Use of the BacT/ALERT MB Mycobacteria Blood Culture System for Detecting ACCEPTED

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1 JCM Accepts, published online ahead of print on December 00 J. Clin. Microbiol. doi:.11/jcm Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Use of the BacT/ALERT MB Mycobacteria Blood Culture System for Detecting Mycobacteria in Sterile Body Fluids Other than Blood ROMANO MATTEI, 1 * ARNALDO SAVARINO, 1 MARCO FABBRI, SARA MONETA, ENRICO TORTOLI Clinical Laboratory, 1 Blood Bank, and Department of Infectios Diseases, Campo di Marte Hospital 0 Lucca, and Regional Reference Center for Mycobacteria, Microbiology and 9 Virology Laboratory, Careggi Hospital, 01 Florence, Italy 11 RUNNING TITLE 1 Detection of mycobacteria in sterile body fluids. 1 1 *Corresponding author. Mailing address: Clinical Laboratory, Campo di Marte Hospital, 0 1 Lucca, Italy. Phone: Fax: r.mattei@usl.toscana.it 1 1

2 The definitive diagnosis of extrapulmonary tuberculosis is made by a positive body fluid culture. Conventional culture methods require centrifugation or filtration of body fluid (peritoneal, pleural, sinovial, pericardial) in order to improve the sensitivity. The aim of the present study was to evaluate the feasibility of direct inoculum, at patient's bedside, of up to ml of uncentrifuged fluid onto BacT/ALERT MB Culture Bottles (biomérieux, Durham, NC).

3 Tuberculosis remains one of the major global public health problems () and often remains undiagnosed. Because of the low sensitivity of conventional techniques to recover Mycobacterium tuberculosis () the diagnosis of extrapulmonary tuberculosis, in many cases, is made on the basis of clinical presentation, history of contacts, positive tuberculin test result, and characteristic abnormalities on chest radiograph. The isolation of mycobacteria from body fluids remains a challenge owing to the low bacterial charge characterizing such samples (1, ). Conventional culture methods require centrifugation or filtration of body fluid (peritoneal, pleural, sinovial, pericardial) in order to improve the sensitivity. Moreover, the diagnosis of tuberculous lymphadenopaty is problematic because of the small volumes of needle aspirates. Therefore, it is very important to evaluate the usefulness of new diagnostic methods (,, ). The isolation of the aetiologic agent is regarded as the gold standard for diagnosing tuberculosis and it is universally accepted that liquid culture media are needed for the sensitive detection of mycobacteria, in particular from samples in which very few bacterial units may be present. In our laboratory automated culture of the BacT/Alert MP Culture Bottles (MP) (biomerieux, Durham, NC) is routinely used for detection of mycobacteria in clinical samples. Since the beginning of 00 until June 00 we processed 9 body fluids (pleural, peritoneal, and biopsy tissues) were processed using both the same MP and BacT/ALERT MB Culture Bottles (MB) In order to evaluate whether the different yield found with the two methods was effective or not, compared the routine culture method (MP bottles) with the direct inoculation into MB blood-culture bottles.

4 MATERIALS AND METHODS This study involves 9 body fluids ( pleural, 11 peritoneal and biopsy tissues) wich are collected from over a period of 0 months from 9 patients (one sample for each) admitted to the Department of Infectious Diseases at the Campo di Marte Hospital, Lucca, Italy. The final diagnosis of these patients are reported in Table 1. In the standard procedure of our laboratory the pleural and peritoneal fluids are collected in sterile containers and transported, in the shortest period of time, to the microbiology laboratory for processing. After centrifugation, the pellet is treated with an equal volume of NaOH-N-acetyl-L- cysteine (NALC-NaOH, NaOH final concentration %), for 1 min at room temperature and neutralized with sterile phosphate buffer (0.0 M, ph.). After centrifugation at,00 g for 1 min, the pellet is resuspended in 1- ml of sterile distilled water which is used for inoculation to MP culture bottles and Lowenstein-Jensen and to perform PCR by the BD ProbeTec ET Mycobacterium tuberculosis complex Direct Detection assay (Becton Dickinson, Sparks, Md.) Beginning from 00 we added to the standard procedure, the direct inoculum, at the patient's bedside, of up to ml of uncentrifuged fluid onto MB Culture Bottles supplemented with the MB/BacT Enrichment Fluid. MB culture bottles, added with the proper enrichment, are designed for recovery of mycobacteria commonly isolated from blood. The medium supports growth of other aerobic organisms, including yeast, fungi, and bacteria. A - ml volume of blood can be inoculated directly into the MB bottle. Aseptically minced and homogenized tissue biopsy specimens were divided into two parts; one of which was digested-decontamined and inoculated in Lowenstein-Jensen and in MP, and processed for PCR, while the other was directly inoculated onto one MB culture bottles. Inoculated bottles were placed into the instrument where they were incubated ( C- C) and continuously monitored for microbial growth.

5 In order to compare the conventional MP and the innovative MB diagnostic methods, we carried out three experiments in which we used the Mycobacterium tuberculosis strain HRv, adjusted with saline to the turbidity of 0. Mac Farland (equivalent to 0.x bacteria/ml). In the first test the 0. Mac Farland M. tuberculosis supension was diluted in saline solution thus obtaining the final concentrations of 1., 0., 0.1 and 0.0 CFU (Table ). From each of the four dilutions, two ml aliquots were taken. The first one was directly inoculated in MB; the other one was decontaminated, using the standard NALC-NaOH procedure, and its sediment was re- suspended to 0. ml with H O, and finally inoculated in MP. In the second experiment the suspensions inoculated in MP were not treated with NALC-NaOH. The dilutions tested are reported in Table. The final experiment was conducted in order to obtain more reliable results by reproducing usual diagnostic conditions and the M. tuberculosis dilutions were prepared in sterile pleural fluid instead of saline solution. (Table ) In the three experiments each dilution was tested in quadruplicate and all the bottles were inoculated up to 0 days before being considered negative. Threshold value was defined as the development of mycobacteria in three out of four replicates. One drop from each saline, or pleural, dilution and from positive MB or MP bottles was plated on blood agar plates and chocolate agar plates in order to verify the sterility. The real number of CFU present in the bacterial dilutions was double-checked, when possible, by seeding 0 µl on agar Middlebrook H11 plates.

6 RESULTS The results of MP/MB/PCR tests conducted on positive specimens (11 out of 9 clinical samples) are reported in Table. Available epidemiological data (collection and hospitalization dates, and absence of known contacts between patients) suggest that mycobacteria isolated represent separate strains. Two pleural fluids, one PCR-positive and the other PCR-negative, remained sterile in Lowenstein Jensen and MP, while M. tuberculosis grew in the MB bottles. The average time required for positive detection in MB and MP was of 19.9 days in 1 samples and for MP of 1. days in 11 samples. In the first experiment all MB culture bottles inoculated with 1, CFU, and three out of four of the ones inoculated with 0. UFC, scored positive on average after.1 and. days respectively. No growth was obtained in any MP bottle (Table ). The results of the second experiment (Table ) showed a CFU/bottle threshold of about 1. for MP and 0.1 for MB; with bacterial concentrations greater than CFU/bottle the average time required for positive detection was 19. for MP and.9 for MB. In the third experiment (pleural fluid dilution) MB and MP (without digestion/decontamination) showed the same sensitivity in the recovery of mycobacteria, the average time required for positive detection was 1. days for MB and 1. days for MP. In the pre-treated samples (NALC-NaOH) inoculated in MP culture bottles the sensitivity was,0 CFU/bottle.

7 DISCUSSION The definitive diagnosis of extrapulmonary tuberculosis is made by a positive culture, but unfortunately the techniques for detecting mycobacteria have limitations for a number of reasons: the paucibacillary nature of the specimens, the concentration of organisms (that can be low despite the centrifugation or filtration), the presence of clot in some samples, while the presence of inhibitors undermines the performance of PCR and the delay in transport of the specimen to the microbiology laboratory. We demonstrates (first experiment) that the decontamination of culture should be avoided, that MB method has a major sensitivity compared to the conventional MP, although the detection time required for positive cultures is slightly higher (second experiment) and that the detection time is slightly higher for MP (third experiment carried out using pleural fluid). Data obtained from clinical and experimental samples suggest that MB allows higher recovery rates than the conventional MP while the detection time was comparable. Moreover MB eliminates the problems linked to the collection and transport of specimens, does not allow the coagulation of the sample times necessary to avoid the clotting) and doesn t require further technical manipulations. In conclusion, we suggest MB without decontamination digestion as a routine method to improve the 1 accuracy and reliability of extrapulmonary tuberculosis diagnosis.

8 REFERENCES Chakravorty, S., M. Dudeja, M. Hanif, and J. S. Tyagi. 00. Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis. J. Clin. Microbiol. : 0.. Chakravorty, S., and J. S. Tyagi. 00. Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J. Clin. Microbiol. :9 0.. Goel M. M., V. Ranjan, t. N. Dhole, a. N. Srivastava, A. Mehrotra, M. R. Kushwaha, A. Jain. 00. Polymerase chain reaction vs. conventional diagnosis in fine needle aspirates of tuberculous lymph nodes. Southeast Asian J. Trop. Med. Public Health. : Merino J. M., T. Alvarez, M. Marrero, S. Ansó, A. Elvira, G. Iglesias, J.B. González Microbiology of pediatric primary pulmonary tuberculosis. Chest. 119:1-1.. Purohit M. R., T. Mustafa, L. Sviland Detection of Mycobacterium tuberculosis by Polymerase Chain Reaction with DNA eluted from aspirate smears of tuberculous lymphadenitis. Pathol. :-1.. Singh K.K., M. Muralidhar, A. Kumar, T. K. Chattopadhyaya, K. Kapila, M. K. Singh, S. K. Sharma, N. K. Jain, J. S. Tyagi Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. Acta Cytol. :-0.

9 . World Health Organization. Global tuberculosis control: surveillance, planning, financing. World Health Organization, Geneva, Switzerland. W.H.O./CDS/TB/

10 TABLE 1: Distribution of patients on the basis of final clinical diagnosis and the type of sample. Patients number Final Clinical diagnosis Specimen type Cirrhosis Peritoneal fluid Hepatic tumor Peritoneal fluid Cancer Tissue biopsy 1 Tubercular spondylodiscitis with multiple abscesses Tissue biopsy Bacterial lymphadenopathy Lymph node biopsy 1 Non-specific lymphadenopathy Lymph node biopsy Superficial tuberculous lymphadenopathy Lymph node biopsy Empiema Pleural fluid Parapneumonic Pleural fluid Lung carcinoma Pleural fluid 1 Non-specific pleuritis Pleural fluid 1 Pleural effusion in cirrhosis Pleural fluid Tubercular pleuritis Pleural fluid Pleuro-pulmonary tuberculosis Pleural fluid

11 TABLE : Comparison of BacT/ALERT MP (MP) and BacT/ALERT MB (MB) culture bottles for the recovery of M. tuberculosis in clinical samples. Collection Time to detection Final diagnosis Specimen type date MB MP Pleuro-pulmonary tuberculosis Pleural fluid 0/0/ Negative Tubercular pleuritis (HIV +) Pleural fluid /0/0 No growth PCR Negative Tubercular pleuritis Pleural fluid 11/0/0 1 0 Negative Pleuro-pulmonary tuberculosis Pleural fluid 1/0/ Negative Pleuro-pulmonary tuberculosis Pleural fluid /0/0 0 Negative Tubercular pleuritis (HIV +) Pleural fluid 0// Positive Superficial tuberculous lymphadenopathy Superficial tuberculous lymphadenopathy Tubercular spondylodiscitis with multiple abscesses Lymph node biopsy Lymph node biopsy 01/09/0 9 0 Positive 0/0/0 9, Positive Tissue biopsy 1/01/0 0 0 Positive Superficial tuberculous Lymph node 9/0/0 1 Positive lymphadenopathy biopsy Pleuro-pulmonary tuberculosis Pleural fluid 1/0/0 1 No growth Positive 11

12 TABLE : Time to detection, in MB and MP, inoculated with M. tuberculosis saline dilutions after treatment of the samples with NALC-NaOH. Bacillary load in the starting CFU/ml CFU/bottle solution a a Double-checked CFU on the agar Middlebrook H11 plates b All specimens were processed and pretreated with NALC-NaOH as previously described c NG: no growth after 0 days MB culture bottles MB culture bottles b Time to detection (days) c Average (days) Time to detection (days) c 0 0, 1, 1,, 0,,,1 NG NG NG NG 00 0,1 0, NG,, 1,0, d NG NG NG NG 00 0,0 0,1 NG NG NG NG - NG NG NG NG 00 0,01 0,0 NG NG NG NG - NG NG NG NG d One out of four bottles showed no growth; the value given is average of three bottles 1

13 TABLE : Time to detection, in MB and MP, of M. tuberculosis saline suspension (without pre- treatment of the samples with NALC-NaOH) Bacillary load in the starting solution a CFU/ml CFU/bottle MB culture bottles Time to detection (days) Average (days) MP culture bottles b Time to detection (days) 0 0 b b b b b c c c Average 00. c NG d e 0 1 c NG 1.0 NG c NG NG NG NG NG c NG NG NG NG NG c NG NG NG NG NG c NG. e NG NG NG NG NG c 0.1 NG NG NG - NG NG NG NG NG c 0.0 NG NG NG NG NG NG NG NG NG NG c 0.0 NG NG NG NG NG NG NG NG NG NG c 0.0 NG NG NG NG NG NG NG NG NG NG a All the samples were centrifuged but not processed with NALC-NaOH (days) b Double-checked CFU on the agar Middlebrook H11 plates c Theoretical CFU d NG: no growth after 0 days e One out of four bottles showed no growth; the value given is average of three bottles 1

14 TABLE : Time to detection, in MB and MP, of M. tuberculosis pleural fluid suspension (with and without pretreatment of the samples with NaLC-NaOH) Bacillary load CFU/ml CFU/bottle in the starting solution a a Double-checked CFU on the agar Middlebrook H11 plates b All the samples were centrifuged but not processed with NALC-NaOH c Specimens were processed and pretreated with NALC-NaOH as previously described d NG: no growth after 0 days MB culture bottles Time to detection (days) Average (days) MP culture bottles b Time to detection (days) 0 1 a a NG d NG a NG NG. NG a NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG Average (days) 1