Unique combined penA/mtrR/porB mutations and NG-MAST strain types associated with ceftriaxone and cefixime MIC increases in a 'susceptible' Neisseria gonorrhoeae population.

OBJECTIVES: To determine which mutations in penA, mtrR and porB are implicated in increasing minimum MICs of ceftriaxone and cefixime in a susceptible gonococcal population and to ascertain associations with gonococcal strain types (STs). METHODS: One hundred and forty-six Neisseria gonorrhoeae isolates formed two extended-spectrum cephalosporin susceptibility groups: group 1 isolates with cefixime and ceftriaxone MICs of 0.0005-0.016 mg/L; and group 2 isolates with cefixime MICs of 0.03-0.125 mg/L (n = 24) and ceftriaxone MICs of 0.03-0.06 mg/L (n = 23). Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB) were ascertained by DNA sequence and bioinformatic analysis. STs were determined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516G and P551L; 50/146, 34.2%), a G45D substitution in MtrR (37.7%) and a wild-type (WT) sequence for PorB (43.2%). Group 2 gonococcal isolates were significantly associated with: penA pattern IX; dual mutations in the promoter (A-) and DNA dimerization domain (H105Y) of MtrR; and G120K;A121D substitutions in PorB. There were 50 combined penA/mtrR/porB mutation patterns, with corresponding patterns I/WT/WT and IX/G45D/G120K;A121D predominating. Gonococci susceptible to ceftriaxone and cefixime were significantly associated with NG-MAST ST 25 (33/36; 92%) and the combined penA/mtrR/porB mutation pattern I/WT/WT. No combined mutation pattern or specific ST was associated with elevated ceftriaxone MICs. NG-MAST ST 3654 was significantly associated with the pattern IX/G45D/G120K;A121D and cefixime group 2 isolates. CONCLUSIONS: Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime.

Classification of Leptospira genomospecies 1, 3, 4 and 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively.

The genus Leptospira currently comprises 16 named species. In addition, four unnamed hybridization groups were designated Leptospira genomospecies 1, 3, 4 and 5. These groups represent valid species-level taxa, but were not assigned names in the original description by Brenner et al. [Int J Syst Bacteriol 49, 839-858 (1999)]. To rectify this situation, it is proposed that Leptospira genomospecies 1, genomospecies 3, genomospecies 4 and genomospecies 5 should be classified as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively, with strains L. alstonii 79601(T) ( = ATCC BAA-2439(T)), L. vanthielii WaZ Holland(T) ( = ATCC 700522(T)), L. terpstrae LT 11-33(T) ( = ATCC 700639(T)) and L. yanagawae Sao Paulo(T) ( = ATCC 700523(T)) as the type strains. The type strains are also available from the culture collections of the WHO Collaborating Centres in Amsterdam, The Netherlands, and Brisbane, Australia.

A comparison of risk factors associated with community-associated methicillin-resistant and -susceptible Staphylococcus aureus infections in remote communities.

In this case-control study, cases [community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), n=79] and controls [community-associated methicillin-susceptible S. aureus (CA-MSSA), n=36] were defined as a laboratory-confirmed infection in a patient with no previous hospital-associated factors. Skin and soft tissue were the predominant sites of infection, both for cases (67.1%) and controls (55.6%). Most of the cases (79.7%) and controls (77.8%) were aged <30 years. Investigations did not reveal any significant statistical differences in acquiring a CA-MRSA or CA-MSSA infection. The most common shared risk factors included overcrowding, previous antibiotic usage, existing skin conditions, household exposure to someone with a skin condition, scratches/insect bites, and exposure to healthcare workers. Similar risk factors, identified for both CA-MRSA and CA-MSSA infections, suggest standard hygienic measures and proper treatment guidelines would be beneficial in controlling both CA-MRSA and CA-MSSA in remote communities.

Evaluation of three automated nucleic acid amplification systems for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in first-void urine specimens.

A total of 500 first-void urine specimens were tested for the presence of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids using ProbeTec ET reagents on a Viper platform (BD Diagnostics, Mississauga, Ontario, Canada), Aptima Combo 2 reagents on a Tigris platform (Gen-Probe, Inc., San Diego, CA), and Abbott RealTime CT/NG reagents on an m2000 platform (Abbott Molecular Diagnostics, Des Plaines, IL). The performance of the three assays for detection of N. gonorrhoeae was comparable, but detection of C. trachomatis by the three assays showed more variation. All three platforms were suitable for the detection of C. trachomatis and N. gonorrhoeae, but additional factors, such as maximum daily specimen throughput, are important in evaluating automated systems for C. trachomatis and N. gonorrhoeae detection in high-volume laboratories.

Heterophilic interference in specimens yielding false-reactive results on the Abbott 4th generation ARCHITECT HIV Ag/Ab Combo assay.

BACKGROUND: False-reactivity in HIV-negative specimens has been detected in HIV fourth-generation antigen/antibody or 'combo' assays which are able to detect both anti-HIV-1/HIV-2 antibodies and HIV-1 antigen. OBJECTIVES: We sought to characterize these specimens and determine the effect of heterophilic interference. STUDY DESIGN: Specimens previously testing as false-reactive on the Abbott ARCHITECT HIV Ag/Ab combo assay and re-tested on a different (Siemens ADVIA Centaur HIV Ag/Ab) assay. A subset of these specimens were also pre-treated with heterophilic blocking agents and re-tested on the Abbott assay. RESULTS: Here we report that 95% (252/264) of clinical specimens that were repeatedly reactive on the Abbott ARCHITECT HIV Ag/Ab combo assay (S/Co range, 0.94-678) were negative when re-tested on a different fourth generation HIV combo assay (Siemens ADVIA Centaur HIV Ag/Ab). All 264 samples were subsequently confirmed to be HIV negative. On a small subset (57) of specimens with available volume, pre-treatment with two different reagents (HBT; Heterophilic Blocking Tube, NABT; Non-Specific Blocking Tube) designed to block heterophilic antibody interference either eliminated (HBT) or reduced (NABT) the false reactivity when re-tested on the ARCHITECT HIV Ag/Ab combo assay. CONCLUSIONS: Our results suggest that the Abbott ARCHITECT HIV Ag/Ab combo assay can be prone to heterophilic antibody interference.

Canadian recommendations for laboratory interpretation of multiple or extensive drug resistance in clinical isolates of Enterobacteriaceae, Acinetobacter species and Pseudomonas aeruginosa.

The goal of this document was to provide Canadian laboratories with a framework for consistent reporting and monitoring of multidrug resistant organisms (MDRO) and extensively drug resistant organisms (XDRO) for common gram-negative pathogens. This is the final edition of the interim recommendations, which were modified after one year of broad consultative review. This edition represents a consensus of peer-reviewed information and was co-authored by the Canadian Public Health Laboratory Network and the Canadian Association of Clinical Microbiology and Infectious Diseases. There are two main recommendations. The first recommendation provides standardized definitions for MDRO and XDRO for gram-negative organisms in clinical specimens. These definitions were limited to antibiotics that are commonly tested clinically and, to reduce ambiguity, resistance (rather than non-susceptibility) was used to calculate drug resistance status. The second recommendation identifies the use of standardized laboratory reporting of organisms identified as MDRO or XDRO. Through the broad consultation, which included public health and infection prevention and control colleagues, these definitions are ready to be applied for policy development. Both authoring organizations intend to review these recommendations regularly as antibiotic resistance testing evolves in Canada.

A quantitative risk assessment of West Nile virus introduction into Barbados.

OBJECTIVE: To present a quantitative risk assessment of West Nile (WNV) virus introduction into Barbados, West Indies. DESIGN AND METHODS: Three possible modes were considered: a) WNV infected mosquitoes via air transport, by city of departure, b) WNV infected mosquitoes via marine transport and c) viraemic migratory, birds. We estimated the number of WNV infected migratory birds as the product of the proportion of migratory birds infected and the number of migratory birds entering Barbados in three taxonomic groups. We further estimated the number of days these birds would be infectious as: [formula: see text]. We then estimated the number (#) of infectious mosquito-days for mosquitoes entering Barbados via air transport as: # infected mosquitoes = (total flights per week/city) x (duration of WNV season) x (number of Culex mosquitoes aboard each flight) x (Culex mosquito WNV infection prevalence) x (vector competence index) x (days infectious). The number of infected mosquitoes entering Barbados via marine transport was estimated using a similar expression as for air transport, except that the number of airplanes and mosquitoes/airplane were substituted with the # of sea containers during a 22-week mosquito season and # of mosquitoes/container. RESULTS: Migratory birds (approximately 69-101 infected birds/year) were associated with the highest introductory risk followed by mode (a) (approximately 2 infected mosquitoes/year) and mode (b) (0. 004 infected mosquitoes/year). CONCLUSIONS: Migratory birds and mosquitoes via air are imminent threats for virus introduction. Impending co-circulation of West Nile virus and four strains of dengue virus may present new challenges for public health.

HIV type 1 envelope sequences from seroconverting patients in Barbados.

PIP: Because of the prevalence of leptospirosis in Barbados, patients who present to the hospital with febrile illnesses are routinely screened for Leptospira infection and their sera are stored for future reference. While the majority of patients are infected with Leptospira, some are not. Since some symptoms of acute HIV-1 illness are similar to those of leptospirosis, patient records were reviewed to identify patients whose clinical symptoms may have been due to HIV-1 infection. 10 HIV-1-positive patients originally hospitalized during 1990-94 were identified whose medical histories suggested the occurrence of acute HIV-1 illness at the time of Leptospira testing. Stored sera from those patients were then tested for the presence of HIV-1 p24 antigen and by Western blotting. Evidence of acute HIV-1 infection was considered to be a positive p24 test or a characteristic Western blot profile occurring at or shortly before the time of seropositivity for HIV-1 antibody. The authors determined the sequence of viral RNA from the 12 remaining sera samples from 8 patients, including paired samples drawn at 3- or 4-day intervals from 4 people. The Barbados patient variants aligned more closely with HIV-1 clade B reference strains than with the other subtypes. 2 variants, however, align separately from the classic B subtype and somewhat closer to variants from clades A and C. The Venezuelan isolate, although different from the patient sequences, is also separate from the other B variants.^ieng

Development and evaluation of a miniaturised method as an aid to the identification of clinically important anaerobic bacteria.

A miniaturised method for the identification of anaerobic bacteria is described which employs microtitre fermentation tests, a spot indole test and a nitrate reduction disc test. The results obtained are directly comparable with those produced by a standard conventional method in use at present.

Detection of Clostridium difficile in faeces by direct gas liquid chromatography.

Stool specimens examined for the presence of Clostridium difficile and its cytotoxin were screened by gas liquid chromatography for the presence of volatile fatty acids and p-cresol. Twenty seven of 110 (25%) stools yielded C difficile or cytotoxin; iso-valeric acid was detected in 63/110 (57%) and iso-caproic acid in 18/110 (16%) stools. Para-cresol was found in 24/71 (34%) stools examined. Iso-valeric acid was detected in 85% of stools positive for C difficile, whereas iso-caproic acid (41%) and p-cresol (52%) were found in much lower numbers of C difficile-positive stools. It is concluded that gas chromatographic detection of volatile fatty acids or p-cresol in faeces are not satisfactory screening tests for the presence of C difficile.

Gas chromatographic identification of Clostridium difficile and detection of cytotoxin from a modified selective medium.

A modification of an existing selective medium for Clostridium difficile is described. Inclusion in the medium of DL nor-leucine and p-hydroxyphenylacetic acid enables identification of C difficile to be made directly from primary isolation plates by gas chromatographic detection of caproic acid and p-cresol. Plugs of agar withdrawn from the selective medium also allow the detection of cytotoxin production in vitro.

Serological survey of leptospirosis in livestock animals in the Lesser Antilles.

A serological survey was performed of 1788 cattle, goats and sheep on 13 islands in the Lesser Antilles. Sera were tested by microscopic agglutination (MAT) using a panel of 22 live antigens. Evidence of past exposure, at a titer of > or = 100, was found in 101 animals (5.6%). Antibodies were more common in cattle and goats (7.2% in each) than in sheep (1.7%). Seroprevalence was highest in cattle in Martinique (20%) and in goats in St. Vincent (23%). The predominant serogroups were Sejroe (largely confined to cattle in Martinique), Autumnalis, Icterohaemorrhagiae, Grippotyphosa, and Cynopteri. Eleven cattle from Martinique and 2 sheep with titers of > or = 800 showed evidence of recent infection.

Assessment of the efficacy of an IgM-elisa and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis.

In a prospective study in Barbados between 1979 and 1989, 321 cases were diagnosed in 638 patients presenting at a hospital with symptoms of leptospirosis. Initial diagnosis was based on patient history and characteristic signs and symptoms. In 92 cases (29%), diagnosis was confirmed by isolation of organisms from the blood, urine, or dialysate fluid; in the remaining 229 cases (71%) diagnosis was confirmed by serology alone. Results of an IgM-ELISA and microscopic agglutination test (MAT) in cases with isolates and in non-leptospirosis cases were used to assess the sensitivity and specificity of the tests. The sensitivity of IgM detection by ELISA was 52% in the first acute-phase specimen, increasing to 89% and 93% in the second acute-phase and convalescent specimens, respectively. The specificity of the IgM-ELISA was high (> or = 94%) in all specimens. The sensitivity of the MAT was low (30%) in the first acute-phase specimen, increasing to 63% in the second acute-phase specimen and 76% in the convalescent specimen. The specificity of the MAT was > or = 97% in all specimens.

Detection of dengue infection in patients investigated for leptospirosis in Barbados.

The annual incidence of leptospirosis in Barbados is approximately 13 severe cases/100,000. The peak incidence occurs in October to December of each year, coinciding with the months of heaviest rainfall. During the second half of 1995, an epidemic of dengue type 1 infection produced almost 1,000 laboratory-confirmed cases. During the same period, leptospirosis mortality was twice the average, suggesting that some cases of leptospirosis were being misdiagnosed and treated inappropriately. Sera from patients investigated for dengue or leptospirosis were analyzed retrospectively to determine the extent of misdiagnosis. During 1995 and 1996, 31 of 139 and 29 of 93 patients, respectively, were confirmed as having leptospirosis. Sera from the remaining leptospirosis-negative patients were tested for IgM antibodies to dengue virus. During 1995 and 1996, 48 of 108 patients and 21 of 64 patients, respectively, were found to have dengue. In 1997, sera from all patients investigated for leptospirosis were also tested prospectively for IgM antibodies to dengue: 38 of 92 leptospirosis-negative patients (41%) were dengue IgM-positive, while 2 of 25 leptospirosis cases also had serologic evidence suggesting acute dengue infection. A second large outbreak of dengue caused by serotype 2 occurred in 1997. During the 1995 and 1997 dengue epidemics in Barbados, dengue cases outnumbered leptospirosis cases investigated in the leptospirosis diagnostic protocol. During 1997, patients investigated but negative for dengue were also tested for anti-leptospiral IgM: 7.3% (19 of 262) were IgM-positive. Substantial misdiagnosis of both dengue and leptospirosis can occur and greater public awareness and clinical suspicion of the similar presentations of these two diseases are necessary.

Radiometric studies on the use of selective inhibitors in the identification of Mycobacterium spp.

Radiometric selective inhibition tests were developed and evaluated for the rapid differentiation of Mycobacterium spp. Both a p-nitrobenzoic acid (PNB) test and a commercially-prepared p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test successfully differentiated M. tuberculosis and M. bovis from "atypical" mycobacteria or mycobacteria other than tubercle bacilli (MOTT). Thiophene-2-carboxylic acid hydrazide (TCH) readily distinguished human M. tuberculosis strains from M. bovis, irrespective of resistance to isoniazid. Both PNB and TCH tests were utilised in a routine radiometric susceptibility testing scheme over a period of 1 year in which 110 isolates of M. tuberculosis, 10 of M. bovis and one isolate of BCG were correctly differentiated from 10 isolates of MOTT. The rapidity, sensitivity and specificity of these radiometric tests can play a useful role in mycobacterial identification.

Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR.

Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. biflexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCR-based typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes.

Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis.

Early diagnosis of leptospirosis is important because severe leptospiral infection can run a fulminant course. The polymerase chain reaction (PCR) was evaluated for the detection of leptospires in clinical samples from patients with acute leptospiral infection. Blood and urine samples from 71 patients with leptospirosis were examined by PCR, culture or serology. Samples from 44 (62%) patients with the diagnosis of leptospirosis were positive by PCR as compared to 34 (48%) by culture. The presence of leptospires was demonstrated by PCR in 13 patients before the development of antibodies, as well as in two patients who were seronegative during their illness and at autopsy. Samples from 16 patients without leptospirosis were seronegative and culture negative, and also negative by PCR. We conclude that PCR is a rapid, sensitive and specific means of diagnosing leptospiral infection, especially during the first few days of the disease.

Effect of temperature and pH on survival of Listeria monocytogenes in coleslaw.

The survival and growth of Listeria monocytogenes in fresh coleslaw, pH 3.9, and in coleslaw adjusted to pH 4.0, 5.0, 6.0 or 7.0 before inoculation was studied at three temperatures (4, 15 and 25 degrees C). L. monocytogenes was not detectable after 5 days incubation in fresh coleslaw nor in coleslaw adjusted to pH 4.0. Coleslaw at pH 5.0 was also inhibitory to L. monocytogenes at all three temperatures studied. A decline in viable numbers of L. monocytogenes in coleslaw at pH 6.0 occurred at 4 degrees C and at 15 degrees C, whereas at 25 degrees C the viable count of L. monocytogenes increased initially and remained high after incubation for 25 days. L. monocytogenes grew rapidly in coleslaw at pH 7.0 at all three temperatures studied, followed by an equally rapid decline in viable count.

Penicillin-resistant Streptococcus pneumoniae isolated in Barbados.

An imported case of pneumonia caused by penicillin-resistant Streptococcus pneumoniae occurred in a tourist, shortly after arriving in Barbados. The isolate was of serogroup 6 and exhibited intermediate resistance to penicillin. This was the first isolation of penicillin-resistant S. pneumoniae in Barbados.

Cerebrospinal fluid shunt infection due to Corynebacterium xerosis.

We report the case of a neonate who developed ventriculitis following insertion of a ventriculoperitoneal shunt. Corynebacterium xerosis was isolated from CSF and from the tip of the catheter after it was removed. The isolate was resistant to multiple antibiotics, but the infant responded to treatment with vancomycin.