Microsatellite analysis of the population structure in Rhizopus arrhizus.

OBJECTIVES: Rhizopus arrhizus is recognized as an emergent agent of superficial and invasive mucormycosis. Despite an increasing number of these infections, the molecular epidemiology of Rhizopus species has not been well studied. MATERIALS AND METHODS: In this study, 43 R. arrhizus strains (25 environmental and 18 clinical isolates) were genotyped using six novel panels of microsatellite markers. RESULTS: Upon the analysis of 43 isolates, 4-8 distinct alleles were detected for each marker. The discriminatory power for the individual markers ranged from 0·522 to 0·830. The combination of all six markers yielded 33 different haplotypes with a high degree of discrimination (0·989 D value). A four-marker combination were selected as the most parsimonious panel achieving D > 0·95. One clinical isolate and one environmental isolate shared the same genotype suggesting the possible nosocomial outbreak of mucormycosis in hospitalized patients. We have noted that the strains isolated from cutaneous mucormycosis were different from the strains isolated from rhino-orbito-cerebral mucormycosis. Then, the hypothesis of particular tropism of infectious strains for a given site is not excluded. The standardized indices of association IA and rBarD were significantly different from zero (P < 0·01), suggesting a prevailing clonal reproduction. The environmental population was significantly differentiated from clinical populations (Fst = 0·2249). CONCLUSIONS: Microsatellite typing method described in our study showed an excellent degree of discriminatory power. It is a promising tool for illuminating the molecular epidemiology of R. arrhizus species, including strain relatedness and transmission pathways.

Prevalence of free living amoeba in the domestic waters reservoirs in Sfax, Tunisia.

Free living amoebas (FLA) are opportunistic pathogen found in different water sources in the environment. The aim of this study was to investigate the prevalence of free living amoeba in different samples of domestic water reserves (DWR) in Sfax region from Tunisia. It was a prospective study dealing with 486 water samples collected from different DWR. After filtration through a cellulose acetate membrane samples were cultured on non-nutrient agar and the FLA were detected and strained with Giesma, Trichrome and red nuclear stain for morphological and morphotypic studies. FLA were found in 62% of samples. The Acanthopodial morphotype was detected in 43%, Polytactic (38%), Monotactic (28%), Fan-shaped (17%), Rugose (11%), Dactilopodial (10%) and Eruptive (9%). These results demonstrate that domestic water reserves are a significant source of the FLA and maintenance of DWR is recommended.

Isolation and molecular identification of Acanthamoeba spp from oasis water in Tunisia.

In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health.

Influence of environmental and seasonal factors on microbial contamination levels in clam production areas in southern Tunisia: Escherichia coli, Salmonella spp., hepatitis A virus and norovirus. / Influence des facteurs environnementaux et saisonniers sur le niveau de contamination des zones de production des palourdes par Escherichia coli, Salmonella spp., le virus de l'hépatite A et les norovirus dans la région de Sfax (Sud tunisien).

To study the influence of environmental and biological factors on levels of contamination by Escherichia coli, Salmonella spp., hepatitis A virus (HAV) and norovirus in clam production areas, an epidemiological study was conducted on 791 samples of live clams (Ruditapes decussatus), 539 of which were sent for bacteriological analysis and 252 for detection of norovirus and HAV. These samples were collected in different production areas in the Sfax region of southern Tunisia over four consecutive years, from March 2013 to December 2016. The prevalence of positive samples was 36% for E. coli, 11% for Salmonella spp., 19% for norovirus and 3% for HAV. There was a significant correlation between contamination by E. coli and by Salmonella spp., as well as between contamination by noroviruses and by HAV and between contamination by noroviruses and by Salmonella spp. Temperature, the presence of migratory birds and tourism are the main factors affecting microbial contamination levels in bivalve molluscs.

Pour étudier l'influence des facteurs environnementaux et biologiques sur le niveau de contamination des zones de production des palourdes par Escherichia coli, Salmonella spp., le virus de l'hépatite A (VHA) et les norovirus, une enquête épidémiologique a été réalisée en utilisant 791 échantillons de palourdes vivantes (Ruditapes decussatus), dont 539 destinés à des analyses bactériologiques et 252 destinés à la détection des norovirus et du VHA. Ces échantillons ont été collectés dans différentes zones de production de la région de Sfax (Sud de la Tunisie) durant quatre années consécutives, du mois de mars 2013 au mois de décembre 2016. La prévalence d'échantillons positifs était respectivement de 36 % pour E. coli, de 11 % pour Salmonella spp., de 19 % pour les norovirus et de 3 % pour le VHA. La corrélation était fortement significative entre la contamination par E. coli et celle par Salmonella spp., ainsi qu'entre la contamination par les norovirus et celle par le VHA et entre la contamination par les norovirus et celle par Salmonella spp. La température, la présence d'oiseaux migrateurs et l'activité touristique sont les principaux facteurs associés au niveau de contamination microbienne des mollusques bivalves.

Los autores describen una investigación epidemiológica encaminada a estudiar la influencia de los factores ambientales y biológicos sobre el nivel de contaminación de zonas de producción de almejas por Escherichia coli, salmonelas, virus de la hepatitis A (VHA) y norovirus. Para ello se llevó a cabo una investigación epidemiológica: en el curso de cuatro años consecutivos (de marzo de 2013 a diciembre de 2016), se obtuvieron en distintas zonas productivas de la región de Sfax (sur de Túnez) 791 muestras de almejas vivas (Ruditapes decussatus), de las que 539 fueron sometidas a análisis bacteriológicos y 252 sirvieron para detectar norovirus y el VHA. La prevalencia de muestras positivas resultó respectivamente del 36% para E. coli, del 11% para Salmonella spp., del 19% para los norovirus y del 3% para el VHA. Había una correlación muy significativa entre la contaminación por E. coli y la contaminación por salmonelas, y también entre la presencia de los norovirus y la del VHA y entre la de los norovirus y la de salmonelas. La temperatura, la presencia de aves migratorias y la actividad turística son los principales factores asociados al nivel de contaminación microbiana de los moluscos bivalvos.

Analysis of virulence factors and in vivo biofilm-forming capacity of Yarrowia lipolytica isolated from patients with fungemia.

Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter-related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU.

Molecular Identification of Malassezia Species in Patients with Malassezia folliculitis in Sfax, Tunisia.

AIM: Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome. PATIENTS AND METHODS: Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm. RESULTS: Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes. CONCLUSION: There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

Fungemia caused by Yarrowia lipolytica.

Yarrowia lipolytica is weakly pathogenic yeast, which is rarely isolated from the blood. We report unusual cases of Y. lipolytica fungemia occurred between October 2012 and June 2014 in the intensive care unit (ICU) of the UH Habib Bourguiba Sfax. During this period, 55 cases of Y. lipolytica septicemia were diagnosed. There were 44 men and 11 women (sex ratio = 4).The median age was 43 years. The broad-spectrum antibiotics (100 %), the catheterization (96 %), and the prolonged hospitalization in ICU (91 %) were the main risk factors. Patients were hospitalized in ICU, mostly, for polytraumatism (45.4 %), pneumopathy (9 %), and post-operative complications (7 %). Fever unresponsive to broad-spectrum antibacterial therapy was the predominant sign of infection (83.6 %). Y. lipolytica was isolated in one or several blood cultures (14.5 %) and in the catheter tip culture of nine patients (16.3 %).Treatment was based on intravenous amphotericin B (58.2 %), fluconazole (45.4 %) and/or removal catheter (69 %). Apyrexia or blood cultures sterilization was obtained for 34 patients (61.8 %). Y. lipolytica candidemia is an opportunistic and emerging human yeast pathogen. It can reach to the bloodstream of immunocompromised or critically ill patients during hospitalization through intravascular catheterization. Further clinical data need to be evaluated for formulating management strategies of seriously ill patients infected with uncommon fungal agents.

Geotrichum capitatum septicemia: case report and review of the literature.

Geotrichum capitatum is an uncommon cause of invasive infections in immunocompromised patients, particularly those with hematological malignancies and severe neutropenia. The aim of this study was to report the cases of invasive geotrichosis in our hospital. It is a retrospective study of invasive geotrichosis diagnosed in the Laboratory of Parasitology-Mycology of the UH Habib Bourguiba, Sfax, from January 2005 to August 2013. Six cases of invasive Geotrichum infections were diagnosed. There were three men and three women. The mean age was 35 years. Five patients have acute myeloid leukemia with a profound neutropenia, and one patient was hospitalized in the intensive care unit for polytraumatism. Clinically, the prolonged fever associated with pulmonary symptoms was the predominant symptom (n = 5). Geotrichum capitatum was isolated in one or more blood culture. Two patients had urinary tract infections documented by multiple urine cultures positive for G. capitatum. Five patients received conventional amphotericin B alone or associated with voriconazole. The outcome was fatal in four cases. Invasive geotrichosis is rare, but particularly fatal in immunocompromised patients. Approximately, 186 cases have been reported in the literature. The prognostic is poor with mortality over 50 %. So, early diagnosis and appropriate management are necessary to improve prognosis.

Polymorphisms in the ITS rDNA regions for differentiating strains of the Trichophyton mentagrophytes complex in Sfax-Tunisia.

The Trichophyton mentagrophytes complex is the main cause of superficial mycoses in humans and animals. Molecular research has provided useful insights into the taxonomy of this complex to overcome the challenges with conventional diagnostics. The aim of this study was to identify, type and differentiate anthropophilic and zoophilic species of the T. mentagrophytes complex. Sixty clinical samples identified as T. mentagrophytes by morphological characteristics were isolated using polymerase chain reaction-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer (ITS) regions. The identification of our strains by conventional methods was confirmed using polymerase chain reaction (PCR) sequencing in 93.34% of the cases. The strains under investigation were recategorised as T. rubrum (Tr2711). In addition, PCR products were independently digested with the restriction endonucleases, MvaI and HinfI, to produce a single dominant profile for T. interdigitale. ITS sequence analysis revealed a polymorphism in the ITS1 and 5.8S regions. Analysis of the consensus sequences distinguished four types of genotypes among our T. interdigitale species. Moreover, ITS type I was the dominant genotype characterising the anthropophilic variant of T. interdigitale. The phylogenetic study showed that only 5% of our strains were zoophilic. PCR sequencing was useful for distinguishing anthropophilic and zoophilic species of T. interdigitale, in which the differentiation is relevant because it helps to prescribe the correct treatment and to identify the surrounding source of infection.

Acanthamoeba T4 genotype associated with keratitis infections in Tunisia.

Acanthamoeba keratitis (AK) is a sight-threatening infection. We report five cases of AK diagnosed from 2005 to 2009 in the Laboratory of Parasitology-Mycology at Habib Bourguiba Sfax Hospital, Tunisia. All were associated with improper care of contact lenses (rinsing of contact lenses with tap water and inappropriate cleaning) and lens storage. The patients displayed different clinical presentations: corneal inflammation, corneal ulceration, and corneal abscess. The diagnosis was made after direct examination, culture, and polymerase chain reaction amplification with specific primers. The genotype classification was based on the highly variable DF3 region in the 18S rRNA gene. This is the first study characterizing Acanthamoeba genotype in Tunisia and North Africa. All Acanthamoeba isolates were associated to the T4 genotype. Three different DF3 sequence types were related to AK infections T4/10, T4/15, and T4/16.

[Pneumocystis: epidemiology and molecular approaches]. / Pneumocystis : épidémiologie et approches moléculaires.

Pneumocystosis is a common opportunistic infection in immunocompromised patients, especially in AIDS patients. The diagnosis of this pneumonia has presented several difficulties due to the low sensitivity of conventional staining methods and the absence of culture system for Pneumocystis. The molecular biology techniques, especially the PCR, have improved the detection of DNA of this fungus in invasive and noninvasive samples, and in the environment which highlighted human transmission and the existence of environmental source of Pneumocystis. In addition, various molecular biology techniques were used for typing of Pneumocystis strains, especially P. jirovecii, which is characterized by a significant genetic biodiversity. Finally, the widespread use of cotrimoxazole for the treatment and prophylaxis of pneumocystosis has raised questions about possible resistance to sulfa drugs in P. jirovecii.

Candida glabrata strain relatedness by new microsatellite markers.

We investigated six microsatellite markers to type 85 unrelated and 118 related isolates of Candida glabrata from 36 patients. Three new markers were selected from the complete sequence of CBS138 and three previously described markers, RPM2, MTI and ERG3 were used. We found a genetic diversity of 0.949 by combining four of them. By applying the new microsatellite markers GLM4, GLM5 and GLM6 we were able to discriminate 29 isolates, originally identified by the more established markers, RPM2, MTI and ERG3. When epidemiologically closely related isolates from 36 patients were typed, 25 patients (72%) exhibited identical or highly related multilocus genotypes. We noted a microvariation in 4 of the patients. This minor change of one locus could be explained by a single step mutation. Since one of these patients had not received antifungal treatment; thus, the relationship between genome variation and antifungal therapy remains controversial. We can conclude from our analysis of these new microsatellite markers that they are highly selective and therefore should be considered as a useful typing system for differentiating related and unrelated isolates of C. glabrata, as well as being able to detect microvariation.

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping.

AIM: To develop and evaluate an in-house reverse hybridization technique for Chlamydia trachomatis genotype identification. METHODS AND RESULTS: The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion-forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases. CONCLUSION: The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique allowed rapid C. trachomatis genotype identification.

Pathogenic free-living amoebae: epidemiology and clinical review.

Free-living amoebae are widely distributed in soil and water. Small number of them was implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitis) while others are non opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis and some cases of Balamuthia encephalitis). Although, the number of infections caused by these amoebae is low, their diagnosis was still difficult to confirm and so there was a higher mortality, particularly, associated with encephalitis. In this review, we present some information about epidemiology, ecology and the types of diseases caused by these pathogens amoebae.

Clinical utility and prognostic value of galactomannan in neutropenic patients with invasive aspergillosis.

UNLABELLED: Invasive aspergillosis (IA) is a major cause of morbidity and mortality in profoundly neutropenic patients. Delayed diagnosis and therapy may lead to poor outcomes. AIMS: The objective of this study was to assess the performance characteristics of the galactomannan (GM) assay in serum and bronchoalveolar lavage specimens for the diagnosis of IA in neutropenic patients with hematological malignancies. We also evaluated the prognostic outcome. PATIENTS AND METHODS: A total of 1198 serum samples and 42 BAL from 235 neutropenic patients were tested with a GM elisa platelia test. We used Cox modeling of time to 6- and 12-week mortality for GM level at the time of diagnosis (GM0) and GM decay in the week following diagnosis in proven and probable IA patients with more than two GM values. RESULTS: There were three proven, 55 probable, and four possible cases of IA. The sensitivity and specificity of the GM test were 96.8% and 82.4% respectively. In BAL samples, sensitivity was 86% and the specificity 93%. BAL GM was more sensitive than microscopy (22.2%) and BAL culture (38.9%). Among patients with proven/probable IA, serum and BAL GM were in agreement for 92.8% of paired samples. The hazard ratio (HR) of GM0 and 1-week GM decay per unit increase in Aspergillus enzyme immunoassay (EIA) was 1.044 (95% CI, 0.738 to 1.476) and 0.709 (95% CI, 0.236 to 2.130) respectively. CONCLUSION: We found good correlation between the GM0 and GM decay combination and outcome of IA patients. The GM is a useful tool for diagnosis and monitoring of IA.

Microsatellite analysis and susceptibility to FCZ of Candida glabrata invasive isolates in Sfax Hospital, Tunisia.

We have noted that, during the last few years, there has been a redistribution of the most common Candida species with an increase in non-C. albicans Candida species, particularly Candida glabrata. In many countries, the high frequency of Candida glabrata shows the highest resistance rates. The main objective of this investigation was to analyze the genotypic variability of invasive C. glabrata isolates recovered over a period of six years and assess their in vitro susceptibility to fluconazole to determine the possible existence of relationships between genotype and susceptibility. We collected 50 invasive C. glabrata isolates (21.4%) from January 2001 to December 2007. The in vitro susceptibility profiles as determined by the E-test method showed that 8.3% of the isolates were resistant to fluconazole. The typing with three microsatellite markers RPM2, MTI and ERG3 demonstrated 12 multilocus genotypes distributed irregularly with a predominance of G1 (38%). A cluster (G9) was found among isolates collected in the same ward, at the same time period, suggesting cross transmission. Eleven of 13 patients who had previously been colonized by C. glabrata, were infected by their colonizing strains. However, we noted after prolonged treatment with fluconazole that there was an increase of the MIC for an isolate from one patient and in another patient, the selection of a more resistant variant. In our study, we didn't find an association between genotype and susceptibility to fluconazole. In conclusion, the predominance of some genotypes could be explained by nosocomial transmission or a selective ecological advantage rather than an emergence of a resistant isolate.

Epidemiological survey of vulvovaginal candidosis in Sfax, Tunisia.

Vulvovaginal candidosis (VVC) is a common infection of the female genital tract affecting 75% women at least once in their lifetime. The aim of this study was to determine the incidence and potential risk factors associated with VVC and recurrent vulvovaginal candidosis (RVVC). A prospective study of women with vaginitis symptoms was conducted over 2 years in the regional clinic of population and family education in Sfax. A discriminant analysis was used to evaluate the association between the incidence of Candida vaginitis and potential risk factors. Sporadic and recurrent VVC were documented respectively in 48% and 6.1%. The most frequent factors associated with positive Candida culture were employed women, uncontrolled diabetes, history of genital infection and intrauterine device contraception. Increased episode numbers of VVC and condom/spermicidal contraception were positively associated with recurrences. Candida albicans was the predominantly isolated species (76.3%) followed by Candida glabrata (19.3%). Infection with C. glabrata occurred in 34% and 17.5% of patients with RVVC and VVC respectively. The discriminant investigation had provided further insights into the basis for prevention and control of RVVC. Increased prevalence of C. glabrata in patients with RVVC and observed risk factors should be taken into consideration to achieve success in the management of this infection.

[Candida dubliniensis: Identification methods and epidemiologic implication]. / Candida dubliniensis : méthodes d'identification et implications épidémiologiques.

Candida dubliniensis was recently described (1995) associated with oral candidiasis in HIV-positive patients. This organism is very closely related to the pathogenic human yeast, Candida albicans, and share a great number of phenotypic and genotypic characters. This great similarity limits the discrimination between these two species. Several phenotypic and molecular methods were developed. The phenotypic methods are simply used in routine discrimination between these two species and depend on the growth at high temperature, sugar assimilation, growth on special mediums and chlamydospore production…; but these methods are insensitive in discrimination between these two species. The molecular biology methods are highly reliable and able to confirm rapidly the identification of this species. In this article, we will review the various studies run out concerning the methods deployed for the identification of C. dubliniensis as well as the epidemiological implication of this new pathogen.

Dihydropteroate synthase gene mutations in Pneumocystis jiroveci strains isolated from immunocompromised patients.

The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase (DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.