Use of the BacT/Alert MB Mycobacterial Blood Culture System for Detection of Mycobacteria in Sterile Body Fluids Other than Blood

The definitive diagnosis of extrapulmonary tuberculosis is madeby a positive body fluid culture result. Conventional culturemethods require centrifugation or filtration of body fluid (peritoneal,pleural, synovial, or pericardial fluid) in order to improvethe sensitivity. The aim of the present study was to evaluatethe feasibility of the direct inoculation, at the patient'sbedside, of up to 5 ml of uncentrifuged fluid onto BacT/AlertMB culture bottles (bioMérieux, Durham, NC).

INTRODUCTION

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INTRODUCTION

Tuberculosis remains one of the major global public health problems(7) and often remains undiagnosed. Because of the low sensitivityof conventional techniques for the recovery of Mycobacteriumtuberculosis (4), the diagnosis of extrapulmonary tuberculosis,in many cases, is made on the basis of the clinical presentation,the history of contacts, a positive tuberculin test result,and characteristic abnormalities on the chest radiograph. Theisolation of mycobacteria from body fluids remains a challengeowing to the low bacterial charge characterizing such samples(1, 2). Conventional culture methods require centrifugationor the filtration of body fluid (peritoneal, pleural, synovial,or pericardial fluid) in order to improve the sensitivity. Moreover,the diagnosis of tuberculous lymphadenopathy is problematicbecause of the small volumes of needle aspirates. Therefore,it is very important to evaluate the usefulness of new diagnosticmethods (3, 5, 6).

The isolation of the etiologic agent is regarded as the "goldstandard" for the diagnosis of tuberculosis, and it is universallyaccepted that liquid culture media are needed for the sensitivedetection of mycobacteria, in particular, from samples in whichvery few bacterial units may be present.

In our laboratory, automated culture of the BacT/Alert MP culturebottles (MP bottles; bioMerieux, Durham, NC) is routinely usedfor the detection of mycobacteria in clinical samples.

From the beginning of 2006 to June 2008, we processed 96 bodyfluid specimens (pleural fluid, peritoneal fluid, and biopsytissue specimens) using both MP bottles and BacT/Alert MB bloodculture bottles (MB bottles).

In order to evaluate whether the different yields found by thetwo methods was effective or not, we compared the results obtainedby the routine culture method (with MP bottles) with those obtainedby direct inoculation into MB bottles.

MATERIALS AND METHODS

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MATERIALS AND METHODS

This study involved 96 body fluid specimens (75 pleural fluid,11 peritoneal fluid, and 10 biopsy tissue specimens) which werecollected over a period of 30 months from 96 patients (1 samplefrom each patient) admitted to the Department of InfectiousDiseases at the Campo di Marte Hospital, Lucca, Italy. The finaldiagnoses for these patients are reported in Table 1.

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TABLE 1. Distribution of patients on the basis of the final clinical diagnosis and the type of sample

According to the standard procedure used in our hospital, thepleural and peritoneal fluids were collected in sterile containersand transported, in the shortest period of time, to the microbiologylaboratory for processing. After centrifugation, the pelletwas treated with an equal volume of NaOH-N-acetyl-L-cysteine(NALC-NaOH; final NaOH concentration, 2%) for 15 min at roomtemperature and neutralized with sterile phosphate buffer (0.067M, pH 6.8). After centrifugation at 4,200 x g for 15 min, thepellet was resuspended in 1 to 2 ml of sterile distilled water,which was used for the inoculation of MP bottles and Lowenstein-Jensenmedium and the performance of PCR by the BD ProbeTec ET Mycobacteriumtuberculosis complex direct detection assay (Becton Dickinson,Sparks, MD).

Beginning in 2006, we added to the standard procedure the directinoculation, at the patient's bedside, of up to 5 ml of uncentrifugedfluid into MB bottles supplemented with the MB/BacT enrichmentfluid. MB bottles, to which the proper enrichment is added,are designed for the recovery of mycobacteria commonly isolatedfrom blood. The medium supports the growth of other aerobicorganisms, including yeast, fungi, and bacteria. A 3- to 5-mlvolume of blood can be inoculated directly into the MB bottle.

Aseptically minced and homogenized biopsy tissue specimens weredivided into two parts; one part was digested-decontaminatedand inoculated in Lowenstein-Jensen medium and MP bottles andprocessed for PCR, while the other part was directly inoculatedinto one MB bottle. The inoculated bottles were placed intothe instrument, where they were incubated (35°C to 37°C)and continuously monitored for microbial growth.

In order to compare the conventional method with MP bottlesand the innovative diagnostic method with MB bottles, we carriedout three experiments in which we used Mycobacterium tuberculosisstrain H37Rv, which was adjusted with saline to the turbidityof a 0.5 McFarland standard (equivalent to 0.6 x 10⁸ bacteria/ml).

In the first test the 0.5 McFarland M. tuberculosis suspensionwas diluted in saline solution, thus obtaining final concentrationsof 1.5, 0.75, 0.15, and 0.075 CFU (see Table 3). Two 5-ml aliquotswere taken from each of the four dilutions. The first one wasdirectly inoculated into MB bottles; the other one was decontaminatedby the standard NALC-NaOH procedure, and its sediment was resuspendedto 0.5 ml of H₂O and finally inoculated into the MP bottles.

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TABLE 3. Time to detection in MB and MP bottles inoculated with M. tuberculosis saline dilutions after treatment of samples with NALC-NaOH

In the second experiment, the suspensions inoculated into MPbottles were not treated with NALC-NaOH (see Table 4 for thedilutions tested).

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TABLE 4. Time to detection in MB and MP bottles of M. tuberculosis saline suspension without pretreatment of the samples with NALC-NaOH

The final experiment was conducted in order to obtain more reliableresults by reproducing the usual diagnostic conditions, andthe M. tuberculosis dilutions were prepared in sterile pleuralfluid instead of saline solution (see Table 5).

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TABLE 5. Time to detection in MB and MP bottles of M. tuberculosis pleural fluid suspension with and without pretreatment of the samples with NALC-NaOH

In the three experiments, each dilution was tested in quadruplicateand all the bottles were inoculated for up to 60 days beforethey were considered negative. The threshold value was definedas the development of mycobacteria in three of four replicates.One drop from each saline or pleural fluid dilution and frompositive MB or MP bottles was plated on blood agar plates andchocolate agar plates in order to verify the sterility.

The real numbers of CFU present in the bacterial dilutions weredouble-checked, when possible, by seeding 100 µl on Middlebrook7H11 agar plates.

RESULTS

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RESULTS

The results of the tests with MP bottles and MB bottles andby PCR conducted with positive specimens (11 of 96 clinicalsamples) are reported in Table 2. The available epidemiologicaldata (collection and hospitalization dates and absence of knowncontacts between patients) suggest that the mycobacteria isolatedrepresent separate strains.

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TABLE 2. Comparison of MP and MB bottles for recovery of M. tuberculosis in clinical samples

Two pleural fluid specimens, one PCR positive and the otherPCR negative, remained sterile in Lowenstein-Jensen medium andthe MP bottles, while M. tuberculosis grew in the MB bottles.The average times required for the detection of a positive resultwere 19.9 days for samples in MB bottles and 21.6 days in samplesin MP bottles.

In the first experiment, all MB bottles inoculated with 1.5CFU and three of four of the bottles inoculated with 0.75 CFUscored positive on average after 33.1 and 36.8 days, respectively.No growth was obtained in any MP bottle (Table 3).

The results of the second experiment (Table 4) showed thresholdsof the numbers of CFU per bottle of about 12.5 for MP bottlesand 0.31 for MB bottles; with bacterial concentrations greaterthan 25 CFU/bottle, the average times required for the detectionof a positive result were 19.3 days for MP bottles and 23.9days for MB bottles.

In the third experiment (pleural fluid dilution) (Table 5),the MB and MP bottles (without digestion-decontamination) showedthe same sensitivity for the recovery of mycobacteria; the averagetimes required for the detection of a positive result were 14.4days for MB bottles and 15.6 days for MP bottles. For the samplespretreated with NALC-NaOH and inoculated into MP bottles, thesensitivity was 5,750 CFU/bottle.

DISCUSSION

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DISCUSSION

The definitive diagnosis of extrapulmonary tuberculosis is madeby a positive culture result, but unfortunately, the techniquesused for the detection of mycobacteria have a number of limitations:the paucibacillary nature of the specimens, the concentrationof organisms (which can be low, despite centrifugation or filtration),the presence of a clot in some samples, the presence of inhibitorsthat undermine the performance of the PCR, and delays in thetransport of the specimen to the microbiology laboratory.

We demonstrated in the first experiment that decontaminationof the culture should be avoided. The second experiment showedthat the method with the MB bottles is more sensitive than theconventional method with MP bottles, although the time requiredfor the detection of positive cultures with MB bottles is slightlylonger. Finally, we showed in third experiment, which was carriedout with pleural fluid, that the time required for the detectionof positive cultures is slightly longer for MP bottles.

The data obtained from studies with clinical and experimentalsamples suggest that the use of MB bottles allows higher ratesof recovery than the conventional method with MP bottles, whilethe detection times were comparable. Moreover, the use of MBbottles eliminates the problems linked to the collection andtransport of specimens, does not allow the coagulation of thesample, and does not require further technical manipulations.In conclusion, we suggest that MB bottles can be used withoutdecontamination-digestion as a routine method to improve theaccuracy and reliability of the diagnosis of extrapulmonarytuberculosis.

FOOTNOTES

* Corresponding author. Mailing address: Clinical Laboratory, Campo di Marte Hospital, Lucca 55100, Italy. Phone: 39 0583970313. Fax: 39 0583970402. E-mail: r.mattei{at}usl2.toscana.it

Published ahead of print on 24 December 2008.

REFERENCES

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