Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin.

In vitro susceptibility of 933 Candida isolates, from 16 French hospitals, to micafungin was determined using the Etest in each center. All isolates were then sent to a single center for determination of MICs by the EUCAST reference method. Overall essential agreement between the two tests was 98.5% at ±2 log2 dilutions and 90.2% at ±1 log2 dilutions. Categorical agreement was 98.2%. The Etest is a valuable alternative to EUCAST for the routine determination of micafungin MICs in medical mycology laboratories.

In vitro combination of voriconazole and miltefosine against clinically relevant molds.

Invasive infections caused by filamentous fungi are a major threat for immunocompromised patients. Innate/acquired resistance to antifungal drugs might necessitate combination therapies. We assessed the potential combination of voriconazole with miltefosine, an original drug with antifungal activity against 33 clinically relevant mold isolates, including both azole-susceptible and -resistant Aspergillus. Using complete inhibition as an endpoint, interactions were indifferent for 32/33 isolates. An alternative 50% inhibition endpoint showed synergistic interactions for 14/33 isolates. Antagonism was absent.

Aspergillus fumigatus harbouring the sole Y121F mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole.

OBJECTIVES: Voriconazole, itraconazole and posaconazole are members of the azole family and widely used for the treatment of aspergillosis. They act by inhibiting the activity of the fungal Cyp51A enzyme. The emergence of environmental azole-resistant Aspergillus fumigatus strains raises major concerns for human health. METHODS: Recently, a new cyp51A-mediated resistance mechanism (namely TR46/Y121F/T289A) was described in clinical samples and patient-frequented environmental sites. In an azole-naive patient, we isolated an A. fumigatus strain that was not susceptible to voriconazole but was susceptible to itraconazole and posaconazole. RESULTS: A molecular analysis indicated a single Y121F substitution without the TR46 or T289A alterations, which to our knowledge has never been reported. Structure modelling and molecular dynamics offered an explanation for the resistance profile consistent with the structural differences between the three azoles. CONCLUSIONS: Taken together, these observations suggest an original mechanism conferring resistance to azoles mediated by cyp51A of environmental origin. This uncommon susceptibility pattern might represent a 'missing link' between the wild-type A. fumigatus and the fully azole-resistant strain harbouring the TR46/Y121F/T289A mutations.

Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy?

Since their introduction in the 2000s, echinocandin drugs have become widely used for the treatment and prophylaxis of invasive fungal infections and, notably, invasive candidiasis. Although cases of breakthrough candidiasis in patients receiving echinocandins have been reported, clinical failure during echinocandin treatment due to the acquisition of resistance by a normally susceptible Candida spp. isolate is considered rare. To date, no publications have been published correlating the use of echinocandins and the emergence of echinocandin resistance among Candida species. So, our goal is to report an initial analysis of echinocandin use in relation to the emergence of resistant Candida isolates. We report here a single-centre experience of the emergence of eight resistant isolates belonging to normally susceptible Candida species in six patients receiving echinocandins. We describe the context and analyse the use of echinocandins over the previous decade. For seven of these isolates, we identified FKS gene mutations involved in decreased susceptibility. Seven isolates were obtained in 2011, on the heels of a ten-fold increase in caspofungin use over the preceding decade. In contrast, in 2012, the use of echinocandins decreased in our institution by 19.5 % and, in that year, only one Candida-resistant isolate was detected, despite the stable global epidemiology of invasive candidaemia. This work underlines the necessity of improving the prescription of antifungal drugs. Improvement in the monitoring of strain susceptibility should also be considered in order to better detect the emergence of resistant or non-susceptible yeast strains.

Rapid emergence of echinocandin resistance during Candida kefyr fungemia treatment with caspofungin.

Echinocandin drugs are widely used for the treatment of candidemia. Resistance is considered rare, and only a few cases of breakthrough candidiasis in patients receiving echinocandin have been reported worldwide. We report here for the first time a Candida kefyr isolate that acquired echinocandin resistance very rapidly after the initiation of caspofungin treatment for candidemia. We characterized the FKS gene mutation responsible for the resistance via the comparison of isolates sampled before and during treatment.

Successful treatment of disseminated Geotrichum capitatum infection with a combination of caspofungin and voriconazole in an immunocompromised patient.

Disseminated Geotrichum capitatum infection is uncommon, and has been reported exclusively in immunocompromised patients. The prognosis is poor with a mortality rate of approximately 50-75%. We report a case of disseminated G. capitatum infection in a severely neutropenic patient who was receiving chemotherapy for acute myeloblastic leukaemia. G. capitatum was isolated from blood cultures, skin lesions, bronchoalveolar lavage fluid, throat swabs and stools. The infection was successfully cured with a combination of voriconazole and caspofungin.

[Caspofungin: mode of action and therapeutic applications]. / La caspofungine: du mécanisme d'action aux applications thérapeutiques.

SUBJECT: Since the last two decades, the incidence of invasive fungal infections has drastically increased. It becomes urgent to enlarge the panel of antifungal drugs with more potent activity and less toxicity. Since the target of all previously available antifungal agents is the synthesis of ergosterol located in the fungal membrane, the fungicidal activity of echinocandins is based on the inhibition of the glucan synthesis. Caspofungin (CAS) (Cancidas MSD France), a cyclic hexapeptide semisynthetic derivative of pneumocandin B, is the first available drug belonging to this new class. MAIN ISSUES: CAS has a fungicidal activity covering a wide range of pathogens, including Candida spp. Data from animal and human studies demonstrate that the drug is 96% plasmatic protein bound and the proposed route of elimination is hepatic. For the treatment of systemic, oesophageal and oropharyngeal candidiasis, CAS has the same efficacy as amphotericin B or as triazoles. Among 50% of patients suffering of invasive aspergillosis with intolerance or resistance to classical treatments, CAS induces a successful response and even more in combination with these drugs. For patients with fever and neutropenia, the efficacy of CAS is non inferior than Ambisome. CAS is generally well tolerated. The most common adverse effects are fever, nausea, vomiting and complication at the infusion point of the vein. CAS has a better tolerability than amphotericin B and a similar one compared to fluconazole (FCZ) but with less drug interactions. PERSPECTIVES: For rare but severe localisations (i.e.: endocarditis, cerebral, arthritis, etc.), CAS combinations with classical antifungal drugs could be tested in order to improve the life time in patients suffering from systemic fungal infections.

Role of a NSAID in the apparent cure of a fungal mycetoma.

UNLABELLED: We report the case of a fungal mycetoma due to Madurella mycetomatis that failed to respond to surgery and antifungal treatment but responded strongly to the addition of a non-steroidal anti-inflammatory drug (NSAID). This African patient was born in Mauritania in 1972. He was a herdsman, living close to the Senegal River. The first nodules appeared on the left foot at the age of 13years (1985). The patient suffered frequent flare-ups with the appearance of black grains and underwent surgery in 1988 and 1992 in Senegal. After remission for several months after surgery, new fistulae occurred. The patient emigrated to France in 1995 and underwent a third surgical intervention in 1996. M. mycetomatis was cultured from the black grains. The patient was otherwise in good health, with no diabetes, and HIV tests were negative. We saw the patient for the first time in 2005, at which time he had flare-ups every two to three months. Imaging disclosed an absence of bone involvement. The patient underwent a fourth operation in October, 2005, and voriconazole treatment was initiated. A new flare-up occurred in February, 2006. CT, MRI, and PET scans revealed calcaneus and tarsal involvement, and posaconazole then replaced voriconazole. Flucytosine was added four months later, due to an absence of improvement. New flares-ups occurred and a fifth surgical intervention was performed in September, 2006. The pain, which had been present for three years, worsened; the patient had to stop working and was no longer able to walk without crutches. Amputation of the foot was considered. Empiric treatment with a NSAID, diclofenac (Voltaren(®); 100mg/day), was added to the antifungal treatment in November 2006, to treat the patient's pain and inflammation. A major improvement was observed within one week. The patient was able to walk without crutches one month later. After two months, clinical examination was normal: no pain, inflammation, nodules or fistulae. Flucytosine was stopped after six months of treatment, in January 2007, diclofenac after 10months, in October 2007, and posaconazole after 18.5months, also in October 2007. No relapse has occurred during the eight years of follow-up since treatment ended. The patient seems to have been cured and has normal CT, MRI, and PET scans. IN SUMMARY: This eumycetoma, which had progressed over 20years despite surgery and antifungal treatments, seems to have been cured by the addition of a NSAID. This observation suggests that inflammation plays a major role in the pathogenesis of fungal mycetoma. Clinical studies of treatments including an NSAID should be conducted to confirm this finding.

Aspergillus PCR in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients.

We evaluated the usefulness of a serum Aspergillus PCR assay for the diagnosis and prognosis of invasive aspergillosis in a study involving 941 patients for a total of 5146 serum samples. Fifty-one patients had proven/probable aspergillosis. We compared galactomannan (GM), PCR and mycologic analysis of pulmonary samples in both neutropenic and nonneutropenic patients. PCR performed in serum yielded 66.7% sensitivity, 98.7% specificity, 75.6% positive predictive value and 98.0% negative predictive value, while the GM index yielded 78.4% sensitivity, 87.5% specificity, 27% positive predictive value and 98.6% negative predictive value. The inclusion of PCR in the European Organization for Research and Treatment of Cancer (EORTC) and the Mycosis Study Group (MSG) mycologic criteria permitted the reclassification of nine other cases from possible to probable aspergillosis and increased the sensitivity to 71.7%. Combining the GM index with serum PCR increased the detection rate of invasive aspergillosis with 88.2% sensitivity. PCR was systematically negative in 16 patients with noninvasive forms of aspergillosis (namely aspergilloma and chronic aspergillosis). Remaining PCR positive after a period of 14 to 20 days of treatment was related to poor outcome at 30 and 90 days. Our results also indicate that, unlike the determination of the GM index, the initial fungus load as determined by PCR was highly predictive of 90-day mortality, with the rate of the latter being 15.8% for patients with <150 copies/mL vs. 73.2% for patients at or above that cutoff (p <0.0001). Therefore, PCR appears to be a powerful and interesting tool for the identification of patients with invasive aspergillosis who might benefit from more intense care.

In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest: results of a French multicentre study.

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

Disseminated Penicillium marneffei infection associated with human immunodeficiency virus: a report of two cases and a review of 35 published cases.

Penicillium marneffei, a dimorphic fungus that is endemic in southeast Asia, causes deep-seated infection in humans and rodents. About 20 cases have been reported among the local populations of China, Thailand, and Hong Kong, and 35 cases have now been described in patients infected with the human immunodeficiency virus (HIV). We present a review of the literature and report two additional cases. Both immunocompromised and apparently immunocompetent hosts tend to develop disseminated, symptomatic infection. HIV-infected patients having travelled to southeast Asia and presenting with fever, skin lesions, hepatomegaly, adenopathies, or lung disease should be investigated for Penicillium marneffei infection. The diagnosis is based on the demonstration of the organism in clinical specimens. Treatment with amphotericin B or itraconazole is generally successful, but maintenance therapy is warranted for patients with an underlying immunodeficiency.

Toxoplasma gondii pneumonia in patients with the acquired immunodeficiency syndrome.

PURPOSE: To perform a retrospective and descriptive study of Toxoplasma gondii pneumonia in patients infected with the human immunodeficiency virus (HIV). Clinical presentation, diagnostic procedures, results of therapy, and hypotheses on pathophysiology are discussed. PATIENTS AND METHODS: The study consisted of 13 HIV-infected patients who had developed T. gondii pneumonia. Eight had acquired immunodeficiency syndrome (AIDS) prior to T. gondii pneumonia and three of them had non-Hodgkin's lymphoma. Mean CD4 cell count was 32 x 10(6)/L. Serum anti-toxoplasma antibody titers were measured by an indirect hemagglutination assay and/or by an indirect immunofluorescence assay. RESULTS: All patients had fever and bilateral pulmonary infiltrates; two of them presented with septic shock. Mean arterial oxygen tension was 47 +/- 12 mm Hg. The diagnosis was established by bronchoalveolar lavage in 10 of 11 cases, open lung biopsy in one case, and postmortem biopsy in two cases. Serologic evidence of past infection was observed in 11 of 12 cases, while one patient presented with acute disseminated disease and absence of serum anti-toxoplasma antibody response. Extrapulmonary involvement was present in seven patients: liver (four), brain (three), bone marrow (two), heart (two), stomach (one). Ten patients recovered from T. gondii pneumonia. CONCLUSION: T. gondii pneumonia must be considered in AIDS patients with severe diffuse bilateral pneumonia, especially when associated with a very low CD4 cell count or non-Hodgkin's lymphoma. In most of these cases, disseminated disease was associated with reactivation of prior latent infection.

Transmission of Plasmodium falciparum by allogeneic bone marrow transplantation.

We describe a case of Plasmodium falciparum infection in a Comorian patient undergoing BMT. The patient's last visit to an endemic area was 1 year prior to BMT. The donor left the Comoro Islands 2 months before marrow harvesting. They had both had previous episodes of malaria and were seropositive for Plasmodium falciparum. At the time of BMT, blood smears were negative in both the donor and recipient. On day 12 post-BMT the patient was asymptomatic but a blood smear revealed 12.5% parasitemia. We consider that donors and recipients at risk pre-BMT should routinely be given specific treatment before marrow harvesting and conditioning, independent of the appearance of blood smears.

A randomized trial of ivermectin versus albendazole for the treatment of cutaneous larva migrans.

In an open study, we compared the efficacy of single doses of oral ivermectin (12 mg) and oral albendazole (400 mg) for the treatment of cutaneous larva migrans. Twenty-one patients were randomly assigned to receive ivermectin (n = 10) or albendazole (n = 11). All patients who received ivermectin responded and none relapsed (cure rate = 100%). All but one patient in the group receiving albendazole responded, but five relapsed after a mean of 11 days (cure rate = 46%; P = 0.017). No major adverse effects were observed. We conclude that a single 12-mg dose of ivermectin is more effective than a single 400-mg dose of albendazole for the treatment of cutaneous larva migrans.

Use of cross-reactive antigens of the microsporidian Glugea atherinae for the possible detection of Enterocytozoon bieneusi by western blot.

The microsporidia Enterocytozoon bieneusi is reported in 10-30% of those infected with the human immunodeficiency virus. The parasite appears to be a cause of gastralgia, malabsorption, and diarrhea. A Western blot technique using another microsporidian species, Glugea atherinae, has demonstrated an antigenic similarity between this parasite and E. bieneusi. Preliminary results show the variability of the antigenic profiles obtained from the sera of immunodeficient patients infected with E. bieneusi and also of the cross-reactivity to Glugea sp. antigens of some sera from patients with cryptosporidiosis. The origin of this cross-reactivity is undetermined. The possibility of coinfection with undetected microsporidia is not excluded. These results raise questions concerning the interpretation of serologic data and of the potential immunodiagnostic value of microsporidian antigens.

Pneumocystis carinii is rare in AIDS in Central Africa.

PIP: Pneumocystis carinii occurs in 60-65% of cases of acquired immunodeficiency syndrome (AIDS) in Europe and North America. Its prevalence in Africa is unknown, however, due to a lack of suitable investigative techniques. Initial studies suggest that this parasitic complication may be lower in Africa. An investigation of AIDS case in Harare, Zimbabwe, revealed pneumocystosis in only 22%. Reported here are the results of a study of 45 Congolese patients with AIDS conducted at the Brazzavile University Hospital. The patients were 24-54 years of age (mean age, 37 years); 20 were female. All patients presented with pulmonary manifestations, but not with open pulmonary tuberculosis. Analysis of bronchoalveolar lavage fluid obtained by bronchofibroscopy revealed the presence of P carinii in only 5 cases (11%). 2 of these patients were women aged 25 and 49 years old and 3 were men aged 31, 35, and 51 years old. 4 presented with interstitial pneumopathy; the 5th had a localized lesion of the right lung.^ieng

Hepatocytes as feeder-layers for in vitro cultivation of Plasmodium falciparum blood-stages.

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.

Treatment of Strongyloides stercoralis infection with ivermectin compared with albendazole: results of an open study of 60 cases.

Ivermectin is highly effective against animal intestinal nematodes and is used in the treatment of onchocerciasis in humans. A study was undertaken to compare the efficacy of the drug with that of albendazole in the treatment of uncomplicated strongyloidiasis. Sixty patients with confirmed Strongyloides stercoralis infection were enrolled in an open randomized study and given either albendazole, 400 mg/d for 3 d or ivermectin, 150-200 micrograms/kg in a single dose. Efficacy and tolerance were evaluated on days 7, 30 and 90. Each visit included a parasitological examination of 3 stool specimens, using saline and Kato smears and formalin-ether and Baermann concentrations. Fifty-three patients were eligible for evaluation. Parasitological cure was obtained in 24 of the 29 patients treated with ivermectin (83%) and in 9 of the 24 patients who were given albendazole (38%); ivermectin was significantly more effective than albendazole (P < 0.01). Clinical and biological adverse reactions were negligible in both treatment groups. The 20 patients who failed therapy were given a second treatment course with 150-200 micrograms/kg of ivermectin in a single dose or on 2 consecutive days. Sixteen patients were cured and the other 4 had only incomplete follow-up. Ivermectin therefore constitutes an acceptable therapeutic alternative for uncomplicated strongyloidiasis.

Morphogenesis of the polaroplast in Enterocytozoon bieneusi Desportes et al., 1985, a microsporidan parasite of HIV infected patients.

The development of the microsporidan Enterocytozoon bieneusi is characterized by the formation of the extrusion apparatus of the spore before the division of the sporogonial plasmodium into sporoblasts. The extrusion apparatus consists of the classical polar tube, polaroplast and anchoring disc reported in the other Microsporida. It is shown that the precursors of the polaroplast are already present in early developmental stages i.e. uninucleate meronts. Each consists of an aggregate of vesicles which originates from the endoplasmic reticulum and develops into stacks of saccules. The saccules, which display the morphologic polarity of a Golgi apparatus, correspond to the future polaroplasts. The nuclear divisions and concomitant production of new aggregates of vesicles result in the formation of plasmodia containing several nuclei, each of them closely associated with a polaroplast. Elongated cisternae of the endoplasmic reticulum, characteristic of the plasmodial cytoplasm, contribute to the differentiation of the polar tubes. The extrusion apparatus is achieved after the association of the polar tubes with the preformed polaroplasts. In most Microsporida the formation of the extrusion apparatus occurs in the sporoblasts, in which the polar tube emerges from a reticular structure. Thus the differentiation of the extrusion apparatus observed in E. bieneusi may be considered as another characteristic of this microsporidan species.