- Volume 63, Issue 7, 2014
Volume 63, Issue 7, 2014
- Pathogenicity and virulence
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Genetic characterization of Shigella spp. isolated from diarrhoeal and asymptomatic children
Phenotypic and genetic characteristics of Shigella spp. isolated from diarrhoeal and asymptomatic children aged up to 5 years were analysed in this study. In total, 91 and 17 isolates were identified from diarrhoeal (case) and asymptomatic (control) children, respectively. All the isolates were tested for antimicrobial resistance, the presence of integrons, plasmid-mediated quinolone resistance (PMQR), virulence-associated genes and Shigella pathogenicity island (SH-PAI). The majority of the Shigella spp. from cases (68.1 %) and controls (82.3 %) were found to be resistant to fluoroquinolones. Integron carriage was detected more in cases (76.9 %) than in controls (35.5 %). Atypical class 1 integron was detected exclusively in Shigella flexneri from cases but not from the controls. PMQR genes such as aac(6′)-Ib-cr and qnrS1 were detected in 82.4 and 14.3 % of the isolates from cases and in 53 and 17.6 % in controls, respectively. Shigella isolates from cases as well as from controls were positive for the invasive plasmid antigen H-encoding gene ipaH. The other virulence genes such as virF, sat, setA, setB, sen and ial were detected in Shigella isolates in 80.2, 49.4, 27.4, 27.4, 80.2 and 79.1 % of cases and in 64.7, 52.9, 17.6, 17.6, 64.7 and 64.7 % of controls, respectively. The entire SH-PAI was detected in S. flexneri serotype 2a from cases and controls. In an isolate from a control child, the SH-PAI was truncated. Integrons, PMQR and virulence-encoding genes were detected more frequently in cases than in controls. In diarrhoea endemic areas, asymptomatic carriers may play a crucial role in the transmission of multidrug-resistant Shigella spp. with all the putative virulence genes.
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Characterization of 17 chaperone-usher fimbriae encoded by Proteus mirabilis reveals strong conservation
More LessProteus mirabilis is a Gram-negative enteric bacterium that causes complicated urinary tract infections, particularly in patients with indwelling catheters. Sequencing of clinical isolate P. mirabilis HI4320 revealed the presence of 17 predicted chaperone-usher fimbrial operons. We classified these fimbriae into three groups by their genetic relationship to other chaperone-usher fimbriae. Sixteen of these fimbriae are encoded by all seven currently sequenced P. mirabilis genomes. The predicted protein sequence of the major structural subunit for 14 of these fimbriae was highly conserved (≥95 % identity), whereas three other structural subunits (Fim3A, UcaA and Fim6A) were variable. Further examination of 58 clinical isolates showed that 14 of the 17 predicted major structural subunit genes of the fimbriae were present in most strains (>85 %). Transcription of the predicted major structural subunit genes for all 17 fimbriae was measured under different culture conditions designed to mimic conditions in the urinary tract. The majority of the fimbrial genes were induced during stationary phase, static culture or colony growth when compared to exponential-phase aerated culture. Major structural subunit proteins for six of these fimbriae were detected using MS of proteins sheared from the surface of broth-cultured P. mirabilis, demonstrating that this organism may produce multiple fimbriae within a single culture. The high degree of conservation of P. mirabilis fimbriae stands in contrast to uropathogenic Escherichia coli and Salmonella enterica, which exhibit greater variability in their fimbrial repertoires. These findings suggest there may be evolutionary pressure for P. mirabilis to maintain a large fimbrial arsenal.
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Identification of Mycobacterium avium genes associated with resistance to host antimicrobial peptides
More LessAntimicrobial peptides are an important component of the innate immune defence. Mycobacterium avium subsp. hominissuis (M. avium) is an organism that establishes contact with the respiratory and gastrointestinal mucosa as a necessary step for infection. M. avium is resistant to high concentrations of polymyxin B, a surrogate for antimicrobial peptides. To determine gene-encoding proteins that are associated with this resistance, we screened a transposon library of M. avium strain 104 for susceptibility to polymyxin B. Ten susceptible mutants were identified and the inactivated genes sequenced. The great majority of the genes were related to cell wall synthesis and permeability. The mutants were then examined for their ability to enter macrophages and to survive macrophage killing. Three clones among the mutants had impaired uptake by macrophages compared with the WT strain, and all ten clones were attenuated in macrophages. The mutants were also shown to be susceptible to cathelicidin (LL-37), in contrast to the WT bacterium. All but one of the mutants were significantly attenuated in mice. In conclusion, this study indicated that the M. avium envelope is the primary defence against host antimicrobial peptides.
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- Diagnostics, typing and identification
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Characterization of culturable vaginal Lactobacillus species among women with and without bacterial vaginosis from the United States and India: a cross-sectional study
Lactobacillus species play an integral part in the health of the vaginal microbiota. We compared vaginal Lactobacillus species in women from India and the USA with and without bacterial vaginosis (BV). Between July 2009 and November 2010, a cross-sectional study was conducted among 40 women attending a women’s health clinic in Mysore, India, and a sexually transmitted diseases clinic in San Francisco, USA. Women were diagnosed with BV using Amsel’s criteria and the Nugent score. Lactobacillus 16S rDNA was sequenced to speciate the cultured isolates. Ten Indian and 10 US women without BV were compared with an equal number of women with BV. Lactobacilli were isolated from all healthy women, but from only 10 % of Indian and 50 % of US women with BV. 16S rDNA from 164 Lactobacillus colonies was sequenced from healthy women (126 colonies) and women with BV (38 colonies). Seven cultivable Lactobacillus species were isolated from 11 Indian women and nine species from 15 US women. The majority of Lactobacillus species among Indian women were L. crispatus (25 .0%), L. jensenii (25.0 %) and L. reuteri (16.7 %). Among US women, L. crispatus (32.0 %), L. jensenii (20.0 %) and L. coleohominis (12.0 %) predominated. L. jensenii and L. crispatus dominated the vaginal flora of healthy Indian and US women. Indian women appeared to have a higher percentage of obligate heterofermentative species, suggesting the need for a larger degree of metabolic flexibility and a more challenging vaginal environment.
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- Antimicrobial agents and chemotherapy
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The calcineurin inhibitor cyclosporin A exhibits synergism with antifungals against Candida parapsilosis species complex
Candida parapsilosis complex comprises three closely related species, C. parapsilosis sensu stricto, Candida metapsilosis and Candida orthopsilosis. In the last decade, antifungal resistance to azoles and caspofungin among C. parapsilosis sensu lato strains has been considered a matter of concern worldwide. In the present study, we evaluated the synergistic potential of antifungals and the calcineurin inhibitor cyclosporin A (Cys) against planktonic and biofilms of C. parapsilosis complex from clinical sources. Susceptibility assays with amphotericin, fluconazole, voriconazole, caspofungin and Cys were performed by microdilution in accordance with Clinical and Laboratory Standards Institute guidelines. Synergy testing against planktonic cells of C. parapsilosis sensu lato strains was assessed by the chequerboard method. Combinations formed by antifungals with Cys were evaluated against mature biofilms in microtitre plates. No differences in the antifungal susceptibility pattern among species were observed, but C. parapsilosis sensu stricto strains were more susceptible to Cys than C. orthopsilosis and C. metapsilosis. Synergism between antifungals and Cys was observed in C. parapsilosis sensu lato strains. Combinations formed by antifungals and Cys were able to prevent biofilm formation and showed an inhibitory effect against mature biofilms of C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. These results strengthen the potential of calcineurin inhibition as a promising approach to enhance the efficiency of antifungal drugs.
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A Galleria mellonella infection model reveals double and triple antibiotic combination therapies with enhanced efficacy versus a multidrug-resistant strain of Pseudomonas aeruginosa
More LessThe aim of this study was to compare the inhibitory effect of antibiotic combinations in vitro with efficacy in Galleria mellonella larvae in vivo to identify efficacious combinations that target Pseudomonas aeruginosa. P. aeruginosa NCTC 13437, a multidrug-resistant strain resistant to β-lactams and aminoglycosides, was used. Susceptibility to cefotaxime, piperacillin, meropenem, amikacin, levofloxacin and colistin alone, or in dual or triple combinations, was measured in vitro via a 24 h time-kill assay. In vitro results were then compared with the efficacy of the same dual or triple antibiotic combinations versus G. mellonella larvae infected with P. aeruginosa. G. mellonella haemolymph burden of P. aeruginosa was determined over 96 h post-infection and treatment with the most potent combination therapies. Many dual and triple combinations of antibiotics displayed synergistic inhibition of multidrug-resistant P. aeruginosa in vitro. There was little correlation between combinations that were synergistic in vitro and those that showed enhanced efficacy in vivo versus infected G. mellonella larvae. The most potent dual and triple combinations in vivo were cefotaxime plus piperacillin, and meropenem plus piperacillin and amikacin, respectively. Fewer combinations were found to offer enhanced therapeutic benefit in vivo compared with in vitro. The therapeutic benefit arising from treatment with antibiotic combinations in vivo correlated with reduced larval burden of P. aeruginosa. This study has identified antibiotic combinations that merit further investigation for their clinical potential and has demonstrated the utility of using G. mellonella to screen for novel antibiotic treatments that demonstrate efficacy in vivo.
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Synergistic effect of fluconazole and doxycycline against Candida albicans biofilms resulting from calcium fluctuation and downregulation of fluconazole-inducible efflux pump gene overexpression
More LessCandida albicans biofilms are intrinsically resistant to antimicrobial agents. Previous work demonstrated that the antifungal activity of fluconazole against C. albicans biofilms is notably enhanced by doxycycline. In order to explore the synergistic mechanism of fluconazole and doxycycline, we investigated the changes of efflux pump gene expression, intracellular calcium concentration and cell cycle distribution after drug intervention in this study. The expression levels of CDR1, CDR2 and MDR1 were determined by real-time PCR, and the results showed that fluconazole alone could stimulate the high expression of CDR1, CDR2 and MDR1, and the combination of doxycycline and fluconazole downregulated the gene overexpression induced by fluconazole. Intracellular calcium concentration was determined using Fluo-3/AM by observing the fluorescence with flow cytometry. A calcium fluctuation, which started 4 h and peaked 8 h after the treatment with fluconazole, was observed. The combined drugs also initiated a calcium fluctuation after 4 h treatment and showed a peak at 16 h, and the peak was higher than that stimulated by fluconazole alone. The cell cycle was measured using flow cytometry. Fluconazole alone and the combined drugs both induced a reduction in the percentages of S-phase cells and an elevation in the percentages of cells in the G2/M phase. The results of this research showed that the synergism of fluconazole and doxycycline against C. albicans biofilms is associated with blockade of the efflux pump genes CDR1, CDR2 and MDR1, and stimulation of high intracellular calcium concentration. The findings of this study are of great significance in the search for new antifungal mechanisms.
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Detection of TEM-, SHV- and CTX-M-type β-lactamase production among clinical isolates of Salmonella species
More LessEnteric fever is a major public health problem in developing countries. Due to the problem of resistance to first-line drugs and fluoroquinolone, cephalosporins are currently used for treatment of enteric fever. Cephalosporin resistance in Salmonella spp. is mainly due to production of extended-spectrum β-lactamases (ESBLs). The majority of ESBLs in Salmonella are derivatives of the TEM and SHV β-lactamase families. The objectives of this study were to detect antibiotic susceptibility patterns, ESBL production and TEM-, SHV- and CTX-M-encoding genes (bla TEM, bla SHV and bla CTX-M) among clinical isolates of Salmonella spp. A total of 134 Salmonella isolates [Salmonella Typhi (n = 101), Salmonella Paratyphi A (n = 31), Salmonella Paratyphi B (n = 1) and Salmonella Typhimurium (n = 1)] were included in this study. Multidrug resistance was seen in 5/134 (3.73 %) isolates, all of which belonged to serotype S. Typhi. A better susceptibility profile was observed for first-line drugs (ampicillin, chloramphenicol, co-trimoxazole and tetracycline) and cephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefixime and cefepime). However, 131 (97.76 %) of the 134 isolates were resistant to nalidixic acid and one (0.75 %) was resistant to ciprofloxacin. TEM-1-type β-lactamase (bla TEM-1) was detected in six (4.47 %) of the 134 isolates, which belonged to the serotype S. Typhi. All six TEM-positive isolates were negative for the bla SHV gene and none of the isolates was positive for the bla CTX-M gene. The presence of the bla TEM gene encoding TEM-1 β-lactamase is believed to confer resistance only to penicillins and early cephalosporins; however, the resistance spectrum of TEM-1 descendants may extend to second-, third- and fourth-generation cephalosporins. The ESBLs derived from TEM-1 differ from their progenitors by as few as 1 aa, and have the ability to hydrolyse third-generation cephalosporins. Therefore, appropriate selection and rotation of antibiotics as well as continuous monitoring of antibiotic susceptibility profiles could help to control the emergence and spread of resistant strains.
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- Epidemiology
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Impact of the London 2012 Olympic and Paralympic Games on demand for microbiology gastrointestinal diagnostic services at the Public Health Laboratory London
More LessPlanning for the London 2012 Olympic and Paralympic Games at the Public Health Laboratory London was based on the requirement to meet potential increased demand with scalable capacity. The aim of this study was to determine the impact on demand for microbiology gastrointestinal diagnostic services during the Games period. Retrospective cross-sectional time-series data analysis was used to assess the number of gastrointestinal specimens received in the laboratory and the number of positive results. There was no increase in the number of gastrointestinal specimens received during the Games period, thus the Games had no impact on demand for microbiology gastrointestinal diagnostic services at the laboratory. There was a decrease in the number of public health specimens received for culture [incidence rate ratio = 0.34, 95 % confidence interval (CI) = 0.13–0.86, P = 0.02] and a decrease in the number of culture positive community specimens (odds ratio = 0.59, 95 % CI = 0.40–0.85, P = 0.005), suggesting a decrease in gastrointestinal illness during the Games period. As previous planning assumptions were not based on actual specimen activity, the results of this study may modify the extent of additional planning for microbiological services required for mass gatherings.
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Prevalence of hepatitis E virus infection in West Bengal, India: a hospital-based study
More LessIndia is an endemic zone for hepatitis E virus (HEV), which is associated with both epidemic and sporadic infections. In West Bengal, only two hepatitis E outbreaks have been studied to date. However, sporadic cases of HEV infection also occur during inter-epidemic periods. The aim of this hospital-based study was to detect the prevalence of HEV infection in patients with acute sporadic hepatitis in West Bengal, India. Blood samples and clinical information were collected from 285 patients of both sexes and different ages with acute viral hepatitis (AVH) at Calcutta Medical College, Kolkata, a tertiary-care centre. Samples were tested for hepatitis B virus (HBV) surface antigen, anti-hepatitis C virus antibodies, anti-hepatitis A virus IgM and anti-HEV antibodies (IgM and IgG) by ELISA. Only those patients with AVH who were in their first week of illness and negative for all hepatotropic viral antibodies were tested for HEV RNA by reverse transcriptase nested PCR. HEV was identified as the most common cause of AVH (41.8 % of patients), followed by HBV (21.4 %), hepatitis A virus (17.2 %) and hepatitis C virus (4.6 %). Co-infections with more than one virus were found in 22 patients, with HBV–HEV the most common co-infection (3.8 %). Only 14.7 % of patients had no viral marker. To the best of our knowledge, this is the first documented epidemiological study of acute sporadic hepatitis with HEV in the state of West Bengal, India, indicating that this state is an endemic zone for HEV infection.
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- Clinical microbiology and virology
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Characterization of the bla KPC-2 and bla KPC-3 genes and the novel bla KPC-15 gene in Klebsiella pneumoniae
Three Klebsiella pneumoniae isolates exhibiting high-level resistance to carbapenem were analysed by PCR, PFGE, gene mapping, plasmid conjugation and Southern blot hybridization using a bla KPC probe. In addition to the frequently reported bla KPC-2 and bla KPC-3 genes, a novel bla KPC-15 gene was identified in one of the isolates. The results of plasmid analysis and Southern blot hybridization revealed that the three bla KPC genes were located on transferable plasmids exhibiting three different patterns. The patterns A, B and C were observed in the genetic makeup of each individual plasmid, and all three structures contained ISKpn6-like and ISKpn8 transposons. The results of the gene mapping and hybridization experiments performed with the bla KPC probe demonstrated that the plasmids harboured the three genes at approximately the 85.0, 54.0 and 73.0 kb positions. The study concluded that carbapenem resistance in the three isolates was primarily due to the production of carbapenem-hydrolysing β-lactamase.
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Synergistic mechanism for tetrandrine on fluconazole against Candida albicans through the mitochondrial aerobic respiratory metabolism pathway
More LessWe found that tetrandrine (TET) can reverse the resistance of Candida albicans to fluconazole (FLC) and that this interaction is associated with the inhibition of drug efflux pumps. Mitochondrial aerobic respiration, which plays a major role in C. albicans metabolism, is the primary source of ATP for cellular processes, including the activation of efflux pumps. However, it was unclear if TET exerts its synergistic action against C. albicans via its impact on the mitochondrial aerobic respiratory metabolism. To investigate this mechanism, we examined the impact of FLC in the presence or absence of TET on two C. albicans strains obtained from a single parental source (FLC-sensitive strain CA-1 and FLC-resistant strain CA-16). We analysed key measures of energy generation and conversion, including the activity of respiration chain complexes I and III (CI and CIII), ATP synthase (CV) activity, and the generation of reactive oxygen species (ROS), and studied intracellular ATP levels and the mitochondrial membrane potential (ΔΨm), which has a critical impact on energy transport. Mitochondrial morphology was observed by confocal microscopy. Our functional analyses revealed that, compared with strains treated only with FLC, TET+FLC increased the ATP levels and decreased ΔΨm in CA-1, but decreased ATP levels and increased ΔΨm in CA-16 (P<0.05). Additionally, CI, CIII and CV activity decreased by 23–48 %. The production of ROS increased by two- to threefold and mitochondrial morphology was altered in both strains. Our data suggested that TET impacted mitochondrial aerobic respiratory metabolism by influencing the generation and transport of ATP, reducing the utilization of ATP, and resulting in the inhibition of drug efflux pump activity. This activity contributed to the synergistic action of TET on FLC against C. albicans.
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Plasma LL-37 correlates with vitamin D and is reduced in human immunodeficiency virus-1 infected individuals not receiving antiretroviral therapy
More LessLow levels of the vitamin D-regulated antimicrobial peptide cathelicidin (LL-37) may negatively impact the immune status of human immunodeficiency virus-1 (HIV-1) infected individuals (HIV+). We compared plasma LL-37 levels in healthy controls (HIV−) and HIV+ individuals on or off antiretroviral therapies (ARTs) (ART+ and ART−, respectively), and evaluated the relationship between vitamin D and LL-37 levels. In this cross-sectional study, levels of LL-37, 25-hydroxycholecalciferol [25(OH)D3] and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were measured from an initial cohort of 18 healthy controls and 10 HIV+/ART− individuals. Because this cohort lacked HIV+/ART+ subjects, LL-37 was also quantified from a second cohort of 10 HIV+/ART− and 13 HIV+/ART+ individuals. LL-37 levels were significantly lower in the HIV+/ART− group compared to the healthy controls (P = 0.01). A direct relationship was observed between LL-37 and both 25(OH)D3 and 1,25(OH)2D3. The level of 25(OH)D3 was predictive of higher LL-37 (P = 0.04) and for any given level of 25(OH)D3, HIV+/ART− subjects averaged 20 % lower LL-37 compared to the healthy controls (P = 0.045). For any given level of 1,25(OH)2D3, HIV+/ART− subjects averaged 25 % lower LL-37 compared to the healthy controls (P = 0.018), although 1,25(OH)2D3 was not predictive of higher LL-37 (P = 0.28). Finally, LL-37 levels were significantly lower in the HIV+/ART− group compared to the HIV+/ART+ group from the second cohort (P = 0.045). Untreated HIV infection may contribute to lower LL-37 levels, independent of vitamin D levels. ART treatment may potentially mitigate this decrease in LL-37 levels.
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- Models of infection
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Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma
Considerable progress has been made in understanding the roles of Helicobacter pylori in inflammation and gastric cancer; however, far less is known about the roles of enterohepatic Helicobacter species (EHS) in carcinogenesis and their zoonotic or pathogenic potential. We determined the prevalence of EHS infection in a cohort of geriatric rhesus monkeys in which intestinal adenocarcinoma (IAC) is common and investigated the association between EHS infection and IAC. The cohort consisted of 36 animals, 14 of which (age 26–35 years) had IAC. Of the 36 rhesus, 35 (97 %) were positive for EHS using PCR or bacterial isolation from faeces, colonic or tumour tissues. Only a single rhesus, which had IAC, was negative for EHS by all detection methods. The EHS identified by 16S rRNA sequencing in this study were from three Helicobacter taxa: Helicobacter macacae (previously rhesus monkey taxon 1), Helicobacter sp. rhesus monkey taxon 2, previously described from strain MIT 99-5507, and Helicobacter sp. rhesus monkey taxon 4, related to Helicobacter fennelliae. Thirteen of 14 monkeys with IAC were positive for either H. macacae (7/13, 54 %), EHS rhesus monkey taxon 4 (4/13, 31 %) or a mixture of the two EHS (2/13, 15 %). These results indicate that EHS are prevalent among aged rhesus macaques with IAC. Using Helicobacter genus-specific florescent in situ hybridization, EHS were detected on the surface of colonic epithelia of infected monkeys. All Helicobacter isolates, including H. macacae, effectively adhered to, invaded, and significantly induced proinflammatory genes, including IL-8, IL-6, TNF-α and iNOS, while downregulating genes involved in the function of inflammasomes, particularly IL-1β, CASPASE-1, NRLP3, NLRP6 and NLRC4 in the human colonic T84 cell line (P<0.0001). These results suggest that EHS may represent an aetiological agent mediating diarrhoea, chronic inflammation, and possibly intestinal cancer in non-human primates, and may play a role in similar disease syndromes in humans. Downregulation of inflammasome function may represent an EHS strategy for long-term persistence in the host and play a role in inducing pathological changes in the host’s lower bowel.
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- Case reports
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First report of rhinosinusitis caused by Neoscytalidium dimidiatum in Iran
The study describes isolation of Neoscytalidium dimidiatum from a case of eosinophilic fungal rhinosinusitis (EFRS). The isolate was identified by routine mycological methods and confirmed by DNA sequencing of the internal transcribed spacer (ITS) region of rDNA. To our knowledge, this is the first report of its kind from Iran.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 62 (2013)
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Volume 61 (2012)
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Volume 60 (2011)
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Volume 59 (2010)
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Volume 58 (2009)
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Volume 57 (2008)
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Volume 56 (2007)
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Volume 55 (2006)
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Volume 54 (2005)
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Volume 53 (2004)
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Volume 52 (2003)
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Volume 51 (2002)
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Volume 50 (2001)
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Volume 49 (2000)
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Volume 48 (1999)
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Volume 47 (1998)
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Volume 46 (1997)
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Volume 45 (1996)
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Volume 44 (1996)
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Volume 43 (1995)
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Volume 42 (1995)
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Volume 41 (1994)
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Volume 40 (1994)
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Volume 39 (1993)
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Volume 38 (1993)
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Volume 37 (1992)
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Volume 36 (1992)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 32 (1990)
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Volume 31 (1990)
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Volume 30 (1989)
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Volume 29 (1989)
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Volume 28 (1989)
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Volume 27 (1988)
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Volume 26 (1988)
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Volume 25 (1988)
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Volume 24 (1987)
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Volume 23 (1987)
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Volume 22 (1986)
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Volume 21 (1986)
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Volume 20 (1985)
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Volume 19 (1985)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 13 (1980)
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Volume 12 (1979)
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Volume 11 (1978)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)