Publications by authors named "Hadis Jafarian"

14 Publications

  • Page 1 of 1

Development a hydrolysis probe-based quantitative PCR assay for the specific detection and quantification of .

Curr Med Mycol 2020 Sep;6(3):50-56

Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background And Purpose: is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of .

Materials And Methods: The internal transcribed spacer 2 regions in the nuclear ribosomal DNA of and other related yeasts were assayed to find a specific PCR target for . A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast.

Results: The qPCR assay was able to identify and quantify with a detection limit of 1 CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R=0.99; slope=-3.42).

Conclusion: The applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic . Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe- based qPCR assay for the screening and identification of .
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http://dx.doi.org/10.18502/cmm.6.3.4665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018816PMC
September 2020

Molecular epidemiology of zygomycosis and their related factors in tertiary referral centers in southern Iran.

J Infect Dev Ctries 2020 12 31;14(12):1424-1430. Epub 2020 Dec 31.

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Introduction: For the best management of the zygomycosis in immunocompromised patients, the present study aims to detect and identify the etiologic agents by DNA sequencing method and their related factors in clinical samples of patients.

Methodology: Clinical samples from 1,058 patients admitted in 11 university hospitals in Shiraz, Southern Iran were collected between July 2015 and July 2018. All samples (bronchoalveolar lavage, sputum, blood, tissue) were examined by routine microscopic and culture tests for zygomycetes. The etiologic agents were identified by the molecular method and sequencing.

Results: Direct microscopic examinations or pathology smear, culture, and PCR were positive in 61 (5.8%), 15 (1.4%), and 103 (9.7%) patients, respectively. According to EORTC/MSG criteria, the rates of proven, probable, and possible zygomycosis were 59.2% (61/103), 14.6% (15/103), and 26.2% (27/103 patients), respectively. The most prevalent etiologic agents according to sequencing were Rhizopus oryzae (44 cases), Rhizopus microsporus (31 cases), Rhizopus stolonifer (15 cases). Twenty-two patients (21.4%) with positive PCR died. There were significant relations between zygomycosis and the underlying disease (p = 0.043) and prior antifungal therapy (p = 0.023). White blood cell count was in the normal range in 14.1% of patients, and the means of erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were 65 mm/hour and 57 mg/L, respectively.

Conclusions: Molecular methods and sequencing may have considered as suitable tools to diagnose zygomycosis. Identification of the etiologic agents may be considered as the future antifungal therapy and management of the respective patients.
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http://dx.doi.org/10.3855/jidc.12997DOI Listing
December 2020

Cerebral and pulmonary aspergillosis, treatment and diagnostic challenges of mixed breakthrough invasive fungal infections: case report study.

BMC Infect Dis 2020 Jul 23;20(1):535. Epub 2020 Jul 23.

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Namazi Hospital, Zand Ave, Shiraz, 7193711351, Iran.

Background: Breakthrough invasive fungal infections (bIFIs) are an area of concern in the scarcity of new antifungals. The mixed form of bIFIs is a rare phenomenon but could be potentially a troublesome challenge when caused by azole-resistant strains or non-Aspergillus fumigatus. To raise awareness and emphasize diagnostic challenges, we present a case of mixed bIFIs in a child with acute lymphoblastic leukemia.

Case Presentation: A newly diagnosed 18-month-old boy with acute lymphoblastic leukemia was complicated with prolonged severe neutropenia after induction chemotherapy. He experienced repeated episodes of fever due to extended-spectrum beta-lactamase-producing Escherichia coli bloodstream infection and pulmonary invasive fungal infection with Aspergillus fumigatus (early-type bIFIs) while receiving antifungal prophylaxis. Shortly after pulmonary involvement, his condition aggravated by abnormal focal movement, loss of consciousness and seizure. Cerebral aspergillosis with Aspergillus niger diagnosed after brain tissue biopsy. The patient finally died despite 108-day antifungal therapy.

Conclusions: Mixed bIFIs is a rare condition with high morbidity and mortality in the patients receiving immunosuppressants for hematological malignancies. This case highlights the clinical importance of Aspergillus identification at the species level in invasive fungal infections with multiple site involvement in the patients on antifungal prophylaxis.
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http://dx.doi.org/10.1186/s12879-020-05162-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376672PMC
July 2020

Investigation of the Physical, Chemical Characteristics and Microbial Contamination of the Indoor Swimming Pools

Turkiye Parazitol Derg 2019 Sep;43(3):130-134

Shiraz University of Medical Sciences, Prof. Alborzi Clinical Microbiology Research Center, Shiraz, Iran

Objective: The aim of this study was to investigate the physical, chemical and microbiological contamination of indoor swimming pools.

Methods: Pool water specimens were collected using a plastic polypropylene sterilized bottle. The physical and chemical qualities of the waters were analyzed in terms of temperature, turbidity, pH, and free residual chlorine, with the standard methods for the examination of water. Bacteriological (routine methods) and parasitological (molecular methods) tests were carried out on pools water.

Results: The mean temperature, pH, and residual chlorine of the indoor pools were 31.2 °C, 7.6 and 1.5 mg/L, respectively. Turbidity was not observed in any of the pools. The pH and temperature values were in standard ranges in 92.3% and 15.4% of the waters of swimming pools, respectively. The prevalence rates of bacterial and amoebic contaminations of the water in the swimming pools were 53.8% and 46.2%, respectively. One pool (7.7%) was contaminated with both bacteria and amoeba. and spp. were isolated from the pool waters.

Conclusion: In this study, some microorganisms were identified from the water pools. Effective management of swimming pools and proper control of the physical, chemical and microbiological property of water pools can produce the healthy recreational activity.
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http://dx.doi.org/10.4274/tpd.galenos.2019.6112DOI Listing
September 2019

Changing face of Candida colonization pattern in pediatric patients with hematological malignancy during repeated hospitalizations, results of a prospective observational study (2016-2017) in shiraz, Iran.

BMC Infect Dis 2019 Aug 30;19(1):759. Epub 2019 Aug 30.

Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Background: Surveillance of current changes in the epidemiology of Invasive Fungal Diseases (IFDs) as an important component of the antifungal stewardship programs (ASP), requires careful regular monitoring, especially in high-risk settings such as oncology centers. This study aimed to examine Candida colonization status and corresponding current changes in children with malignancy during repeated admissions and also investigate the possible epidemiological shifts after the implementation of ASP.

Methods: In this prospective observational study, all eligible patients younger than 18 years were recruited during 2016-2017 at Amir Medical Oncology Center (AMOC) in Shiraz, Iran. Totally, 136 patients were enrolled and 482 samples were collected from different sites (oral/nasal discharges, urine and stool). Weekly regular sampling was carried out during hospitalization. Candida colonization status and epidemiological changes were monitored during repeated admissions. Samples were cultivated on Sabouraud Dextrose agar medium and identified by Polymerase Chain Reaction -Restriction Fragment Length Polymorphism (PCR-RFLP).

Results: Estimated Candida colonization incidence was 59.9% (82/136) in our patients. Candida colonization was found to be higher in oral cavity and rectum than that in nasal cavity. Among those long-term follow ups and repetitive hospitalizations, a significant number of patients exhibited changes in their colonization patterns (37.7%). Candida colonization did not reveal any significant relationship with age, sex, oncologic diseases and degree of neutropenia. C. albicans (72.0%) was found as the most common Candida species in colonized patients, followed by C. krusei, C. kefyr, C. glabrata and C. parapsilosis.

Conclusion: Given the high incidence of Candida infections in children with cancers, close monitoring of epidemiologic changes is essential for judicious management, based on local surveillance data and improvement of overall quality of care in high risk patients.
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http://dx.doi.org/10.1186/s12879-019-4372-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717378PMC
August 2019

Antifungal effect of the bark and root extracts of on oral isolates.

Curr Med Mycol 2018 Dec;4(4):20-24

Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background And Purpose: Oral candidiasis is one of the most common fungal infections in humans. The treatment and prophylaxis of the patients suffering from this infection require the identification of new anti- agents with no side effects or toxicity like medicinal plants. The present study was conducted to compare the antifungal activities of the aqueous, ethanolic, and methanolic extracts of the bark and roots of with those of two routine antifungal agents (i.e., fluconazole and nystatin) on oral strains isolated from liver transplant recipients.

Materials And Methods: Minimum inhibitory concentrations (MICs) of the ethanolic, methanolic, and aqueous extracts of the bark and root of against and isolated from oral cavities were evaluated according to the CLSI M27-A3. All data were analyzed in SPSS (version 16.0) by pairwise comparison and Kruskal-Wallis test.

Results: The MIC50 and MIC90 values for the methanolic and ethanolic extracts of the bark and root of against were both obtained as 0.05 mg/ml with the geometric mean (GM) of 0.07. Furthermore, the MIC90 values for the aqueous extracts of bark and root were estimated as 0.05 and 0.2 mg/ml, respectively. With regard to the MIC50 and MIC90 values for the methanolic and ethanolic extracts of the bark and root were 0.05 mg/ml. However, the MIC90 value for the aqueous extract against this species was obtained as 25 mg/ml. The GM values for the aqueous extracts of the bark and root were 9.49 and 0.32, respectively.

Conclusion: As the findings indicated, the methanolic and ethanolic extracts of the bark and root of had anti- activities. Therefore, they can be considered as mouthwash or toothpaste to prevent and treat infections in the oral cavity.
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http://dx.doi.org/10.18502/cmm.4.4.382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386507PMC
December 2018

Comparison of anti- activities of the ancient plants and with antifungal drugs in species isolated from oral cavity.

J Conserv Dent 2018 Jul-Aug;21(4):359-362

Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Context: Medicinal plants have reportedly fewer side effects, compared to antifungal agents.

Aims: This study was designed to compare the anti- activity of two medicinal plants with two routine antifungal agents, used in the clinics, on species isolated from the oral cavity of the liver transplant patients.

Subjects And Methods: Anti- activities of ethanol extracts of leaf and unripe and ripe fruit (collected from south of Iran) versus nystatin and fluconazole were evaluated using CLSI M27-A3 standard method.

Results: The minimum inhibitory concentration (MIC) 90 values for , ripe, and unripe fruits in were 0.1 μg/mL, 25 μg/mL, and 0.1 μg/mL, and in were 0.05 μg/mL, 25 μg/mL, and 0.05 μg/mL, respectively. The MIC90 value for nystatin in both species was 0.035 μg/mL, but MIC90 value for fluconazole in was 0.5 μg/mL and in was 2 μg/mL.

Conclusion: Unripe fruits of and were found more effective than fluconazole on species. unripe fruit and leaf have potential anti- activity and can be considered as a new mouthwash agent to prevent and treat infections. Further studies regarding pharmacokinetics and toxicities of these extracts are needed for their use in humans.
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http://dx.doi.org/10.4103/JCD.JCD_291_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080183PMC
August 2018

Antifungal susceptibility patterns of colonized species isolates from immunocompromised pediatric patients in five university hospitals.

Iran J Microbiol 2017 Dec;9(6):363-371

Department of Pediatrics, Besat Tertiary Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.

Background And Objectives: Colonization of species is common in pediatric patients admitted to hematology-oncology wards. The aim of this study was to identify colonized species and their susceptibility patterns in hematologic pediatric patients.

Materials And Methods: Samples were collected from mouth, nose, urine and stool of the patients admitted to five university hospitals and cultured on sabouraud dextrose agar. The isolates were identified by API 20 C AUX system and their susceptibility patterns were evaluated by CLSI M27-A3 and S4.

Results: From 650 patients, 320 (49.2%) were colonized with 387 species. was the most prevalent isolated species, followed by and . The epidemiological cut off value (ECV) for all species to amphotericin B was ≤0.25 μg except (4 μg). The resistance rate to fluconazole in this study in was 4.9% with ECV 8 μg/ml, followed by 8.8% with ECV 0.5 μg/ml. Voriconazole and posaconazole were effective antifungal agents for all isolates. The ECV of and for itraconazole were 0.5, 0.25, 0.5, 1 and 2 μg, respectively. The resistant and intermediate rates of species to caspofungin in this study were 2.9%, 5.9%, 18.8%, 47.9%, 0.0% and 16.7% in and respectively.

Conclusion: was the most prevalent species in pediatric colonized patients. New azole agents like voriconazole and posaconazole are effective against non-albicans species. Increase in intermediate species is alarming to future emerging resistant species.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825937PMC
December 2017

Antifungal susceptibility testing of Candida species isolated from the immunocompromised patients admitted to ten university hospitals in Iran: comparison of colonizing and infecting isolates.

BMC Infect Dis 2017 11 21;17(1):727. Epub 2017 Nov 21.

Department of Pediatrics, Besat Tertiary Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.

Background: Antifungal susceptibility testing is a subject of interest in the field of medical mycology. The aim of the present study were the distributions and antifungal susceptibility patterns of various Candida species isolated from colonized and infected immunocompromised patients admitted to ten university hospitals in Iran.

Methods: In totally, 846 Candida species were isolated from more than 4000 clinical samples and identified by the API 20 C AUX system. Antifungal susceptibility testing was performed by broth microdilution method according to CLSI.

Results: The most frequent Candida species isolated from all patients was Candida albicans (510/846). The epidemiological cutoff value and percentage of wild-type species for amphotericin B and fluconazole in Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei were 0.5 μg/ml (95%) and 4 μg/ml (96%); 1 μg/ml (95%) and 8 μg/ml (95%); 0.5 μg/ml (99%) and 19 μg/ml (98%); and 4 μg/ml (95%) and 64 μg/ml (95%), respectively. The MIC90 and epidemiological cutoff values to posaconazole in Candida krusei were 0.5 μg/ml. There were significant differences between infecting and colonizing isolates of Candida tropicalis in MIC 90 values of amphotericin B, and isolates of Candida glabrata in values of amphotericin B, caspofungin, and voriconazole (P < 0.05).

Conclusions: Our findings suggest that the susceptibility patterns of Candida species (colonizing and infecting isolates) in immunocompromised patients are not the same and acquired resistance was seen in some species.
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http://dx.doi.org/10.1186/s12879-017-2825-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697407PMC
November 2017

Prevalence of colonization and mitochondrial large subunit rRNA mutation of among Iranian children.

Iran J Microbiol 2016 Oct;8(5):326-330

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background And Objectives: is an important causative agent of pneumonia. During childhood, exposure to occurs and antibody was built in early childhood. The aim of this study was to describe the molecular epidemiology of in children without any respiratory syndrome and survey the distribution of different mitochondrial large subunit, ribosomal ribonucleic acid (mtLSU- rRNA) genotypes.

Materials And Methods: Mini-bronchoalveolar lavage (mini-BAL) fluids from pediatric patients with no history of lung disorders were obtained during a 14-month period. colonization was confirmed by immunofluorescence staining, nested PCR and sequencing. Genotypic characterization at the mitochondrial large subunit rRNA gene was performed by direct sequencing.

Results: Of 172 BAL specimens from patients, with mean age of 4.9 years, the prevalence of colonization was 3.5% (6 samples). The results of sequencing revealed the two polymorphisms; 85/A; 248/C in 3 cases, and 85/T; 248/C in other cases. One sample also showed a mutation replacement at position 258 (T-to-C change), which was not reported previously.

Conclusion: Colonized person as an environmental reservoir might play an important role in the progression of infection in immunocompromised patients. Diagnosis of the reservoir and genotyping can be essential for the prevention of nosocomial infections.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5277602PMC
October 2016

Antifungal Effects of Common Mouthwashes on Candida Strains Colonized in the Oral Cavities of Liver Transplant Recipients in South Iran in 2014.

Hepat Mon 2016 Jan 30;16(1):e31245. Epub 2016 Jan 30.

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

Background: Among the opportunistic microorganisms, fungi, particularly Candida, play an important role in the mortality of transplant recipients. Thus, controlling and preventing fungal colonizations in various parts of the body, including the oral cavity, can reduce the possibility of post-transplant invasive fungal infections. This can be done simply by using mouthwashes.

Objectives: The current study aimed to determine the prevalence of fungal species of Candida within the oral cavities of liver transplant recipients, and to evaluate the effects on Candida colonization of different exposure times to common mouthwashes.

Patients And Methods: Specimens were taken from the oral cavities of 101 liver transplant recipients who were referred to our clinic for their first monthly examination. After cultivation and DNA extraction, yeast strains were identified with the RFLP technique. Each strain's susceptibility to 0.2% chlorhexidine, Vi-One, Oral-B, Nanosil D1, and Nystatin mouthwashes was determined based on the CLSI M27-A2 standard method.

Results: The obtained data were analyzed using SPSS. Out of 101 samples from liver transplant recipients, 68 cases showed fungi growing within the culture media (67.4%). C. albicans and C. glabrata, respectively, were the first and second most frequent types. Mouthwash susceptibility tests revealed that their antifungal effects over 60 seconds were significantly higher than with an exposure time of 30 seconds. At both 30 and 60 seconds, chlorhexidine was significantly the most efficient.

Conclusions: Chlorhexidine mouthwash with an exposure time of 60 seconds or more is suggested as an effective antifungal agent to be included in the medication regimen of liver transplant patients pre- and postoperatively, in order to prevent fungal colonization and subsequent systemic infections.
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http://dx.doi.org/10.5812/hepatmon.31245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834379PMC
January 2016

Evaluation of nested PCR in diagnosis of fungal rhinosinusitis.

Iran J Microbiol 2015 Feb;7(1):62-6

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background And Objective: Given the importance of rapid diagnosis for fungal rhinosinusitis, this study aimed to evaluate the use of nested PCR to identify Aspergillus and Mucor species in clinical samples from patients with suspected fungal rhinosinusitis.

Methods: Functional endoscopic sinus surgery specimens were collected from 98 patients with rhinosinusitis from 2012 to 2013. All samples were ground and cultured on sabouraud dextrose agar. The isolated fungi were identified based on their macroscopic and microscopic features. Fungal DNA was extracted from the tissue samples and nested PCR was performed with two sets of primers for Mucor and Aspergillus.

Results: Direct microscopic showed that 5.1% contained fungal components and 9.2% exhibited growth of fungi in culture. The most common agents isolated were Aspergillus fumigatus (n= 3), Aspergillus flavus (n=2), Penicillium sp (n=3) and Alternaria sp. (n=1). Mucor sp. was identified in the pathology smear from 1 patient. Positive results for fungal rhinosinusitis were obtained for a total of 10.2% by culture or pathology smear. Positive PCR results were obtained in 72 samples for Aspergillus and 31 samples for Mucor.

Conclusion: Our results suggest that endoscopic sinus surgery specimens are not suitable for nested PCR, probably because of the accumulation of fungi that contaminate the environmental air. This drawback is a limiting factor for diagnosis with nasal cavity specimens. Therefore, molecular methods and conventional culture techniques are helpful complementary diagnostic methods to detect fungal rhinosinusitis and determine appropriate management for these patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670470PMC
February 2015

Yeast colonization and drug susceptibility pattern in the pediatric patients with neutropenia.

Jundishapur J Microbiol 2014 Sep 1;7(9):e11858. Epub 2014 Sep 1.

Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

Background: Pediatric patients with neutropenia are vulnerable to invasive Candida infections. Candida is the primary cause of fungal infections, particularly in immunosuppressed patients. Candida albicans has been the most common etiologic agent of these infections, affecting 48% of patients.

Objectives: The aim of this study was to identify Candida spp. isolated from children with neutropenia and determine the antifungal susceptibility pattern of the isolated yeasts.

Patients And Methods: In this study 188 children with neutropenia were recruited, fungal surveillance cultures were carried out on nose, oropharynx, stool, and urine samples. Identification of Candida strains was performed using germ tube and chlamydospore production tests on an API 20 C AUX system. Susceptibility testing on seven antifungal agents was performed using the agar-based E-test method.

Results: A total of 229 yeasts were isolated. Among those, C. albicans was the most common species followed by C. krusei, C. parapsilosis, C. glabrata, C. tropicalis, C. famata, C. dubliniensis, C. kefyr, and other Candida species. C. glabrata was the most resistant isolated yeasts, which was 70% resistant to fluconazole and 50% to itraconazole, 7.5% to amphotericin B and 14% to ketoconazole. All the tested species were mostly sensitive to caspofungin.

Conclusions: Knowledge about the susceptibility patterns of colonized Candida spp. can be helpful for clinicians to manage pediatric patients with neutropenia. In this study, caspofungin was the most effective antifungal agent against the colonized Candida spp. followed by conventional amphotericin B.
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http://dx.doi.org/10.5812/jjm.11858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255375PMC
September 2014

Comparison of histopathological analysis, culture and polymerase chain reaction assays to detect mucormycosis in biopsy and blood specimens.

Iran J Microbiol 2013 Dec;5(4):406-10

Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background And Objectives: The aim of this study was to compare direct microscopic examination with culture and PCR for the diagnosis of Mucorales infection in blood and tissue specimens.

Material And Methods: Blood samples and tissue specimens were obtained from 28 patients (total 58 samples) with suspected invasive fungal infection and cultured on proper media. Direct smear of tissue samples was done with potassium hydroxide, hematoxylin and eosin, and methenamine silver staining. DNA extracted from blood and tissue specimens were used for semi-nested PCR targeting 18S rDNA of Mucorales species.

Results: Mucormycosis was documented in 7/28 (25%) of tissue specimens with positive findings by direct smear, of which PCR and culture were positive in 6 (86%) and 5 (70%) specimens, respectively. The etiologic agents were Mucor spp. and Rhizopus spp. However, culture and PCR results for all blood specimens were negative.

Conclusions: As the orders of Mucorales do not have well growth in culture media, PCR with tissue specimens is more sensitive than tissue or blood culture methods. Unfortunately, there is no alternative method for direct smear, which is an invasive method. Molecular methods may be helpful in these cases.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385169PMC
December 2013