Nocardia Isolation by Paraffin Baiting Technique

Nocardia isolation from clinical samples with paraffin baiting technique
Abstract

Background: 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 polymicrobial specimens. Several methods have been reported for Nocardia isolation from clinical samples. In current study, we used of three methods such as paraffin baiting technique, paraffin agar, and conventional media for Nocardia isolation of various clinical specimens from Iranian patients.

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Methods: In this study, we collected five hundred and seventeen from various clinical specimens including sputum of patients with suspected tuberculosis, bronchoalveolar lavage, sputum of patients withcystic fibrosis, trachea, cutaneous and subcutaneous abscess, cerebrospinal fluid, dental abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. Smears of all clinical specimens were investigated with Gram stain, partially acid fast and kinyoun stain. All collected specimens were cultured on to carbon free broth tube (paraffin baiting technique), paraffin agar, sabouraud dextrose agar, sabouraud dextrose agar with cycloheximide and incubated at 35°C.

Results: In direct microscopy, partially acid fast and Gram staining were seen positive for five and three clinical specimens respectively and the kinyoun stain were negative for all isolates. Seven isolates of clinical specimens were isolated 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.

Key words: Nocardia, Paraffin baiting technique, Paraffin agar, Sabouraud dextrose agar

Introduction

Nocardia spp. are group aerobic actinomycetes, gram positive rods, 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 around the natural environments. This bacterium is not part of normal microbialflora in human body and animals [1, 4, 5] as well as, there is no report of person to person transmission [5]. This microorganism first introduced by Edmond in 1888 [1, 6]. Nocardia species are cause serious infections in different parts of the body especially lung and skin [6]. Nocardial infections acquired via inhalation of aerosolsor skin damage [7]. In recent years, nocardiosis increased in immune disorder diseases such as Pemphigus disorder, Behcet’s 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. Standard 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 pulmonary specimens such as sputum 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 two 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 isolate 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 better assess the prevalence of this bacteria.

Methods
Sample collection

Five 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 were examined with direct microscopy. The first, clinical specimens were homogenized and were centrifuged in 10000 rpm 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 media

Sediment 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- Agar and Distilled water) and McClung’s 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 35°C for one month with daily controlled [14, 17].

Results

Examination of stained smears in direct microscopic, were detected three specimens with Gram staining and five specimens with partially acid fast staining and all smears were negative for Kinyoun stain. In McClung’s carbon-free broth, colonies similar 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 medium. 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 better 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) and 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.

Discussion

In scientific resource, recommended the use of paraffin baiting technique for isolation Nocardia from polymicrobial flora such as sputum [18]. Nocardia spp. utilized of paraffin wax 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 range of nocardiosis occurs in Immunocompromised and immunosuppressive patients [8-10]. Mycobacterium tuberculosis mimicking pulmonary nocardiosis so isolation and identification Nocardia is very important because treatment in two organisms is difficult. In a study by Mishra and colleagues in 1969, they investigated 555 clinical specimens such as sputum, BAL and Gastric lavage and were positive respectively 10, 1 and 1 about Nocardia spp. [19]. Singh et al surveyed 1510 sputum specimens and results showed paraffin baiting method has higher efficacy 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 increasing in Iranian patients. The reports showed paraffin baiting technique is more selective and effective than usual medium and paraffin agar.

Conclusions

We recommended that the be used of paraffin baiting technique for Nocardia isolation in clinical laboratories. due to aging and autoimmune or immune disorders in Iranian patients, isolation Nocardia spp is very necessary for treatment.

Acknowledgments

This study was supported by Tehran University of Medical Sciences, Deputy of Research.

References

1.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 fatal 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. Iran J Public Health 2001(3-4); 30: 99-102.

5.Stevens DA, Pier AC, Beaman BL, et al. Laboratory evaluation of an outbreak of nocardiosis in immunocompromised hosts. Am J Med 1981; 71(6): 928-934.

6.Brown-Elliott BA, Brown JM, Conville PS, et al. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 2006; 19(2): 259-282.

7.Patel MP, Kute VB, Gumber MR, , et al. Successful treatment of Nocardia pneumonia with cytomegalovirus retinitis coinfection in a renal transplant recipient. Int Urol Nephrol 2012; 45: 581-5.

8.Poonwan N, Kusum M, Mikami Y, , et al. Pathogenic Nocardia isolated from clinical specimens including those of AIDS patients in Thailand. Eur J Epidemiol 1995; 11(5): 507-512.

9.Srifuengfung S, Poonwan N, Tribuddharat C, et al. Prevalence of Nocardia species isolated from patients with respiratory tract infections at siriraj hospital, Thailand. J Infect Dis Antimicrob Agents 2007; 24: 1-6.

10.Sahathevan M, Harvey FA, Forbes G, et al. 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 1994;5(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 specimens. J Clin Pathol 2003; 56(12): 966-969.

13.Gupta N, Srinivasan R, Kumar R, et al. Two cases of nocardiosis diagnosed by fineaˆ?needle aspiration cytology: Role of special stains. Diagn Cytopathol 2011; 39(5): 363-364.

14.Shawar RM, Moore DG, LaRocco MT. Cultivation 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. Selective 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. Application 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 rod

Figure 2. Nocardia grown on nutrient agar medium

Table1. Comparative results of paraffin baiting technique and conventional technique of direct culture on sabouraud dextrose agar, sabouraud dextrose agar(SDA) with cycloheximide and Paraffin agar(PA) in the isolation of Nocardia spp from various clinical specimens.

Clinical samples

Number of cases

Direct microscopy

Paraffin baiting method

PA

SDA

SDA+ cycloheximide

Partially acid fast

Gram stain

Acid fast

Sputum

238

2

1

0

4

2

1

1

CF sputum

53

0

0

0

0

0

0

0

BAL

143

1

1

0

2

2

1

1

cutaneous Abscess

45

2

1

0

1

1

1

1

CSF

1

0

0

0

0

0

0

0

Mycetoma

2

0

0

0

0

0

0

0

dental abscess

1

0

0

0

0

0

0

0

Trachea

31

0

0

0

0

0

0

0

Wound

1

0

0

0

0

0

0

0

Bone marrow biopsy

1

0

0

0

0

0

0

0

Gastric lavage

1

0

0

0

0

0

0

0

1

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