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Volume 56,
Issue 9,
2007
Volume 56, Issue 9, 2007
- Pathogenicity And Virulence
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Nontypeable Haemophilus influenzae and Streptococcus pneumoniae bind respiratory syncytial virus glycoprotein
More LessRespiratory syncytial virus (RSV) infection is associated with secondary bacterial infections caused by nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. The pathogenesis of these complications is not completely understood; however, viral infection of respiratory epithelial cells promotes colonization by these bacteria. In the present study, RSV virions associated with NTHi and pneumococci in an inoculum-dependent manner in a fluid-phase binding assay. Adherence of NTHi and S. pneumoniae to epithelial cells transiently expressing RSV G glycoprotein was 2- and 2.2-fold higher, respectively, than adhesion to cells transfected with the vector alone (P <0.01). Furthermore, 4.6- and 6.2-fold larger numbers of NTHi and pneumococci bound to cells expressing a membrane-bound full-length RSV G protein than to cells expressing a truncated non-membrane-bound protein (P ≤0.005). Pre-incubating cells expressing membrane-bound G protein with blocking anti-RSV G antibodies reduced bacterial adherence by 78–84 % (P ≤0.005). These studies demonstrate that RSV G protein is a receptor for both NTHi and S. pneumoniae. Strategies to prevent this interaction may reduce the incidence of secondary bacterial complications of RSV infection.
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Susceptibility of gnotobiotic transgenic mice (Tgϵ26) with combined deficiencies in natural killer cells and T cells to wild-type and hyphal signalling-defective mutants of Candida albicans
Germfree transgenic epsilon 26 mice (Tgϵ26), deficient in natural killer cells and T cells, were colonized (alimentary tract) with Candida albicans wild-type or each of two hyphal transcription factor signalling mutant strains (efg1/efg1, efg1/efg1 cph1/cph1). Each Candida strain colonized the alimentary tract, infected keratinized gastric tissues to a similar extent, and induced a granulocyte-dominated inflammatory response in infected tissues. Both wild-type and mutant strains formed hyphae in vivo and were able to elicit an increase in cytokine [tumour necrosis factor alpha, interleukin (IL)-10 and IL-12] and chemokine (KC and macrophage inflammatory protein-2] mRNAs in infected tissues; however, administration of the wild-type strain was lethal for the Tgϵ26 mice, whereas the mice colonized with the mutant strains survived. Death of the Tgϵ26-colonized mice appeared to be due to occlusive oesophageal candidiasis, and not to disseminated candidiasis of endogenous origin. In contrast, the mutant strains exhibited a significantly reduced capacity to infect (frequency and severity) oro-oesophageal (tongue and oesophagus) tissues. Therefore, the two hyphal signalling-defective mutants were less able to infect oro-oesophageal tissues and were non-lethal, but retained their ability to colonize the alimentary tract with yeast and hyphae, infect keratinized gastric tissues, and evoke an inflammatory response in orogastric tissues.
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Internalization by HeLa cells of latex beads coated with mammalian cell entry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis
More LessThe mammalian cell entry (Mce) operon 3 (mce3) is one of four homologous mce operons of Mycobacterium tuberculosis, encoding six (Mce3A–F) invasin-like membrane-associated proteins. Previous studies have shown that recombinant expression of Mce1A encoded by the mce1 operon in Escherichia coli allows this non-pathogenic bacterium to invade and survive inside macrophages, and latex beads coated with Mce1A are internalized by non-phagocytic HeLa cells. However, the role of other mce1 operon proteins (Mce1B–F) and proteins encoded by the operons mce2–4 in facilitating the internalization of M. tuberculosis in mammalian cells has not been studied. This study was carried out to determine whether Mce proteins encoded by the mce3 operon also facilitated the internalization of latex beads by HeLa cells. Recombinant pure Mce3A and lipoprotein LprM (Mce3E) were expressed and purified from E. coli cells. Mce1A expressed as a fusion protein with glutathione S-transferase (GST–Mce1A) and GST alone, purified similarly from E. coli cells, were used as control proteins. Fluorescent latex beads coated with purified proteins were used to study their uptake by HeLa cells using fluorescence microscopy, flow cytometry and electron microscopy. Fluorescence microscopy and flow cytometry showed an association of HeLa cells with beads coated with both Mce3A and LprM, whilst GST–Mce1A and GST yielded the expected results. Transmission electron microscopy confirmed the uptake of beads coated with Mce3A or LprM by HeLa cells. The data showed that Mce3A encoded by the mce3 operon facilitated the uptake and internalization of latex beads by HeLa cells. The data also showed, for the first time, the role of another Mce protein (LprM/Mce3E) in facilitating the interaction and internalization of M. tuberculosis by mammalian cells.
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- Diagnostics, Typing And Identification
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Amplified fragment length polymorphism analysis of Mycobacterium avium complex isolates recovered from southern California
More LessFine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprinting 159 patient and environmental MAC isolates from southern California. AFLP analysis accurately identified strains belonging to M. avium and Mycobacterium intracellulare and differentiated between strains within each species. The method was also able to differentiate strains that were presumed to be genetically identical in two previous studies using large RFLP analysis with PFGE, or PCR-amplification of DNA segments located between insertion sequences IS1245 and IS1311. For M. avium, drinking-water isolates clustered more closely with each other than with patient or food isolates. Patient isolates were more genetically diverse. None of the environmental isolates shared identical AFLP patterns with patient isolates for either species. There were, however, environmental isolates that shared identical patterns, and patient isolates that shared identical patterns. A subset of the isolates, which are referred to as MX isolates due to their ambiguous identification with the Gen-Probe system, produced AFLP patterns similar to those obtained from M. intracellulare isolates. Sequence analysis of 16S rDNA obtained from the MX isolates suggests that they are strains of M. intracellulare that were not correctly identified by the M. intracellulare AccuProbe from Gen-Probe.
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Serodiagnosis of Salmonella enterica serovar Typhi and S. enterica serovars Paratyphi A, B and C human infections
The aim of this study was to evaluate an immunoassay for the detection of human serum antibodies to the LPS and flagellar antigens of Salmonella Typhi and Salmonella Paratyphi A, B and C, and to the Vi capsular polysaccharide of S. Typhi and S. Paratyphi C. A total of 330 sera were used; these originated from 15 patients who were culture-positive for S. Typhi and 15 healthy controls, together with 300 sera submitted to the Laboratory of Enteric Pathogens for Salmonella serodiagnosis. By SDS-PAGE/immunoblotting, all 15 sera from culture-positive patients had serum antibodies to the 9,12 LPS antigens and 10 had antibodies to the ‘d’ flagellar antigens. Of the 300 reference sera, 22 had antibodies to the 9,12 LPS antigens, one to the 1,4,5,12 LPS antigens and 12 to the 6,7 LPS antigens. Only two sera had antibodies to flagellar antigens, one of which bound to the ‘b’ and the other to the ‘d’ antigen. An ELISA was developed that successfully detected serum antibodies to the Vi capsular polysaccharides, but because of the kinetics of serum antibody production to the Vi, these antibodies may be of limited value in the serodiagnosis of acute infection with S. Typhi and S. Paratyphi C. The immunoassays described here provide a sensitive means of detecting serum antibodies to the LPS, flagellar and Vi antigens of S. Typhi and S. Paratyphi, and constitute a viable replacement for the Widal assay for the screening of sera. The Salmonella serodiagnosis protocols described here are the new standard operating procedures used by the Health Protection Agency's National Salmonella Reference Centre based in the Laboratory of Enteric Pathogens, Colindale, UK.
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The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species
More LessIn view of the growing incidence and the high mortality of invasive aspergillosis and candidiasis, adequate diagnostic techniques permitting timely onset of treatment are of paramount importance. More than 90 % of all invasive fungal infections in immunocompromised individuals can be attributed to Candida and Aspergillus species. To date, standardized techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically most relevant fungi are lacking. In the present report, a real-time quantitative PCR assay, developed for the detection of the most common pathogenic Candida and Aspergillus species, is described. The single-reaction PCR assay targets a judiciously selected region of the 28S subunit of the fungal rDNA gene. The unique design of the universal primer/probe system, including a pan- Aspergillus and pan- Candida (Pan-AC) hydrolysis probe, facilitates the detection of numerous Aspergillus species (e.g. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor and Aspergillus nidulans) and Candida species (e.g. Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida kefyr, Candida guilliermondii, Candida lusitaniae and Candida dubliniensis). The assay permits highly reproducible detection of 10 fg fungal DNA, which corresponds to a fraction of a fungal genome, and facilitates accurate quantification of fungal load across a range of at least five logs. Upon standardization of the technique using cultured fungal strains, the applicability in the clinical setting was assessed by investigating a series of clinical specimens from patients with documented fungal infections (n=17). The Pan-AC assay provides an attractive and economic approach to the screening and monitoring of invasive aspergillosis and candidiasis, which is readily applicable to routine clinical diagnosis.
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Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces
The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections.
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Sequential multiplex PCR for identifying pneumococcal capsular serotypes from south-Saharan African clinical isolates
A serial multiplex PCR approach was reformulated for pneumococcal serotyping to test 153 clinical isolates from children in Mozambique. This approach identified serotypes in 139 (90.8 %) of 153 isolates; 126 (82.4 %) were identified within two reactions. This approach in developing countries would require minimal training and could provide useful serotype information without requiring transport of specimens.
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Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children
More LessCapsular serotype surveillance of clinical isolates of Streptococcus pneumoniae is essential for evaluation of the potential impact of introducing multivalent capsular serotype-based vaccines in Latin America. Here, a previously described sequential multiplex PCR method was revised for optimal targeting of prevalent serotypes in Latin America. The revised protocol successfully serotyped 139/147 pneumococci (94.6 %) from Brazilian children, demonstrating a labour-efficient, accurate method requiring only conventional PCR capability.
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- Antimicrobial Agents And Chemotherapy
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Prevalence and regional variation in meticillin-resistant Staphylococcus aureus (MRSA) in the USA and comparative in vitro activity of tigecycline, a glycylcycline antimicrobial
More LessThe Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) is a surveillance study established in 2004 to monitor the activity of tigecycline, the first glycylcycline, and comparator agents [β-lactams (including penicillins, cephalosporins and carbapenems), glycopeptides, tetracyclines, fluoroquinolones and oxazolidinones] against Gram-positive and Gram-negative pathogens worldwide. This report examines 1692 isolates of Staphylococcus aureus collected in the continental United States between January 2004 and September 2005. Meticillin-resistant S. aureus (MRSA) accounted for 52.0 % of isolates. Prevalence of MRSA by state ranged from 12.5 % in New Hampshire to 100 % in Kentucky. All isolates were susceptible to tigecycline, linezolid and vancomycin. In vitro, tigecycline was potent against both meticillin-susceptible S. aureus (MSSA) (MIC50 and MIC90=0.12 μg ml−1) and MRSA (MIC50=0.12 μg ml−1; MIC90=0.25 μg ml−1). Only a single isolate was resistant to three or more antimicrobial classes. Ninety-six isolates (5.7 %) were susceptible to the complete antimicrobial panel.
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Racemoside A, an anti-leishmanial, water-soluble, natural steroidal saponin, induces programmed cell death in Leishmania donovani
Leishmaniasis remains a major health problem of the tropical and subtropical world. The visceral form causes the most fatalities if left untreated. Dramatic increases in the rates of infection and drug resistance and the non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents. This study reports that racemoside A, a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, is a potent anti-leishmanial molecule effective against antimonial-sensitive (strain AG83) and -unresponsive (strain GE1F8R) Leishmania donovani promastigotes, with IC50 values of 1.15 and 1.31 μg ml−1, respectively. Incubation of promastigotes with racemoside A caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. This compound exerts its leishmanicidal effect through the induction of programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide, loss of mitochondrial membrane potential culminating in cell-cycle arrest at the sub-G0/G1 phase, and DNA nicking shown by deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL). Racemoside A also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a 7–8-fold lower dose, with IC50 values of 0.17 and 0.16 μg ml−1, respectively, and was non-toxic to murine peritoneal macrophages up to a concentration of 10 μg ml−1. Hence, racemoside A is a potent anti-leishmanial agent that merits further pharmacological investigation.
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Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus
More LessAspergillus fumigatus is an increasingly prevalent opportunistic fungal pathogen of various immunocompromised individuals. It has the ability to form filaments within the lungs, producing dense intertwined mycelial balls, which are difficult to treat. The aim of this study was to develop a suitable model of A. fumigatus to examine the effects of antifungal challenge on these intertwined filamentous communities. A. fumigatus NCPF 7367 growth conditions were optimized on both Thermanox coverslips and on flat-bottomed microtitre plates to establish optimal conidial seeding densities. Isolates were treated with itraconazole, voriconazole, amphotericin B and caspofungin and their overall killing efficiency was measured using an XTT formazan metabolic dye assay. This was compared with the CLSI (formerly NCCLS) methodology of broth microdilution of moulds (standard M38-A). It was shown that 1×105 conidia ml−1 in RPMI 1640 was the optimum concentration of spores for biofilm formation. Filamentous growth characteristics were not observed until 10 h incubation, followed by an exponential increase in the biofilm biomass (hyphae and extracellular material) and cellular activity (metabolism). When susceptibility testing of biofilms was compared with that of planktonic cells by CLSI broth microdilution testing, all antifungal drugs were at least 1000 times less effective at reducing the overall metabolic activity of 90 % of the cells. Overall, this study showed that A. fumigatus has the ability to form coherent multicellular biofilm structures that are resistant to the effects of antifungal drugs.
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Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes
A major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an established anti-malarial compound that showed anti-leishmanial activity in both promastigotes and amastigotes, with IC50 values of 160 and 22 μM, respectively, and, importantly, was accompanied by a high safety index (>22-fold). The leishmanicidal activity of artemisinin was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, in situ labelling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G0/G1 phase. Taken together, these data indicate that artemisinin has promising anti-leishmanial activity that is mediated by programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.
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- Epidemiology
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Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China
More LessMycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis, and may represent a feasible approach to study M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating M. tuberculosis strains, isolates from 105 patients in Wuhan City, China, were genotyped by this method as compared to spoligotyping. MIRU typing identified 55 types that defined 21 clusters and 34 unique isolates. The discriminatory power was high [Hunter–Gaston discriminatory index (HGDI), 0.97]. Spoligotyping showed that 86 (81.9 %) of 105 isolates belonged to the Beijing family genotype. For Beijing family and non-Beijing strains, the discriminatory power of MIRU was high (HGDI, 0.95 and 0.98, respectively). Among the alleles of the MIRU loci for the Beijing family, only locus 26 was highly discriminative, but for non-Beijing strains, loci 10, 16 and 26 were highly discriminative. MIRU typing is a simple and fast method which may be used for preliminary screening of M. tuberculosis isolates in China.
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Molecular characterization of serogroup C Neisseria meningitidis isolated in China
An increase in the number of serogroup C meningococcal disease cases occurred in China from September 2003 to January 2006 as a result of several successive outbreaks. In addition, the proportion of serogroup C Neisseria meningitidis isolates from sporadic cases and carriers has also increased. In this study, 113 serogroup C meningococcal isolates were characterized by multilocus sequence typing (MLST) and PorA typing. These isolates comprised those from outbreak cases and their close contacts, the national carriage survey conducted during the same period and some historical isolates from 1966–2002. Twenty MLST sequence types (STs) and 21 PorA variable region (VR) types were identified in the collection. The ST-4821 complex, a newly identified lineage, was the most prevalent lineage (95/113). These data also showed a high level of diversification of serogroup C isolates, as indicated by the number of variants of the ST-4821 clone and the VR types present. There were ten PorA VR types among the ST-4821 isolates, and certain VR types (P1.7-2,14, P1.12-1,16-8) were associated with isolates from outbreak cases. The results of this study allow us to draw a profile of the molecular characteristics of serogroup C strains in China. These data are helpful for monitoring the spread of virulent strains and will provide valuable information for the prevention of bacterial meningitis in China.
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- Clinical Microbiology And Virology
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Endotoxin contamination in the dental surgery
More LessDental waterlines contain large numbers of Gram-negative bacteria. Endotoxin, a component of such organisms, has significant health implications. Paired samples of dental unit water and the aerosols generated during dental procedures were collected, and assayed for bacteria and endotoxin levels, using heterotrophic plate counts and the Limulus amoebocyte lysate test. Consistent with published studies, the extent of bacterial contamination in the dental waters sampled for this investigation surpassed the levels associated with potable water, with counts in excess of 2.0×106 c.f.u. ml−1 in some samples. Correspondingly high concentrations of endotoxin [up to 15 000 endotoxin units (EU) ml−1] were present in the water. A statistically significant Spearman correlation coefficient of ρ=0.94 between endotoxin (EU ml−1) and bacterial load (c.f.u. ml−1) was demonstrated. All of the aerosol samples contained detectable endotoxin. Further studies of the consequences of dental endotoxin exposure, and evaluation of means to prevent exposure, are warranted.
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- Models Of Infection
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Spiroplasma spp. from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants
Spiroplasma, small motile wall-less bacteria, are linked by molecular and serological studies to the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, chronic wasting disease (CWD) in deer and Creutzfeldt–Jakob disease in humans. In this study, two experiments were undertaken to determine the role of spiroplasma in the pathogenesis of TSE. In experiment 1, Spiroplasma mirum, a rabbit tick isolate that had previously been shown to experimentally induce spongiform encephalopathy in rodents, was inoculated intracranially (IC) into ruminants. S. mirum-inoculated deer manifested clinical signs of TSE after 1.5 to 5.5 months incubation. The deer, as well as sheep and goats, inoculated with S. mirum developed spongiform encephalopathy in a dose-dependent manner. In experiment 2, spiroplasma closely related to S. mirum were isolated from TSE-affected brains via passage in embryonated eggs, and propagated in cell-free M1D media. Spiroplasma spp. isolates from scrapie-affected sheep brain and from CWD-affected deer brain inoculated IC into sheep and goats induced spongiform encephalopathy closely resembling natural TSE in these animals. These data show spiroplasma to be consistently associated with TSE, and able experimentally to cause TSE in ruminant animal models, therein questioning the validity of studies that have concluded the prion, a miss-folded protease-resistant protein that builds up in TSE brains during the course of the disease, to be the sole causal agent. The spiroplasma infection models reported here will be important for investigating factors involved in the pathogenesis of TSE since ruminants are the natural hosts.
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- Case Reports
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Fatal post-traumatic zygomycosis in an immunocompetent young patient
Zygomycosis, a relatively uncommon infection, usually occurs among immunocompromised individuals. It has been reported only rarely in trauma patients. A fatal case is reported of pulmonary and rapidly progressive cutaneous zygomycosis in a young, otherwise healthy farmer, with multiple bone fractures, wounds and soft tissue injuries after an accident with an agricultural machine in the field. Rhizopus spp. was isolated from both cultures of bronchial washings and wound samples. The diagnosis was confirmed by histopathological examination of tissue specimens from a large wound. Despite systemic antifungal therapy and surgical debridement, the patient's condition deteriorated and he died from refractory septic shock.
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Invasive infection with Streptococcus iniae in Taiwan
More LessStreptococcus iniae, a common fish pathogen, rarely infects humans. In this report, a case of invasive S. iniae infection in a 51-year-old woman with diabetes mellitus and hepatitis C-related liver cirrhosis is described. The isolate was identified by 16S rDNA sequencing. The patient recovered after 1 week of treatment with ampicillin.
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A case of bilateral microsporidial keratitis from Bangladesh – infection by an insect parasite from the genus Nosema
More LessAn HIV-negative patient from Bangladesh with bilateral keratitis was found to be infected with a microsporidian parasite belonging to the genus Nosema. Significantly, the patient had bathed in a rural pond 7 days prior to the development of ocular symptoms. Nosema parasites are common insect parasites and the source of this microsporidial infection was possibly from mosquito larvae developing in the pond in which the patient bathed. The reduced temperature of the human eye and its immune status may have allowed a poikilothermic insect parasite to establish infection in the cornea of a homeothermic human host. This case highlights the opportunistic potential of insect microsporidial parasites to infect immunocompetent humans as well as those who are immunodeficient.
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Volumes and issues
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Volume 74 (2025)
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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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