Pasteur Institute of Iran

Various adjuvants in combination with different antigens have been utilized as a vaccine candidate against leishmaniasis. However, the search for ideal adjuvants is still pursued due to the inefficiencies of current compounds. In the present study, the effect of imiquimod, as an adjuvant, is studied with soluble Leishmania antigens (SLA) in BALB/c mice. Four groups of mice were immunized with SLA, SLA plus imiquimod, SLA plus BCG, and PBS as control. Immunized mice received a boosting dose of SLA after 15 days. All groups were challenged with Leishmania major (L. major) promastigotes 2 weeks after the booster immunization. Our results showed that strong TH1 responses were induced in groups of SLA plus imiquimod as well as SLA plus BCGafter immunization. These responses included smaller footpad thickness, lower parasite load in lymph node, and higher proliferative response of lymph node cells to SLA, higher levels of interferon &gamma in culture supernatant of lymph node cells, and higher levels of IgG, and IgG2a in sera. The data supports the possibility of using imiquimod as a suitable adjuvant in leishmania vaccination.

  Biofilm of Pseudomonas aeruginosa, an opportunistic pathogen, can cause serious health problems, such as chronic infections, especially in immunocompromised patients. Many studies have suggested administration of new generation of antibiotics, as P. aeruginosa biofilms have developed high resistance to antimicrobial drugs. This study reports the inhibitory effect of three medicinal plant extracts and an essential oil on biofilm formation by a clinical isolate of P. aeruginosa. In this study biofilm formation of P. aeruginosa strain 214 was determined in presence of three plant extracts, Cyclamen coum, Dianthus orieltalis and Origanum majorana, and Zataria multiflora Bio essential oil. Minimum Biofilm Inhibitory Concentrations (MBICs) were determined by microdilution techniques and XTT assay. The C. coam extract and Z. multiflora Bio essential oil inhibited biofilm formation completely at concentrations<0.062 mg/ml and 4 µl/ml, respectively. The D. orientalis and O. majorana extracts did not inhibit biofilm formation at the used concentrations (0.003 – 8 mg/ml). The results of this study indicate that some plant extracts at low concentrations may provide a complementary medication for biofilm-associated infections. Further evaluations are required to validate the antibiofilm effect of these medicinal plants.

  Pseudomonas aeruginosa is among the most important pathogens in the nosocomial infections. A genetic mobile element, the integron, is one of the major agents involved in dissemination of multi-drug resistance among gram negative bacteria. During a descriptive study from October 2009 to August 2010, some 130 P. aeruginosa clinical isolates were collected from different wards of three hospitals in Tehran. The Minimal inhibitory concentration (MIC) of 4 antibiotics conventionally used in clinical settings against the isolates was determined by E-test method. Also, the existence of integron classes and metallo-β-lactamases (bla_VIM-1, bla_IMP-1, and bla_VIM-2) were investigated by PCR assay. Out of 130 isolates, 74 (56.9%) carried class 1 integron. None of the isolates harbored integrons class 2 and 3. Also, the bla_VIM-1 gene was detected in 10 (13.3%) high level ceftazidime and imipenem- resistant isolates that carried class 1 integrons. The bla_IMP-1 and bla_VIM-2 genes were not detected in any isolates. In the present study, the antibiotic resistance rates in class 1 integron-positive isolates of P. aeruginosa were significantly higher than those lacking this integron , e.g., 82.6% resistance versus 17.3% sensitivity to ceftazidime. Also, 13.3% of ceftazidime and imipenem resistant isolates was metallo-β-lactamase producer. This indicates that all metallo-β-lactamase genes are correlated with class 1 integrons. These results imply that the bla_VIM-1 gene has been presumably dispersed into P. aeruginosa isolates with the help of class 1 integron element.

  Introduction : The decreased level of immunity in Human Immunodeficiency Virus (HIV) infected patients increases their vulnerability to various opportunistic fungal infections. Oral candidiasis has been found to be the most common fungal infection among HIV infected patients. The present study was conducted to evaluate the spectrum of various opportunistic fungal infections and their correlation with CD4+ counts. Methods: A total of 163 clinically suspected cases of fungal infections with HIV seropositive status were studied. Results: The most common infections observed were oropharyngeal candidiasis (39.26%) followed by cryptococcal meningitis (6.74%). The study showed opportunistic fungal infections in 46.6% of HIV infected patients with CD4+ counts ≤200 cell/ µl. Conclusion: Early diagnosis and prompt antifungal treatment is necessary to decrease the morbidity and mortality associated with the infections to increase the survival of HIV infected patients. J Med Microbiol Infec Dis, 2014, 1 (2): 4 pages.

  Introduction : Haemophilus influenzae is a gram-negative bacterium causing a variety of respiratory infections in developing countries, especially in children. Nasopharynx carriers of H. influenzae are the prominent source and transitional vectors of invasive diseases. As very limited information on H. influenzae carriage rate in Iran was available, an evaluation on prevalence of this bacterium in children ≤ ‌ 6 years old seems crucial. Methods: Totally 533 mucus samples were collected using nasopharyngeal swabs from children ≤ ‌ 6 years old who lived in 4 nursery centers in Tehran or refereed to the Children's Medical Center of Tehran, Iran, from August 2011 to October 2012. The samples were transported in Stuart transport medium to the Microbiology Laboratory of Pasteur Institute Tehran, Iran, and were cultured on chocolate agar containing bacitracin antibiotic. The initial diagnosis for detection of H. influenzae was performed by standard biochemical tests, and confirmation was achieved by PCR assay targeting outer membrane protein (omp) P6 gene. Results: Based on primary cultures and biochemical tests, out of 533 samples, 182 (33%) showed to be H. influenzae positive, but PCR assay confirmed presence of H. influenzae in 153 (28%) isolates 56(37%) belonged to girls and 97 (63%) to boys. The prevalence of H. influenzae in three different age groups: ≤ ‌ 24, 25-48, and 49-72 month-old children were 31 (20%), 69 (45%), and 53 (35%), respectively. Conclusion : Our results showed a high rate of H. influenzae carriers among children ≤ ‌ 6 years old, which is similar to those of other unvaccinated countries. H. influenzae carriage rate was associated to age and respiratory infection diseases. The children aged 25-48 months showed a higher rate and the rate reduced with increase in age. Further investigation including molecular studies is required to obtain the carriage rate throughout the country. J Med Microbiol Infec Dis, 2014, 1 (2): 5 pages.

Introduction : This study was aimed to investigate the effects of risk factors, and environmental and climatic factors a ffecting the occurrence of Crimean-Congo hemorrhagic fever (CCHF) in Iran. We used temporal modeling to predict the future occurrence of the disease in the country . Methods : We analyzed the data of 165 CCHF patients from all over Iran (e xcept the districts Zabol and Zahedan in Eastern Iran ) during 2000 to 2006. In this study, 130 districts with at least one reported case patient, and 780 districts with no reported case patient, as the control group, were included in the model. Logistic regression was used to design the temporal model of the disease at the district-month level nationwide with the purpose of predicting the occurrence of CCHF disease with in one month in a district. Results: The designed model indicated that the history of previous reports of the disease in a district increased the risk of further reports of the disease (odds ratio: 2.53 (95% CI: 1.61, 3.97), (P ‌ <0.001)). Moreover, w ith each one-million increase in the urban population, the odds of a report of the disease increased 20% (P ‌ =0.003). The odds of the occurrence of the disease were reduced by 9% w ith the increase in e ach degree of latitude (P ‌ =0.028). The odds of the occurrence of the disease increased 6.25 times w ith the increase in e ach kilometer of altitude (P ‌ <0.001). T he disease had a decreasing s ecular trend so that the occurrence of the disease was reduced by 10 ‌ % each year (P ‌ =0.008). Conclusion : Our findings showed that based on the history of CCHF in districts, and population and geographical features, hot zones may be defined with some acceptable accuracy. J Med Microbiol Infec Dis, 2014, 1 (2): 7 pages.

  Introduction : Rabies is a disease that has been known since antiquity. It is a highly fatal acute disease of the central nervous system caused by a lyssavirus. Prior to the discovery of the rabies vaccine, rabies-infected individuals fell victim to the delusions and superstitions associated with this disease. Though it has been neglected in many regions of the world, rabies remains one of the most feared diseases in many developing countries, where it takes the majority of its victims. The virus circulates mainly in domestic and wild carnivores, taking 60,000 human lives worldwide every year and inflicting significant financial damage. It can, however, be well controlled due to the availability of effective Post-Exposure Prophylaxis (PEP) protocols. Pasteur Institute of Iran has had a significant role in the establishment of current PEP protocols in the world. In spite of the availability of effective PEP protocols, preventive vaccination would be preferable in endemic regions. Annually, a considerable number of exposures to animal bites occur in Iran. The current situation in the country is well-controlled by virtue of a robust surveillance system and efficient PEP treatments, resulting in considerably low death incidences from rabies. High quality vaccines recommended by the World Health Organization (WHO) are expensive and unaffordable in developing countries, where the need for rabies vaccination is greatest. Therefore, there is an increasing need to develop new cost-effective and efficient vaccines requiring fewer injections and providing longer-lasting immunity. J Med Microbiol Infec Dis, 2014, 1 (2): 10 pages.

Introduction: Natural products have been the most successful source of potential drug leads. One of them is lichens, which have been used since antiquity as natural sources of drugs. The aim of this study was to investigate the antibacterial and antifungal activity of different extracts of the Iranian lichens in Ilam Province. Methods: The aqueous, acetone, and methanol extracts of 6 lichen species, including Caloplaca variabilis, Fulgensia subbracteata, Lecanora muralis, Physcia adscendens, Psora decipiens, and Megaspora verrucosa, were produced using Soxhlet extractor. Then, antibacterial and antifungal activities of them against 6 standard strains of bacteria, including Enterococcus faecalis ATCC 2321, Escherichia coli ATCC 1652, Proteus mirabilis ATCC 2601, Salmonella typhi ATCC 1679, Staphylococcus aureus ATCC 1885, and Staphylococcus epidermidis ATCC 2405 and two fungi, including Fusarium moniliforme and Verticillium dahliae, were evaluated using the disc-diffusion method and the minimal inhibitory concentration was determined by broth tube dilution. Streptomycin (10 µg/ml) and ketoconazole (10 µg/ml) were used as positive controls for bacteria and fungi, respectively. Sterile distilled dimethyl sulfoxide (DMSO) was used as negative control. Results: Methanol extract from F. subbracteata and L. muralis lichens showed relatively high antibacterial activities (p<0.01), whereas aqueous extracts showed no activity against the microorganisms and only acetone extract of L. muralis showed antibacterial activity against the S. epidermidis (MIC=500). Methanol extracts of F. subbracteata and L. muralis had higher antifungal activities than others. Conclusion: The methanol extracts of L. muralis and F. subbracteata showed the highest activity against the bacteria and fungi and it seems that they have some antibacterial and antifungal properties.

Introduction: Hydatid cyst disease is caused by the protoscolices of Echinococcus granulosus. Alkaline phosphatase (ALP) enzyme is required for metabolism, physiology, immunology, and nutrients absorption in parasite. The aim of this study was to compare the level of ALP activity (as a pathological biomarker) in hydatid cyst protoscolices (HCP) with that of sheep liver tissue and to determine the effect of cystic infection on the enzyme activity. Methods: HCPs were collected from sheep livers with hydatid cysts at a local abattoir and washed 3 times with PBS buffer. HCP samples were freeze-thawed and sonicated, while the collected liver tissues were homogenized. Then, extract solutions were centrifuged and stored at -20°C. ALP activity was measured in the extract solutions of HCP and healthy and infected sheep liver tissue samples. The amounts and bands of protein samples were detected using Bradford method and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. To determine the significant difference between the two groups, independent two samples t-test was used. Results: The mean values of ALP-specific activity of healthy and infected livers and HCP were estimated 0.019, 0.175, and 1.28 U/ml/mg, respectively. Higher ALP activity level was observed in cystic liver compared to healthy liver (p<0.05). T-test analysis showed higher ALP enzyme activity for HCP compared to healthy liver (p<0.05). SDS-PAGE demonstrated a protein band with molecular weight of 59 kDa in HCP samples, which was identified as ALP. Conclusion: ALP activity in HCP and healthy liver indicates the importance of this enzyme in comparative biochemistry of liver and parasite. Higher level of ALP enzyme activity in cystic liver in comparison with healthy liver could be considered as a pathological biomarker for diagnosis of hydatid cyst disease with other hydatid disease parameters.

Introduction: Pseudomonas aeruginosa is a serious challenge for antimicrobial therapy, due to chromosomal mutations or intrinsic resistance to various antimicrobial agents, such as Metallo-β-Lactams (MBL). This study aimed to investigate the prevalence of β-lactamases encoding genes among P. aeruginosa strains isolated from intensive care unit (ICU) patients by phenotypic and multiplex PCR methods. Methods: A total of 48 non-duplicate strains of P. aeruginosa were collected from different clinical specimens of patients hospitalized in ICU wards of a teaching hospital in Isfahan, Iran. Susceptibility test was performed by disk diffusion method. All meropenem resistant strains were subjected to modified Hodge test (MHT) for detection of carbapenemases. Multiplex PCRs were performed to detect β-lactam-resistant P. aeruginosa isolates. Results: In disk diffusion method, P. aeruginosa strains showed the most (97.9%) resistance against imipenem and meropenem and the least (45.8%) against colistin. Thirty-six (75%) out of the 48 isolates were multidrug resistant. PCR amplification of β-lactamase genes showed the presence of bla_VIM genes in 7 (14.6%) and bla_IMP in 15 (31.3%) strains. Also, bla_{SME, SPM, GIM, AIM and NDM} genes were not observed in any of the strains. We only found a statistically significance difference between the presence of bla_IMP gene and multidrug-resistant (MDR) positivity and source of specimen (p=0.009 and 0.002, respectively). Conclusion: Rapid and reliable identification of MBLs appears to be necessary for effective treatment of related infections. Besides, our results may provide useful perception to make a more appropriate choice of antibiotics, which may put a stop to carbapenem-resistant infections.

Introduction: Most B-cell malignancies are diagnosed based on morphologic and immunohistochemical criteria. Some cases, however, still present a challenge for the pathologist to discriminate between reactive hyperplasia and neoplastic disorders. Molecular techniques can be used as a helpful diagnostic tool in these cases. In this study, we assessed the value of polymerase chain reaction (PCR) technique in determination of monoclonality of immunoglobulin heavy chain gene rearrangements for the diagnosis of large B-cell non-Hodgkin's lymphoma (NHL) in paraffin embedded tissue samples. Methods: DNA was extracted from paraffin embedded tissues of 44 diffuse large B-cell lymphoma (DLBCL) cases and 20 samples of reactive lymphoid tissues from appendix and tonsils as control. Framework 3 and the joining region (FR3/JH) of the variable segment of the immunoglobulin heavy chain gene were amplified using degenerated primers. PCR products from each sample were analyzed on 8% polyacrylamide gels following AgNO3 staining. Results: Monoclonal rearrangements were identified in 33 of 44 cases (75%) of DLBCL using FR3/JH primers. Monoclonal IgH gene rearrangements were not detected in any of the reactive lymphoid hyperplasic samples. All these control cases showed polyclonal pattern. Conclusion: Through PCR analysis, using degenerated primers, monoclonality was demonstrated in 75% of DLBCL cases. This technique can thus be used as a sensitive, reliable and valuable diagnostic supplement to conventional morphologic examination and immunohistocytochemical evaluation of lymphoproliferative disorders, particularly in cases with restrictions in amount or type of analytic material.

Introduction: Some snails play an important role in the transmission of helminthes, mainly trematodes of medical and veterinary importance. There seems to be no information on the freshwater snails of Gahar Lake in Lorestan province of Iran. The present study aimed to identify medically important snails of this lake. Methods: Samples were collected from ten localities around Gahar Lake in April 2015 by hand. The snails were classified according to shells morphology. The data were then analyzed using descriptive statistics. Results: A total of 6 snail species were collected from all localities. Four medically important snail species including: Lymnaea truncatula, Lymnaea peregra, Melanoides tuberculata and Melanopsis spp. were detected. M. tuberculata was seen in all sampling sites. Physa acuta and Melanopsis spp. were observed in five sampling sites. Planorbis intermixtus, L. peregra and L. truncatula were found in four, three and two sampling sites, respectively. Conclusion: Presence of medically important snails in Gahar Lake could be a source of trematode infections for visitors. Therefore control measures, especially biological ones should be applied to the lake. 

Introduction: This study is aimed to compare phenotypic test methods and determine antibiotic susceptibility pattern of AmpC beta-lactamase producing uropathogenic Escherichia coli and Klebsiella pneumoniae in clinical isolates. Method: E. coli and K. pneumoniae were identified by standard microbiological procedures. Screening of AmpC beta-lactamase production was done by using cefoxitin disc (30 µg) showing inhibition zone diameter of <18 mm. Then, screen-positive isolates were subjected to Disc Approximation Test (DAT) and three dimensional extract test (3-DET) methods. Antibiotic susceptibility testing was performed by Kirby Bauer Disc diffusion technique. Results: A total of 120 Gram Negative Rods (GNRs) were included in the study. Amongst them cefoxitin resistant isolates were 68.33% (n=82/120). In these 82 isolates, E. coli were n=57 (69.51%) and K. pneumoniae were n=25 (30.48%). DAT identified 52.43% of AmpC beta-lactamase producing isolates, sensitivity of DAT was found to be 88% with 92% specificity, Positive Predictive Value of 92.68%, Negative Predictive Value of 87.80%, and Diagnostic Accuracy of 90.24%. Antibiotic susceptibility testing by Kirby Bauer Disc diffusion technique showed that carbapenems (meropenem) and tigecycline were of higher therapeutic effects against these resistant pathogens. Conclusion: Introducing simple tests like DAT in the laboratories can control the spread of AmpC beta-lactamase harboring organisms. Carbapenems (meropenem) and tigecycline are of suitable therapeutic effect against these resistant pathogens.

Introduction: There is not much data on parasitic infections of laboratory animals that are kept in conventional conditions in Iran. The present study was designed to investigate intestinal helminths infections in laboratory colonies of rats and mice. Methods: Droppings from 110 mice and 110 rats (each animal one dropping) belonging to experimental and breeding groups in four animal houses were collected. Experimental groups were being used in biomedical researches and breeding groups were not under any experiment. The droppings were preserved in formaldehyde 10% individually and examined by microscopy with 10x magnification. Results: Out of 220 droppings examined, 96 (43.6%) harbored helminths eggs; 53 (48.1%) belonged to mice and 43 (39.09%) to rats. Four helminthes species including, Syphacia obvelata, Syphasia muris, Hymenolepis nana, and Hetrakis spumosa were identified in the both animals, while Aspicularis tetraptera was merely seen in mice. H. nana was the most frequent helminth infection in mice and rats and infection with H. spumosa and A. tetraptera, showed the lowest rates in droppings of mice and rats, respectively. Mixed infections with ≥ two species was observed in 21 (9.5%) of 220 droppings, 14 (12.7%) belonged to mice and 7 (6.3%) to rats. Conclusion: The present results emphasizes more careful monitoring in laboratory animal houses, such as improving the cleaning and ventilating systems as well as adopting therapeutic measures, when required.

Introduction: During the past few years, an increasing body of evidence has suggested a possible role for human herpes virus 6 (HHV-6) in Multiple Sclerosis (MS) pathogenesis. Despite the many reports supporting the relationship between HHV-6 and MS, this association has not been definitely proved or refuted, and the matter remains unresolved. The current study was aimed to investigate any relation between HHV-6 viremia and classic MS in patients in Guilan province, Northern Iran. Method: HHV-6 viremia was certified by molecular detection in study group (n=46) and control group (n=46) using nested-PCR. Data were analyzed by using three statistical tests (Chi square, odd ratio, and Relative Risk). Results: HHV-6 genomic sequences were found totally in 28 out of 46 (60.8%) plasma DNA samples of patients with MS, but were not found in rest of them. It was also found in 13 out of 46 (28.2%) control group. The difference in prevalence of HHV-6 DNA in blood between patients with MS and control group was statistically significant (P=0.0027 and odd ratio=0.277). Conclusion: The data of our study showed that HHV-6 can be implicated in the development of MS. We strongly support the need for further, objective, evidence-based examination of the relationship between HHV-6 infection and MS.

Introduction: To determine an indicator for Triclabendazole (TCBZ) efficacy, Alanine aminotransferase (ALT) activity in Fasciola hepatica (Iranian isolate) parasite in presence and absence of TCBZ was evaluated by an in vitro cultivation method. Also, ALT enzyme activity between none and parasitized-infected sheep liver tissues was assessed. Method:  The sheep livers were collected and transferred immediately to the Department of Parasitology. Adult living parasites were recovered, washed and divided into two groups, treatment and control groups with 10 parasites in each. We added 15 μg TCBZ to the treatment group; then incubated both groups for 4 h at 37ºC. The parasites, infected and parasite free liver tissues were ground and homogenized by a Mortar and pestle, centrifuged, and supernatants were recovered. Protein concentration and ALT enzyme activity were measured in the supenatants. Results: The results of ALT enzyme activity assay showed 0.03 U/ml/mg protein for treated F. hepatica and 0.01 U/ml/mg protein for untreated samples, the mean values of difference was not significant (p>0.05). The difference between ALT activity in none and parasitized-infected liver was not significant (p>0.05). However, two-sample T-test analysis showed higher ALT activity in treated and untreated parasite in comparison with none and parasitized-infected liver specimens (p<0.05). In addition to ALT protein band for parasite and liver tissue, Cathepsin enzyme (proteases) was detected for parasite by SDS-PAGE analysis. Conclusion: ALT activity cannot be considered as a useful marker for TCBZ efficacy in F. hepatica treatment. However, ALT enzyme showed comparable activities in parasite and its host liver tissue.

Introduction: Aloe Vera compounds have inhibitory activity on fungi, bacteria, and viruses. This study examines the antibacterial activity of A. Vera purified extracts including gel, boiled skin, boiled gel, and distilled extract against pathogenic bacteria, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Klebsiella pneumonia and Pseudomonas aeruginosa were elucidated. Method: The bacterial strains were collected from veterinary hospital. Freshly collected A. vera leaves were used for the juice extraction of gel, skin and distilled extracts. Antibacterial effects of various A. Vera extracts were evaluated using broth microdilution method. The crude polysaccharides of boiled skin extract were purified by phenol method; and fractionated by anion exchange chromatography. For each bacterium, minimum inhibitory concentration of various A. Vera extracts was determined. The protein expression changes of treated bacteria were detected by SDS-PAGE electrophoresis. Results: The distillate extract exhibited more antibacterial effects than other extracts. Out of seven-carbohydrate fractions of the skin extract, the fractions 6 and 7 had antibacterial effects on S. aureus and MRSA at 0.089 and 0.134 mg/ml, respectively; also fraction 5 showed antibacterial effects on MRSA at 0.113 mg/ml concentration. The protein profiles of these strains before and after treatment with A. Vera showed significant differences at 175, 60, 200 and 70 kDa protein bands of S. aureus, MRSA, P. aeruginosa and K. pneumonia, respectively. Conclusion: This finding showed that the distillate extract despite the minimal amount of carbohydrate and protein was more efficient against both Gram-positive and Gram negative bacteria.