A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Aspergillus flavus genetic structure at a turkey farm.

BACKGROUND: The ubiquitous environmental fungus Aspergillus flavus is also a life-threatening avian pathogen. OBJECTIVES: This study aimed to assess the genetic diversity and population structure of A. flavus isolated from turkey lung biopsy or environmental samples collected in a poultry farm. METHODS: A. flavus isolates were identified using both morphological and ITS sequence features. Multilocus microsatellite genotyping was performed by using a panel of six microsatellite markers. Population genetic indices were computed using FSTAT and STRUCTURE. A minimum-spanning tree (MST) and UPGMA dendrogram were drawn using BioNumerics and NTSYS-PC, respectively. RESULTS: The 63 environmental (air, surfaces, eggshells and food) A. flavus isolates clustered in 36 genotypes (genotypic diversity = 0.57), and the 19 turkey lung biopsies isolates clustered in 17 genotypes (genotypic diversity = 0.89). The genetic structure of environmental and avian A. flavus populations were clearly differentiated, according to both F-statistics and Bayesian model-based analysis' results. The Bayesian approach indicated gene flow between both A. flavus populations. The MST illustrated the genetic structure of this A. flavus population split in nine clusters, including six singletons. CONCLUSIONS: Our results highlight the distinct genetic structure of environmental and avian A. flavus populations, indicative of a genome-based adaptation of isolates involved in avian aspergillosis.

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

In Vivo Efficacy of Voriconazole in a Galleria mellonella Model of Invasive Infection Due to Azole-Susceptible or Resistant Aspergillus fumigatus Isolates.

Aspergillus fumigatus is an environmental filamentous fungus responsible for life-threatening infections in humans and animals. Azoles are the first-line treatment for aspergillosis, but in recent years, the emergence of azole resistance in A. fumigatus has changed treatment recommendations. The objective of this study was to evaluate the efficacy of voriconazole (VRZ) in a Galleria mellonella model of invasive infection due to azole-susceptible or azole-resistant A. fumigatus isolates. We also sought to describe the pharmacokinetics of VRZ in the G. mellonella model. G. mellonella larvae were infected with conidial suspensions of azole-susceptible and azole-resistant isolates of A. fumigatus. Mortality curves were used to calculate the lethal dose. Assessment of the efficacy of VRZ or amphotericin B (AMB) treatment was based on mortality in the lethal model and histopathologic lesions. The pharmacokinetics of VRZ were determined in larval hemolymph. Invasive fungal infection was obtained after conidial inoculation. A dose-dependent reduction in mortality was observed after antifungal treatment with AMB and VRZ. VRZ was more effective at treating larvae inoculated with azole-susceptible A. fumigatus isolates than larvae inoculated with azole-resistant isolates. The concentration of VRZ was maximal at the beginning of treatment and gradually decreased in the hemolymph to reach a Cmin (24 h) between 0.11 and 11.30 mg/L, depending on the dose. In conclusion, G. mellonella is a suitable model for testing the efficacy of antifungal agents against A. fumigatus.

Case Report: Allergic Bronchopulmonary Aspergillosis Revealing Asthma.

Allergic bronchopulmonary aspergillosis (ABPA) is an immunological pulmonary disorder caused by hypersensitivity to Aspergillus which colonizes the airways of patients with asthma and cystic fibrosis. Its diagnosis could be difficult in some cases due to atypical presentations especially when there is no medical history of asthma. Treatment of ABPA is frequently associated to side effects but cumulated drug toxicity due to different molecules is rarely reported. An accurate choice among the different available molecules and effective on ABPA is crucial. We report a case of ABPA in a woman without a known history of asthma. She presented an acute bronchitis with wheezing dyspnea leading to an acute respiratory failure. She was hospitalized in the intensive care unit. The bronchoscopy revealed a complete obstruction of the left primary bronchus by a sticky greenish material. The culture of this material isolated Aspergillus fumigatus and that of bronchial aspiration fluid isolated Pseudomonas aeruginosa. The diagnosis of ABPA was based on elevated eosinophil count, the presence of specific IgE and IgG against Aspergillus fumigatus and left segmental collapse on chest computed tomography. The patient received an inhaled treatment for her asthma and a high dose of oral corticosteroids for ABPA. Her symptoms improved but during the decrease of corticosteroids, the patient presented a relapse. She received itraconazole in addition to corticosteroids. Four months later, she presented a drug-induced hepatitis due to itraconazole which was immediately stopped. During the monitoring of her asthma which was partially controlled, the patient presented an aseptic osteonecrosis of both femoral heads that required surgery. Nine months after itraconazole discontinuation, she presented a second relapse of her ABPA. She received voriconazole for nine months associated with a low dose of systemic corticosteroid therapy with an improvement of her symptoms. After discontinuation of antifungal treatment, there was no relapse for one year follow-up.

High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis.

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

Prevalence of healthcare-associated infections at a Tunisian onco-hematology ward.

INTRODUCTION: Healthcare-associated infections (HAIs) are with high rates of mortality and an additional cost, in onco-hematology patients. AIM: To assess the prevalence trends of HAIs in the onco-hematology ward of the Tunisian National Bone Marrow Transplant Center (NBMTC), and to determine the principal associated risk factors. METHODS: Six repeated point prevalence surveys were conducted, from May 2018 to March 2019, using a two months interval. All patients hospitalized in the day of the survey were included. Risk factors of HAIs were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). They were assessed using a logistic regression model. RESULTS: Nineteen patients out of a total of 74 patients have been diagnosed with 19 HAIs, representing a prevalence of 25.7%. No significant downward or upward trend of prevalence was revealed over time (p=0.3). The most common HAI was respiratory tract infection (57.9%) with a prevalence of 14.9%. Multiple logistic regression analysis revealed that HAI was significantly associated with neutropenia (Adjusted OR: 14; 95% CI: 1.5-127; p=0.01) and duration of central venous catheter (Adjusted OR: 1.1; 95% CI: 1-1.2; p=0.005). CONCLUSION: High prevalence of HAIs in our center with a high rate of mortality, requiring identifying potential problems in infection control practices.

Galleria mellonella for the Evaluation of Antifungal Efficacy against Medically Important Fungi, a Narrative Review.

The treatment of invasive fungal infections remains challenging and the emergence of new fungal pathogens as well as the development of resistance to the main antifungal drugs highlight the need for novel therapeutic strategies. Although in vitro antifungal susceptibility testing has come of age, the proper evaluation of therapeutic efficacy of current or new antifungals is dependent on the use of animal models. Mammalian models, particularly using rodents, are the cornerstone for evaluation of antifungal efficacy, but are limited by increased costs and ethical considerations. To circumvent these limitations, alternative invertebrate models, such as Galleria mellonella, have been developed. Larvae of G. mellonella have been widely used for testing virulence of fungi and more recently have proven useful for evaluation of antifungal efficacy. This model is suitable for infection by different fungal pathogens including yeasts (Candida, Cryptococcus, Trichosporon) and filamentous fungi (Aspergillus, Mucorales). Antifungal efficacy may be easily estimated by fungal burden or mortality rate in infected and treated larvae. The aim of the present review is to summarize the actual data about the use of G. mellonella for testing the in vivo efficacy of licensed antifungal drugs, new drugs, and combination therapies.

Multidisciplinary exams in medical studies : Interest of docimologic analysis.

Docimology has allowed the development of evaluative processes assuring valid, reliable and objective assessments. It was adopted within the faculty of Medicine of Tunis since  2007. The aim of this study was to analyze the docimological survey results of hematology-oncology exams, to evaluate the interest of this analysis in the elaboration of exams and the construction of an item bank, and propose some corrections  in order to improve assessment.     Methods :We have analyzed the hematology-oncology exams of SCMS1 (Second cycle of Medical Studies 1) from educational year 2008-2009 to 2013-2014. The data input was already done with Excel. The test includes 4 disciplines (Hematology, Oncology, Genetics and the Anatomic Pathology). We have calculated docimological parameters allowing global analysis, by discipline and by item. Results : A total of 3281 papers and 1004 questions were analyzed. The average success rate per year was 91,54% ± 7,12. The highest average success rate was found in hematology (80,51% ± 10,18). The lowest rate was found in the anatomic pathology (51,61% ± 23,76). The average rate of students succeeding the test without having average note in hematology was 5,36%. It was 42,29% in the anatomic pathology. Average difficulty index was 0,57 ± 0,05. Items analysis showed that 38,04% were easy and 19,02% were difficult. Average discrimination index was 0,25 ± 0,02. Discrimination was very good in 20,51% of items and good in 17,13%. Useless and bad discrimination items were about 40,53%. The average of Cronbach Alpha coefficient was 0,84 ± 0,03, showing a good internal-consistency. Conclusion :This study allowed an objective evaluation of "contributive disciplines" in multidisciplinary evaluation and showed the interest of integrating questions. Question analysis with teachers would be important to reevaluate and improve these items.

Post transfusion malaria: current state in Tunisia (new case reported in 2016).

Since the elimination of indigenous transmission of malaria in Tunisia in 1979, almost all the cases observed are imported cases related to travel. We report a recent case of highly probable post-transfusion malaria (PTM) in a 27-year-old Tunisian who has never left Tunisia. He has been allografted and has received of the globular pellets and the platelet units along with his hospitalization. The evolution was marked by the appearance of a fever resistant to antibiotics 15 days later. On day 11 of fever, a thick drop (TD) and a blood smear (BS) showed trophozoites of Plasmodium falciparum with 20% parasitaemia. The evolution was favorable under quinine. The epidemiological survey concluded that among blood donors an African donor from Ivory Coast, in Tunisia for 2 months, had a TD, a BS, a rapid test and a nested PCR for P. falciparum species were negative, only the serology was positive by indirect immunofluorescence (1/20). Real-time PCR was positive for P. falciparum, and the diagnosis of highly probable PTM was retained. Blood transfusion is a transmission pathway for Plasmodium and contamination can occur with a very few parasites. As a result, the PTM must be considered for any unexplained fever arising in the aftermath of a blood transfusion that and establish strict prevention recommendations for PTM in our country.

Plasmodium falciparum infection transmitted by transfusion: A cause of hemophagocytic syndrome after bone marrow tranplantation in a non-endemic country.

A 27-year-old man with severe aplastic anemia underwent bone marrow transplantation from his HLA identical brother in July 2016. Conditioning included ATGAM 30 mg/kg for 3 days and Cyclophosphamide 50 mg/kg for 4 days. The patient received several platelet and red blood cell transfusions before and after the conditioning. The patient received broad spectrum antibiotics and caspofungin because persistant febrile neutropenia without bacteriological or mycological documentation. Hemophagocytic syndrome was diagnosed on day +12. Steroids at 1 mg/kg were started on day +12. Fever resolved the same day but resumed 3 days later associated to intravascular hemolysis with no schizocytes on blood smears and negative DAT. Thick blood film smears performed on day +26 revealed Plasmodium falciparum parasites (parasitemia = 20%). Except the level of parasitemia, there were no signs of gravity. Quinine was started on day 26 at a loading dose of 15 mg/kg followed by 8 mg/kg three times a day for 20 doses. Fever vanished after 2 days. Parasitemia cleared in 3 days and remained negative thereafter. Investigations revealed that the patient was transfused by a red cell unit harvested in a voluntary donor native of a malaria endemic country. PCR for P. falciparum performed in this donor in the frame of investigations was positive. The patient is alive with a normal blood count 1 year after BMT.

Airport malaria: report of four cases in Tunisia.

Four cases of airport malaria were notified for the first time in Tunisia during the summer of 2013. All patients were neighbours living within 2 km of Tunis International Airport. They had no history of travel to malarious countries, of blood transfusion or of intravenous drug use. Although malaria transmission had ceased in Tunisia since 1980, autochthonous infection by local Anopheles mosquitoes was initially considered. However, this diagnostic hypothesis was ruled out due to negative entomological survey and the absence of additional cases.All cases were caused by Plasmodium falciparum. Clinical presentation was severe (important thrombocytopaenia and parasitaemia), because of relatively important delay in diagnosis (average of seven days). This indicates the need to consider malaria while examining airport employees or people living near international airports presenting with fever of unknown origin. It also stresses the need for effective spraying of aircrafts coming from malarious areas.

[Intestinal parasitosis among non-permanent resident students in Tunisia: a review of 23 years of monitoring in the department of Parasitology-Mycology at the Rabta Hospital of Tunisia]. / Les parasitoses intestinales chez les étudiants non-residents permanents en Tunisie: Bilan de 23 ans de surveillance au laboratoire de Parasitologie-Mycologie à l'hôpital la Rabta de Tunis.

BACKGROUND: In order to fight digestive parasitism in Tunisia, a national program of surveillance of non-permanent resident students in Tunisia has been found to detect these parasitosis in this target population. OBJECTIVE: To determine the prevalence of intestinal parasitosis among non-permanent resident students in Tunisia, to identify the different parasitic species founded and to show the interest of this screening. METHODS: During a period of 23 years (1990-2012), 7386 parasitological examinations of stools has been made among students essentially from or had visited tropical Africa, Maghreb and Middle-East, at the laboratory of Parasitology-Mycology at the Rabta Hospital of Tunis. RESULTS: The prevalence of intestinal parasitism found was 34.45% (i.e. 2545 infested students). Among the protozoa that have been isolated in the majority of cases (78.75%), amoebae were most frequently found (86.4%) represented mainly by Entamoeba coli and Endolimax nanus in respectively, 25.62 and 23.33% of parasites isolated; while Entamoeba histolytica/dispar, only pathogenic Amoeba was found in 8.05% of the total of parasites isolated. Regarding helminths, found in 21.25% of parasites isolated, Ankylostome was predominant (34.5%) represented by the species of Necator americanus. A single case of Ancylostom duodenale has been isolated. Among the identified parasite species, 38.7% were known parasitic pathogens for humans. CONCLUSIONS: These results note the interest of the control of the non-permanent resident students in Tunisia. The precocious tracking and treatment of affected subjects permits to avoid the introduction and the dissemination of parasites already rare and virulent strains in our country.

[Risk factors for Toxoplasma gondii and immune status of pregnant women: cause and effect?]. / Facteurs de risque pour Toxoplasma gondii et statut immunitaire des femmes parturientes: relation de cause à effet.

BACKGROUND: Human infection with the protozoan parasite Toxoplasma gondii is found world-wide. The differences in prevalence across the countries may be ascribed to variations in hygiene and eating habits. AIM: To identify preventable risk factors for Toxoplasma gondii infection in pregnancy through a transversal study achieved in the laboratory of Parasitology Mycology of Rabta including all pregnant women referred to the laboratory between March 2010 and February 2011 to serological testing for toxoplasmosis methods: The age, the stage of the current pregnancy and exposures to potential risk factors associated with Toxoplasma infection were collected by a questionnaire. An analysis with SPSS and chi-square was used to analyse exposure frequencies for each risk factor variable. RESULTS: A total of 2351 women were included. The median age of the pregnany women was 29.4 years (range, 16 - 48 years). In all, 47.7% of them were seropositive. Seropositivity for toxoplasmosis was significantly associated with eating undercooked meat (p = 0.002) and eating miswashed vegetables (p = 0.003). However, the other factors (contact with cats, cleaning the cat litter box, washing the hands after preparation of raw meat, contact with the ground) were found to be independently associated with seropositivity toxoplasmosis. CONCLUSION: In Tunisia, there is still no prevention program toxoplasmosis well structured. Health education on avoidance of maternal infection is an important aspect of any program for prevention of congenital toxoplasmosis.

Pulsed Field Gel Electrophoresis types of Candida albicans isolates from an intensive care unit in a Tunisian hospital.

Candida albicans is the most important cause of fungal infections in intensive care units. The aim of this work was to compare the profiles of C. albicans in order to specify their genetic polymorphism and to determine the origin of these infections. Thirty-five C. albicans strains were collected from different clinical samples of 12 patients and three health-workers in an intensive care unit (ICU) in Rabta hospital of Tunisia, between August 2007 and April 2008. After digestion with BssHII, the isolates were typed by pulsed field gel electrophoresis (PFGE). The PFGE profiles were analyzed using a visual method, which showed three PFGE types (A, B and C) and the dendrogram generated three clusters (clusters I to III). An average similarity coefficient of 0.83, suggests that isolates are related.

[Keratomycosis in the area of Tunis: epidemiological data, diagnostic and therapeutic modalities]. / Les kératites fongiques dans la région de Tunis: caractéristiques épidémiologiques, modalités diagnostiques et thérapeutiques.

Fungal keratitis is a serious disease involving the visual prognosis. This pathology is not well known in Tunisia. The aim of our study is to determine epidemiological data and clinical and mycological characteristics of fungal keratitis in the area of Tunis (North of Tunisia) and discuss its therapeutic modalities. This is a retrospective study including 19 cases of fungal keratitis collected over a period of 11 years (January 1998-December 2008). The diagnosis of keratomycosis was based on clinical and mycological data. Mycological examination interested corneal scraping including direct examination and culture. The cases of fungal keratitis concerned 13 men and 6 women with a mean age of 48.7 years. The most common risk factors was corneal trauma (47.4%). The mean delay between the first ophthalmic signs and consultation was 17.7 days. Most frequently fungal isolated fungi were Candida albicans (6 cases), followed by Aspergillus spp (5 cases) and Fusarium spp (4 cases). All patients received topical and systemical antifungal therapy. The evolution was favourable in six cases. Three patients retained corneal scars. The surgery was necessary in 7 cases, consisting of a penetrating keratoplasty (5 cases), an enucleating (1 case) and amniotic membrane transplantation (1 case). In conclusion, despite the improvement of diagnosis and treatment of fungal keratitis, its prognosis remains pejorative. This prognosis depends on early diagnosis and choice of antifungal therapy.

[Subcutaneous dirofilariasis of the upper lip in Tunisia]. / Dirofilariose sous-cutanée en Tunisie : une observation à localisation labiale.

INTRODUCTION: Dirofilariasis is a rare anthroponotic disease caused by Dirofilaria, the principal reservoir of which is the dog. The first case of subcutaneous dirofilariasis in Tunisia was reported in 1990. CASE REPORT: We report a case involving a 40-year-old woman living in northeastern Tunisia who presented with a subcutaneous lesion of the upper lip resembling a sebaceous cyst. Excisional biopsy released a worm identified on morphologic examination as Dirofilaria repens. Only excision allows simultaneous diagnosis and treatment. CONCLUSION: This is the 14th case of dirofilariasis reported in Tunisia. This parasitic disease is probably underestimated in our country. It should be considered for all subcutaneous nodules, regardless of localisation.

[Acanthamoeba keratitis: diagnosis to consider]. / La kératite amibienne: il faut y penser.

INTRODUCTION: Acanthamoeba keratitis is a rare but severe disease that can cause blindness. The objective of this study is to call attention to its severity and conditions of development and to emphasize the importance of early treatment. CASE REPORT: We report a case of a 27-year-old woman. After wearing soft contact lenses for a month, she consulted for redness, pain and poor visual acuity in her left eye. The ophthalmological examination showed an epithelial corneal abscess measuring 2 mm in diameter in the left eye and very tiny epithelial corneal abscesses in the right eye. Microscopic examination and culture of samples from the contact lenses and the contact lens solution found Acanthamoeba spp. Rapid anti-amoeba treatment led to disappearance of the corneal abscesses and improvement of her vision. CONCLUSION: This case confirmed that the prognosis of Acanthamoeba keratitis depends on rapid diagnosis and early treatment.