- Volume 58, Issue 3, 2009

Current data suggest that functional URA3 genes are necessary for the full pathogenesis of Candida albicans. Herein it is shown that a putatively avirulent URA3/URA3 null mutant of C. albicans (CAI-4) can colonize the murine alimentary tract, invade oro-oesophageal and gastric tissues with yeasts and hyphae, evoke a granulocyte-dominated inflammatory response, and kill transgenic mice that are deficient for both natural killer cells and T cells. Because C. albicans-colonized (gnotobiotic) mice lack a viable prokaryotic microbiota, this study also demonstrates that the gut microbiome is not required to supply the mutant's nutritional needs. The gnotobiotic murine model described herein can be used to assess the capacity of C. albicans mutants to colonize and infect cutaneous, mucosal and systemic tissues and kill the susceptible host via a clinically common, natural route of infection; namely the alimentary tract.

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A–F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A–C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

Dried blood spots (DBSs) on filter paper are being used increasingly in population-based human immunodeficiency virus (HIV) studies. This study evaluated the application of a BED enzyme immunoassay (EIA) on DBSs to estimate HIV incidence in a population-based study in India. The Calypte HIV-1 BED Incidence EIA was performed on 224 HIV-1-positive DBS samples, after screening 12 617 individuals from a population-based sample in Guntur district in the southern Indian state of Andhra Pradesh. The number of recently infected HIV cases was identified using this BED assay and was used to estimate the annual HIV incidence rate based on calculations and adjustment formulae suggested by the Centers for Disease Control and Prevention (CDC). The updated BED data management software provided by the CDC was used for analyses. Of the 224 HIV-1 antibody-positive DBS samples, 29 (12.95 %) were estimated by the BED HIV-1 assay to have been infected within the past 155 days. After adjusting for age, gender and rural/urban distribution of the population, the annual incidence rate of HIV-1 infection was estimated to be 0.32 % (95 % confidence interval 0.20–0.44 %). This annual incidence was 18.6 % of the HIV prevalence of 1.72 % in this study. Thus, the BED assay revealed a higher incidence of HIV in this study than was expected from the prevalence. Correlation of the BED assay with panel testing and longitudinal incidence data in the Indian population is needed to calibrate it for use in India.

Stenotrophomonas maltophilia KH has two acid β-lactamases with isoelectric points (pIs) of 4.6 and 5.4, and several basic β-lactamases (pIs >7.0) that produce a ladder-shaped pattern by IEF. An isogenic L2 mutant, KHL2xylE, was constructed by gene replacement. From IEF and native PAGE zymograms of strains KH and KHL2xylE, it was demonstrated that the basic β-lactamases and the acid β-lactamase with pI 5.4 are encoded by the same L2 gene and that the active types of these L2 charge variants were dependent on the buffer pH. The β-lactamase activities of these L2 charge variants in phosphate buffer at pH 7.0 and 8.0 were 1075±29 and 1114±81 U mg−1, respectively. These results indicate that L2 charge variants give S. maltophilia a better chance of adapting and surviving in response to changes in the environment.

Eighty per cent of the cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) have an infective aetiology, atypical bacteria including Mycoplasma pneumoniae accounting for 5–10 % of these. However, the importance of association of M. pneumoniae with episodes of AECOPD still remains doubtful. The present study was therefore undertaken to delineate the extent of involvement of M. pneumoniae in patients with AECOPD at a referral hospital in Delhi, India. Sputum samples and throat swabs from a total of 100 AECOPD patients attending the Clinical Research Center of Vallabhbhai Patel Chest Institute, Delhi, were collected during a 2-year period (January 2004–June 2006). The samples were investigated for the presence of aerobic bacterial pathogens and M. pneumoniae. Diagnosis of infection with M. pneumoniae was based on culture, serology, direct detection of M. pneumoniae specific antigen and PCR. Bacterial aetiology could be established in 16 of the 100 samples studied. Pseudomonas spp. were recovered from eight cases, Streptococcus pneumoniae from four and Klebsiella spp. from two cases. Acinetobacter sp. and Moraxella catarrhalis were isolated from one case each. Serological evidence of M. pneumoniae infection and/or detection of M. pneumoniae specific antigen were seen in 16 % of the cases. One case with definite evidence of M. pneumoniae infection also had coinfection with Pseudomonas spp. However, no direct evidence of M. pneumoniae infection was found in our study population as defined by culture isolation or PCR. In conclusion, although the serological prevalence of M. pneumoniae infection in our study population was significantly higher than in the control group, there was no direct evidence of it playing a role in AECOPD.

Acriflavine resistance in the clinical meticillin-resistant Staphylococcus aureus isolate KT24 was found not to be mediated by multidrug efflux pumps encoded by qacA/B, smr, qacE, qacG, qacH, qacJ or norA. Early uptake and accumulation of ethidium bromide in MRSA KT24 was significantly lower than that in a susceptible strain, although the efflux rates were similar. Therefore, a permeability barrier in MRSA KT24 may be the conceivable mechanism of acriflavine resistance. Interestingly, it was found that MRSA KT24 had a significantly thickened cell wall, and that cell-wall thickness increased gradually during bacterial growth. In contrast, cell size and surface area in MRSA KT24 were not different from those in the susceptible strain. Moreover, MRSA KT24 exposure to sub-MIC concentrations of acriflavine resulted in a thicker cell wall. These results indicate that cell-wall thickness may be responsible for acriflavine resistance in S. aureus.

Antimicrobial resistance in Salmonella spp. is of grave concern, more so in quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing isolates that cause complicated infections. The MIC of azithromycin, ciprofloxacin, cefixime, cefepime, ceftriaxone, gatifloxacin, imipenem, levofloxacin, meropenem and ofloxacin (E-test strip) and tigecycline and faropenem (agar dilution) against 210 Salmonella spp. was determined. MIC90 (defined as the antimicrobial concentration thatinhibited growth of 90 % of the strains) of the carbapenems (imipenem and meropenem) for Salmonella Typhi and Salmonella Paratyphi A was 0.064 μg ml−1. MIC90 of faropenem was 0.25 μg ml−1 for S. Typhi, S. Paratyphi A and Salmonella Typhimurium. The MIC90 of azithromycin for all Salmonella spp. ranged from 8 to 16 μg ml−1. Tigecycline showed an MIC90 of 2 μg ml−1 for S. Typhi, 1 μg ml−1 for S. Paratyphi A and 4 μg ml−1 for S. Typhimurium. We concluded that tigecycline and the carbapenems are likely to have roles in the final stage of treatment of quinolone-resistant and ESBL-producing multidrug-resistant salmonellae.

Determination of the avidity of specific IgG antibodies has become a generally accepted diagnostic aid for dating Toxoplasma infection. In this study, the Labsystems, VIDAS and EUROIMMUN Toxoplasma IgG avidity assays were compared on a series of 133 Toxoplasma IgG- and IgM-positive sera from symptomatic patients (n=28), from pregnant (n=43) and non-pregnant (n=26) women, and on 18 IgG-positive and IgM-negative sera from chronically infected patients. The results showed excellent concordance between the Labsystems and VIDAS tests in both the IgM-positive (r=0.82, κ=0.771) and IgM-negative (κ=0.609) sera, whilst the agreement of the EUROIMMUN assay with both the Labsystems and VIDAS tests in the IgM-positive sera was moderate (κ=0.575 and κ=0.525, respectively) and in the IgM-negative sera was poor (κ=0.000). Analysis of the kinetics of the maturation of avidity in 13 patients in whom follow-up sera were available showed that, despite a general trend of maturation, in two patients the avidity did not become high during 6 and 11 months of follow-up. In view of the clinical setting, in the symptomatic patients, despite one case of complete discrepancy and five cases of partial discrepancy, the Labsystems and VIDAS tests were in almost perfect agreement (κ=0.812), whilst the agreement in pregnant and non-pregnant women was substantial (κ=0.754 and κ=0.708, respectively). In conclusion, the Labsystems and VIDAS tests are equally reliable for the measurement of Toxoplasma IgG avidity; the choice of test should depend on the laboratory set-up. The EUROIMMUN test may be an acceptable alternative in resource-limited settings, but should be used prudently.

Nowadays, influenza antigen detection test kits are used most frequently to detect influenza A or B virus to establish the diagnosis of influenza rapidly and initiate appropriate therapy. This study was conducted to evaluate the performance of the actim Influenza A&B test (Medix Biochemica). Overall, 473 respiratory specimens were analysed in the actim Influenza A&B test and the results were compared with those from an RT-PCR assay; 461 of these samples originated from paediatric patients aged 7 weeks to 6.5 years either with influenza-related symptoms or from the intensive care unit, and 12 samples originated from adults with underlying lung or haematological diseases. Diagnosis of influenza A or B virus could be established using the actim Influenza A&B test (9/473 samples for influenza A virus and 6/473 for influenza B virus). RT-PCR revealed 23 patients with influenza virus (13/473 for influenza A virus and 10/473 for influenza B virus). The sensitivity and specificity of the actim Influenza A&B test were 65 and 100 % compared with the RT-PCR assay. However, 32 external quality assessment samples containing seven different strains of influenza A subtypes H1N1 and H3N2 and the avian H5N1 were detected correctly by the actim Influenza A&B test. No cross-reactivity to a range of bacterial, fungal and other viral pathogens was observed. In conclusion, the actim Influenza A&B test is reliable for positive results due to its high specificity. Nevertheless, negative results from this test need to be confirmed by a more sensitive assay because of the low sensitivity observed with diagnostic samples.

Here, we describe the first report of post-operative endophthalmitis due to Comamonas testosteroni in an elderly diabetic patient after complicated cataract surgery. The isolate was identified by using Mini API strips. The patient was successfully treated with intravitreal ceftazidime and oral ciprofloxacin.

We report a patient who developed a meticillin-resistant Staphylococcus aureus (MRSA) central venous catheter infection complicated by infective endocarditis. The patient was initially treated with glycopeptides, which led to the development of heterogeneous glycopeptide resistance, the detection of which required the use of a macro Etest screening test. Subsequently, the causative strain, confirmed by PFGE as a UK epidemic MRSA-15, was treated with daptomycin, and again resistance developed in vivo. The development in vivo of resistance to both these agents suggests that the resistance mechanisms may be associated. We suggest that the clinician managing MRSA infection should anticipate daptomycin resistance when reduced glycopeptide susceptibility is detected.

We report to our knowledge the first case of human infection with Ophiostoma piceae. This Sporothrix schenckii-related fungus caused disseminated infection involving the lung and the brain in a patient with lymphoblastic lymphoma. The case emphasizes the significance of molecular techniques for identification of rare fungi in the clinical microbiology laboratory.

Candida dubliniensis is an emerging fungal pathogen, especially in immunodeficient patients. We report what is to the best of our knowledge the first case of multifocal osteomyelitis following disseminated infection in a patient after haematopoietic stem cell transplantation. PFGE for typing of C. dubliniensis was developed and the necessity of long-term antifungal therapy is discussed.

Characteristic morphological and phylogenetic analyses demonstrated the presence of Aspergillus fumigatus and Aspergillus lentulus as the aetiological agents in a case of probable invasive aspergillosis (IA). This is believed to be the first report of an A. lentulus strain isolated from a patient with probable IA in Argentina.