- Volume 55, Issue 11, 2006
Volume 55, Issue 11, 2006
- Review
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Treatment of plague: promising alternatives to antibiotics
More LessPlague still poses a significant threat to human health, and interest has been renewed recently in the possible use of Yersinia pestis as a biological weapon by terrorists. The septicaemic and pneumonic forms are always lethal if untreated. Attempts to treat this deadly disease date back to the era of global pandemics, when various methods were explored. The successful isolation of the plague pathogen led to the beginning of more scientific approaches to the treatment and cure of plague. This subsequently led to specific antibiotic prophylaxis and therapy for Y. pestis. The use of antibiotics such as tetracycline and streptomycin for the treatment of plague has been embraced by the World Health Organization Expert Committee on Plague as the ‘gold standard’ treatment. However, concerns regarding the development of antibiotic-resistant Y. pestis strains have led to the exploration of alternatives to antibiotics. Several investigators have looked into the use of alternatives, such as immunotherapy, non-pathogen-specific immunomodulatory therapy, phage therapy, bacteriocin therapy, and treatment with inhibitors of virulence factors. The alternative therapies reported in this review should be further investigated by comprehensive studies of their clinical application for the treatment of plague.
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- Pathogenicity And Virulence
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Mutation of luxS affects motility and infectivity of Helicobacter pylori in gastric mucosa of a Mongolian gerbil model
Helicobacter pylori is associated with gastric disorders in humans and some experimental animals, and possesses the luxS/type 2 autoinducer (AI-2) system. The effects of a specific luxS mutation on the characteristics of H. pylori were examined. On 0.3 % agar medium, motility of H. pylori HPKY08 (luxS : : cat) was significantly lower than that of wild-type H. pylori TK1402. The luxS-complemented strain HPKY21 exhibited motility comparable to that of H. pylori TK1402. It was shown that the luxS/AI-2 system plays an important role in H. pylori motility. The luxS mutant exhibited a reduced infection rate relative to the wild-type parent strain TK1402 in a Mongolian gerbil model. At 3 months after oral inoculation, lower numbers of H. pylori were detected by semi-quantitative real-time reverse transcription PCR (qRT-PCR) in luxS − mutant-infected gerbils than in TK1402-infected gerbils. Gastric inflammation and increased antibody titre for H. pylori were observed in TK1402-infected gerbils only.
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Cytolethal distending toxins in Shiga toxin-producing Escherichia coli: alleles, serotype distribution and biological effects
More LessTo assess the prevalence of cytolethal distending toxin (CDT) among Shiga toxin-producing Escherichia coli (STEC), 202 STEC strains were investigated using PCRs targeting various cdt alleles (cdt-I to cdt-V). Seven of the 202 strains contained cdt-III and an additional seven contained cdt-V. All 14 cdt-positive strains produced biologically active CDT, as demonstrated by a progressive distension of cultured Chinese hamster ovary cells. The CDT-positive STEC belonged to eight different serotypes, including sorbitol-fermenting O157 : NM (non-motile). The data demonstrate that CDT is present in some STEC serotypes only. However, more studies are required to evaluate whether CDT presence is associated with severe disease.
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- Diagnostics, Typing And Identification
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Detection of enteroaggregative Escherichia coli in faecal samples from patients in the community with diarrhoea
The aim of this study was to assess the usefulness of a multiplex PCR assay targeting the aat, aaiA and astA genes for the detection of typical and atypical enteroaggregative Escherichia coli (EAEC) in bacterial cultures from faecal samples from patients with community-acquired diarrhoea. The isolates harbouring these genes were also tested using the HEp-2 cell-adhesion assay to clarify their EAEC status. aat, aai or astA was found in E. coli faecal isolates from 39 (7.8 %) of 500 patients, and 20 of these strains adhered to HEp-2 cells in a pattern characteristic of EAEC. Eight isolates carrying the aai or astA gene but not the aat gene were shown to be HEp-2 cell test positive, although 12 strains with this genotype were HEp-2 cell test negative. Using the HEp-2 adhesion assay as the gold standard, the addition of primers detecting aaiA and astA to the aat PCR increased the number of EAEC isolates detected, but identified strains of E. coli that were not EAEC. The variety of genotypes exhibiting aggregative adherence highlights the problems associated with developing a molecular diagnostic test for EAEC. This PCR assay detects a variety of strains exhibiting characteristics of the EAEC group, making it a useful tool for identifying both typical and atypical EAEC.
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Antigenicity of borrelial protein BBK32 fragments in early Lyme borreliosis
More LessRecombinantly produced borrelial BBK32 protein fragments originating from Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii were evaluated as antigens in the serology of Lyme borreliosis (LB). In ELISA, a mid-portion hydrophilic fragment reacted with LB patient sera. Of the 23 patients with culture- or PCR-positive erythema migrans (EM), 43 % at diagnosis and 52 % at convalescence were positive for at least one Borrelia species-specific variant BBK32 fragment antigen. In parallel ELISAs with BBK32 whole proteins from the three borrelial subspecies as antigens, 17 % at diagnosis and 26 % at convalescence were positive. These results suggest that BBK32 protein fragments may improve the early IgG serology of LB compared to the BBK32 whole protein.
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Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases
An increase in the incidence of fungal infections has highlighted the need for rapid and precise detection and identification methods in clinical mycology. This report describes the data obtained on corneal samples from 24 patients with suspected keratomycosis using a conventional cultural approach in parallel with PCR amplification and sequencing of the internal transcribed spacers (ITSs) of the rDNA regions. Using the cultural approach, seven samples (58.3 % of the 12 samples positive for an infectious pathogen) tested positive for a fungal aetiology, with final identification taking a mean time of more than 5 days. In two cases, diagnosis required 10 days. Using the ITS-based molecular approach, a direct diagnosis was obtained in only five of the seven fungus-positive cases (71.4 %) starting from the clinical samples, but identification was still possible in all seven cases within 24 h (by using 16 h cultures for the two remaining cases). Despite the less-than-optimal sensitivity when working directly on clinical samples, the obtained data indicate that the molecular strategy used in this study is a useful complement to the conventional diagnostic approaches used for keratomycosis and, in particular, allows precise and fast fungal identification, in response to the clinical requirements. Similar studies on larger panels of patients and on different clinical samples are required for further investigation of the clinical potential of ITS-based approaches in the diagnosis of mycotic infections.
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- Antimicrobial Agents And Chemotherapy
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Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support
More LessA porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC90 determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2–3 h culture. Additionally, a trimethoprim-resistant subpopulation (10 % of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1 %) after 3.5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.
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Insertion sequence ISEcp1-like element connected with a novel aph(2″) allele [aph(2″)-Ie] conferring high-level gentamicin resistance and a novel streptomycin adenylyltransferase gene in Enterococcus
More LessEnterococcus casseliflavus HZ95 is an enterococcus with high-level resistance to aminoglycosides. Nine genes responsible for high-level aminoglycoside resistance, including aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, aph(3′)-IIIa, aac(6′)-Ii, ant(3′)-Ia, ant(4′)-Ia and ant(6′)-Ia, were not detected in HZ95. An 8 kb fragment from unconjugative plasmids of HZ95 was cloned, and expressed gentamicin resistance in Escherichia coli DH5α. The genetic structures (∼8 kb DNA fragment) containing these aminoglycoside-modifying enzyme genes in Ent. casseliflavus HZ95 were determined. The deduced amino acid sequence of the novel aph(2″) allele, aph(2″)-Ie, had 93.7 % amino acid identity with APH(2″)-Id. The aph(2″)-Ie gene was bracketed upstream by an insertion sequence (IS)Ecp1-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80.3 % amino acid identity with the protein encoded by aadE. The plasmid of ∼16 kb could hybridize with a PCR-generated aph(2″)-Ie intragenic probe. The ISEcp1-like element had 91 % identity with ISEcp1. ISEcp1, which commonly acts as a key factor in the dissemination of CTX-M-type β-lactamase genes in Gram-negative bacteria, has not been reported in Enterococcus.
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Molecular characterization of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from the USA
More LessDrug-resistant tuberculosis poses a significant problem for treatment. The mechanisms of resistance to the front-line drug isoniazid (INH) are complex and can be mediated by katG, inhA and other unknown genes. To identify the percentage of INH-resistant strains with no katG or inhA mutation, this study characterized a panel of 28 clinical isolates of Mycobacterium tuberculosis and five mutants derived from H37Rv resistant to INH. Seventeen of 33 resistant strains (51 %) had katG mutations with 12 of the 17 strains having the most common KatG Ser315Thr mutation. Three of the 17 strains with the KatG 315 mutation had an additional mutation in the inhA promoter and were resistant to a high level of INH. Seventeen of the 33 INH-resistant strains (51 %) had inhA mutations. The most common inhA promoter mutation was −15C→T and was present in 13 of the 17 inhA mutations. This promoter mutation occurred alone without katG mutations and was associated with a low level of INH and ethionamide resistance. However, other inhA mutations were associated with katG mutations. No mutations were found in the ndh gene. Three of 33 strains (9 %) had no mutations in katG, inhA or ndh, indicating that their resistance was due to a new mechanism of resistance. Detection of the KatG Ser315Thr mutation and the −15C→T inhA mutation accounted for 76 % (25/33) of the INH-resistant strains and should be useful for rapid detection of INH-resistant strains by molecular tests.
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High level of ciprofloxacin resistance and its molecular background among Campylobacter jejuni strains isolated in the United Arab Emirates
More LessThe antibiotic sensitivity and the serotype and molecular type (MT) distribution of 41 Campylobacter jejuni strains isolated from individual patients in Tawam Hospital, Al Ain, United Arab Emirates, were investigated. While all strains were sensitive to erythromycin (MIC 0.5–4 mg l−1), 35 isolates (85.4 %) exhibited resistance to ciprofloxacin (MIC 8–64 mg l−1). All resistant strains carried the Thr-86 to Ile mutation in the gyrase A (gyrA) gene, as shown by mismatch amplification mutation assay (MAMA) and confirmed by sequencing. Based on the partial sequences of gyrA, resistant isolates carried 10 distinct alleles, eight of them representing new variants. Strains were assigned to 30 MTs based on the combined results of PFGE and flaA PCR-RFLP typing. Eight of the 35 ciprofloxacin-resistant strains, isolated over a period of more than 1 year, represented the largest MT, also carrying the same allelic variant of the gyrA gene. These results show that the local incidence of fluoroquinolone resistance among C. jejuni is one of the highest reported worldwide. It was also demonstrated that stable MTs could persist for a relatively long time among the clonally unrelated antibiotic-resistant isolates of C. jejuni. The data also emphasize the need to replace fluoroquinolones as empirical therapy for diarrhoea of undiagnosed aetiology.
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- Epidemiology
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Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil
More LessMolecular typing and virulence markers were used to evaluate the genetic profiles and virulence potential of 106 Yersinia enterocolitica strains. Of these strains, 71 were bio-serotype 4/O : 3, isolated from human and animal clinical material, and 35 were of biotype 1A or 2 and of diverse serotypes, isolated from food in Brazil between 1968 and 2000. Drug resistance was also investigated. All the strains were resistant to three or more drugs. The isolates showed a virulence-related phenotype in the aesculin, pyrazinamidase and salicin tests, except for the food isolates, only two of which were positive for these tests. For the other phenotypic virulence determinants (autoagglutination, Ca++ dependence and Congo red absorption), the strains showed a diverse behaviour. The inv, ail and ystA genes were detected in all human and animal strains, while all the food isolates were positive for inv, and 3 % of them positive for ail and ystA. The presence of virF was variable in the three groups of strains. The strains were better discriminated by PFGE than by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A higher genomic similarity was observed among the 4/O : 3 strains, isolated from human and animal isolates, than among the food strains, with the exception of two food strains possessing the virulence genes and grouped close to the 4/O : 3 strains by ERIC-PCR. Unusually, the results revealed the virulence potential of a bio-serotype 1A/O : 10 strain, suggesting that food contaminated with Y. enterocolitica biotype 1A may cause infection. This also suggests that ERIC-PCR may be used as a tool to reveal clues about the virulence potential of Y. enterocolitica strains. Furthermore, the results also support the hypothesis that animals may act as reservoirs of Y. enterocolitica for human infections in Brazil, an epidemiological aspect that has not been investigated in this country, confirming data from other parts of the world.
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An improved multiple-locus variable number of tandem repeats analysis for Leptospira interrogans serovar Australis: a comparison with fluorescent amplified fragment length polymorphism analysis and its use to redefine the molecular epidemiology of this serovar in Queensland, Australia
More LessIn this study, an improved multiple-locus variable number of tandem repeats analysis (MLVA) method based upon a previously published method is described. Improvements to the method included redesigned primers and PCR conditions, combined with pooled capillary electrophoresis using multicolored dyes. Allele sizes were converted into an allele string, and each unique allele string was assigned a numerical MLVA type (MVT). The improved MLVA method was then applied to 96 previously characterized Leptospira interrogans serovar Australis isolates from human and animal sources. The improved MLVA was found to have between six and 13 alleles at each locus, compared with three to eight in the original. The mean Hunter–Gaston diversity index (HGDI) for the improved MLVA method was 0.654, compared with 0.599 in the original; this increase in diversity was largely due to changes in the analysis of the variable number of tandem repeat (VNTR) data. When the improved MLVA method was compared with the fluorescent amplified fragment length polymorphism (FAFLP) method, there was a high level of concordance between the profiles; however, the MLVA method produced an additional four unique profiles amongst the subset of 30 isolates tested. Given that the improved MLVA method was found to be superior to the original MLVA method, it was subsequently used to redefine the molecular epidemiology of L. interrogans serovar Australis in Queensland, Australia. Using cluster analysis, the authors were able to demonstrate clonal links amongst rodent isolates, rodent and human isolates, and rodent and canine isolates. These results highlight the role of rodents in the disease, and also the potential role of MLVA in defining the molecular epidemiology of L. interrogans.
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- Clinical Microbiology And Virology
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Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji
Five strains of Vibrio cholerae O1, one each from an Australian and a New Zealand tourist with gastrointestinal illness returning from an island resort in Fiji and the remaining three from water sources located in the same resort, were extensively characterized. Conventional phenotypic traits that are used for biotyping of O1 V. cholerae categorized all five strains as belonging to the El Tor biotype. Genetic screening of 11 regions that are associated with virulence in V. cholerae showed variable results. The absence of genes comprising Vibrio seventh pandemic island-I (VSP-I) and VSP-II in all the strains indicated that these strains were very similar to the pre-seventh pandemic V. cholerae O1 El Tor strains. The ctxAB genes were absent in all strains whereas orfU and zot were present in four strains, indicating that the strains were non-toxigenic. Four strains carried a truncated CTX prophage. Although epidemiological and molecular studies suggested that these strains did not cause cholera amongst tourists at the resort, their similarity to pre-seventh pandemic strains, their prior association with gastrointestinal illness and their presence in the island resort setting warrant more attention.
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Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes
The Matlab variants of Vibrio cholerae O1, defined as hybrids between the classical and El Tor biotypes, were first isolated from hospitalized patients with acute secretory diarrhoea in Matlab, a rural area of Bangladesh. These variants could not be categorized as classical or El Tor biotypes by phenotypic and genotypic tests, and had representative traits of both the biotypes. A number of virulence-associated genes and/or gene clusters were screened by PCR and DNA sequencing. El Tor-specific gene clusters, Vibrio seventh-pandemic islands (VSP)-I and -II and repeat toxin (RTX) were present in the genome of these variants, indicating their El Tor lineage, whereas the nucleotide-sequence-derived CtxB amino acid sequence of these strains grouped them under the classical biotype. Matlab variants possessed all the necessary genes to initiate pandemics. The genetic relatedness of Matlab variants to the V. cholerae strains recently isolated in Mozambique is another important observation of this study, which underscores the epidemiological significance of Matlab variants.
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An outbreak of psittacosis due to Chlamydophila psittaci genotype A in a veterinary teaching hospital
An outbreak of psittacosis in a veterinary teaching hospital was recognized in December 2004. Outbreak management was instituted to evaluate the extent of the outbreak and to determine the avian source. Real-time PCR, serologic testing and sequencing of the ompA gene of Chlamydophila psittaci were performed. Sputum samples from patients, throat-swab samples from exposed students and staff, and faecal specimens from parrots and pigeons were tested. In this outbreak, 34 % (10/29) of the tested individuals were infected. The clinical features of the infection ranged from none to sepsis with multi-organ failure requiring intensive-care-unit admission. C. psittaci genotype A was identified as the outbreak strain. Parrots, recently exposed to a group of cockatiels coming from outside the teaching facility, which were used in a practical class, appeared to be the source of the outbreak. One of the tested pigeons harboured an unrelated C. psittaci genotype B strain. The microbiological diagnosis by real-time PCR on clinical specimens allowed for rapid outbreak management; subsequent genotyping of the isolates identified the avian source. Recommendations are made to reduce the incidence and extent of future outbreaks.
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- Models Of Infection
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Arthroconidia production in Trichophyton rubrum and a new ex vivo model of onychomycosis
More LessThe dermatophyte fungus Trichophyton rubrum often produces arthroconidia in vivo, and these cells are thought to be involved in pathogenesis, and, in shed skin scales, to act as a source of infection. The purpose of this study was (i) to examine the environmental and iatrogenic factors which affect arthroconidiation in T. rubrum in vitro, (ii) to look at arthroconidia formation in a large number of clinical isolates of T. rubrum and (iii) to develop a new model for the study of arthroconidia formation in nail tissue. Arthroconidia production was studied in T. rubrum grown on a number of media and under conditions of varying pH, temperature and CO2 concentration. The effect of the presence of antifungals and steroids on arthroconidia formation was also examined. Nail powder from the healthy toenails of volunteers was used as a substrate for arthroconidial production. On Sabouraud dextrose agar in the presence of 10 % CO2 plus air, arthroconidial formation occurred optimally at 37 °C and pH 7.5, and was maximal at 10 days. Most isolates of T. rubrum showed a similar level of arthroconidial production, and only two out of 50 strains were unable to produce arthroconidia. Subinhibitory levels of some antifungals and betamethasone resulted in the stimulation of arthroconidia formation. Arthroconidial production in ground nail material also occurred under the same optimal conditions, but took longer to reach maximal levels (14 days). These in vitro and ex vivo results provide a useful basis for the understanding of arthroconidium formation in vivo in infected tissues such as nails.
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- Case Reports
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Canine dermatophytosis caused by an anthropophilic species: molecular and phenotypical characterization of Trichophyton tonsurans
More LessMicrosporum canis is the most common species isolated from canine and feline dermatophytosis in the world. However, this study reports a rare case of canine dermatophytosis caused by the anthropophilic dermatophyte Trichophyton tonsurans in the city of Fortaleza, Ceará, Brazil. The fungal characterization was performed by classical mycological examination and by genotypical analysis using the restriction enzymes Sau3A, RsaI, DdeI and EcoRI. The phenotypical characteristics were compatible with T. tonsurans. The results obtained in the genotypical analysis were similar to the digestion pattern of the ITS sequences for T. tonsurans strains. In addition, an antifungal susceptibility test was performed with griseofulvin, ketoconazole and itraconazole. The MICs were 0.5 μg ml−1 for griseofulvin, 0.25 μg ml−1 for ketoconazole and 1 μg ml−1 for itraconazole. This study emphasizes the adaptability of anthropophilic fungi such as T. tonsurans to animal conditions.
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Intractable colitis associated with chronic granulomatous disease
The case of a 20-year-old Japanese man, diagnosed as having autosomal recessive chronic granulomatous disease (CGD), who was being treated with corticosteroids for intractable unclassified colitis, is described. He died from multiple organ failure following disseminated intravascular coagulation secondary to disseminated varicella-zoster virus (VZV) infection. He was diagnosed as an index case of CGD when 2 years old, was inoculated against VZV at the age of 5 years and had had an unremarkable course for 19 years. He was admitted to hospital because of a third episode of recurrent bloody diarrhoea. Clinical remission for each episode was achieved by intravenous corticosteroid therapy. Unclassified colitis associated with CGD was diagnosed based on a colonic biopsy demonstrating characteristic macrophages with lipofuscin deposits. From a treatment viewpoint, idiopathic inflammatory bowel disease (IBD) should be differentiated from secondary IBD occurring in CGD, in which immunosuppressive drugs including corticosteroids, still the mainstay of IBD treatment, should be avoided.
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Enterohepatic Helicobacter species isolated from the ileum, liver and colon of a baboon with pancreatic islet amyloidosis
More LessMicroaerobic bacteria were isolated from a baboon with pancreatic islet amyloidosis and hepatitis. Phenotypic and molecular analyses identified two distinct helicobacters. Analyses of 16S rRNA demonstrated ‘Helicobacter macacae’ in the ileum and liver, and Helicobacter cinaedi in the colon. To the best of the authors' knowledge, this is the first report describing the isolation of enterohepatic Helicobacter species from a baboon.
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Central nervous system borreliosis mimicking a pontine tumour
More LessIn childhood, facial nerve palsy and headache are typical symptoms of second and third stage neuroborreliosis. While focal demyelination is occasionally observed on MRI scans, the appearance of a tumorous lesion is extremely rare. The case of a 10-year-old girl with neuroborreliosis mimicking a space-occupying lesion in the brainstem, without any previously recognized manifestations of borreliosis, is reported.
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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 58 (2009)
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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 50 (2001)
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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 35 (1991)
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Volume 34 (1991)
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Volume 33 (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 21 (1986)
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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)