Gary Lum

Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis

From a prospective melioidosis study commencing in 1989 at Royal Darwin Hospital, 170 initial isolates of Burkholderia pseudomallei were available for susceptibility testing. Of these 163 (96%) were susceptible to meropenem/imipenem, ceftazidime, trimethoprim-sulphamethoxazole (SMX/TMP) and doxycycline. Seven (4%) showed primary resistance; three had low-level resistance to SMX/TMP, one to ceftriaxone and amoxycillin/clavulanate (AMOX/CA) and three to doxycycline. Of 167 patients who survived their initial presentation, seven (4%) had culture positive infections which persisted for greater than 3 months after start of therapy. All ultimately cleared carriage of B. pseudomallei though three required changing to SMX/TMP after development of doxycycline resistance. Nineteen (11%) of the initial survivors clinically relapsed and 17 of these had repeat isolates available for testing. Four of these had acquired resistance: one to doxycycline, one to AMOX/CA and ceftazidime, one to SMX/TMP and one to both SMX/TMP and doxycycline. Molecular typing using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis showed all but one relapse isolate to be the same as the original strain. These data are similar to published data from Thailand. As melioidosis has a high mortality (21% in this series) these results emphasize the need for prolonged eradication therapy and regular clinical and microbiological monitoring so that the emergence of resistance can be detected early and appropriate treatment modifications made.

Epidemiology of community‐acquired and nosocomial bloodstream infections in tropical Australia: a 12‐month prospective study

Objectivesu2003 To define the relative incidence of organisms causing blood stream infections in a tropical setting with a very low prevalence of human immunodeficiency virus infection (<1%).

Failure of azithromycin therapy in gonorrhea and discorrelation with laboratory test parameters.

Background: Azithromycin is efficacious in the treatment of chlamydial genital tract infection but less so in gonorrhea. However, MICs of azithromycin for gonococci from previously reported azithromycin treatment failures were consistently below the ‘susceptible’ MIC level of 2 mg/L. Goal of this Study: To examine gonococci not eliminated with 1 g azithromycin therapy to establish treatment outcome/MIC correlates in gonorrhea. Study Design: The MICs and phenotypes of gonococci isolated from five cases of treatment failure after 1 g azithromycin therapy were determined and compared with the MICs of a systematic sample of routine isolates. Results: Azithromycin MICs of gonococci from five cases of failed 1 g azithromycin treatment were 0.125 or 0.25 mg/L, well within the current ‘susceptible’ MIC range. None of the isolates were of the mtr phenotype. The MIC90 of a systematic sample of 219 gonococcal isolates was 0.25 mg/L. Conclusion: The antibiotic MIC/treatment outcome correlates that are usually found in gonorrhea do not apply for azithromycin. Current MIC criteria do not accurately define susceptibility or resistance of gonococci to azithromycin and by themselves do not predict the likely outcome of therapy. Pharmacokinetic factors may decrease the predictive value of MIC data.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

A cluster of culture positive gonococcal infections but with false negative cppB gene based PCR

Objectives: To describe the prevalence and characteristics of isolates of Neisseria gonorrhoeae grown from urine samples that produced negative results with nucleic acid amplification assays (NAA) targeting the cppB gene. Methods: An initial cluster of culture positive, but cppB gene based NAA negative, gonococcal infections was recognised. Urine samples and suspensions of gonococci isolated over 9 months in the Northern Territory of Australia were examined using cppB gene based and other non-cppB gene based NAA. The gonococcal isolates were phenotyped by determining the auxotype/serovar (A/S) class and genotyped by pulsed field gel electrophoresis (PFGE). Results: 14 (9.8%) of 143 gonococci isolated were of A/S class Pro−/Brpyut, indistinguishable on PFGE and negative in cppB gene based, but not other, NAA. Conclusions: This cluster represents a temporal and geographic expansion of a gonococcal subtype lacking the cppB gene with consequent loss of sensitivity of NAA dependent on amplification of this target. Gonococci lacking the cppB gene have in the past been more commonly associated with the PAU-/PCU- auxotype, a gonococcal subtype hitherto infrequently encountered in Australia. NAA based on the cppB gene as a target may produce false positive as well as false negative NAA. This suggests that unless there is continuing comparison with culture to show their utility, cppB gene based NAA should be regarded as suboptimal for use either as a diagnostic or supplemental assay for diagnosis of gonorrhoea, and NAA with alternative amplification targets should be substituted.

Further Evaluation of a Rapid Diagnostic Test for Melioidosis in an Area of Endemicity

ABSTRACT Immunochromatographic test (ICT) kits for the rapid detection of immunoglobulin G (IgG) and IgM antibodies to Burkholderia pseudomallei were compared to the indirect hemagglutination (IHA) assay. In 138 culture-confirmed melioidosis cases, sensitivities were 80, 77, and 88% for IHA, ICT IgG, and ICT IgM, respectively. In a prospective study of 160 consecutive sera samples sent for melioidosis serology, respective specificities were 91, 90, and 69, positive predictive values were 41, 32, and 18, and negative predictive values were 99, 98, and 100%. ICT IgM kits are unreliable for diagnosis of melioidosis, but ICT IgG kits may be useful for diagnosing travelers presenting with possible melioidosis who return from regions where melioidosis is endemic.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target

Aims: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. Methods: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. Results: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. Conclusions: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

Age-Specific Cluster of Cases of Serotype 1 Streptococcus pneumoniae Carriage in Remote Indigenous Communities in Australia

ABSTRACT Seven-valent pneumococcal conjugate vaccination commenced in 2001 for Australian indigenous infants. Pneumococcal carriage surveillance detected substantial replacement with nonvaccine serotypes and a cluster of serotype 1 carriage. Our aim was to review Streptococcus pneumoniae serotype 1 carriage and invasive pneumococcal disease (IPD) data for this population and to analyze serotype 1 isolates. Carriage data were collected between 1992 and 2004 in the Darwin region, one of the five regions in the Northern Territory. Carriage data were also collected in 2003 and 2005 from four regions in the Northern Territory. Twenty-six cases of serotype 1 IPD were reported from 1994 to 2007 in the Northern Territory. Forty-four isolates were analyzed by BOX typing and 11 by multilocus sequence typing. In the Darwin region, 26 children were reported carrying serotype 1 (ST227) in 2002 but not during later surveillance. Scattered cases of serotype 1 carriage were noted in two other regions. Cocolonization of serotype 1 with other pneumococcal serotypes was common (34% serotype 1-positive swabs). In conclusion, pneumococcal carriage studies detected intermittent serotype 1 carriage and an ST227 cluster in children in indigenous communities in the Northern Territory of Australia. There was no apparent increase in serotype 1 IPD during this time. The rate of serotype 1 cocolonization with other pneumococcal serotypes suggests that carriage of this serotype may be underestimated.

Cryptic-Plasmid-Free Gonococci May Contribute to Failure of cppB Gene-Based Assays To Confirm Results of BD ProbeTEC PCR for Identification of Neisseria gonorrhoeae

We note the recent article by Koenig et al. ([3][1]) which describes discordance between results obtained with the BD ProbeTEC ET System (BDPT) and a cppB gene-based PCR assay for the detection of Neisseria gonorrhoeae . Overall, 22.6% of BDPT-positive assays were not confirmed by the cppB gene-

Supplemental testing is still required in australia for samples positive for Neisseria gonorrhoeae by nucleic acid detection tests

The introduction and widespread application of nucleic acid detection tests (NADT) for Neisseria gonorrhoeae (i.e., gonococci [GC]) in Australia has enhanced diagnostic capacity and public health control of gonococcal disease. However, the well-recognized problems with the use and interpretation of GC NADT in the difficult and widely varying conditions in Australia led to a formal request for a specially convened expert reference group (ERG) to develop guidelines relevant to these different situations (9). We therefore noted with some concern conclusions by Lowe et al. (4) suggesting that supplemental testing for diagnosis of gonorrhea using GC NADT is not required in Australia when initial testing is by the Gen-Probe APTIMA Combo 2 (ACT) assay. This assertion contradicts the recommendations contained in the national consensus guidelines that were based on a review of the published literature and the extensive experience obtained over many years in the diverse test conditions experienced here. The ERG guidelines are consistent with another recent review of GC NADT testing in Australia (11) and the current National Pathology Accreditation Advisory Council of Australia (NPAAC) guidelines (7). The latter supersedes advice contained in the earlier NPAAC (6) guidelines on which Lowe et al. (4) relied. n nThere were a number of important factors that were subject to formal consideration by the Australian ERG seemingly ignored by Lowe et al. (4). First, this ERG (9) pointed to problems with GC NADT sensitivity that may result from the capacity of Neisseria gonorrhoeae to repeatedly recombine/alter its genome through acquisition or loss of genetic sequences. These alterations result in gain or loss of important characteristics not only for NADT-based diagnosis but also for culture-based analyses (3, 8). These subpopulations may vary quite substantially as a proportion of the total gonococcal population in different patient subgroups and over time and place (3, 5). The test numbers mentioned by Lowe et al. (4) suggest that their study was limited to a sample obtained over approximately 1 month only, which is insufficient to properly assess any GC NADT. n nSecond, the Australian guidelines (9) also emphasized that cross-reactions with non-gonococcal Neisseria strains remains a significant limitation of GC NADT and that specificity may also vary depending on the patient population. Lowe et al. (4) mention that the AC2 “does not cross-react with other Neisseria spp.” To our knowledge, this has not been extensively investigated in Australia or elsewhere. Hence, the high clinical specificity of the AC2 observed by Lowe et al. (4) may not necessarily reflect the performance of the AC2 in other patient populations, either within or outside Australia. Additionally, the numbers of assays positive for GC were very low (n = 39), not all four GC NADT were used in all instances introducing a bias into the “resolved” data, and the population (not specifically defined) was seemingly a low-prevalence group, further compromising the assessment. n nThird, experience in Australia has shown us that commercial GC NADT satisfying existing FDA standards and the earlier NPAAC guidelines (6) but using limited prerelease evaluation have had to be subsequently withdrawn from use in Australia (9). Some “in-house” assays (5) were similarly withdrawn after more extensive and complete “in-use” appraisals revealed substantial deficiencies in performance. The relatively small number of samples examined by Lowe et al. (4) would not reliably detect these problems. n nAlso in an Australian context, Lowe et al. relied on a cppB-based supplemental assay as their final “confirmatory” test (4), despite the fact that the use of this assay as both a screening and supplemental assay has been associated with false-negative and false-positive results both in Australia and elsewhere (1, 3, 9, 10). n nIt is important that GC NADT is subjected to rigorous, extensive, and continuing evaluation before being endorsed as being suitable for use as a single definitive diagnostic test. It is our view that Lowe et al. (4) failed to satisfy the principles and details required for proper appraisal of this GC NADT, particularly in an Australian context. Data such as those presented may be relevant to some situations and do contribute to the wider appraisal that we recommend. However, an incomplete evaluation performed at one geographic location at one time point on small numbers of positive cases is essentially meaningless if it is to be used, as implied by Lowe et al., as a basis for universal test validation for a particular GC NADT. n nIn the opinion of both the Australian ERG (9) and other national bodies (7), a modification of the Centers for Disease Prevention and Control approach (2) that includes supplemental testing provides a reasonable and pragmatic basis for reliable reporting of results of GC NADT in Australian conditions. It was also the considered opinion of the ERG (9) that the available local data on AC2 assays were to date incomplete and that total reliance on manufacturers data to remedy this deficiency may be misplaced. The additional information supplied by Lowe et al. (4) does not alter these views.