Detection of azole-susceptible and azole-resistant Aspergillus coinfection by cyp51A PCR amplicon melting curve analysis.

BACKGROUND: The AsperGenius® assay is a multiplex real-time PCR test that allows the simultaneous detection of Aspergillus species and identification of the most common mutations in the Aspergillus fumigatus cyp51A gene conferring resistance (TR34/L98H and TR46/T289A/Y121F) by using melting curve analysis. Mixed infections with azole-resistant and susceptible A. fumigatus have rarely been described. METHODS: The AsperGenius® multiplex real-time PCR assay (PathoNostics, Maastricht, the Netherlands) was used on bronchoalveolar lavage (BAL) samples of 91 consecutive patients with a suspected invasive Aspergillus infection at the Erasmus MC University Medical Center, Rotterdam. RESULTS: In three cases the AsperGenius® assay indicated the simultaneous presence of WT and mutant genes (two patients with TR34/L98H mutation and one patient with TR46/T289A/Y121F mutation) and therefore mixed infections with azole-susceptible and -resistant isolates. In one of the three cases, the mixed infection was confirmed by phenotypic antifungal testing of multiple A. fumigatus colonies. CONCLUSIONS: The use of a dedicated A. fumigatus cyp51A resistance PCR allowed the detection of mixed infections with azole-resistant and -susceptible Aspergillus strains. These mixed infections may remain undiagnosed with conventional phenotypic susceptibility testing.

PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay® in 201 patients with haematological disease suspected for invasive aspergillosis.

OBJECTIVES: In patients with invasive aspergillosis (IA), fungal cultures are mostly negative. Consequently, azole resistance often remains undetected. The AsperGenius® multiplex real-time PCR assay identifies clinically relevant Aspergillus species and four resistance-associated mutations (RAMs; TR34/L98H/T289A/Y121F) in the Cyp51A gene. This multicentre study evaluated the diagnostic performance of this assay on bronchoalveolar lavage (BAL) fluid and correlated the presence of RAMs with azole treatment failure and mortality. METHODS: Stored BAL samples from patients with haematological diseases with suspected IA were used. BAL samples that were galactomannan/culture positive were considered positive controls for the presence of Aspergillus. Azole treatment failure and 6 week mortality were compared in patients with and without RAMs that had received ≥5 days of voriconazole monotherapy. RESULTS: Two hundred and one patients each contributed one BAL sample, of which 88 were positive controls and 113 were negative controls. The optimal cycle threshold cut-off value for the Aspergillus species PCR was <38. With this cut-off, the PCR was positive in 74/88 positive controls. The sensitivity, specificity, positive predictive value and negative predictive value were 84%, 80%, 76% and 87%, respectively. 32/74 BAL samples were culture negative. Azole treatment failure was observed in 6/8 patients with a RAM compared with 12/45 patients without RAMs (P = 0.01). Six week mortality was 2.7 times higher in patients with RAMs (50.0% versus 18.6%; P = 0.07). CONCLUSIONS: The AsperGenius® assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure.

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

Invasive fungal infections are relatively common opportunistic infections in immunocompromised patients and are still associated with a high mortality rate. Furthermore, these infections are often complicated by resistance or refractoriness to current antimicrobial agents. Therefore, an urgent need exists for new therapeutic strategies based on the identification of new microbial targets and novel antimicrobial agents. One such hypothetical therapeutic strategy may involve the use of mycoviruses that are able to selectively infect fungi. Current knowledge of mycoviruses of human pathogenic fungi and the scope for using (recombinant) mycoviruses as future biological control agents are reviewed here.

[Methicillin-resistant Staphylococcus aureus (MRSA) in the community]. / Meticillineresistente Staphylococcus aureus (MRSA) in de gemeenschap.

Methicillin-resistant Staphylococcus aureus (MRSA) infections have been confined to healthcare centres for decades. However, MRSA infections are increasingly seen in young healthy individuals with no exposure to healthcare centres. These community-acquired MRSA (CA-MRSA) strains differ from healthcare-associated MRSA (HA-MRSA) strains in various ways. For example, CA-MRSA is strongly associated with the staphylococcal cassette chromosome mec (SCCmec) type IV and the toxin Panton-Valentine leukocidin. CA-MRSA spreads relatively easily but often remains susceptible to non-3-lactam antibiotics. Given the epidemic potential of CA-MRSA strains, there is a high probability that the number of CA-MRSA infections will increase in The Netherlands. In order to prevent and control CA-MRSA outbreaks in the community successfully, the restrictive Dutch antibiotic policy must be followed with strict infection prevention measures.

Interspecies discrimination of A. fumigatus and siblings A. lentulus and A. felis of the Aspergillus section Fumigati using the AsperGenius® assay.

The AsperGenius® assay detects several Aspergillus species and the A. fumigatus Cyp51A mutations TR34/L98H/T289A/Y121F that are associated with azole resistance. We evaluated its contribution in identifying A. lentulus and A. felis, 2 rare but intrinsically azole-resistant sibling species within the Aspergillus section Fumigati. Identification of these species with conventional culture techniques is difficult and time-consuming. The assay was tested on (i) 2 A. lentulus and A. felis strains obtained from biopsy proven invasive aspergillosis and (ii) control A. fumigatus (n=3), A. lentulus (n=6) and A. felis species complex (n=12) strains. The AsperGenius® resistance PCR did not detect the TR34 target in A. lentulus and A. felis in contrast to A. fumigatus. Melting peaks for L98H and Y121F markers differed and those of the Y121F marker were particularly suitable to discriminate the 3 species. In conclusion, the assay can be used to rapidly discriminate A. fumigatus, A. lentulus and A. felis.

Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice.

Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) play an important role in the recruitment of PMNs at the site of invasive Candida infection. In the absence of either TNFalpha or IL-6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G-CSF (rG-CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicans from the organs of rG-CSF-treated mice is significantly decreased. Treatment with the combination of rG-CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candida infection, rG-CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life-threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candida infection.

Modulation of LPS-, PHA- and M. tuberculosis-mediated cytokine production by pentoxifylline and thalidomide.

Pentoxifylline and thalidomide have been used to downregulate the production of TNF-alpha in several disease entities including mycobacterial infections and autoimmune disorders. These drugs inhibit the production of TNF-alpha by different mechanisms, but little is known about possible synergism and modulation of other cytokines. Pentoxifylline and thalidomide inhibited the in vitro stimulated production of TNF-alpha, IL-1 beta and IFN-gamma in blood mononuclear cells. No significant modulation of antiinflammatory cytokines was found. When used together, these agents demonstrated additive inhibition, but no synergism. Modulation of cytokine response was similar when different stimuli were used, including M. tuberculosis in tuberculin-positive individuals. Therefore, the balance between efficacy and toxicity may be more favourable when pentoxifylline and thalidomide are used together instead of either drug alone. Clinical studies are needed to establish this advantage when anti-cytokine strategies are considered.

Increased voluntary exercise in mice deficient for tumour necrosis factor-alpha and lymphotoxin-alpha.

BACKGROUND: The endogenous mediators playing a role in the sensing of fatigue and cessation of exercise are yet to be characterized. We hypothesized that proinflammatory cytokines, in particular tumour necrosis factor-alpha (TNFalpha) and lymphotoxin-alpha (LT) transmit signals leading to fatigue. MATERIALS AND METHODS: Mice were placed in a cage with a freely rotating exercise wheel and allowed to adapt for 24 h. The running distance was measured for two additional periods of 24 h. The effects of the administration of intravenous anti-TNF antibodies, intracerebral recombinant TNF, or intravenous lipopolysaccharide (LPS) were also determined. RESULTS: Compared to normal littermates, the voluntary daily running distance was 1.8-fold greater in mice with a disruption of the gene for TNFalpha, and 3-fold greater in mice with a gene disruption for both TNFalpha and LT. Intravenous administration of a monoclonal antibody against murine TNFalpha did not affect the running distance of wild-type mice, whereas administration of TNF intracerebrally reduced by 4-fold the voluntary running distance of the animals. This demonstrates that fatigue is mediated by TNFalpha expressed in the central nervous system (CNS) and not by increased peripheral TNFalpha concentrations. TNFalpha and LT are strong inducers of prostaglandins, but mice with disrupted prostaglandin or prostacyclin receptors exhibited running distances not significantly different from their wild-type littermates. Thus, signalling molecules other than prostaglandins mediate the effect of TNFalpha and LT on exercise capacity. CONCLUSIONS: Our finding that exercise capacity is controlled by TNFalpha is the first to define the endogenous mediators of fatigue, and may have important implications for diseases with impaired exercise tolerance.

Limited efficacy of thalidomide in the treatment of febrile attacks of the hyper-IgD and periodic fever syndrome: a randomized, double-blind, placebo-controlled trial.

Hyper-IgD and periodic fever syndrome (HIDS) is an autosomal recessive disorder featured by recurrent febrile attacks. Previous unpublished experience (J. van der Meer and R. Powell) suggested that thalidomide may prevent febrile attacks. Six HIDS patients (5 male and 1 female) who had at least one febrile attack every 6 weeks, entered a randomized, double-blind, placebo-controlled crossover trial to explore the efficacy of a daily 200-mg thalidomide dose in the treatment of recurrent febrile attacks of HIDS. The patients received either thalidomide, 200-mg daily, or placebo for 16 weeks, followed by a 4-week washout period and another 16-week treatment (crossover) with either thalidomide or placebo. Patients completed a weekly diary card noting attacks and side effects. During the study, C-reactive protein (CRP), serum amyloid A (SAA), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-1 receptor antagonist, soluble TNF receptor p55 and p75, and lipopolysaccharide-stimulated IL-1 beta and TNF-alpha production were measured at six different points, whereas urine neopterin levels were measured weekly. During the active treatment with thalidomide, there were 10 attacks compared with 13 attacks with placebo. Thalidomide resulted in a nonsignificant decrease of CRP and SAA, but the concentrations of other inflammatory mediators, including urine neopterin, remained unchanged. One patient developed sensory polyneuropathy, but this resolved when thalidomide administration was stopped. The effect of thalidomide in HIDS is limited to a decrease in acute phase protein synthesis without an effect on the attack rate.

Modulation of the pro- and anti-inflammatory cytokine balance by amphotericin B.

Amphotericin B is an antifungal drug associated with side effects such as fever and chills, symptoms which may be mediated by pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha). We assessed the capacity of amphotericin B to modulate production of these pro-inflammatory cytokines as well as the anti-inflammatory IL-1 receptor antagonist (IL-1ra), induced by LPS, heat-killed Candida albicans or Staphylococcus aureus. The results of the present study show that amphotericin B slightly increased the production of pro-inflammatory cytokines by human mononuclear cells (PBMC), whereas the production of the anti-inflammatory cytokine IL-1ra was significantly inhibited. This results in a shift towards pro-inflammatory cytokine production, as indicated by a decreased IL-1ra/IL-1beta ratio. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) indicated that levels of IL-1beta and TNFalpha mRNA were increased. In conclusion, amphotericin B is able to cause a shift towards pro-inflammatory cytokine production by human PBMC. This may explain the side effects, such as fever and chills, observed after treatment of patients with amphotericin B.