Jonathan R. Iredell

Critical care services and 2009 H1N1 influenza in Australia and New Zealand

BACKGROUND Planning for the treatment of infection with the 2009 pandemic influenza A (H1N1) virus through health care systems in developed countries during winter in the Northern Hemisphere is hampered by a lack of information from similar health care systems. METHODS We conducted an inception-cohort study in all Australian and New Zealand intensive care units (ICUs) during the winter of 2009 in the Southern Hemisphere. We calculated, per million inhabitants, the numbers of ICU admissions, bed-days, and days of mechanical ventilation due to infection with the 2009 H1N1 virus. We collected data on demographic and clinical characteristics of the patients and on treatments and outcomes. RESULTS From June 1 through August 31, 2009, a total of 722 patients with confirmed infection with the 2009 H1N1 virus (28.7 cases per million inhabitants; 95% confidence interval [CI], 26.5 to 30.8) were admitted to an ICU in Australia or New Zealand. Of the 722 patients, 669 (92.7%) were under 65 years of age and 66 (9.1%) were pregnant women; of the 601 adults for whom data were available, 172 (28.6%) had a body-mass index (the weight in kilograms divided by the square of the height in meters) greater than 35. Patients infected with the 2009 H1N1 virus were in the ICU for a total of 8815 bed-days (350 per million inhabitants). The median duration of treatment in the ICU was 7.0 days (interquartile range, 2.7 to 13.4); 456 of 706 patients (64.6%) with available data underwent mechanical ventilation for a median of 8 days (interquartile range, 4 to 16). The maximum daily occupancy of the ICU was 7.4 beds (95% CI, 6.3 to 8.5) per million inhabitants. As of September 7, 2009, a total of 103 of the 722 patients (14.3%; 95% CI, 11.7 to 16.9) had died, and 114 (15.8%) remained in the hospital. CONCLUSIONS The 2009 H1N1 virus had a substantial effect on ICUs during the winter in Australia and New Zealand. Our data can assist planning for the treatment of patients during the winter in the Northern Hemisphere.

Identification of Bacteria in Blood Culture Broths Using Matrix-Assisted Laser Desorption-Ionization Sepsityper™ and Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturers specified bacterial identification criteria (spectral scores ≥1.700–1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Identification of Yeasts Is Contingent on Robust Reference Spectra

Background Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods. Methods MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster. Principal Findings Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results. Conclusions MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.

Comparison of a Rapid Antigen Test with Nucleic Acid Testing during Cocirculation of Pandemic Influenza A/H1N1 2009 and Seasonal Influenza A/H3N2

ABSTRACT The rapid diagnosis of influenza is critical in optimizing clinical management. Rapid antigen tests have decreased sensitivity in detecting pandemic influenza A/H1N1 2009 virus compared to seasonal influenza A subtypes (53.4% versus 74.2%, P < 0.001). Nucleic acid tests should be used to detect pandemic influenza virus when rapid antigen tests are negative.

Multiplex Tandem PCR: a Novel Platform for Rapid Detection and Identification of Fungal Pathogens from Blood Culture Specimens

ABSTRACT We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-α (EF1-α), and β-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans. The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (<2 h), sensitive, and specific simultaneous detection and identification of fungal pathogens directly from blood culture specimens.

Characterization of the Natural Population of Bartonella henselae by Multilocus Sequence Typing

ABSTRACT Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Dominance of blaCTX-M within an Australian Extended-Spectrum β-Lactamase Gene Pool

ABSTRACT bla CTX-M genes, particularly blaCTX-M-15, are the dominant extended-spectrum β-lactamase (ESBL) genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sydney, Australia, where we also found one example of blaCTX-M-62, encoding a novel enzyme conferring ceftazidime resistance. ESBL genes were present in diverse community isolates and in a variety of associated conjugative plasmids.

blaIMP-4 in Different Genetic Contexts in Enterobacteriaceae Isolates from Australia

ABSTRACT The IMP-4 metallo-β-lactamase, originally recognized in Acinetobacter spp. from Hong Kong, more recently appeared simultaneously in isolates of the family Enterobacteriaceae from Sydney and Melbourne, Australia. The blaIMP-4-qacG2-aacA4-catB3 cassette array was found in isolates from both cities, but in different wider genetic contexts and on different plasmids, suggesting movement of this array by homologous recombination.

Recombination in IS26 and Tn2 in the Evolution of Multiresistance Regions Carrying blaCTX-M-15 on Conjugative IncF Plasmids from Escherichia coli

ABSTRACT CTX-M-15 now appears to be the dominant extended-spectrum β-lactamase worldwide, and a number of different factors may contribute to this success. These include associations between blaCTX-M-15 and particular plasmids (IncF) and/or strains, such as Escherichia coli ST131, as well as the genetic contexts in which this gene is found. We previously identified blaCTX-M-15 as the dominant ESBL gene in the western Sydney area, Australia, and found that it was carried mainly on IncF or IncI1 plasmids. Here, we have mapped the multiresistance regions of the 11 conjugative plasmids with one or more IncF replicons obtained from that survey and conducted a limited comparison of plasmid backbones. Two plasmids with only an IncFII replicon appear to be very similar to the published plasmids pC15-1a and pEK516. The remaining nine plasmids, with multiple IncF replicons, have multiresistance regions related to those of pC15-1a and pEK516, but eight contain additional modules previously found in resistance plasmids from different geographic locations that carry a variety of different resistance genes. Differences between the multiresistance regions are largely due to IS26-mediated deletions, insertions, and/or rearrangements, which can explain the observed variable associations between blaCTX-M-15 and certain other resistance genes. We found no evidence of independent movement of blaCTX-M-15 or of a large multiresistance region between different plasmid backbones. Instead, homologous recombination between common components, such as IS26 and Tn2, appeared to be more important in creating new multiresistance regions, and this may be coupled with recombination in plasmid backbones to reassort multiple IncF replicons as well as components of multiresistance regions.

Pathogen profiling for disease management and surveillance

The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen profiling integrates microbial genomics data into communicable disease control by consolidating phenotypic identity-based methods with DNA microarrays, proteomics, metabolomics and sequence-based typing. Sharing data on pathogen profiles should facilitate our understanding of transmission patterns and the dynamics of epidemics.