Geoff Hogg

Multilocus Sequence Typing Scheme for Enterococcus faecium

ABSTRACT A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Invasive central nervous system aspergillosis: cure with liposomal amphotericin B, itraconazole, and radical surgery--case report and review of the literature.

Invasive aspergillosis of the central nervous system is a rare but well-described disease. There have been only a few reported survivors, and mortality exceeds 95% in the immunosuppressed host. We present a 2-year-old boy with acute lymphatic leukemia and multiple Aspergillus brain abscesses who was successfully treated with liposomal amphotericin B, itraconazole, and surgical excision of the abscesses. Liposomal amphotericin B is a new preparation that safely allows the attainment of significantly higher tissue levels with less toxicity than standard amphotericin B. The treatment of patients with invasive central nervous system aspergillosis is reviewed.

Newly emerging clones of Bordetella pertussis carrying prn2 and ptxP3 alleles implicated in Australian pertussis epidemic in 2008-2010.

Australia is experiencing a prolonged epidemic of pertussis that began in 2008. A total of 194 Bordetella pertussis isolates collected from 2008 through 2010 were typed by single-nucleotide polymorphism (SNP) analysis, by multilocus variable number tandem repeats analysis, and by fim3, prn, and ptxP sequence analyses. Strains with 2 closely related SNP profiles carrying prn2 and ptxP3 from the recently emerged SNP cluster I predominated. The data suggest increasing selection among the B. pertussis population in Australia in favor of strains carrying prn2 and ptxP3 under the pressure of acellular vaccine-induced immunity.

Rapid Increase in Pertactin-deficient Bordetella pertussis Isolates, Australia

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008–2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.

Invasive Central Nervous System Aspergillosis

Invasive aspergillosis of the central nervous system is a rare but well-described disease. There have been only a few reported survivors, and mortality exceeds 95% in the immunosuppressed host. We present a 2-year-old boy with acute lymphatic leukemia and multiple Aspergillus brain abscesses who was successfully treated with liposomal amphotericin B, itraconazole, and surgical excision of the abscesses. Liposomal amphotericin B is a new preparation that safely allows the attainment of significantly higher tissue levels with less toxicity than standard amphotericin B. The treatment of patients with invasive central nervous system aspergillosis is reviewed.

Classification of Cryptosporidium Species from Patients with Sporadic Cryptosporidiosis by Use of Sequence-Based Multilocus Analysis following Mutation Scanning

ABSTRACT In the present study, we analyzed genetic variation in Cryptosporidium species from humans (n = 62) with clinical cryptosporidiosis in South Australia. Sequence variation was assessed in regions within the small subunit of nuclear rRNA (p-SSU), the 70-kDa heat shock protein (p-hsp70), and the 60-kDa glycoprotein (p-gp60) genes by employing single-strand conformation polymorphism analysis and sequencing. Based on the analyses of p-SSU and p-hsp70, Cryptosporidium hominis (n = 38) and Cryptosporidium parvum (n = 24) were identified. The analysis of p-gp60 revealed eight distinct subgenotypes, classified as C. hominis IaA17R1 (n = 3), IbA9G3R2 (n = 14), IbA10G2R2 (n = 20), and IfA12G1R1 (n = 1), as well as C. parvum IIaA18G3R1 (n = 15), IIaA20G3R1 (n = 6), IIaA22G4R1 (n = 2), and IIcA5G3R2 (n = 1). Subgenotypes IaA17R1 and IIaA22G4R1 are new. Of the six other subgenotypes, IbA10G2R2, IIaA18G3R1, IIaA20G3R1, and IIcA5G3R2 were reported previously from the state of Victoria. This is the fourth record in Australia of C. parvum subgenotype IIaA18G3R1 from humans, which, to date, has been isolated only from cattle in other countries. This subgenotype might be a significant contributor to sporadic human cryptosporidiosis and may indicate a greater zoonotic contribution to the infection of humans in the area of study. Comparative analyses revealed, for the first time, the differences in the genetic makeup of Cryptosporidium populations between two relatively close, major metropolitan cities.

Prospective Study of a Real-Time PCR That Is Highly Sensitive, Specific, and Clinically Useful for Diagnosis of Meningococcal Disease in Children

ABSTRACT Due to the early administration of antibiotics, meningococcal disease is increasingly difficult to diagnose by culturing. Laboratory studies have shown PCR to be sensitive and specific, but there have been few clinical studies. The objectives of this study were to determine the diagnostic accuracy and clinical usefulness of meningococcal PCR through a prospective comparison of real-time PCR, nested PCR, and standard culturing of blood and cerebrospinal fluid (CSF). The setting was a tertiary-care pediatric hospital in Australia, and the participans were 118 children admitted with possible septicemia or meningitis. The main outcome measures—sensitivity, specificity, and positive and negative predictive values—were compared to a “gold standard ” fulfilling clinical and laboratory criteria. For 24 cases of meningococcal disease diagnosed by the gold standard, culturing of blood or CSF was positive for 15 (63%), nested PCR was positive for 21 (88%), and real-time PCR was positive for 23 (96%). The sensitivity, specificity, and positive and negative predictive values of real-time PCR (the most sensitive test) for all specimens were, respectively, 96% (95% confidence interval, 79 to 99%), 100% (95% confidence interval, 96 to100%), 100% (95% confidence interval, 85 to 100%), and 99% (95% confidence interval, 94 to 100%). Of 54 patients with suspected meningococcal disease at admission, 23 had positive PCR results. Only one PCR specimen was positive in a patient thought unlikely to have meningococcal disease at admission. Blood PCR remained positive for 33% of patients tested at up to 72 h. Real-time PCR has high positive and negative predictive values in this clinical setting, with better confirmation of cases than nested PCR. Targeting patients for PCR based on admission criteria appears to be practical, and the test may remain useful for several days after the start of antibiotic administration.

Failure of 500 mg of ceftriaxone to eradicate pharyngeal gonorrhoea, Australia

been occurring since admission. On day 14, the patient developed leucocytosis and blood cultures were drawn, which grew MRSA. The MIC of vancomycin increased to 2 mg/L, with that of daptomycin remaining at 0.38 mg/L. Since the patient continued to be symptomatic, irrigation and debridement was scheduled, but due to surgeon availability issues this was not performed until hospital day 19. A sample was obtained during the procedure and cultured, and on day 22, after .2 weeks of treatment with daptomycin, MRSA grew with a daptomycin MIC of 3 mg/L [daptomycin-non-susceptible S. aureus (DNSSA)]. The primary cause of the daptomycin failure was most likely a delay in incision and drainage of the abscesses. Experimental osteomyelitis models have shown selective resistance with daptomycin monotherapy, 2 which possibly contributed to the resistance in this case. Daptomycin was discontinued and, unfortunately, vancomycin was restarted over the weekend. After the patient became bacteraemic on day 14, we recommended a TEE, which was ordered but not performed until day 23. Lesions were found on the left coronary cusp (LCC) and the tricuspid valve, consistent with endocarditis. Vancomycin was discontinued, since current expert opinion states alternative anti-microbials should be considered when the vancomycin MIC is ≥2 mg/L, due to treatment failures. 3,4 Considering this and the severity of infection in this patient, we turned to salvage therapy options and ceftaroline (600 mg intravenously every 12 h), which had an Etest MIC of 0.5 mg/L, was chosen. Ceftaroline is a fifth-generation cephalosporin with activity against MRSA. Ceftaroline was chosen for a number of reasons. First, it is rapidly bactericidal. Ho et al. 5 reported sterilization in 13 days in a case of endocarditis. Also, the incidence of thrombo-cytopenia with long-term use is low, which was particularly important since our patient had a history of thrombocytopenia secondary to hepatitis. Finally, a literature search revealed a series of case reports showing successful use of ceftaroline in the treatment of MRSA endocarditis 5 and an experimental model demonstrating potential use in osteomyelitis. 6 Subsequent blood cultures drawn on days 29 and 32 were negative. On day 32, a repeat irrigation and debridement was performed. After 37 consecutive days of ceftaroline treatment, a TEE showed resolution of echodensities at the base of the tri-cuspid valve and a stable, fibronodular lesion on the LCC. Ceftaro-line was continued for a total of 44 days, with no further episodes of bacteraemia, leucocytosis or fever, …

Staphylococcus aureus infections in Australasian neonatal nurseries

Objective: To study the incidence and outcome of systemic infections with methicillin sensitive (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) infections in Australasian neonatal nurseries. Methods: Prospective longitudinal study of systemic infections (clinical sepsis plus positive cultures of blood and/or cerebrospinal fluid) in 17 Australasian neonatal nurseries. Results: The incidence of early onset sepsis with S aureus, mainly MSSA, was 19 cases per 244 718 live births or 0.08 per 1000. From 1992 to 1994, MRSA infections caused only 8% of staphylococcal infections. From 1995 to 1998, there was an outbreak of MRSA infection, in two Melbourne hospitals. The outbreak resolved, after the use of topical mupirocin and improved handwashing. Babies with MRSA sepsis were significantly smaller than babies with MSSA sepsis (mean birth weight 1093 v 1617 g) and more preterm (mean gestation 27.5 v 30.3 weeks). The mortality of MRSA sepsis was 24.6% compared with 9.9% for MSSA infections. The mortality of early onset MSSA sepsis, however, was 39% (seven of 18) compared with 7.3% of late onset MSSA infection presenting more than two days after birth. Conclusions:S aureus is a rare but important cause of early onset sepsis. Late onset MRSA infections carried a higher mortality than late onset MSSA infections, but babies with early onset MSSA sepsis had a particularly high mortality.