Our main object was to use a rapid and cheap molecular method for monitoring of Aspergillus infections and epidemiological approaches. Different molecular methods such as restriction fragment length polymorphism based on amplification of ribosomal RNA have been employed to identify Aspergilli in the level of species. The subject of our study was a group of hospitalized patients with clinical and subclinical signs of infection. All of the collected clinical specimens were transported to the medical mycology lab and examined for Aspergillus identification. Environmental specimens were collected from air and surfaces inspected for the Aspergillus hospital sources. A morphological study was firstly performed including; growth characteristics and microscopic features of Aspergillus species on mycological media for the identification. For the confirmation of Aspergillus isolates which similarly found in clinical and environmental sources, molecular method polymerase chain reaction/restriction fragment length polymorphism was carried out. From the mentioned specimens, 110 fungal isolates included Candida spp., Aspergillus spp. and other fungi. Among the clinical isolates of Aspergilli; Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.6%) were the most frequent species respectively, and also environmental Aspergillus isolates were Aspergillus niger (43.7%), Aspergillus flavus (41.8%), Aspergillus fumigatus (14.7%). Because of facility in use, speed and high sensitivity of diagnosis, the polymerase chain reaction/restriction fragment length polymorphism with a single restriction enzyme was very useful in identification of medically important Aspergillus spp

DIBA, Kambiz -

Eslamloo, N F

Makhdoomi, khadijeh H

Rahimirad, Mohamad H

ATHMSI Journals

Diba et al., Afr. J. Infect. Dis. (2014) 8(1): 1 – 4.11i8v.ajid/.431410/org.doi.dx://http1ASPERGILLUS MONITORING PROJECT IN A LARGE EDUCATIONAL HOSPITAL USING MOLECULARASSAYDiba K*1, Rahimirad MH2, Makhdoomi KH3, Eslamloo NF41Cellular and Molecular Research Center, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.2Department of Lung Diseases, Imam educational hospital, Urmia University of Medical Sciences, Urmia, Iran(mrahimirad@umsu.ac.ir) .3Department of Nephrology, Imam educational hospital, Urmia University of Medical Sciences, Urmia, Iran(kmakhdoomi@umsu.ac.ir)4Department of foreign languages, Urmia University of Medical Sciences, Urmia, Iran.(eslamlufn@gmail.com)*Email: Kambiz37diba@gmail.comAbstractBackground: It is important to find reliable and accessible methods for the diagnosis and identification of fungal species causing hospitalacquired infections. Our main objective was using a rapid and accessible molecular method for the monitoring of Aspergillus infections andidentification of causing agents in the level of species.Material and Methods: The study subjects were primarily clinical specimens collected from suspected HAI patients with clinical symptomsafter hospitalization. Also some environmental specimens were collected from air and instruments of health care facilities for the investigation ofAspergillus sources in a university hospital of UMSU, Urmia. All specimens were transported to Medical Mycology Center for the detection andidentification of Aspergillus species using morphological methods. Also molecular method, PCR-RFLP using single restriction enzyme as a rapidand available method was performed to investigate environmental sources of Aspergillus infections.Results: Total of 110 clinical fungal isolates included Candida and Aspergillus species and some other opportunistic fungi. Among the clinicalAspergillus findings, Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.6%) were the most frequent speciesrespectively and also Aspergillus niger (43.7%), Aspergillus flavus (41.8%), Aspergillus fumigatus (14.7%) were isolated as the most frequentspecies from environmental sources.Conclusion: Because of accessibility, speed and high sensitivity of diagnosis, the PCR-RFLP was very useful for the identification of medicallyimportant Aspergillus species and epidemiological approaches.Key words: Aspergillus, identification, molecular, hospital.IntroductionAspergillus species as opportunistic fungi are found in soil, water and decaying vegetation. They are also isolated from unfiltered air,ventilation systems, false ceiling dust, contaminated dust dislodged during hospital renovation and construction. Catheters and implants might becolonized by Aspergillus species (Warris and Verweij, 2005). There is plenty of evidence supporting the role of these fungi as agents of hospitalacquired infections (HAIs) (Dancer et al., 2009). The primary route of acquiring Aspergillus infection is by inhalation of the fungal spores. Inseverely immunosuppressed patients, pneumonia results from local lung tissue invasion. In addition, the fungus may disseminate through thebloodstream to deep organs. Patients with severe, prolonged, granulocytopenia, especially bone-marrow transplant recipients, are at the highestrisk. A. fumigatus and A. flavus are the most frequently isolated species in patients with proven aspergillosis (Verweij et al., 2002). The presenceof invasive disease may be inferred by the clinical symptoms and signs, and images obtained by CT scanning etc.It is important to find reliable and accurate methods for the identification of fungal agents of HAIs and epidemiological approaches aswell. Aspergillus infections are difficult to diagnose at early stages and conventional microbiological, serologic or imaging techniques are ofteninsufficient to ensure the early diagnosis and identifications. Tissue invasion, as demonstrated by examination of biopsy material, providesunequivocal evidence of invasive disease, and blood cultures are usually negative (Healy et al., 2004).Recently, some immunological and molecular tests were used for the identification of Aspergillus species causing HAIs (Verweij et al.,2002; Healy et al., 2004). Different molecular methods such as restriction fragment length polymorphism (RFLP) based on amplification ofribosomal RNA have been employed to identify Aspergilli in the level of species (Moody and Tyler, 1990). Because of facility in use, speed,availability and high sensitivity of diagnosis, PCR-RFLP using single restriction enzyme was studied in this study for the identification ofAspergillus species isolated from the HAI patients and the hospital indoor environments (Mirhendi and Diba, 2007).Materials and MethodsClinical and environmental specimensOur main subjects were clinical specimens including; sputum, bronchoalveolar lavage (BAL), sinus discharge, urine and synovial fluidcollected from HAI suspected patients with clinical symptoms after hospitalization. The presence of invasive disease was inferred by the clinicalsymptoms and signs, and images obtained by CT scanning etc. and fungal colonization was suggested by frequently isolation of organisms fromcultures while there are no clinical symptoms. All specimens were transported to Medical Mycology Center, UMSU, Urmia. The morphologicaldiagnosis was performed using growth characteristics on Sabouraud glucose agar 4% and microscopic features for the identification ofAspergillus species (Raper and Fennell, 1965). Also some environmental specimens were collected from air and instruments of health carefacilities including: surfaces of the walls, carpets, beds and blankets, trolleys, air condition, medical devices and also finger touch samples ofDiba et al., Afr. J. Infect. Dis. (2014) 8(1): 1 – 4.11i8v.ajid/.431410/org.doi.dx://http2cases, health care workers (HCWs) and visitors. Air samples were collected by using Sabouraud glucose agar (SGA4%) plates which leftuncapped expose to blowing air and the other samples were taken with sterile swabs and inoculated on a transport medium like SGA4% (Diba etal., 2007). Also the molecular method, PCR-RFLP using single restriction enzyme as a rapid and available method was performed to investigateenvironmental sources of Aspergillus infections.DNA extractionIt was necessary to prepare Aspergillus mycelia mass which is filtered and purified from the 12-24 hours fungal liquid cultures.Genomic DNA was extracted from Aspergillus mycelia mass by glass beads in a lysis buffer (1mM EDTA, 1% SDS, 100mM NaCl, 10mM Tris-HCl, 2% Triton X-100, PH: 8.0) followed by conventional phenol-chloroform method. The extracted DNA was checked by using agarose gelelectrophoresis (Loeffler et al., 2002).PCR amplificationThe PCR assay was performed using 5 µl of the DNA template in a total reaction volume of 50 µl (consisting of PCR buffer (20 mMTris- HCL at pH 8.0), 50 mM KCL, 0.1 mM each of forward (ITS: 5-TCC GTA GGT GAA CCT GCG G-3) and reverse (ITS-4 5-TCC TCCGCT TAT TGA TAT GC-3) primers for ITS regions of rDNA (purchased from Mirhendi Molecular Biology Center, TUMS), and 1.5 U of TaqDNA polymerase. We used universal primers for the amplification of Aspergillus ITS regions (Forward Primer: 5'- TCC GTA GGT GAA CCTGCG G - 3' Reverse Primer: 5'- TCC TCC GCT TAT TGAT TAT GC - 3'). All reactions were performed in a Thermocycler model XL (Bioer,China). Thermal program included an initial DNA denaturation at 95 °C for 5 min that followed by 30 cycles. Each cycle consisted of adenaturation step at 95 °C for 30 s, an annealing step at 55°C for 30 s, and an extension step at 72°C for 1 min, with a final extension at 72 °C for5 min following the last cycle. For all PCRs, we had a negative control and a positive control as well. Double deionized water (DDW, Merck,Germany) was used as negative control and DNA template extracted (Boiling, Phenol-Chloroform method) from standard Candida strain: C.albicans (ATCC 10261), was employed as positive control. The DNA fragments were length separated by electrophoresis through 1.5% agarosegels in Tris Borate EDTA buffer and 0.50 mg ethidium bromide per ml. Results were documented by using a tarns illuminator, Gel Doc System(Figure 1).Digestion of PCR products using RFLP methodThe method of restriction fragment length polymorphism was used to make differential pattern for the identification of Aspergillus spp.(Martínez-Culebras and Ramón, 2007). Digestion of amplified ITS fragments with our restriction enzyme, MwoI at 37 °C enabled us todifferentiate some medically relevant Aspergillus species. It was performed on all clinical and environmental Aspergillus isolates. For therestriction digestion, 13 µl of each PCR product was directly digested by 5 U (0.5 µl) of the restriction enzyme, 1.5 µl of the enzyme buffer, andincubated at 37 °C for 180 min (Almirante et al., 2005). Digested PCR products were subjected to electrophoresis in a 2% agarose gel andvisualized with gel doc system. Molecular identification of Aspergillus species was performed comparing the electrophoresis bands withdifferential patterns (Figure 2).Figure 1: Agarose gel electrophoresis of amplified ITS fragments of Aspergillus species. , Lane I: A.niger, Lanes 2-5: A. flavus , Lanes: 6,7: A.fumigatus and Lane 8: A frequently tested A. fumigatus as positive control and M: 100 bp DNA ladder. As it shown, approximately allspecies make the same size PCR bands on agarose gel.ResultsThe results of experimental studies on 198 clinical specimens showed totally 93 (47%) fungal or bacterial infections. Among allpositive cases, 54 (58%) had a fungal infection, supposing that all of the isolated fungi were causing agents of nosocomial infections with clinicalsymptoms after hospitalization. The isolated fungi included, 36 Candida spp. (66.6%) and 17 Aspergillus spp. (31.4%). Among the clinicalDiba et al., Afr. J. Infect. Dis. (2014) 8(1): 1 – 4.11i8v.ajid/.431410/org.doi.dx://http3isolated Aspergillus species; A. flavus (47%), A. fumigatus (29.4%) and A. niger (23.6%) were the most frequent species respectively (Table 1).The black filamentous mold ‘Alternaria alternata’ was the only non Candida, non Aspergillus isolated fungus in this study which was isolatedfrom synovial sample of a patient with septic arthritis. All identifications findings were confirmed by PCR-RFLP method (Figure 2). Among allenvironmental specimens, 256 specimens were collected from finger touch and body surface samples of patients, personnel and visitors, bed,floor, walls, trolleys, sink, medical devices and air samples (Table 2) which made up 110 fungal isolates including Candida, Aspergillus spp. andother fungi as saprophytic molds: Alternaria, Saccharomyces, Mucorals, Penicillium, Cladosporidium and Pheohyphomycetes. Among allisolates, Candida spp. 35(31.5%), Aspergillus spp. 48(43.2%) and other fungi 28(20.3%) were included. Environmental Aspergillus isolates were:A. niger (43.7%), A. flavus (41.8%) and A. fumigatus (14.7%) (Table 1).Figure 2: Agarose gel electrophoresis of ITS PCR products from standard Aspergillus species after digestion with the restriction enzyme MwoI.Lanes ST: standard Aspergillus species as positive control, lanes 1, 3, 5, 6: A. flavus, lanes 2, 4, 7: A. fumigatus, Lane M: 100 bp DNAladder.Table 1: Identification of Aspergillus species isolated from clinical and environmental sources by RFLP method aswell as their frequencies.TotalEnvironmental specimensClinical specimens%No.%No.%No.43.12841.720478A. flavus38.52543.72123.54A. niger18.41214.6729.55A. fumigatus100651004810017TotalTable 2: The frequency of opportunistic fungi isolated from air, instruments, indoor /outdoor surfaces (number of isolations) and finger touch ofpatients, HCWs and visitors. The last specimens are very important in the transmission of Candida spp.* Health care workerDiscussionThere is plenty of evidence supporting the role of opportunistic fungi as important agents of HAIs. During the period 1980-90Aspergillus species emerged as causing agents of life-threatening infections in immuno-compromised patients (Martínez-Culebras and Ramón,2007). Recently, some molecular techniques were used for the detection and identification of opportunistic fungi including Aspergillus spp. SusanF. Moody and co-worker (Moody and Tyler, 1995) used the PCR-RFLP for the analysis of inter species variations of Aspergillus group flavi: A.flavus, A. parasiticus, A. nomius. Also Dendis his group (Dendis et al., 2003) performed this method for identifying some pathogenic fungiisolated from febrile neutropenic patients. Molecular method PCR-RFLP with single restriction enzyme was used by Mirhendi et al. (Mirhendi etEnvironmentalspecimensCase HCW Visitor Carpet Walls Bed &BlanketSink Trolleys MedicaldevicesAir AirconditionerOutdoorContaminantsCandida 1 5 1 2 4 4 3 1 0 0 0 2Aspergillus 3 1 0 9 7 7 2 5 1 5 3 8Other 0 0 0 3 5 4 1 4 6 4 1 0Total 13 6 1 14 16 15 6 10 7 9 4 10Diba et al., Afr. J. Infect. Dis. (2014) 8(1): 1 – 4.11i8v.ajid/.431410/org.doi.dx://http4al., 2006), for the identification and differentiation of pathogenic fungi such as Candida spp. The PCR-RFLP method using a single restrictionenzyme, MwoI has also been used for the identification of Aspergillus spp. A whole process of molecular identification including, isolation offungal agent from a short incubated culture, a profile of DNA extraction, PCR and RFLP, for 6-10 samples takes a long day (12-18 hours)approximately. It should absolutely be more rapid than classic or morphologic methods of identifications. In the present study the rapid andreliable method of PCR-RFLP employing MwoΙ enabled us to identify Aspergillus spp. isolated from clinical specimens. Our results of Aspergillus isolation showed that A. fumigatus and A. flavus were the most frequent Aspergillus species isolated from cases with provenaspergillosis. Likewise our findings of environmental isolates showed that more than 50% of all samples were positive for at least anopportunistic fungus. From all of the tested samples, carpets, walls and beds were the most contaminated surfaces by the above fungi.Considering the frequent and long time contacts between the above surfaces and cases, personnel and visitors, It could be a reasonableexplanation for that result. It is important the most hospital yeast isolates were obtained from cases and personnel finger touch samples (Table 2).Aspergillus species (the second group of medically important fungi causing HAIs) are obtained from air and indoor/outdoor hospital environment.The most contaminated hospital indoor places in our study were the carpets, walls and also air samples of rooms (Dancer et al., 2009). Theprimary route of acquiring Aspergillus spp. is inhalation of the spores within aerosols suspended in air. Hospital renovations or constructions arecapable of liberating large numbers of fungal spores into the health care units (Warris and Verweij, 2005). Although isolation of Aspergillusspores from air samples was not considered in this study.Comparing various differential methods for the identification of Aspergillus species including conventional and molecular tests,because of it's accessibility, speed and high sensitivity of diagnosis, the PCR-RFLP was very useful for the identification of medically importantAspergillus species and epidemiological approaches.References1. Warris, A. and Verweij, P. (2005). Clinical implications of environmental sources for Aspergillus. Med. Mycol., 43: S59-65.2. Dancer, S. J. (2009). The role of environmental cleaning in the control of hospital-acquired infection. J. Hosp. Infect., 73: 378-385.3. Verweij, P. E., Te Dorsthorst, D. T. A., Rijs, A. J. M. M., De Vries-Hospers, H. G. and Meis, J. F. G. M. (2002). Nationwide survey of Invitro activities of Itraconazole and voriconazole against clinical Aspergillus fumigatus isolates cultured between 1945 and 1998. J. Clin.Microbiol., 40: 2648-2650.4. Healy, M., Reece, K., Walton, D., Huong, J., Shah, K. and Kontoyiannis, D. P. (2004). Identification to the species level and differentiationbetween strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR. J. Clin. Microbiol., 42: 4016-4024.5. Moody, S. F. and Tyler, B. M. (1990). Use of nuclear DNA restriction fragment length polymorphisms to analyze the diversity of theAspergillus flavus group: A. flavus, A. parasiticus, and A. nomius. Appl. Environ. Microb., 56: 2453-2461.6. Mirhendi, H., Diba, K., Kordbacheh, P., Jalalizand, N. and Makimura, K. (2007). Identification of pathogenic Aspergillus species by a PCR-restriction enzyme method. J. Med. Microbiol., 56: 1568-1570.7. Raper, K. and Fennell, D. (1965). The Genus Aspergillus (Baltimore: Williams & Wilkins).8. Diba, K., Kordbacheh, P., Mirhendi, H., Rezaie, S. and Mahmoudi, M. (2007). Identification of Aspergillus species using morphologicalcharacteristics. Pak. J. Med. Sci., 23: 867-872.9. Loeffler, J., Kloepfer, K., Hebart, H., Najvar, L., Graybill, J. R., Kirkpatrick, W. R., Patterson, T. F., Dietz, K., Bialek, R. and Einsele, H.(2002). Polymerase chain reaction detection of Aspergillus DNA in experimental models of invasive aspergillosis. J. Infect. Dis., 185: 1203-1206.10. Martínez-Culebras, P. V. and Ramón, D. (2007). An ITS-RFLP method to identify black Aspergillus isolates responsible for OTAcontamination in grapes and wine. Int. J. Food Microbiol., 113: 147-153.11. Almirante, B., Rodríguez, D., Park, B. J., Cuenca-Estrella, M., Planes, A. M., Almela, M., Mensa, J., Sanchez, F., Ayats, J., Gimenez, M.,Saballs, P., Fridkin, S. K., Morgan, J., Rodriguez-Tudela, J. L., Warnock, D. W. and Pahissa, A. (2005). Epidemiology and Predictors ofMortality in Cases of Candida Bloodstream Infection: Results from Population-Based Surveillance, Barcelona, Spain, from 2002 to 2003. J.Clin. Microbiol., 43: 1829-1835.12. Dendis, M., Horváth, R., Michálek, J., Růzicka, F., Grijalva, M., Bartos, M. and Benedík, J. (2003).  PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia. Clin. Microbiol. Infect., 9: 1191-1202.13. Mirhendi, H., Makimura, K., Khoramizadeh, M. and Yamaguchi, H. (2006). A one-enzyme PCR-RFLP assay for identification of sixmedically important Candida species. Nippon Ishinkin Gakkai Zasshi, 47: 225-229.

English

ASPERGILLUS MONITORING PROJECT IN A LARGE EDUCATIONAL HOSPITAL USING MOLECULAR

https://journals.athmsi.org/index.php/AJID/article/download/1519/1746/

ASPERGILLUS MONITORING PROJECT IN A LARGE EDUCATIONAL HOSPITAL USING MOLECULAR

Abstract

Similar works

Full text

Available Versions

ATHMSI Journals