Nocardia Isolation by Paraffin Baiting Technique

Nocardia Isolation by Paraffin Baiting proficiencyNocardia isolation from clinical samples with alkane baiting proficiencyAbstract accentuate The genus Nocardia is cause infection in lung, skin, brain, cerebrospinal, eyes, joints and kidneys. This bacterium is slow-growing and it is difficult to isolate of poly microbial specimens. some(prenominal) methods have been reported for Nocardia isolation from clinical samples. In current believe, we utilize of three methods much(prenominal) as alkane baiting proficiency, paraffin nutrient agar-agar, and naturalized media for Nocardia isolation of various clinical specimens from Iranian patients.Methods In this study, we collected phoebe bird hundred and cardinal from various clinical specimens including lethargy of patients with suspected renderrculosis, bronchoalveolar lavage, languor of patients withcystic fibrosis, trachea, cutaneous and subcutaneous abscess, cerebrospinal fluid, dental consonant abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. Smears of all clinical specimens were investigated with gee stain, part venomous fast and kinyoun stain. any collected specimens were culture on to carbon free broth tube (paraffin baiting proficiency), paraffin agar, sabouraud dextrose agar, sabouraud dextrose agar with cycloheximide and incubated at 35C.Results In direct microscopy, partially acid fast and Gram patch were seen positive for five and three clinical specimens respectively and the kinyoun stain were banish for all isolates. Seven isolates of clinical specimens were quarantined with paraffin baiting technique. In our study, paraffin baiting technique is more effective than conventional media and paraffin agar for Nocardia isolation.Conclusions In the present study, shown that use of paraffin baiting technique is more effective of other methods for Nocardia isolation of various clinical specimens. advert words Nocardia, Paraffin baiting technique, Paraffin agar, Sabou raud dextrose agar substructureNocardia spp. argon group aerobic actinomycetes, gram positive poles, partially acid fast, non-motile, filamentous branches, catalase positive and methenamine silver-positive 1-3. The genus Nocardia is opportunistic pathogens 2 that are found almost the natural environments. This bacterium is not part of normal microbialflora in homo body and animals 1, 4, 5 as well as, there is no report of mortal to person transmission 5. This micro electronic organism first introduced by Edmond in 1888 1, 6. Nocardia species are cause serious infections in different parts of the body oddly lung and skin 6. Nocardial infections acquired via inhalation of aerosolsor skin damage 7. In recent years, nocardiosis change magnitude in immune disorder diseases such as Pemphigus disorder, Behets disease, malignancy, organ transplantation 8-10. Todate, isolation and identification of Nocardia is improved from clinical specimens 11, 12. Clinical diagnosis in nocardiosis is controversial and clinical signs are not specific for this bacterium. received tool in Nocardia infections identification are including isolation and pure culture, Gram stain and partially acid-fast 12, 13. Nocardia species are slow growing bacteria and isolation this bacterium of polymicrobial specimens is difficult in clinical microbiology laboratory 14. Decontamination of pulmonic specimens such as lethargy is toxic for the genus Nocardia. Paraffin baiting technique was reported for Nocardia and mycobacteria isolation of soil 15. Paraffin baiting technique was reported that is successfully for Nocardia isolation of various clinical specimens especially poly microbial specimens such as sputum 3,14, 16. The aim of this study is unique for devil reasons The first aim of this study were comparison of paraffin baiting technique with other methods such as conventional media including sabouraud dextrose agar, sabouraud dextrose agar with cycloheximide and paraffin agar to isolat e Nocardia from various clinical specimens such as bronchoalveolar lavage (BAL), sputum of patients with suspected tuberculosis, sputum of patients with cystic fibrosis, cutaneous abscess, cerebrospinal fluid(CSF), dental abscess, mycetoma, trachea, wound, bone marrow biopsy and gastric lavage. The second aim was to estimate the prevalence of Nocardia infection in Iranian patients. There are few numbers of case reports of Nocardia infection and there is no comprehensive database of nocardiosis, therefore, it is essential to let on assess the prevalence of this bacteria.MethodsSample collectionFive hundred and seventeen various clinical specimens such as sputum of patients with suspected tuberculosis, sputum of patients with cystic fibrosis, BAL, cutaneous and subcutaneous abscess, CSF, dental abscess, mycetoma, wound, bone marrow biopsy, gastric lavage and trachea were collected between February 28, 2011 through March 8, 2013 (Table 1).Direct microscopy all clinical specimens wer e examined with direct microscopy. The first, clinical specimens were homogenized and were centrifuged in 10000 rev for 10 minutes and the supernatant was discarded. The smears were prepared from the sediments and were stained with Gram stain, partially acid fast and Kinyoun stain.Culture on different mediaSediment of specimens were inoculated on sabouraud dextrose agar (Merck- Germany), sabouraud dextrose agar with cycloheximide(cycloheximide-Sigma-Aldrich-USA), paraffin agar (KH2PO4, K2HPO4, NH4Cl, NH4NO3, MgSO4 .7H20, ZnSO4, FeSO4, MnSO4, Bacto- nutrient agar and Distilled water supply) and McClungs carbon-free broth tube (MgSO4 7H2O 0.5 g, ZnSO4 2 mg, FeCl3 10 mg, MnCl2. 4H2O 8 mg, K2HPO4 0.8 g, NaNO3 2 g, Distilled water 1lit, pH 7.2) with paraffin coated glass rod placed. Tubes were incubated at 35C for one month with daily overtopled 14, 17.ResultsExamination of stained smears in direct microscopic, were detected three specimens with Gram staining and five specimens with pa rtially acid fast staining and all smears were negative for Kinyoun stain. In McClungs carbon-free broth, colonies correspondent cream to white-colored appearing on the paraffin-coated glass rod ( figure 1). Colonies similar to the genus Nocardia were cultured on nutrient agar and were purified (Figure 2). Colonies were stained with Gram positive and partially acid fast and were negative for Kinyoun stain. All clinical isolates were grown in lysozyme broth speciality. Seven specimens were positive for the genus Nocardia (1.3%) with paraffin baiting technique as compared with sabouraud dextrose agar and sabouraud dextrose agar with cycloheximide and paraffin agar (Table 1). In comparative to various media, paraffin baiting technique was damp in the isolation Nocardia, so this technique is effective and specific for Nocardia isolation of various clinical specimens especially poly microbial samples (Table 1). Prevalence of nocardiosis in sputum (238 specimens), BAL (143 specimens) a nd cutaneous abscess (45 specimens) were 1.6%, 1.3%, 2.2%, respectively. In our study, we isolated one Nocardia spp. from cutaneous abscess of patient with Pemphigus disorder. banterIn scientific resource, recommended the use of paraffin baiting technique for isolation Nocardia from polymicrobial flora such as sputum 18. Nocardia spp. utilized of paraffin climb as the sole carbon source 15, 19. It has been reported different medium containing antibiotic for isolation this organism such as chloramphenicol with sabouraud dextrose agar. Some species of the genus Nocardia are susceptible to chloramphenicol 16. A wide tramp of nocardiosis occurs in Immunocompromised and immunosuppressive patients 8-10. Mycobacterium tuberculosis mimicking pulmonary nocardiosis so isolation and identification Nocardia is genuinely important because treatment in cardinal organisms is difficult. In a study by Mishra and colleagues in 1969, they investigated 555 clinical specimens such as sputum, BAL an d Gastric lavage and were positive respectively 10, 1 and 1 almost Nocardia spp. 19. Singh et al surveyed 1510 sputum specimens and results showed paraffin baiting method has higher skill of sabouraud dextrose agar 17. Another study by Venugopal et al were examined 350 sputum, BAL, pleural fluid, pus , biopsy specimens and isolated 15 strains of Nocardia 20. A study in 2001 from Iran, Eshraghi et al surveyed 142 sputum specimens and was positive 1 isolate (0.7%) but in our study was positive 4 isolates (1.3%) of 291 sputum of patients with suspected tuberculosis. The results show that Nocardia infection is increase in Iranian patients. The reports showed paraffin baiting technique is more selective and effective than usual medium and paraffin agar.ConclusionsWe recommended that the be used of paraffin baiting technique for Nocardia isolation in clinical laboratories. due to develop and autoimmune or immune disorders in Iranian patients, isolation Nocardia spp is very necessary fo r treatment.AcknowledgmentsThis study was supported by Tehran University of Medical Sciences, police lieutenant of Research.References1.Eshraghi SS Molecular typing of Nocardia species. J Med Bacteriol 2012 1(1) 38-45.2.Budzik JM, Hosseini M, Mackinnon AC Jr, et al. Disseminated Nocardia farcinica literature review and foreboding(a) outcome in an immunocompetent patient. Surg. Infect 2012 13(3) 163-170.3.Hollick GE. Nocardiosis. clinical microbiology newsletter 1988 10(14) 105-109.4.Eshraghi S, Amin M. Nocardia asteroides complex in patient with symptomatic pulmonary nocardiosis in a patient with bronchiectasis. 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Epidemiology, bacteriology and control of an outbreak of Nocardia asteroides infection on a liver unit. J Hosp 1991 18 473-480.11.Das D. Actinomycosis in fine needle aspiration cytology. Cytopathology 19945(4) 243-250.12.Wada R., Itabashi C, Nakayama Y, et al. Chronic granulomatous pleuritis caused by Nocardia PCR based diagnosis by nocardial 16S rDNA in pathological s pecimens. J Clin Pathol 2003 56(12) 966-969.13.Gupta N, Srinivasan R, Kumar R, et al. cardinal cases of nocardiosis diagnosed by fineneedle aspiration cytology Role of special stains. Diagn Cytopathol 2011 39(5) 363-364.14.Shawar RM, Moore DG, LaRocco MT. close of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens. J Clin Microbio l 1990 28(3) 508-512.15.Narang P, Dey S, Mendiratta D. Paraffin slide culture technique for Baiting Non-Tuberculous Mycobacteria. Indian. J. Tuberc 2000 47(4) 219-222.16.Garrett M, Holmes H, Nolte F. discriminating buffered charcoal-yeast extract medium for isolation of Nocardiae from mixed cultures. J Clin Microbiol 1992 30(7) 1891-1892.17.Singh M, Sandhu RS, Randhawa HS. Comparison of paraffin baiting and conventional culture techniques for isolation of Nocardia asteroides from sputum. J Clin Microbiol 1987 25(1) 176-177.18.Yu C T, Chua JA. Nocardiosis. PJMID 2001 30 56-61.19.Mishra S, Randhawa H. Appl ication of paraffin bait technique to the isolation of Nocardia asteroides from clinical specimens. Appl Microbiol 1969 18(4) 686-687.20.Venugopal PV, Taralakshmi VV, Subramanian S, , et al. Nocardia species from bronchopulmonary infections and mycetomas. Sabouraudia 1980 18(1) 11-18.Figure 1. Isolation and growth Nocardia on paraffin coated glass rodFigure 2. Nocardia grown on nutrient agar medium1