Absence of dihydropteroate synthase gene mutations in Pneumocystis jirovecii isolated from Swedish patients.

Pneumocystis jirovecii remains an important cause of pneumonia in the immunocompromised host, with the largest group of patients at risk for P. jirovecii pneumonia (PCP) in Sweden being those with haematological diseases. Widespread prophylaxis and treatment for P. jirovecii with sulfa-containing drugs have effectively decreased the incidence of PCP, but concerns have been raised about the possible emergence of P. jirovecii isolates that are resistant to these drugs. Two point mutations in the gene coding for the dihydropteroate synthase enzyme (DHPS) in P. jirovecii have been shown to be associated with prior exposure to sulfa drugs. We retrospectively studied the occurrence of P. jirovecii DHPS mutations in isolates recovered from 103 Swedish patients. The DHPS gene, including the polymorphic positions 165 and 171, were amplified and sequenced by pyrosequencing technology. All the clinical specimens showed a wild-type pattern indicating that the occurrence of P. jirovecii DHPS mutations in Sweden is very low or absent.

Echinocandin susceptibility testing of Candida isolates collected during a 1-year period in Sweden.

The susceptibilities of Candida isolates to the echinocandins anidulafungin, caspofungin, and micafungin were determined by using the recently revised CLSI breakpoints and Etest on 238 clinical bloodstream Candida isolates collected between September 2005 and August 2006. The isolates represent approximately 95% of all non-albicans Candida bloodstream infections and one-third of Candida albicans bloodstream infections during this 1-year period in Sweden. The collection included 81 C. albicans, 81 C. glabrata, 36 C. parapsilosis, 14 C. dubliniensis, 8 C. tropicalis, 8 C. lusitaniae, 5 C. krusei, 2 C. guilliermondii and 2 C. inconspicua isolates as well as 1 C. pelliculosa isolate. The MICs were largely consistent with the global epidemiology of bloodstream Candida isolates. All C. albicans and C. glabrata isolates were susceptible to all 3 echinocandins (MIC ≤ 0.016 µg/ml in all instances). Resistance (MIC ≥ 8 µg/ml) to anidulafungin alone was observed for 4 (11.1%) C. parapsilosis isolates and for 1/2 C. guilliermondii isolates. Intermediate susceptibility to caspofungin alone was observed for 2/5 C. krusei isolates. One of the eight C. tropicalis isolates was classified as being intermediately susceptible to micafungin (MIC, 0.5 µg/ml) and as being resistant to anidulafungin and caspofungin (MIC ≥ 1 µg/ml). This isolate harbored a heterozygous FKS1 hot spot mutation (S80P) known to confer echinocandin resistance. This first study to apply the revised CLSI breakpoints for Etest endpoints showed that the breakpoints worked successfully in detecting an isolate with a hot spot mutation. Acquired echinocandin resistance is rare in Sweden. Echinocandin MICs against C. parapsilosis and C. guilliermondii were lowest for micafungin.

Genotypic characterization of Acanthamoeba spp. causing ocular infections in Swedish patients: identification of the T15 genotype in a case of protracted keratitis.

The genus Acanthamoeba represents free-living amoebae typically widespread in soil and water. It consists of more than 20 known species representing 15 genotypes of different pathogenicity and virulence. The aim of the study was the genotypic characterization of Acanthamoeba spp. isolated from human keratitis cases in Sweden. Thirteen amoeba isolates obtained from contact lens users with suspected Acanthamoeba keratitis (AK) were subjected to polymerase chain reaction amplification and subsequent sequencing of the SSU rRNA gene fragment. Sequence analysis identified 4 different genotypes in the studied material. The majority of samples (92%) represented sequences of T3, T4 and T11, all belonging to a cluster of related genotypes frequently described in AK cases. Similar to other reports, genotype T4 was the most common finding in our material (77% of samples). Interestingly, an uncommon genotype, T15, mostly reported from environmental sources, was found in a sample from a patient suffering from a protracted keratitis.

Rapid identification of bacteria from positive blood culture bottles by MALDI-TOF MS following short-term incubation on solid media.

Rapid identification of bacteria from blood cultures enables early initiation of appropriate antibiotic treatment in patients with bloodstream infections (BSI). The objective of the present study was to evaluate the use of matrix-associated laser desorption ionization-time of flight (MALDI-TOF) MS after a short incubation on solid media for rapid identification of bacteria from positive blood culture bottles. MALDI-TOF MS was performed after 2.5 and 5.5 h plate incubation of samples from positive blood cultures. Identification scores with values ≥ 1.7 were accepted as successful identification if the results were confirmed by conventional methods. Conventional methods included MALDI-TOF MS, Vitek 2, and diverse biochemical and agglutination tests after overnight culture. In total, 515 positive blood cultures with monomicrobial bacterial growth representing one blood culture per patient were included in the study. There were 229/515 (44.5%) and 286/515 (55.5%) blood culture bottles with Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively. MALDI-TOF MS following short-term culture could accurately identify 300/515 (58.3%) isolates at 2.5 h, GNB being identified in greater proportion (180/229; 78.6%) than GPB (120/286; 42.0%). In an additional 124/515 bottles (24.1%), identification was successful at 5.5 h, leading to accurate identification of bacteria from 424/515 (82.3%) blood cultures after short-term culture. Interestingly, 11/24 of the isolated anaerobic bacteria could be identified after 5.5 h. The present study demonstrates, in a large number of clinical samples, that MALDI-TOF MS following short-term culture on solid medium is a reliable and rapid method for identification of bacteria from blood culture bottles with monomicrobial bacterial growth.

A limited number of ITS haplotypes defines the diversity of Pneumocystis jirovecii strains in Sweden.

The infectious fungus Pneumocystis jirovecii remains an important cause of pneumonia (PCP) in the immunocompromised host. To study the biodiversity of P. jirovecii in Sweden the internal transcribed spacers (ITS) of the rDNA locus were amplified, cloned and sequenced from a set of diagnostic respiratory specimens obtained from 64 patients with P. jirovecii infection during the years 1996-2003. The analysis of 408 cloned sequences from amplified products resulted in the identification of 10 ITS1 and 12 ITS2 established genotypes. Twelve ITS haplotypes (combinations of ITS1 and ITS2) were identified of which nine were found to recur during the time span of the study. Haplotype Eg was the most common, followed by Ne, Bi and Eb. A new ITS2 genotype denoted v was identified in specimens from four patients. There was no association between ITS haplotype and patient age, sex, underlying disease or geographical origin. Shannon and Simpson index analysis revealed no difference in diversity in Sweden compared to other countries studied and no changes in diversity were found during the study period in Sweden. Criteria were defined to enable the discrimination of genuine ITS types and a more accurate assessment of the previously overestimated genetic diversity of P. jirovecii populations. A model depicting the phylogenetic and genealogic relationships in a revised set of global types of this fungus is presented.

Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children.

BACKGROUND: Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A-H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission. METHODOLOGY/PRINCIPAL FINDINGS: Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B. CONCLUSIONS/SIGNIFICANCE: This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.

Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens.

BACKGROUND: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). PRINCIPAL FINDINGS: In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. SIGNIFICANCE: Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.