MONOFLUO™ Pneumocystis jirovecii (P. carinii) IFA Test Kit 32515

Immunofluorescent Antibody Test Kit for the Detection and Identification of Pneumocystis jirovecii (P. carinii) in Respiratory Tract Specimens

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References/Bibliographie/Referencias/Literatur/Bibliografia/Referencer/Referenser ...........................................................111

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INTENDED USEThe MONOFLUO™ Pneumocystis Immunofluorescent AntibodyTest Kit is to be used for the detection of Pneumocystis jirovecii(P. carinii) cysts and trophozoites in specimens collected fromthe respiratory tract.

SUMMARY AND EXPLANATIONPneumocystis jirovecii (P. carinii) is a unicellular, eucaryoticorganism that is present in the lungs of many mammalianspecies, including man. The Pneumocystis organisms found inhumans were originally referred to as P. carinii f. sp. hominis,the subspecies name used to distinguish the Pneumocystisorganisms found in humans from Pneumocystis organismsfound in other mammals. Recently the Pneumocystis organismfound in humans was recognized as a distinct species andrenamed Pneumocystis jirovecii.1 The organism is spread byairborne routes, usually causing asymptomatic infection.Exposure during childhood may result in mild or subclinicalcases, with the organism existing in latent state throughadulthood. In individuals with compromised immune systems,the organism becomes opportunistic, causing diffuse,interstitial pneumonia.2,3

Individuals at risk for Pneumocystis jirovecii include prematureinfants, individuals with immune deficiency disorders (such asacquired immunodeficiency syndrome), and those receivingimmunosuppressive drugs such as corticosteroids. Pneumo-cystis jirovecii is the most common opportunistic infectiousagent in AIDS patients, with almost 80% suffering from theinfection.4,5,6,7

Morphological studies of the organism have revealed threeforms in the life cycle of Pneumocystis jirovecii: cysts, sporozo-ites and trophozoites. The thick-walled cysts (4-7 μm in diame-ter) usually contain eight comma-shaped sporozoites, whichthen mature into larger, pleomorphic trophozoites (2-8 μm indiameter) before encysting to repeat the cycle.2,7 The cysts, withtheir distinct morphologic characteristics, are most frequentlyassociated with presence of Pneumocystis jirovecii.

Rapid clinical diagnosis of Pneumocystis jirovecii is of utmostimportance as the organisms quickly infiltrate lung tissue caus-ing dyspnea, fever, and cough.8 Early detection can facilitate

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initiation of appropriate treatment and may improve chances ofpatient survival.9,10

At present, diagnosis is accomplished through radiologic tech-nique and identification of Pneumocystis jirovecii cysts and tro-phozoites by histologic staining of respiratory specimens.Common staining methods (toluidine blue O, Gomori’s methe-namine silver nitrate or Giemsa) reveal cyst walls and/or tropho-zoites/sporozoites.7,11 Because of the nonspecific nature ofthese stains, proper identification of the organism may be diffi-cult. A more invasive technique for specimen collection such asbronchoalveolar lavage is often required to ensure the presenceof Pneumocystis jirovecii.12

The use of a direct, fluorescent-antibody procedure withinduced sputum, bronchial wash or bronchoalveolar lavagesamples provides a simple, highly-specific procedure for detec-tion of Pneumocystis jirovecii.13,14,15,16 The monoclonal antibod-ies in the MONOFLUO™ Pneumocystis jirovecii (P. carinii)Immunofluorescence Test Kit are chemically linked to fluores-cein isothiocyanate (FITC) to produce a highly specific, direct,immunofluorescent reagent with a low level of background fluo-rescence. In addition to binding with Pneumocystis jiroveciicysts, the monoclonal antibodies also bind specifically to Pneu-mocystis jirovecii trophozoites, sporozoites, and the extracellu-lar matrix. The test kit is used for the rapid identification ofPneumocystis jirovecii cysts and trophozoites directly in patientspecimens.

PRINCIPLES OF THE PROCEDUREThe MONOFLUO™ Pneumocystis jirovecii Staining Reagentcontains murine monoclonal antibodies labeled with fluoresceinisothiocyanate (FITC). These antibodies react with all Pneumo-cystis jirovecii forms (cysts, sporozoites and trophozoites) andalso with the extracellular matrix present in infected specimens

Slides are prepared for microscopy from clinical specimens asdescribed in the Preparation of Clinical Specimens Prior to Flu-orescence Staining section. Following fixation, the slides arestained with the MONOFLUO™ Pneumocystis jirovecii StainingReagent. The Staining Reagent binds to any Pneumocystis jir-ovecii organisms present in the specimen. A subsequent rinsestep removes unbound Staining Reagent from the specimen.

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The slides are then mounted with the MONOFLUO™ MountingMedium included in the kit. The Mounting Medium contains ananti-quencher which increases the length of time slides may beexamined before the fluorescence fades.

When viewed with a fluorescence microscope, infected speci-mens fluoresce bright apple-green. Stained cysts appear aslarge, round-to-elliptical structures found both individually andin clusters. Sporozoites and trophozoites may also be stained,appearing as relatively small, crescent-shaped or pleomorphicstructures. In addition, the amorphous, extracellular matrixshould also fluoresce brightly. Other organisms and cellularmaterial from respiratory specimens are counterstained red toorange-red or gold without characteristic fluorescence of Pneu-mocystis jirovecii cysts.

PRODUCT DESCRIPTION

* Volume sufficient for staining 24 individual test wells. **0.1 % sodium azide; ***0.8 % formaldehyde.

MATERIALS REQUIRED BUT NOT PROVIDED• Acetone, Reagent Grade, (store in a tightly closed con-

tainer to prevent absorption of water).• Deionized water or tap water.• Slide staining dishes with racks.• Humidified chamber (such as a covered petri dish con-

taining a moistened paper towel).• Coverslips, 24 X 40 mm, #1.

Table 1: Materials ProvidedStore reagents at 2-8°C. Slides should be stored at 2-28°C.Component Contents Preparation

R1 • Pneumocystis jirovecii Staining Reagent*1 Dropper bottle(2.2 mL)

• FITC-labeled monoclonal antibodies (murine)

• Counterstain (Evans Blue)• Sodium azide**• Protein-stabilized buffer

Mix gently before use.

R2 • Mounting Medium1 Dropper bottle (3.5 mL)

• Buffered glycerol• Formaldehyde***• Anti-quencher

Ready to use as supplied.

R3 • Fluorescence Microscopy Slides24 (2 wells each)

• Fluorescence microscopy slides

Wipe with lint-free tissue before use.

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• Pasteur pipettes.• Centrifuge or microfuge.• 15 mL centrifuge tubes or 1.5 mL microfuge tubes.• 37±1°C incubator.• Biological safety cabinet (recommended).• Fluorescence-quality immersion oil, if needed.• Fluorescence microscope with 200-250X and 400-630X

magnification, equipped with a filter system for fluores-cein isothiocyanate (FITC). A wide variety of filter sets forreading FITC are available from different manufacturers.Wide band (420-490 nm) or narrow band (470-490 nm)excitation wavelengths with a 515 nm dichromatic mirrorand a 515 nm barrier filter is advisable. The narrow bandexcitation wavelength will diminish background fluores-cence. Variations in the wattage, intensity and alignmentof the bulb and differences in type of illuminator and filtersmay affect the performance of the test.

• Control slides containing Pneumocystis jirovecii organ-isms (see Preparation of Control Slides section).

• Paper toweling or aspiration system.

For Induced Sputum Specimens• Dithiothreitol (DTT): A liquid preparation of 0.1% (6.5x10-3

moles/L). For example, Stat-Pak™ Sputolysin availablefrom Caldon Biotech; Use as directed.

• Phosphate Buffered Saline (PBS).

PRECAUTIONSFor in vitro diagnostic use only.

For professional use only.

1. This test should only be performed by personnel who areproperly trained in the handling of clinical specimens thatmay contain HIV-1. The slides should be examined by per-sonnel who have experience in reading immunofluorescenceassays.

2. Handle all clinical specimens as potentially infectiousmaterial.

3. Procedures that create aerosols should be conducted in abiological safety cabinet. All materials should be disinfectedafter use or prior to disposal.

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4. Use good laboratory technique when handling specimens orthe MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immu-nofluorescence Test Kit reagents. Do not eat, drink or smokein the laboratory. Do not mouth pipette. Employ Standardand Universal Precautions; handle these reagents, all humanblood and specimens as if capable of transmitting infectiousdisease, in a Biosafety Level 2 laboratory, applying theguidelines from the current CDC/NIH Biosafety in Microbio-logical and Biomedical Laboratories21 or the WHO LaboratoryBiosafety Manual20 or equivalent local, regional and nationalregulations. Avoid contaminating the reagents with microbesor other substances.

5. The Staining Reagent contains Evans Blue, which is an irri-tant. Avoid spilling or splashing the Staining Reagent on skinor clothing. Flush any areas which may have come in contactwith the Staining Reagent thoroughly with water.

6. The Mounting Medium has ≤ 2% formalin that contains≤ 0.8% formaldehyde, which, according to the United StatesNational Toxicity Program (NTP), may be reasonably antici-pated to be carcinogenic. However, existing data do notconclusively establish the carcinogenicity of formaldehyde atthe concentrations used in this kit (< 1%). Avoid spilling orsplashing the Mounting Medium on skin or clothing. Flushany areas which may have come in contact with the Mount-ing Medium thoroughly with water. ≤ 2% Formalin (≤ 0.8%Formaldehyde [HCHO]):

7. The Staining Reagent contains sodium azide. Sodium azidemay react with lead and copper plumbing to form metalazides that are highly explosive. If the Staining Reagent is

H317: May cause an allergic skin reaction.P280: Wear protective gloves/protective clothing/

eye protection/face protection.P302 + P352: IF ON SKIN: Wash with plenty of soap and

water.P333 + P313: If skin irritation or rash occurs: Get medical

advice/attention.

WARNINGH317*

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disposed of in the sink, flush plumbing with a large volume ofwater to prevent azide buildup. 0.1% Sodium Azide [NaN3]:

The Safety Data Sheet is available at bio-rad.com and uponrequest.

8. Dispose of product packaging and waste in accordance withall applicable local, regional, national, and international regu-lations.

9. Clean all reusable items thoroughly between uses.

10.Mix Staining Reagent gently before each test.

11.Use a separate slide for each patient specimen to preventany cross-contamination of Pneumocystis jirovecii organ-isms. It is also recommended that each slide be washedseparately.

12.Slides should be prepared using acetone fixation. Formalinfixed slides or ethanol fixed slides are not recommended forthis test.

REAGENT STABILITY AND STORAGEDo not use the kit or any of the components beyond the expira-tion date. Store Staining Reagent in the dark at 2-8°C.

Store reagents at 2-8°C. Slides should be stored at 2-28°C. Ifstored at the specified temperatures, opened or unopenedreagents are stable for the printed shelf life.

PNEUMOCYSTIS jirovecii TEST PROCEDURE

Specimen CollectionClinical specimens should be freshly collected using standardmethods for obtaining induced sputum, bronchial wash orbronchoalveolar lavage.17,18 The specimen should be processedimmediately. Reserve a portion of the specimen for culture, ifneeded.

WARNINGH303: May be harmful if swallowed.H313: May be harmful in contact with skin.P312: Call a POISON CENTER or doctor/physician if you feel

unwell.

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Preparation of Clinical Specimens Prior to Fluorescence Staining

Induced Sputum Specimens

1a.Put 2 to 3 mL of sputum in a 15 mL centrifuge tube. Add anequal volume of dithiothreitol reagent. Vortex the tube andincubate for 5-10 minutes at 37°C or 15 minutes at roomtemperature (RT=15-30°C).

- OR -

1b.If the sample is too viscous to easily transfer to a centrifugetube, add an estimated equal volume of dithiothreitol reagentdirect to the collection vessel, stir vigorously to mix, andincubate for 5-10 minutes at 37°C or 15 minutes at roomtemperature (RT=15-30°C). Transfer to a centrifuge tube assoon as possible during the incubation and vortex beforeproceeding.

2. Add a volume of approximately 5 to 6 mL of PBS pH 7.2,equal to that of the sputum plus DTT combined, to the tubeand thoroughly vortex.

3. Centrifuge at 1500 x G for 5 minutes.

4. Carefully decant or aspirate the supernatant, leaving 0.5 mLor less of the pellet and supernatant in the tube. Steps 2, 3and 4 may be repeated, if necessary, to remove all traces ofmucus.

5. Resuspend the pellet thoroughly. Using a pipettor, apply onedrop (approximately 25-50 μL) onto a microscope slide pro-vided with the kit. Spread the drop evenly over the well withthe side of a pipet tip taking care not to scratch the slide.Allow to air dry. A slide warmer at 37°C can be used to has-ten the specimen drying.

6. Fix for approximately 10 minutes in acetone. Slides may beprocessed immediately or frozen at -20°C for up to onemonth before staining.

Bronchial Wash and Bronchoalveolar Lavage Specimens

Non-mucoid samples such as bronchial washes or bronchoal-veolar lavages will probably not require the mucolytic proce-

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dure. For these samples, the protocol can begin withcentrifugation, step 3 above, in a 15 mL centrifuge tube or1.5 mL microfuge tube.

NOTE: Alternatively, a cytospin centrifuge may be used to pre-pare slides from induced sputum, bronchial wash or BAL speci-mens.13 When the cytospin procedure is followed, controlsshould be processed in the same manner. Slides prepared witha cytospin centrifuge are fixed for ten minutes in acetone andstained as described below in the Fluorescence Staining Proce-dure section.

PREPARATION OF CONTROL SLIDESA positive control slide should be included each time the test isperformed. Bronchial wash and bronchoalveolar lavage speci-mens are the most suitable for use as a positive control. Thespecimens chosen should previously have been confirmed aspositive for Pneumocystis jirovecii by histological stain. Followthe procedures outlined in the Preparation of Clinical Speci-mens Prior to Fluorescence Staining section. After acetone fixa-tion, the slides may be frozen at -20°C for up to one year.Alternately, use a known positive sample as a positive control.For the test to be considered valid, the control slide must stainappropriately and be interpreted as described in the Evaluationof Test Results section.

FLUORESCENCE STAINING PROCEDURE1. If the slides have been frozen, allow them to reach room

temperature before proceeding with the staining procedure.

2. Place the slide in a humidified chamber.

3. Add sufficient amount of Pneumocystis jirovecii StainingReagent to each well containing a specimen (2-3 drops) tocover the specimen, keeping within the boundary of the well.

4. Incubate for 30-35 minutes at 37±1°C. Do not allow thePneumocystis jirovecii Staining Reagent to dry on the slideduring the procedure.

5. Remove excess Pneumocystis jirovecii Staining Reagent bydraining the slide onto clean paper toweling or by aspiration.

6. Wash the slide by placing it in a rack and soaking it in deion-ized water or tap water for one minute with gentle, intermit-

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tent agitation. Remove excess water by draining the slideonto clean paper toweling or by aspiration.

7. Dry the slide thoroughly by air drying or with a slide warmerat 37±1°C.

8. Apply one to two drops of Mounting Medium to the slide andapply a coverslip, being careful to avoid the formation of airbubbles.

9. Examine the slides within 2-3 hours of staining. After read-ing, the slides may be stored overnight in the dark at 2-8°C,or for up to 6 months in the dark at -20°C or lower. Slidesstored in the cold must be allowed to reach room tempera-ture before reading to prevent condensation from obscuringthe sample.

10.For negative results to be valid, confirm by light microscopythat specimen is present on the slide.

EXAMINATION OF STAINED SLIDESScan each well at 200X or greater magnification. If fluorescenceis observed, use 400X or 630X (with immersion oil) magnifica-tion to confirm the characteristic staining patterns described inthe Evaluation of Test Results section. Confirm that specimen ispresent on each slide after the completion of the assay.

EVALUATION OF TEST RESULTS1. Specimens infected with Pneumocystis jirovecii contain large

round-to-elliptical shaped cysts, found both individually andin clusters, which will be stained bright apple-green. Two ormore well-defined, fluorescing cysts must be observedfor the specimen to be considered positive.

2. Sporozoites and trophozoites may also be stained. Theyappear as small, crescent-shaped or pleomorphic struc-tures. Brightly staining, amorphous, extracellular matrix mayalso be present. Specimens containing only brightly-stain-ing, amorphous material or typical sporozoite and trophozo-ite forms should prompt active searching for cysts before adefinite diagnosis can be made. In the absence of cysts, thespecimen should be suspected as positive for Pneumocys-tis jirovecii, and another specimen should be obtained forstaining.

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3. Other organisms and cellular material from respiratory speci-mens should be counterstained red to orange-red or gold.

4. A specimen is considered negative if characteristic fluores-cence, as described above, is not observed.

5. A negative result on an induced sputum should be verifiedon a subsequent specimen collected by bronchial wash orbronchoalveolar lavage.

LIMITATIONS OF THE PROCEDUREA negative result does not exclude the possibility of Pneumo-cystis jirovecii infection in the patient. Sample collection andpreparation are critical steps in the testing procedure. Collect-ing the sample at an improper time during the course of dis-ease, by an inappropriate method or with improper handling ofthe specimen, or misusing the reagents provided can result infailure to detect Pneumocystis jirovecii. Diagnosis should bemade in conjunction with clinical symptoms.

Run only one specimen on each slide. Care must be taken toprocess, fix, stain, and wash each specimen separately to pre-vent any possible wash-over of Pneumocystis jirovecii organ-isms to other slides.

Excess mucus in specimens may prevent adequate staining.Certain thick sputum smears may prevent adequate staining.Care must be taken to make smears as thin as possible. Non-specific trapping of the Pneumocystis jirovecii Staining Reagentmay occur if the specimen is not adequately washed.

In order for negative results to be considered valid, the pres-ence of specimen on the slide must be confirmed by lightmicroscopy.

If the bronchial wash or bronchoalveolar lavage specimen isnegative, but the patient continues to exhibit symptoms associ-ated with respiratory illness, other etiologic agents should besuspected and appropriate diagnostic procedures (includingculture) should be performed.

PERFORMANCE CHARACTERISTICSThree investigators participated in a clinical trial to evaluate theMONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunofluo-rescence test. Clinical specimens were collected from patientswho were suspected of having Pneumocystis jirovecii pneumo-

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nia. An induced sputum specimen was collected from eachpatient and was tested in parallel with the MONOFLUO™ (MF)test and by histologic stain (reference method). Two histologicstains were used: a Wright-Giemsa stain [Diff-Quik™ (DQ)] andtoluidine blue O (TBO).

If the induced sputum was positive with the reference method,the result was considered indicative of infection with P. jirovecii.If the result with the reference method was negative on theinduced sputum specimen, a bronchoalveolar lavage (BAL)specimen was obtained and tested. If the BAL was positive thepatient was positive for P. jirovecii. If the BAL was negative thepatient was considered negative for P. jirovecii. A total of 100patients were included in the analysis. The results obtained areshown in Table 2.

**These six were positive on induced sputum with the MONOFLUO™ test and were also positive on the BAL. BALs were obtained on the six patients because the TBO result on the sputum was negative.

The number of patients classified as positive and negative withthe MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunoflu-orescence Test Kit in comparison to the reference test is pre-sented in Table 3.

Table 2: Patient Results

Sites 1&2 Site 3

Specimen DQ MF TBO MF

Patients Positive for P. jirovecii 62 63 14 14

• by Induced Sputum 53 53 8 14• by BAL 9 10 6 6**

Table 3: Comparison of the Result Obtained Using Histologic Stain with the Result Obtained with the MONOFLUO™ Immunofluorescence Test (Includes both Induced Sputum and BAL)

Histological Stain

+ –

Monofluo™+ 76 1

– 0 23

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Of the 100 patients tested, 76 were positive for P. jirovecii and24 were negative for P. jirovecii as determined by histologicstaining. The MONOFLUO™ test correctly identified all 76 ofthe positive patients and was negative on 23 patients who werenegative for P. jirovecii. One induced sputum specimen wasnegative by both the MONOFLUO™ test and the reference test,but the BAL from this patient was negative by the reference andpositive with the MONOFLUO™ test.

Based on these clinical data, the performance characteristics ofthe MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunoflu-orescence Test Kit were:

Sensitivity = 100%Specificity = 95.8%

13

CROSS-REACTIVITYThe following bacterial and fungal cultures from culture isolatesand from specimens were tested for cross-reactivity with theMONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunofluo-rescence Test Kit and were found to be nonreactive:

Bacteria

Fungi

Acinetobacter calcoaceticusActinomyces israeliiBacteroides spp.Bifidobacterium dentiumCampylobacter sputorumCardiobacterium hominisCorynebacterium ulceransEggerthella lentaEnterobacter cloacaeEscherichia coliFusobacterium nucleatumHaemophilus spp.Klebsiella pneumoniaeLactobacillus catenaformisLegionella pneumophilaLeptotrichia buccalisMicrococcus luteusMoraxella lacunataMoraxella (Branhamella) catarrhalis

Mycobacterium tuberculosisMycobacterium kansasiiMycobacterium avium-intracellu-lareNeisseria spp.Nocardia asteroidesPepto streptococcus Proteus mirabilisPseudomonas spp.Rothia dentocariosaSelenomonas sputigenaStaphylococcus aureusStaphylococcus spp.Streptococcus spp. Streptococcus pneumoniae Streptococcus, Groups A,B,C,F,GTreponema denticolaVeillonella parvula

Aspergillus nigerAspergillus flavusAspergillus fumigatusAspergillus terreusAspergillus versicolorCandida albicansCandida kruseiCandida guillermondii

Candida parapsilosisCandida pseudotropicalisCandida tropicalisCryptococcus neoformansHistoplasma capsulatumSaccharomyces cerevisiaeTorulopsis glabrataTrichosporon spp.

111

REFERENCES/BIBLIOGRAPHIE/REFERENCIAS/LITERATUR/BIBLIOGRAFIA/REFERENCER/REFERENSER

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9. Brenner M, Ognibene FP, et al: Prognostic factors and lifeexpectancy of patients with acquired immunodeficiencysyndrome and Pneumocystis carinii pneumonia. Am RevRespir Dis 136:1199-1206, 1987.

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11.Chandra P, Delaney MD, Tuazon CU: Role of special stainsin the diagnosis of Pneumocystis carinii infection frombronchial washing specimens in patients with the acquiredimmune deficiency syndrome. Acta Cytol 32(1):105-108,1988.

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14.Lim SK, Eveland WC, Porter RJ: Direct fluorescent-antibodymethod for the diagnosis of Pneumocystis cariniipneumonitis from sputa or tracheal aspirates from humans.Appl Microbiol 27(1):144-149, 1974.

15.Kovacs JA, Gill V, et al: Prospective evaluation of amonoclonal antibody in diagnosis of Pneumocystis cariniipneumonia. Lancet 2(8497):1-3, July 5, 1986.

16.Gill VJ, Evans G, et al: Detection of Pneumocystis carinii byfluorescent-antibody stain using a combination of threemonoclonal antibodies. J Clin Microbiol 25(10):1837-1840,1987.

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18.Broaddus C, Dake MD, Stulberg MS, Blumenfeld W, HadleyWK, Golden JA, Hopewell PC: Bronchoalveolar lavage andtransbronchial biopsy for the diagnosis of pulmonaryinfections in the acquired immunodeficiency syndrome. AnnIntern Med 102:747-752, 1985.

19.Medrano F, Montes-Cano M, Conde M, de la Horra C,Respaldiza N, Gasch A, Perez-Lozano MJ, Varela JM,Calderon EJ: Pneumocystis jirovecii in general population.Emerg Infect Dis 11(2):245-50,2005.

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21.US Department of Health and Human Services. Biosafety inMicrobiological and Biomedical Laboratories. 5th ed.Washington, DC: US Government Printing Office, HHSPublication No. (CDC) 21-1112. December 2009.

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