effect.

Bacterial wound infection is one of the most common nosocomial infections. The unnecessary employment of antibiotics led to raising the growth of antibiotic-resistant bacteria. Accordingly, alternative armaments capable of accelerating wound healing along with bactericidal effects are urgently needed. Considering this, we fabricated chitosan (CS)/polyethylene oxide (PEO) nanofibers armed with antibacterial silver and zinc oxide nanoparticles. The nanocomposites exhibited a high antioxidant effect and antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Besides, based on the results of the cell viability assays, the optimum concentration of ZnONPs and AgNPs in the nanofibrous mats is 0.2% w/v and 0.08% w/v respectively and had no cytotoxicity on fibroblast cells. The scaffold also showed good blood compatibility according to the effects of coagulation time. As well as significant fibroblast migration and proliferation on the wound margin, according to wound-healing assay. All in all, the developed biocompatible, antioxidant, and antibacterial Ag-ZnO NPs incorporated CS/PEO nanofibrous mats showed their potential as an effective wound dressing.

Correlation between clarithromycin resistance, virulence factors and clinical characteristics of the disease in Helicobacter pylori infected patients in Shahrekord, Southwest Iran.

The purpose of this study was to determine the mutations associated with clarithromycin resistance in Helicobacter pylori strains isolated from biopsy samples that were collected from the endoscopic ward of Shahrekord Hajar teaching Hospital and also to study the frequency of virulence factor and their correlation and pathological findings with clarithromycin resistance during the years 2019-2020. In this cross-sectional descriptive study, 152 patients with Helicobacter pylori infection were considered, and then, two common A2142G and A2143G mutations in the 23SrRNA gene associated with resistance were analyzed by Real-time PCR (Taq man). The presence of vacA, iceA1, iceA2, cagA, babA2, and oipA virulence genes was investigated by PCR and electrophoresis in 8% polyacrylamide gel. Then, data were analyzed using the relevant statistical tests. In this study, the frequency of Helicobacter pylori was 76% and the frequency of mutant isolates was 57.2%. The frequencies of A2142G and A2143G point mutations were 42.1% and 28.3%. There was a significant correlation among oipA, vacA, and iceA1 virulence factors, type of disease, chronic inflammatory score, and glandular atrophy with the antibiotic resistance to clarithromycin. There was no significant correlation between the age and sex of the patients with antibiotic resistance. According to the results of this study, it seems that the use of clarithromycin to combat this bacterium should be limited.

Determination of CagA EPIYA motif in Helicobacter pylori strains isolated from patients with digestive disorder.

This study was conducted to identify patterns of cagA EPIYA motifs in H. pylori strains isolated from patients with gastrointestinal diseases in Hospitals of Shahrekord, and investigate the association between these biomarkers and clinical outcomes of gastrointestinal diseases due to H. pylori. In this study, 253 patients with gastrointestinal diseases were studied within 1395-1396. Histopathological investigations and urease test showed that 207 isolates were H. pylori-positive. Then, screening using a molecular technique, PCR, confirmed that 159 isolates had cagA. Finally, the pattern and prevalence of the motifs were determined by PCR and identified a number of motifs were sequenced. Results of this study showed that the pattern of motifs was as follows: ABC (140 isolates) (93/7%), ABCC (6 isolates) (3/77%), ABCCC (4 isolates) (2/5%), AB (7 isolates) (4/4%), AC (1 isolate) (0/6%), and BC (1 isolate) (0/6%). Sequencing results showed the presence of changed EPIYA motif in some isolates. CM motif sequence was also seen in all isolates. In this study, no significant association was seen between the prevalence rate of different patterns and clinical symptoms (p = 0.71). There is a slight association between the presence of ABC motifs and the type of digestive disorder (p = 0.056). Results indicated that ABC was the most frequently seen pattern however, in such that positive cases of ABC motifs were more common in gastritis. All isolates had kinase phosphorylation region, and the observed pattern in this region was a generally western type (ABC).

Phenotypic and genotypic detection of metallo-ß-lactamases in A. baumanii isolates obtained from clinical samples in Shahrekord, southwest Iran.

OBJECTIVE: Acinetobacter baumanii is a pathogenic bacterium that is the cause of many nosocomial infections. This study aimed to determine metallo-ß-lactamases (MBL) produced by the A. baumanii isolates obtained from clinical samples in Shahrekord, southwest Iran. RESULTS: A total of 100 A. baumanii were isolated from 250 clinical samples between June 2013 and June 2014. Then, the isolates were identified by biochemical tests, and MBL screening was conducted by the phenotypic tests modified Hodge, EDTA-disk synergy (EDS), combined disk (CD) and AmpC disc after antibiotic sensitivity test. Using PCR technique the bla genes were detected. Eighty-five (85%) isolates were resistant to meropenem and imipenem. Phenotypic tests showed that out of the 100 isolates, 46, 59, 50, 65 and 65 isolates were positive: AmpC disk, CD, EDS, Modified Hodge and E-test MBL respectively. Gene detection by PCR showed that 23 isolates carried the VIM-1 gene and only three isolates carried the IMP-1 gene. The prevalence of metallo-ß-lactamases isolates containing A. baumanii is increasing. Furthermore, the coexistence of various carbapenemases is dominantly act as a major problem. Continuous monitoring of the infections related to these bacteria should be considered to plan an alternative and new therapeutic strategies.

Agr typing of Staphylococcus aureus species isolated from clinical samples in training hospitals of Isfahan and Shahrekord.

OBJECTIVE: As an opportunistic pathogen, Staphylococcus aureus is associated with serious nosocomial infections and growing antimicrobial resistance against beta-lactams among S. aureus strains has become a global challenge. The current study was designed to investigate the presence of agr genes among S. aureus strains recovered from clinical samples in university hospitals of Isfahan and Shahrekord. RESULTS: A total of 150 S. aureus isolates were screened by Disk diffusion method (DDM) and conventional PCR. The minimum (17.3%) and maximum (46%) antibiotic resistance rates were found in vancomycin and cefoxitin, respectively. The majority of our isolates were classified as agr type I followed by type II, type IV, and type III. The statistical analysis showed a significant correlation between agr type I and antibiotic resistance against cefoxitin and erythromycin (p = 0.04 and p = 0.03, respectively). Based on our findings, the agr typing could be considered an effective approach for molecular tracking of S. aureus infections.

Prevalence and distribution of adhesins and the expression of fibronectin-binding protein (FnbA and FnbB) among Staphylococcus aureus isolates from Shahrekord Hospitals.

OBJECTIVE: One of the most important causes of nosocomial infections is Staphylococcus aureus. The aim of this study was to determine the frequency of these genes and the rate of expression of these genes during nasal colonization among the personnel of Kashani and Hajar hospitals. RESULTS: In this Analytical-descriptive study, 240 nasal swab specimens were collected from personnel of different departments of Kashani and Hajar hospitals in Shahr-e-kord. Nasal specimens were cultured and 110 Staphylococcus strains were isolated. Based on the results, 110 carriers of Staphylococcus aureus were identified. The frequency of clfA, clfB, fnbA and fnbB genes were 36.3%, 86.3%, 7.2% and 43.6% respectively. It was also observed that the fnbA gene showed no expression, but of 95 clfB-positive samples, 73 isolates (76.8%) were expressed clfB gene. This study showed that the abundance of these genes varies in nasal colonization. It was also observed that clfB gene with a high frequency and high expression rate has an important role in nose colonization. These results not only provide insight into the factors involved in S. aureus colonization but also provide potential therapeutic targets.

The highly conserved domain of RND multidrug efflux pumps in pathogenic Gram-negative bacteria.

RND (Resistance-Nodulation-Division) family transporters have a vital role in both intrinsic and acquired multi-drug resistance in Gram-negative bacteria. It is important to find a conserved domain in the RND family between different pathogenic bacteria for diagnostic and therapeutic purpose. Total sequences of three-component system RND efflux pumps were retrieved from NCBI nucleotide and protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. The phylogenetic tree for main transporters was drawn and the three-dimensional structure was also evaluated. From the sequence conservation analysis, highly conserved residues with 282 base pair (94 amino acid) long were identified. The location of the highly conserved domain is positioned in the domain 1 crystallographic structure of AcrB Escherichia coli and MexB Pseudomonas aeruginosa. The main transporter component phylogenetic tree shows the clusters of different genotypes and their evolutionary association.  Each of three components of RND proteins is crucial for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. Therefore, this highly conserved region can be used to disable the RND multidrug efflux pumps. In addition, this highly conserved can also be used for diagnostic aspects.

Evaluation of cellular immunogenicity of recombinant cytochrome p450 cyp141 protein of Mycobacterium tuberculosis in human and mouse model.

Mycobacterium tuberculosis (Mtb) is still considered one of the unsolved problems for the World Health Organization Identifying and selecting an immunogenic antigen capable of generating specific immune responses is generally the goal of all studies being carried out in to designing new vaccines. Accordingly, the present study was conducted to evaluate the immunogenicity of a M. tuberculosis recombinant protein which exist in the regions of the bacterium genome and may be an immunogenic protein. Immunogenicity of purified proteins was measured by PBMC and mouse spleen lymphocytes culturing methods using ELISA after an appropriate amount of time of incubation with Recombinant cytochrome P450 CYP141 protein. Cellular immune responses were determined and compared by measuring IFN-γ and IL4 in human, and mouse groups. The results revealed a high level of IFN-γ in PPD + individuals and the mice immunized with protein and adjuvant. Recombinant cytochrome P450 CYP141 protein proved capable of generating an immune response in mice and people with a history of previous encounters with Mycobacterium tuberculosis bacteria. It, could be considered a tuberculosis vaccine candidate in order to induce a specific effective immune response in both mice and humans.

Prevalence of Class D Carbapenemases among Extended-Spectrum ß-Lactamases Producing Escherichia coli Isolates from Educational Hospitals in Shahrekord.

INTRODUCTION: Extended-spectrum ß-lactamases (ESBLs) are a set of plasmid-borne, various and quickly evolving enzymes that are a main therapeutic issue now-a-days for inpatient and outpatient treatment. AIM: The aim of this study was to determine multi-drug resistance (MDR) and ESBLs producing E. coli strains, prevalence of class D Carbapenemases among ESBLs producing Escherichia coli isolates from educational hospitals in Shahrekord, Iran. MATERIALS AND METHODS: Uropathogenic Escherichia coli strains were isolated from patients with Urinary Tract Infections (UTIs). The agar disc diffusion test was used to characterize the antimicrobial sensitivity of the E. coli isolates. The ESBL positive strains were identified by phenotypic double-disk synergy test, by third-generation cephalosporin in combination with or without clavulanic acid. Multiplex PCR was carried out for detection of the three families of OXA-type carbapenamases including OXA-23, OXA-24, and OXA-48 in E. coli strains. RESULTS: All bacterial isolates were susceptible to meropenem. Ninety isolates produced ESBL, 55 E. coli isolates from inpatients, and 35 isolates from outpatients, with a significant association (p< 0.05). The prevalence of OXA-23, OXA-24, and OXA-48 in the ESBLs producing isolates was respectively 21%, 18%, and 11% for inpatients, and 10%, 8%, and 6% for outpatients. CONCLUSION: ESBL-producing E. coli isolates are also a major threat in the clinical setting. The findings of this study indicated the high occurrence of ESBLs and multiple antibiotic resistance in E. coli isolates.

Prevalence of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates in Nosocomial and Community-Acquired Urinary Tract Infections.

BACKGROUND: Klebsiella pneumoniae is a family member of Enterobacteriaceae. Isolates of K. pneumoniae produce enzymes that cause decomposition of third generation cephalosporins. These enzymes are known as extended-spectrum beta-lactamase (ESBL). Resistance of K. pneumoniae to beta-lactamase antibiotics is commonly mediated by beta-lactamase genes. OBJECTIVES: The aim of this study was to identify the ESBL produced by K. pneumoniae isolates that cause community-acquired and nosocomial urinary tract infections within a one-year period (2013 to 2014) in Kashani and Hajar university hospitals of Shahrekord, Iran. PATIENTS AND METHODS: From 2013 to 2014, 150 strains of K. pneumoniae isolate from two different populations with nosocomial and community-acquired infections were collected. The strains were then investigated by double disk synergism and multiplex polymerase chain reaction (PCR). RESULTS: The study population of 150 patients with nosocomial and community-acquired infections were divided to two groups of 75 each. We found that 48 of the K. pneumoniae isolates in the patients with nosocomial infection and 39 isolates in those with community-acquired infections produced ESBL. The prevalence of TEM1, SHV1 and VEB1 in ESBL-producing isolates in nosocomial patients was 24%, 29.3% and 10.6%, and in community-acquired patients, 17.3%, 22.7% and 8%, respectively. CONCLUSIONS: The prevalence of ESBL-producing K. pneumoniae isolate is of great concern; therefore, continuous investigation seems essential to monitor ESBL-producing bacteria in patients with nosocomial and community-acquired infections.

Altered Th17 Cytokine Expression in Helicobacter pylori Patients with TLR4 (D299G) Polymorphism.

Helicobacter pylori (H. pylori) is associated with gastric ulcer and gastric adenocarcinoma. Polymorphisms in the host genes coding for Toll-like receptors (TLRs) may influence the innate and adaptive immune response to the infection, affecting the susceptibility to H. pylori or the disease outcomes. However, the details and association with different polymorphism and clinical expression of infection remain unclear. A case-control study consisting of 58 patients with H. pylori infection and 44 H. pylori uninfection was conducted. Genomic DNA was extracted and genotypes of TLR4 Asp299Gly polymorphism were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mucosal cytokines expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. The expression of IL-6, IL-17, IL-21, IL-23 and TGF-ß1 was significantly higher in patients with D299G polymorphism in TLR4. But the expression of IL-18 between patients with single-nucleotide polymorphisms (SNPs) in TLR4 and patients with the wild-type allele was not significant. In H. pylori-infected patients with gastritis, SNPs in TLR4 may alter cytokine expression toward Th17 immune response in the gastric mucosa and may have increased risk for the development of peptic ulcer.

Expression and Purification of the Recombinant Cytochrome P450 CYP141 Protein of Mycobacterium Tuberculosis as a Diagnostic Tool and Vaccine Production.

BACKGROUND: Tuberculosis (TB) is regarded as a health problem worldwide, particularly in developing countries. Mycobacterium tuberculosis (M. tuberculosis) is the cause of this disease. Approximately two billion people worldwide are infected by M. tuberculosis and annually about two million individuals die in consequence. Forty million people are estimated to die because of M. tuberculosis over the next 25 years if the measures for controlling this infection are not extensively developed. In the vaccination field, Bacillus Calmette-Guérin (BCG) is still the most effective vaccine but it shows no efficacy in adult pulmonary patients. One of the other problems regarding TB is its appropriate diagnosis. OBJECTIVES: In this experimental study, the recombinant cytochrome P450 CYP141 protein of M. tuberculosis was expressed and purified to be used as a vaccine candidate and diagnostic purpose in subsequent investigations. MATERIALS AND METHODS: The optimization of the cytochrome P450 CYP141 protein expression was evaluated in different conditions. Then, this protein was purified with a resin column of nickel-nitrilotriacetic acid and investigated via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting. RESULTS: The highest expression of the cytochrome P450 CYP141 protein was obtained by the addition of 1 mM of isopropyl ß-D-1-thiogalactopyranoside (IPTG) to the bacterial culture grown to an optical density at 600 nm (OD600) of 0.6, 16 hours after induction. This protein was subsequently purified with a purification of higher than 80%. The results of Western Blotting indicated that the purified protein was specifically detected. CONCLUSIONS: In this experimental study, for the first time in Iran the expression and purification of this recombinant protein was done successfully. This recombinant protein could be used as a vaccine candidate and diagnostic purpose in subsequent investigations.

The FimH Gene in Uropathogenic Escherichia coli Strains Isolated From Patients With Urinary Tract Infection.

BACKGROUND: Urinary tract infections (UTIs) are one of main health problems caused by many microorganisms, including uropathogenic Escherichia coli (UPEC). UPEC strains are the most frequent pathogens responsible for 85% and 50% of community and hospital acquired UTIs, respectively. UPEC strains have special virulence factors, including type 1 fimbriae, which can result in worsening of UTIs. OBJECTIVES: This study was performed to detect type 1 fimbriae (the FimH gene) among UPEC strains by molecular method. MATERIALS AND METHODS: A total of 140 isolated E. coli strains from patients with UTI were identified using biochemical tests and then evaluated for the FimH gene by polymerase chain reaction (PCR) analysis. RESULTS: The UPEC isolates were identified using biochemical tests and were screened by PCR. The fimH gene was amplified using specific primers and showed a band about 164 bp. The FimH gene was found in 130 isolates (92.8%) of the UPEC strains. Of 130 isolates positive for the FimH gene, 62 (47.7%) and 68 (52.3%) belonged to hospitalized patients and outpatients, respectively. CONCLUSIONS: The results of this study indicated that more than 90% of E. coli isolates harbored the FimH gene. The high binding ability of FimH could result in the increased pathogenicity of E. coli; thus, FimH could be used as a possible diagnostic marker and/or vaccine candidate.

Inflammatory myofibroblastic tumor: report of a rare case in kidney.

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm mostly seen in the lungs, but also in extrapulmonary sites. The most common genitourinary site of IMT is the bladder, but it may rarely be seen in the kidneys. We report a case of a 15-year-old girl presented with flank pain and hematuria, in which computed tomography scan revealed a mass in the left kidney. The patient underwent left nephrectomy for a diagnosis of Wilms tumor. Further assessment of the tissue demonstrated a pathologic diagnosis of IMT. Despite improvements in imaging technology, the preoperative diagnosis of IMT remains difficult and surgery is the only way for the diagnosis and treatment. Considering the role of the pathologic examination in making the definite diagnosis of IMT, we should be aware of this entity and it must be considered in the differential diagnoses.

Phenotypic and Molecular Characterization of Extended-Spectrum ß-Lactamase Produced by Escherichia coli, and Klebsiella pneumoniae Isolates in an Educational Hospital.

BACKGROUND: Extended-spectrum beta-lactamases (ESBLs) are a group of enzymes that hydrolyze antibiotics, including those containing new cephalosporins, and they are found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains. With the widespread use of antibiotics, difficulties with infection therapy caused by drug resistant organisms, especially those that have acquired resistance to beta-lactams, such as broad-spectrum cephalosporins, have amplified the above-mentioned organisms. OBJECTIVES: This study was conducted to characterize ESBLs among E. coli and K. pneumonia isolates by molecular and phenotypic methods. MATERIALS AND METHODS: Different strains of E. coli and K. pneumonia were collected from patients with urinary tract infections. The ESBL phenotype was determined by a double disk diffusion test (DDDT). In addition, polymerase chain reaction (PCR) analysis specific for ß-lactamase genes of the TEM and SHV family was carried out. The PCR products were run on agarose and examined for DNA bands. RESULTS: A total of 245 E. coli and 55 K. pneumonia strains were isolated from different samples. In total, 128 of the 300 isolates were confirmed as potential ESBLs producers as follows: 107 (43.67%) E. coli and 21 (38.18%) K. pneumonia. ESBLs genes were found in 24 isolates (18.75%): 21 E. coli and 3 K. pneumonia isolates. The TEM gene was present in 13 (12.14%) E. coli strains, but it was not detected in K. pneumonia. In addition, the SHV gene was present in 8 (7.47%) E. coli and 3 (14.28%) K. pneumonia isolates. Five (4.67%) of the E. coli isolates harbored both TEM and SHV genes. All isolates (100%) were susceptible to imipenem. The lowest rates of resistance to other antibiotics were observed for; piperacillin-tazobactam (6.25%), amikacin (12.5%) and gentamicin (14.84%). The rates of resistance to other antibiotics were as follow: nitrofurantoin (16.4%), nalidixic acid (23.43), co-trimoxazole (25%), cefepime (32%), ciprofloxacin (55.46%), ampicillin (69.53%), ceftazidime (100%), and cefotaxime (100%). CONCLUSIONS: The results of this study indicate the widespread prevalence of ESBLs and multiple antibiotic resistance in E. coli and K. pneumoniae. Therefore, beta-lactam antibiotics and beta-lactamase inhibitors or carbapenems should be prescribed based on an antibacterial susceptibility test.

Cloning of the Recombinant Cytochrome P450 Cyp141 Protein of Mycobacterium tuberculosis as a Diagnostic Target and Vaccine Candidate.

BACKGROUND: Tuberculosis has been announced as a global emergency by World Health Organization and the second infectious agent of mortality worldwide. The general policy in the development of new vaccines is to develop some vaccines with higher efficiency not only for infants but also for adults compared with the Bacillus Calmette-Guerin vaccine. Recently, cytochrome P450 cyp141 has been introduced as a new target for detecting Mycobacterium tuberculosis from clinical samples. OBJECTIVES: The aim of this study was to clone this gene in order to pave the way for more evaluation. MATERIALS AND METHODS: M. tuberculosis H37Rv DNA was extracted by a standard phenol-chlorophorm protocol. After designing the specific primers, P450 cyp141 gene was replicated by PCR. The purified PCR products were then subcloned into the pTZ57R/T plasmid vector. After extraction, enzyme digestion, and recombinant pTZ57R/T-cyp141 plasmid vector sequencing, the aforementioned products were cloned into a pET-26b plasmid vector. Then, the recombinant pET26b-cyp141 plasmid molecules were transformed to Escherichia coli strain BL21 (DE3) using the transformation method. Next, the recombinant pET26b-cyp141 plasmids were purified and evaluated by the enzyme digestion analysis. RESULTS: The cloning of P450 cyp141 gene was confirmed by the enzyme digestion and sequencing of the recombinant pTZ57R/T-cyp141 and pET26b-cyp141 plasmid vectors. CONCLUSIONS: The results of this study demonstrated that the P450 cyp141 gene was successfully cloned into a pET26b plasmid vector as an expression vector. In this paper, for the first time in Iran, this gene was cloned for more purposes, including the expression and purification of the recombinant cytochrome P450 cyp141 protein.

Development of an indirect sandwich ELISA for detection of urinary antigen, using Legionella pneumophila PAL protein.

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease.