Revista de Microbiologia (1997) 28:183-189ISSN 0001-3714

AN ALTERNATIVE BIPHASIC CULTURE SYSTEM FOR RECOVERY OFMYCOBACTERIA AND FOR DIFFERENTIA TION OF SPECIES OTHER THAN

M. TUBERCULOSIS COMPLEX FROM BLOOD SPECIMENS

Maria Conceiíão Martins1 *, Suely Yoko Mizuka Uekil, Maria Cecilia de Almeida Palharesf, DavidJamil Hadad , Maria Alice Silva Telles I, Anna Luiza Nunes Placco2, Lucilaine Ferrazolil, Melissa

Curcio I, Áquila Maria Lourenço Gomes I, Moisés Palaci I

Ilnstituto Adol fo Lutz, Seção de Bacteriologia, São Paulo, SP, Brasil; 2Centro de Referência e Treinamento,DST/AIDS, São Paulo, SP, Brasil

ABSTRACT

Mycobacteremia is an increasingly frequent cornplication of late-stage infection withlhe Hurnan Immunodeficiency Virus (I-IIV). Several different pracedures have been usedto detect and characterize mycobacteria in blood. I-Iowever, most of these are expensiveand time consuming. Our study addressed the application of an alternative biphasic bloodculture system containing modified Middlebraok 71-19 broth and Lowenstein Jensenmédium (mod 7H9/LJ) deveJoped for direct recovery of mycobacteria from blood.Additionally, we evaluated the possibility of rapid discrimination of Mycobacterium otherthan tubercle bacilli (MOTT) organisms by differential growth after simultaneousinoculation into control media and media altered by the addition of p-nitrobenzoic acid. Inthe first part of this study rnod 7H9/LJ was compared with conventional 7H9/LJ.Mycobacterial growth curves were generated for M. tuberculosis and M. intracellulare inboth control and test medi um. rn the second part of this study rnycobacteriawere recoveredfrom 64 of 537 cultured specimens (11.9%). Growth was detected in 64 (l00.0%) of the71-19/LJ, and 62 (96.9%) of the mod 7H9/LJ biphasic bottles.The mean time tomycobacterial detection in the two systems was the sarne. For the third part of the study,a total of \09\ blood specimens were cultured in mod 7H9/LJ and in mod 7H9/LJcontaining 500 ug/rnl of p-nitrobenzoic acid. A total of 72% of ali Mvcobacterium aviumcomplex (MAC) isolated from blood were presumptively identified correctly as MOTTwithin 27 days. This study indicates that the alternative biphasic culture system has greatpotential for use under laboratory conditions that prevail in developing countries.

Key words: Mycobacteria, blood, detection, mycobacterernia diagnosis

INTRODUCTION tuberculous infection, until the advent of the H1Vpandemic, mycobacteremia was rarely detected (1,4).

Though historically it has been possible to recoverM. tuberculosis from the blood of patients with miliarydisease or during the initial dissemination phase of

The frequency of both silent and symptomaticmycobacteremia in Acquired ImmunodeficiencySyndrome (AIDS) patients and the range of species

* Corresponding author. Mailing address: Instituto Adolfo Lutz, Seção de Bacteriologia, Av. Dr. Arnaldo, ~51, 9º andar, CEP 01246-902,São Paulo, SP, Brasil

M.C. Martins et ai.

encountered makes the culture of blood and bonemarrow for mycobacteria an essential diagnostic toolfor the modem laboratory (2.3.4,12).

The clinical demand for the diagnosis ofdisseminated mycobacterial disease in AIOS has ledto the developrnent of a number of comrnercial andnon-cornmercial systems aimed at the detection ofmycobacteria in sterile body fluids including Iysiscentrifugation, radiornetric broth culture, and nucleicacid amplification and detection (1,10,16,17,20,21).Though these tools have proven sensitive andclinically useful, they are sometimes cumbersome ortime-consuming and moreover, are toa expensive tobe used routinely by laboratories in the developingworId. We sought to develop a simplified altemativemethod that would avoid practical problemsassociated with other systems and that would be easilyaffordable. This report describes lhe laboratory anddi nical evaluation of a biphasic system containingmodified Middlebrook 7H9 bro th anelLowenstein-Jensen medium.

The study was carried out in three phases: 1) lhelaboratory evaluation of an altemative inexpensiveenrichment method for Middlebrook 7H9 broth, 2) aclinical study comparing the sensitivity of detection oftwo biphasic culture systerns, and 3) the evaluation ofa method for direct species differentiation ofmycobacteria growing from blood cultures.

MA TERIAL AND METHODS

Laboratory Evaluatíon of Modified MiddlebrookMedia. Middlebrook 7H9 broth medium was enrichedaccording to a standard protocol by the addition ofADC - bovine albumin fraction V, glucose andcatalase (Difco Laboratories, Detroit, Michigan,USA) 10% by volume. A modified Middlebrook 71-19broth was prepared by replacing the MiddlebrookADC enrichment with bovine serum (heat inactivatedfor 30 minutes at 56°C) to a final concentration of 10%and pH 7,2. The ability of the two mediumpreparations to support mycobacterial growth wascompared by inoculation with pedigreed strains anelserial quantitative subculture. Standard inocula of M.tuberculosis 1-137Rv (ATCC 25177) and M.intracellulare (ATCC \3950) were prepared in thefollowing manner. Each strain was harvested inlog-phase growth from 15 day cultures onLowenstein-Jensen slants and placed in a tubecontaining 5 mJ of Middlebrook 71-19broth and glassbeads. A homogenous suspension was prepared by

184

shaking in a Vortex mixer for 3 minutes and theresulting suspension was allowed to sit while largerc1umps settled to the bottom of the tube. Thesupematant was then placed in a second tube and leftundisturbed for 15 minutes to allow further settling 01'clumped organisms. The supematant fram this smoothsuspension was then transferred to another tube aneladjusted to a density equal to a 0,1 nm by the additionof 7H9 broth using a nephelometer. M. tuberculosisand M. intracellulare standardized inocula (0,5 1111each) were added to culture tubes containing 4,5 1111ofstandard or modified Middlebrook 7H9 broth. Thecapped tubes were incubated at 37°C and growthcurves were constructed by performing quantitativoculture of the test media on Lowenstein-Jensen sianrsat days 0,3,6,9, and 12 after inoculation.Clinical Evaluation of Modified MiddlebrookMedia. Biphasic culture systems were prepared ing l as s bottles of 50 ml capacity usingLowenstein-Jensen (U) medium for the solicl phaseand either standard or modified Middlebrook 71-19broth for the liquid phase. The U mediurn wasinspissated as an 8 ml slant and a 20 mJ volume ofenriched 71-19broth was added that left roughly half ofthe LJ slant submerged and half exposed. The biphasicsystem using standard Middlebrook 7H9 broth withADC (71-19/LJ) was used as control in a cornparisonwith an identical system replacing the modi·fied 71-19broth described above (mod7H9/LJ). Both brothpreparations were supplemented with 0.025% sodiumpolyanetholsulfonate to prevent coagulation. anelan li bi oti cs (20 ug/rnl tri methopri m, 50 ug/mlcarbenicillin, 10 ug/ml amphotericin B) to prevenicontamination.

A total of 537 blood specimens were obtained frompatients with AIDS with suspected disserninatedmycobacterial infection hospitalized at the AIOSTraining and Reference Center (CRT A) of São Paulo.Brazil, fromJanuary 1991 toJuly 1993. Ten millilitersof blood collected by venipuncture withoutanticoagulant after ioelophor disinfection of the skinwas divided into two 5 ml aliquots and immediatelyinoculated into the control (71-19) anel te st ,(mod71-19/LJ) media. The cultures were incubateelupright at 37°C anel observed daily for growth for upto 8 weeks. Bottles were inverted daily during the firstweek for reinoculation of the exposed solid media.Positive cultures were those demonstrating visiblegrowth in either the solid or liquid meelium, confirrnedto be mycobacterial by Ziehl-Neelsen (Z-N) staining.Broth from the cultures remaining negative after 8

CI

weeks of incubatian was Z-N stained and examinedfor acid-fast bacilli (AFB) before being discarded torule out growth not discovered by visual inspection.Evaluation of a System for Direct MycobacterialSpecies Differentiation. A total of 1091 bloodspecimens were obtained from AIOS patients withsuspicion of disserninated mycobacterial infectionhospitalized at the CRTA during the period fromSeplember 1993 to April 1995. Ten milliliters ofbloodcallected by venipuncture without anticoagulant afteriodophor disinfection ofthe skin was divided into two5 rnl aliquots for inoculation into each oftwo biphasicmod 7H9/LJ médium bottles prepared as detailedabove. One of lhe bottles also contai ned 500 ug/ml ofp-nitrobenzoi c acid (PNB). The cultures wereincubated upright at 3rc and inspected for growthdaily for the first two weeks, at which time they wereinverted for reinoculation of the expcsed solidmedium. and then weekly for an additional 6 weekswithout further inversion. Bacterial growth detectedby visual in s pe ct io n was confirmed to bemyc obact er ial or contaminant on lhe basis ofmicroscopic exami narion or stained preparationsusing lhe Z-N method. For all positive cultures,mycobacterial growth was cornpared in the paired

CFUlmI

Mycobactcria írom blood xpccimcn-

bottles and cultures growing in mediurn containingPNB were presumptively classified as MOTT.

ldentification of Mycobacterial lsolates. Mtuberculosis isolates were identi fied on the basis ofp-nitrobenzoic acicl susceptibility.2-thiophenecarboxyJic acid hydrazide resistance,niacin production, nitrate reduction and colonymorphology. (5,11) MOTT were identified at lhespecies levei using standard methods.

Statistical Methods. The cletection sensitivity of thetwo biphasic systems was cornpared using the Kappaagreernent coefficient (8).

RESULTS

This stucly was carriecl out in three phases. ln thefirst phase a modified Micldlebrook 7H9 brothenrichment method was tested, replacing with bovineser um the more expensive Mielellebrook ADCenr ichment. Mycobacterial growth curves weregenerateel for M. tuberculosis anel Miintracellulare inboth control and test médium by quantitativesubculture at 3-day intervals. As shown in Fig. I.similar logarithmic growth (7) was seen in both media .

o 3 6

TIME (days)

9

. - -.- - -7H9 - M.tuberculosis

- - -.- - - Mod 7H9 - M.tuberculosis••• 7H9 - M.intracellulare

• Mod 7H9 - M.intracellulare

12

~'~ure I - l.ogarithmic growth orM. IlIheTCII/o.\"i.\· and M. intraccllnlnrc in Middlebrook 7H9 broth and Modificd Middlebrook 7H9mcdiulll.

,

I 185

M.C. Martins et ai.

In second phase of the study, 537 blood cu/tureswere performed using a biphasic medium preparedwith Lowenstein-Jensen and Middlebrook 7H9 broth.The cultures were performed in duplicate to comparemycobacterial recovery and contamination rates inbottles with standard ADC-enriched broth 7H9/LJ) tothose of bottles with the modified broth describedabove (mod 7H9/LJ). Mycobacteria were recoveredfrorn 64 of the 537 cultures (11.9%), 64 of which(IOO.O%) were detected by the 7H9/U bottles and 62(96.9%) by the mod 7H9/LJ bottles. Kappa coefficientcalculation showed excellent agreernent (91.0%)between systems.

Of lhe 64 isolates, 37 (57.8%) were MAC and 24(37.5%) were M. tuberculosis cornplex organisms,Three isolates could not be identified at the specieslevei because of contaminating overgrowth or laek ofgrowth on subculture. Primary contaminationoccurred in I I (2.0%) and 8 (1.5%) ofthe 7H9/LJ and1110d7H9/LJ biphasic systems, respectively (Table I).

The number of days required for detection ofpositive eultures did not differ between the twosystems (Table 2). Myeobacterial growth was detectedby inspection ofboth the liquid and solid phases oftheculture systern.

In the final phase of the study a method of speciesdifferentiation directly from the inoculated culture

Tahle I - Rccovcry 01"rnycobacteria and conramination rales Irom~:nhlood spccimcns in two biphasic culturc systcrns

Biphasic systcrnPositive cultures

n=64Contaminatcd

culturesn=12MAC MT NI

7H9/LJ"

Mod.7H9/LJh.'i I

2 2oO

4

7H9/1J andMod. 7H9/L.I 12 )'-.' 7

a Middlebrook 7H9 broth and Lowcnstciu Jcnscnh Modificd Middlcbrook 7H9 broth and Lowcnstcin JcnscnMAC - Mycobacterium avium cornplcxMT - Mycobactcrium ruberculosis cornplcxNI - not idcnti licd spccics

medium was evaluated. Pairs of biphasic 7H9/L.Jbottles, one containing PNB, a speci fie inhibitor of M.tuberculosis, were inoculated with 5 ml ofblood frornAroS patients with suspicion of disserninatertmycobacterial infection. Of 1091 cultures, 58 (5.3%)were positive for mycobacteria. Of the 43 isolatesgiven a final idenfication of MAC, 31 (72.1 %) eoulelbe presurnptively identified as MOTT over an averageof 27 days on the basis of growth in t hcPNB-containing bottle. The 12 MAC isolates thatfailed to grow in the PNB-containing systerns were alisubsequently shown to be PNB-resistant.Misidentification of M. tuberculosis did not occur anelali of these 15 isolates were growth inhibitecl by theselective biphasie bottle (Table 3).

Five isolates could not be identfiecl at the speciesleveI because of either contaminating overgrowth 01'

lack of growth on subculture. Primary contaminationoccurred in 12 (2.23%) biphasic systems.

DISCUSSION

The well-documentecl association of AIOS withDisserninated Mycobacterial Infection prornpted theinvestigation of methods for the detection andidentification of these organisms from bloodspecimens. Although biphasic blood cultures arecommonly used for lhe isolation of bacteria anel fungi.they are seldom used for the isolation of mycobacteriaThe design of our study addressed the application ofan alternative blood culture system for diagnosis andfor monitoring the efficaey of therapy under lheprevailing laboratory eonditions in developingeountries.

In this phase of our study, the simple replacementof ADC supplement with bovine serum to a finalconcentration of 10% in Middlebrook 71-19 médiumdid no! affect the growth of M. tuberculosis or M.intracellulare (Fi g , I). This may be a usefulsubstitution for containing eosts (9), although, as withany other in-house media supplemented with serum orblood, factors may be present that possibly affect

Organisrn N° or isolares"

Tahle 2 - Number 01"days rcquired for dctection of lhe M. tubcrculosis and M. ({I'il/I/I complcx organisms.

Mod 7H9/ 1...17H9 / L.I

M.{l/hel"C/({osis ~J.:\

27.1

Mcan Range

~ 1.:1

27.2

Mcan Range

MACh 32

" Excludin!! 10 isolares that could not bc identifiedh Mycohoc;crillln avium complcx

186

18-6()

14-60

17-60

15-60

s::q

'fable 3 - Scrcening for MOTT in 1091 blood specimcns dircctlyanelsilllultancollslv inoculatcd into lhe mod 7H9/LJ biphasic systcm(canlrol) anel into lhe 7H9/IJ biphasic sysicm conlaining PNFl

(PN81

PN8 (+) PNB (-)Idenli [ication Control Control Control Conlrol TOlal

(+) r- ) (+) (-)

M. tuberculosis O O 1'1 1076 1091

MAC 29 2 12 104~ 1091

Mod 7H9/LJ - Modificd Middlebrook 7H9 / Lowcnstcin Icnscnbiphasic systcmPNB - -nitrobcnzoic acid(+) mycobaclerial growth(_) abscucc of mycobactcrial growthMOTT - Mycobacrerium othcr Ihan tubcrclc hacilliMAC - Mvcobactcrintn aviuin complcx

bacterial growth ( 14). Several methods have been usedto recover mycobacteria frorn blood cultures. Forexample. Agy et al. (I), using the isolator systern,recovcred mycobacteria from 29 of 180 bloodspecimens submitted to culture (16.1 %). Salfinger etal. ( 16) detected mycobacteria in 17.6% of the bloodsamples examined using sodiurn deoxycholatesolution-lysis-centrifugation as the concentrationtechnique, followed by inoculation of the sedimentsinto radiornetric BACTEC (7HI3) (14). Strand et al.(18) reported that BACTEC 13A médium revealed thepresence of mycobacteria in 63 of 1848 blood culture

I (3.4%). ln the present study carried out for médiumII evaluation with clinical sarnples, mycobacteria wereI recovered from 64 of the 537 cultures (12.1 %),64 of: which (100,0%) were detected with the 7H9/LJ bottles

and 62 (96.9%) with the mod 7H9/LJ bottles. It shouldbe noted also that our biphasic médium containedsrnaller amounts of liquid medi um than usuallyrecomrnended (3,6, \1).

It is important to emphasize that factors outside thelaboratory such as therapeutícs and clínicianindications for mycobacterial cultures may greatlyinfluence lhe rate of recovery of mycobacteria frornblood in these studies (1,4).

W it h respect to the ti me needed to detectmycobacterial growth, there was no significantdifference between the biphasic systems studied. Thewide range of growth detection times observed wasprobably due to the variable nurnber of bacilli in thebJooel speci mens. As demonstrated by Ber! in et al. (3),lhe time of recovery is inversely proportional to thenumber of organisms inoculated.

The ability of mycobacteria to grow 011 mediacontaining differentially inhibitory substances has

Mycobaclcria Irom blood spccirnens

repeatedly been shown to be of value for ielentificationpurposes (13,19).

The M tuberculosis complex is inhibited by PNBat a concentration of 500 g/ml, whereas MOTT areresistant to this concentration. The latter, however,may present variation in sensitivity, with the existenceof a small percentage of strains sensitive to certaindrug concentrations, as proposeel by Rastogi et al, (15)and Tsukamura et ai. (19). ln the present study. after arnean growth of 27 days in medium containing PNB.31 strains (72.1 %) were considered to be MOTT. Thismeans that the laboratory can supply a presumptiveMOTT result on the basis ofthe growth ofthese strainson medium containing PNB. Ali of these strains werelater confirmed to be MAC on the basis of the resultsof biochemical tests.

With respect to cultures presenting growth only oncontrol rnedium, i.e., strains sensitive to PNB, it wasnot possible to provide a presumptive result since 15(100%) were identified as M tuberculosis and 12(27.9%) as MAC. One of the tests used for MOTTidentification is resistance of the strain to 500 gim]PNB. Resistance to this PNB concentration wasobserved inl2 MAC st r a in s during theiridentification, suggesting that the bacillary loadinoculated into the two flasks (contraI medium anelmedium modo 7J9/LJ with PNB) was so low that it wasinhibited by this drug con~entration.

Two strains that presented growth inPNB-containing medi um did not grow on controlmedium (with no PNB), suggesting that the sampleswere paucibacillary, permitting the occurrence of adifference between the inocula added to the twomedia.

For analysi s of the agreement between methods forthe presumptive identification ofMOTT by growth onmedium 7H9/LJ containing PNB and by MACidentification using biochemical methods, the Kappacoefficient was calculated with a 95% confidenceinterval, giving a value of 0.799 (0.696-0.90\). Thisvalue may be considered a measure of the relativeefficacy of the method of presumptive identificationsince it anticipates the definitive results obtained bythe biochemical tests with reasonable concordance.

The differentiating ability of PNB-containingbiphasic media appears to be sufficiently rapid andspecific to be useful in a clinical laboratory, sinceidenti fication of mycobacteria by conventionalmethods may take up to I month.

In conclusion, the alternative biphasic systern iscompact, self-contained, simple to use and

I f)7

M.C. Martins e! al.

inexpensive. Ali of these advantages make it suitablefor use under the prevailing laboratory conditions ofdeveloping countries.

ACKNOWLEDGMENTS

We thank Dr. Mark Perkins for valuablesuggestions and Dr. Jose Leopoldo Ferreira Antunesfor statistical analysis.

RESUMO

Sistema alternativo bifásico de cultura para oisolamento de micobactéria e triagem de espéciesoutras que não as do complexo Msuõerculosis a

partir de espécimes de sangue

Micobacteremia constitui uma das manifestaçõesoportunísticas mais frequentes dos estágios avançadosda infecção pelo virus da imunodeficiência humana.Diversas metodologias tem sido propostas para o seudiagnóstico, no entanto, a maioria destas apresentamcusto elevado ou complexidade de operação técnica.Baseados nestes fatos nos propusemos a avaliar umsistema alternativo de cultura bifásico contendoMiddlebrook 7H9 modificado Lowenstein Jensen(rnod 7H9/L.n. especificamente adaptados para (i) oisolamento de micobacterias, (ii) triagem de outrasespécies que não as do complexo Mycobacteriumtuberculosis (MOTT) através da adição de ácidop-nitrobenzoico aos meios (mod 7H9/LJ+PNB). Paraa primeira etapa do estudo comparou-se os meios mod7H9/LJ e 7H9/LJ convencional, realizando-se a curvade crescimento das espécies M. tuberculosis e M.intracellulare em ambos os meios. Na segunda etapado estudo isolou-se um total de 64 cepas demicobacterias a partir de 537 espécimes de sangue(11.9%), das quais 64 000,0%) em 7H9/LJ e 62(96,9%) em mod 7H9/LJ. Verificou-se um temposemelhante de detecção destas micobacterias emambos os sistemas. Para a terceira etapa do estudo,cultivou-se um total de 1091 espécimes de sangue nossistemas mod 7H9/LJ e 7H9 mod/LJ contendo 500ug/rnl de ácido p-nitrobenzoico, constatando-se queum total de 72% dos organismos isolados epertencentes ao complexo Mycobacterium aviumpuderam ser presuntivamente identificados comoMOTI em 27 dias. Estes resultados, associados asimplicidade e baixo custo destes sistemas bifásicosconstituem elementos potenciais para sua

Illg

aplicabilidade na rotina diagnóstica em países emdesenvolvimento.

Palavras-chave: Micobactéria, sangue, detecção.diagnóstico, micobacteremia

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