IntroductionCutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among the most neglected infectious diseases. While they are non-fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving lifelong unsightly scars; they are combined with psychological distress and social stigma. The efficiency of treatment highly depends on the infecting Leishmania species. Diagnosis is mainly based on microscopic direct examination (DE) of Giemsa-stained smears needing experienced microscopists. It can be laborious and time-consuming when the parasite load is low. DE is poorly sensitive and does not identify Leishmania species. So far, only DNA assays accurately identify the species. Despite their wide use for generic detection, PCR methods also require equipment and additional steps to identify causal Leishmania species. L. major is hyperendemic in many countries in Africa, the Middle East, and Asia, where other species co-occur with different endemicity levels according to the situations. This complicates disease management and treatment, particularly as distribution and epidemiology of leishmaniases remain poorly understood. Here, we aimed for a simple and rapid molecular diagnostic test to detect and identify L. major, a predominant CL causal species, which could be prone to become a control tool at the point of care, in endemic areas, using isothermal recombinase DNA amplification (recombinase polymerase amplification, RPA, or recombinase aided amplification, RAA) coupled to detection by the lateral flow (LF) chromatography on a PCRD cassette.MethodsTo develop an L. major species-specific RPA-LF assay, computational analysis of 70 Leishmania DNA targets, identified through bibliography and database searches, selected five targets. We designed and tested 7 primer pairs/probe sets to specifically amplify L. major DNAs. First, the primers were tested for species specificity and sensitivity using basic RPA chemistry. Then, to develop RPA-coupled LF detection, we shifted to the nfo chemistry.ResultsThis way, we retained one set for further investigation, which confirmed it is L. major species-specific. Tested on 86 human cutaneous samples, this selected set was able to detect 100% of L. major infections in confirmed CL patients. We did not observe any cross-reactivity with lesions due to L. infantum or L. tropica.

Introduction Invasive candidiasis (IC) is a widespread infection in intensive care. As culture-based diagnostic techniques take several days before positivity and leaks of sensitivity. (1,3)-β-D-glucan (BDG) was proposed as a mycological criterion for IC diagnosis in selected patients. Aim: To determine the performance of BDG assay in the early diagnosis of IC in non-neutropenic critically ill patients Methods: We conducted a prospective evaluative study. All adults who were hospitalized in La Rabta Tertiary Hospital intensive care unit from January to June 2023 and at risk of IC were screened on a weekly basis. A true positive status corresponded to confirmed or highly probable IC and a positive BDG test (>80 pg/mL). Results: A total of 123 BDG tests were performed on 85 patients with a median age of 58 years [41.5-67.5] and a median SOFA score=3 [2-5.5]. The median colonization index was 0.16 [0-0.33], and Candida albicans was the most common species isolated (71%). The median Candida score was 0.9 [0-2.9]. IC was retained in 30 cases. The median BDG level was 98 pg/mL [24-275]. Sixty-one patients had a positive BDG test, in whom only 21 had an IC. The performance of the BDG test in the diagnosis of IC was moderate (AUC/ROC=0.68 [0.575-0.788], p=0.003). The discriminatory power was better with the negative prediction (PNV=85.5%). Conclusion: The major benefit of BDG test in intensive care seems to lie in its NPV allowing to roll out the invasive candidiasis diagnosis then withhold or interrupt antifungal therapy.

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.

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.

Abstract 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.

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.

Candida albicans colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus , and contributes to disease severity. We serially recovered 160 C. albicans clinical isolates over a period of 30 months from the sputum of 23 pediatric and 2 adult antifungal-naive CF patients at Children’s Hospital Tunis and characterized the genotype and phenotype of a subset of strains using multilocus sequence typing (MLST) and growth assays on multiple stress-, filamentous growth- and biofilm-inducing media. Out of 16 patients regularly sampled for at least 9 months, 8 and 4 were chronically and transiently colonized with C. albicans , respectively. MLST analyses of 56 strains originating from 15 patients indicated that each patient was colonized with a single strain, while 8 patients (53%) carried isolates from clade 4 known to be enriched with strains from Middle East-Africa. A subset of these isolates with the same sequence type and colonizing 3 unrelated patients displayed altered susceptibility to cell wall-perturbing agents, suggesting changes in cell wall structure/function during growth in the CF lung. We also observed differential ability to filament and/or form biofilms in a set of identical isolates from clade 10 sampled over a period of 9 months in a pediatric CF patient, suggesting alterations in phenotypes associated with virulence. Our findings will rely on future whole-genome sequencing analyses to identify polymorphisms that could explain the emergence of new traits in C. albicans strains thriving in the CF host environment.

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.

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.

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.

Despite the changes in their epidemiology, and the improving level of hygiene of the population, tinea capitis is still considered a public health problem in our country, and is the most common type of dermatophytosis in our country. The aim of our study was to evaluate the epidemiological, clinical and mycological features of tinea capitis in children encountered in the Tunis region. A retrospective study concerned 1600 children aged 6 months to 15 years suspected to have tinea capitis was conducted in Parasitology-Mycology laboratory, Rabta hospital, over a 10-years period (2005-2014). Dermatophyte infections were confirmed using scalp scrapings examinated with direct microscopy using potash at 30% and/or culture on Sabouraud medium agar. Tinea capitis diagnosis was confirmed in 947 cases (59.18%). The sex ratio was 2.61 and the average age of 6.28 years with predominance in the age group of 4 to 8 years (52.27%). The most common clinical presentation was ringworm (87.65%). Ringworm large plaque was predominant (65.9%). Direct examination was positive in 884 cases (93.35%). Microsporic tinea was the most frequent (63.25%) followed by trichophytic tinea (29.78%). Positive cultures of dermatophytes were obtained in 912 cases (96.30%). The following dermatophyte species were isolated: Microsporum canis (67%), Trichophyton violaceum (31.68%), Trichophyton mentagrophytes (0.66%), Microsporum audouinii (0.22%), Trichophyton schoenleinii (0.22%) and Microsporum gypseum (0.22%). M. canis is currently the most frequently incriminated species in tinea capitis in Tunisia. This change is related to a change in behavior of our population, in fact the cat; main reservoir of M. canis cohabiting increasingly with Tunisian families.