Fakhri Jeddi - Academia.edu (original) (raw)

Papers by Fakhri Jeddi

Medical Mycology, 2016

In cases of fungal infection of the bloodstream, rapid species identification is crucial to provi... more In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol.

The American journal of tropical medicine and hygiene, Jan 21, 2016

We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic dive... more We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic diversity of Plasmodium falciparum in Mali. Genetic diversity was assessed via multiple loci variable number of tandem repeats analysis (MLVA). We performed DNA extraction from 104 positive and 30 negative used mRDTs that had been stored at ambient temperature for up to 14 months. Extracted DNA was analyzed via quantitative polymerase chain reaction (qPCR), and MLVA genotyping was then assessed on positive qPCR samples. Eighty-three of the positive mRDTs (83/104, 79.8%) and none of the negative mRDTs were confirmed P. falciparum positive via qPCR. We achieved complete genotyping of 90.4% (75/83) of the qPCR-positive samples. Genotyping revealed high genetic diversity among P. falciparum populations in Mali and an absence of population clustering. We show that mRDTs are useful to monitor P. falciparum genetic diversity and thereby can provide essential data to guide malaria control programs.

The American Journal of Tropical Medicine and Hygiene, 2009

PLOS Neglected Tropical Diseases, 2016

Medical Mycology, 2015

The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and ... more The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and capable of detecting mixed infections with species exhibiting a distinct antifungal susceptibility profile. In this study, we report the performance of a procedure combining the detection of mixed yeast cultures with a chromogenic medium and MALDI-TOF identification of the colonies. We then evaluated the impact that (i) the isolation medium and (ii) lowering the identification log score (LS) threshold value have on yeast identification performance in the routine laboratory.Among 15,661 clinical samples analyzed, 5,671 tested positive and 6,192 yeasts of 42 distinct species were identified. Overall, 6,117 isolates (98.79%) were identified on the first or second MALDI-TOF Mass Spectrometry (MS) attempt, yielding an average yeast species identification turnaround time of 0.346 days (95% CI [0.326 to 0.364]). The 75 remaining isolates were identified via nucleotide sequencing. Mixed infections accounted for 498 (8.78%) of the positive samples. The MALDI-TOF MS identification procedure performed well, regardless of the culture media tested. Lowering the recommended 2.0 LS threshold value to 1.8 would reduce the number of required (i) second MALDI-TOF MS identification attempts (178 vs. 490) and (ii) ITS2 and D1-D2 sequence-based identifications (17 vs. 75), while achieving an adequate identification rate (6,183/6,192, 99.85%).In conclusion, we propose applying a 1.8 LS threshold combined with chromogenic medium subculture to optimize the yeast identification workflow and detect mixed infection in the clinical laboratory.

The American journal of tropical medicine and hygiene, 2009

We report a case of drug hypersensitivity syndrome (drug reaction with eosinophilia and systemic ... more We report a case of drug hypersensitivity syndrome (drug reaction with eosinophilia and systemic symptoms [DRESS]) induced by parenteral meglumine antimoniate (Glucantime) in a 40-year-old man who traveled to Bolivia and was treated for mucocutaneous leishmaniasis. Two weeks after starting therapy, the patient had fever, joint pain, a cutaneous eruption, and hypereosinophilia (1,358 cells/mm(3)). These symptoms resolved after drug withdrawal but reappeared upon reintroduction of the drug. Pentavalent antimonials should be definitively withdrawn in patients with hypereosinophilia > 1,000 cells/mm(3) accompanied by systemic manifestations consistent with DRESS.

The American journal of tropical medicine and hygiene, 2009

Visceral leishmaniasis (VL) cases in children less than five years of age were recorded from 1996... more Visceral leishmaniasis (VL) cases in children less than five years of age were recorded from 1996 through 2006 from Tunisian pediatric departments. Mean incidence rates were calculated for each of the 215 districts in the study area. Averages of annual rainfall and extreme values of low temperatures in winter and high temperatures in summer were used to characterize the climate of each district according to its continentality index and bioclimatic zone. A geographic information system and a local indicator of spatial association were used to summarize the spatial properties of VL distribution. Poisson spatial regression was performed to study the relationship between VL incidence rates and climatic parameters. We identified one hot-spot region of 35 inland districts located mostly in the semi-arid bioclimatic zone and two cold-spots located in coastal regions of the northeastern sub-humid zone and the southeastern arid zone. The incidence rate of VL was positively correlated with me...

Médecine et Maladies Infectieuses, 2009

Reçu le 31 octobre 2008 ; accepté le 28 août 2009 Disponible sur Internet le 30 septembre 2009 Ré... more Reçu le 31 octobre 2008 ; accepté le 28 août 2009 Disponible sur Internet le 30 septembre 2009 Résumé Objectifs. -La leishmaniose viscérale (LV) représente en Tunisie un problème de santé publique. L'objectif de ce travail est d'apprécier l'évolution de l'incidence et de la distribution géographique de la maladie et d'actualiser le profil des populations touchées. Patients et méthodes. -Ce travail est une enquête rétrospective exhaustive de recensement des cas de LV diagnostiqués en Tunisie entre 1996 et 2006. La base de recrutement est représentée par les registres nationaux des maladies à déclaration obligatoire et par les dossiers de la majorité des services hospitaliers de pédiatrie du pays.

Journal of Tropical Medicine, 2011

Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 millio... more Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases. There are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions. However, resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules. In this paper, we reviewed the studies carried out on antimonial resistance inLeishmania. Several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related. Afterwards, phenotypic determination of resistance is described, then confronted to clinical outcome. Finally, we detail molecular mechanisms and targets involved in resistance and already identifiedin vitrowithin selected mutant strains or in clinical isolates.

PLOS Neglected Tropical Diseases, 2015

Since cholera appeared in Africa during the 1970s, cases have been reported on the continent ever... more Since cholera appeared in Africa during the 1970s, cases have been reported on the continent every year. In Sub-Saharan Africa, cholera outbreaks primarily cluster at certain hotspots including the African Great Lakes Region and West Africa.

Antimicrobial agents and chemotherapy, 2014

Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in mo... more Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in most areas where the disease is endemic, and increasing cases of therapeutic failure associated with parasite resistance have been reported. In this study, we assessed the molecular status of 47 clinical isolates of Leishmania causing visceral and cutaneous leishmaniasis from Algeria, Tunisia, and southern France. In total, we examined 14 genes that have been shown to exhibit significant variations in DNA amplification, mRNA levels, or protein expression with respect to resistance to antimonials. The gene status of each clinical isolate was assessed via qPCR and qRT-PCR. We then compared the molecular pattern against the phenotype determined via an in vitro sensitivity test of the clinical isolates against meglumine antimoniate, which is considered the reference technique. Our results demonstrate significant DNA amplification and/or RNA overexpression in 56% of the clinical isolates with t...

Blood, Jan 24, 2011

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as ... more Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deforma...

Parasite (Paris, France), 2013

During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic ... more During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells....

Malaria Journal, 2010

Development of an attenuated sporozoite vaccine to prevent and eliminate Plasmodium falciparum ma... more Development of an attenuated sporozoite vaccine to prevent and eliminate Plasmodium falciparum malaria Peter F Billingsley

Journal of Clinical Microbiology, 2013

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocomp... more Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidiumpositive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Clinical Microbiology and Infection, 2013

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now... more Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

Medical Mycology, 2016

In cases of fungal infection of the bloodstream, rapid species identification is crucial to provi... more In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol.

The American journal of tropical medicine and hygiene, Jan 21, 2016

We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic dive... more We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic diversity of Plasmodium falciparum in Mali. Genetic diversity was assessed via multiple loci variable number of tandem repeats analysis (MLVA). We performed DNA extraction from 104 positive and 30 negative used mRDTs that had been stored at ambient temperature for up to 14 months. Extracted DNA was analyzed via quantitative polymerase chain reaction (qPCR), and MLVA genotyping was then assessed on positive qPCR samples. Eighty-three of the positive mRDTs (83/104, 79.8%) and none of the negative mRDTs were confirmed P. falciparum positive via qPCR. We achieved complete genotyping of 90.4% (75/83) of the qPCR-positive samples. Genotyping revealed high genetic diversity among P. falciparum populations in Mali and an absence of population clustering. We show that mRDTs are useful to monitor P. falciparum genetic diversity and thereby can provide essential data to guide malaria control programs.

The American Journal of Tropical Medicine and Hygiene, 2009

PLOS Neglected Tropical Diseases, 2016

Medical Mycology, 2015

The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and ... more The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and capable of detecting mixed infections with species exhibiting a distinct antifungal susceptibility profile. In this study, we report the performance of a procedure combining the detection of mixed yeast cultures with a chromogenic medium and MALDI-TOF identification of the colonies. We then evaluated the impact that (i) the isolation medium and (ii) lowering the identification log score (LS) threshold value have on yeast identification performance in the routine laboratory.Among 15,661 clinical samples analyzed, 5,671 tested positive and 6,192 yeasts of 42 distinct species were identified. Overall, 6,117 isolates (98.79%) were identified on the first or second MALDI-TOF Mass Spectrometry (MS) attempt, yielding an average yeast species identification turnaround time of 0.346 days (95% CI [0.326 to 0.364]). The 75 remaining isolates were identified via nucleotide sequencing. Mixed infections accounted for 498 (8.78%) of the positive samples. The MALDI-TOF MS identification procedure performed well, regardless of the culture media tested. Lowering the recommended 2.0 LS threshold value to 1.8 would reduce the number of required (i) second MALDI-TOF MS identification attempts (178 vs. 490) and (ii) ITS2 and D1-D2 sequence-based identifications (17 vs. 75), while achieving an adequate identification rate (6,183/6,192, 99.85%).In conclusion, we propose applying a 1.8 LS threshold combined with chromogenic medium subculture to optimize the yeast identification workflow and detect mixed infection in the clinical laboratory.

The American journal of tropical medicine and hygiene, 2009

We report a case of drug hypersensitivity syndrome (drug reaction with eosinophilia and systemic ... more We report a case of drug hypersensitivity syndrome (drug reaction with eosinophilia and systemic symptoms [DRESS]) induced by parenteral meglumine antimoniate (Glucantime) in a 40-year-old man who traveled to Bolivia and was treated for mucocutaneous leishmaniasis. Two weeks after starting therapy, the patient had fever, joint pain, a cutaneous eruption, and hypereosinophilia (1,358 cells/mm(3)). These symptoms resolved after drug withdrawal but reappeared upon reintroduction of the drug. Pentavalent antimonials should be definitively withdrawn in patients with hypereosinophilia > 1,000 cells/mm(3) accompanied by systemic manifestations consistent with DRESS.

The American journal of tropical medicine and hygiene, 2009

Visceral leishmaniasis (VL) cases in children less than five years of age were recorded from 1996... more Visceral leishmaniasis (VL) cases in children less than five years of age were recorded from 1996 through 2006 from Tunisian pediatric departments. Mean incidence rates were calculated for each of the 215 districts in the study area. Averages of annual rainfall and extreme values of low temperatures in winter and high temperatures in summer were used to characterize the climate of each district according to its continentality index and bioclimatic zone. A geographic information system and a local indicator of spatial association were used to summarize the spatial properties of VL distribution. Poisson spatial regression was performed to study the relationship between VL incidence rates and climatic parameters. We identified one hot-spot region of 35 inland districts located mostly in the semi-arid bioclimatic zone and two cold-spots located in coastal regions of the northeastern sub-humid zone and the southeastern arid zone. The incidence rate of VL was positively correlated with me...

Médecine et Maladies Infectieuses, 2009

Reçu le 31 octobre 2008 ; accepté le 28 août 2009 Disponible sur Internet le 30 septembre 2009 Ré... more Reçu le 31 octobre 2008 ; accepté le 28 août 2009 Disponible sur Internet le 30 septembre 2009 Résumé Objectifs. -La leishmaniose viscérale (LV) représente en Tunisie un problème de santé publique. L'objectif de ce travail est d'apprécier l'évolution de l'incidence et de la distribution géographique de la maladie et d'actualiser le profil des populations touchées. Patients et méthodes. -Ce travail est une enquête rétrospective exhaustive de recensement des cas de LV diagnostiqués en Tunisie entre 1996 et 2006. La base de recrutement est représentée par les registres nationaux des maladies à déclaration obligatoire et par les dossiers de la majorité des services hospitaliers de pédiatrie du pays.

Journal of Tropical Medicine, 2011

Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 millio... more Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases. There are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions. However, resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules. In this paper, we reviewed the studies carried out on antimonial resistance inLeishmania. Several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related. Afterwards, phenotypic determination of resistance is described, then confronted to clinical outcome. Finally, we detail molecular mechanisms and targets involved in resistance and already identifiedin vitrowithin selected mutant strains or in clinical isolates.

PLOS Neglected Tropical Diseases, 2015

Since cholera appeared in Africa during the 1970s, cases have been reported on the continent ever... more Since cholera appeared in Africa during the 1970s, cases have been reported on the continent every year. In Sub-Saharan Africa, cholera outbreaks primarily cluster at certain hotspots including the African Great Lakes Region and West Africa.

Antimicrobial agents and chemotherapy, 2014

Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in mo... more Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in most areas where the disease is endemic, and increasing cases of therapeutic failure associated with parasite resistance have been reported. In this study, we assessed the molecular status of 47 clinical isolates of Leishmania causing visceral and cutaneous leishmaniasis from Algeria, Tunisia, and southern France. In total, we examined 14 genes that have been shown to exhibit significant variations in DNA amplification, mRNA levels, or protein expression with respect to resistance to antimonials. The gene status of each clinical isolate was assessed via qPCR and qRT-PCR. We then compared the molecular pattern against the phenotype determined via an in vitro sensitivity test of the clinical isolates against meglumine antimoniate, which is considered the reference technique. Our results demonstrate significant DNA amplification and/or RNA overexpression in 56% of the clinical isolates with t...

Blood, Jan 24, 2011

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as ... more Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deforma...

Parasite (Paris, France), 2013

During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic ... more During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells....

Malaria Journal, 2010

Development of an attenuated sporozoite vaccine to prevent and eliminate Plasmodium falciparum ma... more Development of an attenuated sporozoite vaccine to prevent and eliminate Plasmodium falciparum malaria Peter F Billingsley

Journal of Clinical Microbiology, 2013

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocomp... more Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidiumpositive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Clinical Microbiology and Infection, 2013

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now... more Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.