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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WARNING ATTENTION ATENCIÓN ACHTUNG ATTENZIONE ATENÇÃO ADVARSEL VARNING INTENDED USE The MONOFLUO™ Pneumocystis Immunofluorescent Antibody Test Kit is to be used for the detection of Pneumocystis jirovecii (P. carinii) cysts and trophozoites in specimens collected from the respiratory tract. SUMMARY AND EXPLANATION Pneumocystis jirovecii (P. carinii) is a unicellular, eucaryotic organism that is present in the lungs of many mammalian species, including man. The Pneumocystis organisms found in humans were originally referred to as P. carinii f. sp. hominis, the subspecies name used to distinguish the Pneumocystis organisms found in humans from Pneumocystis organisms found in other mammals. Recently the Pneumocystis organism found in humans was recognized as a distinct species and renamed Pneumocystis jirovecii.1 The organism is spread by airborne routes, usually causing asymptomatic infection. Exposure during childhood may result in mild or subclinical cases, with the organism existing in latent state through adulthood. In individuals with compromised immune systems, the organism becomes opportunistic, causing diffuse, interstitial pneumonia.2,3 Individuals at risk for Pneumocystis jirovecii include premature infants, individuals with immune deficiency disorders (such as acquired immunodeficiency syndrome), and those receiving immunosuppressive drugs such as corticosteroids. Pneumo- cystis jirovecii is the most common opportunistic infectious agent in AIDS patients, with almost 80% suffering from the infection.4,5,6,7 Morphological studies of the organism have revealed three forms 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, which then mature into larger, pleomorphic trophozoites (2-8 μm in diameter) before encysting to repeat the cycle.2,7 The cysts, with their distinct morphologic characteristics, are most frequently associated with presence of Pneumocystis jirovecii. Rapid clinical diagnosis of Pneumocystis jirovecii is of utmost importance as the organisms quickly infiltrate lung tissue caus- ing dyspnea, fever, and cough.8 Early detection can facilitate

1 initiation of appropriate treatment and may improve chances of patient 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 of these stains, proper identification of the organism may be diffi- cult. A more invasive technique for specimen collection such as bronchoalveolar lavage is often required to ensure the presence of Pneumocystis jirovecii.12 The use of a direct, fluorescent-antibody procedure with induced sputum, bronchial wash or bronchoalveolar lavage samples 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 jirovecii cysts, 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 of Pneumocystis jirovecii cysts and trophozoites directly in patient specimens. PRINCIPLES OF THE PROCEDURE The MONOFLUO™ Pneumocystis jirovecii Staining Reagent contains murine monoclonal antibodies labeled with fluorescein isothiocyanate (FITC). These antibodies react with all Pneumo- cystis jirovecii forms (cysts, sporozoites and trophozoites) and also with the extracellular matrix present in infected specimens Slides are prepared for microscopy from clinical specimens as described in the Preparation of Clinical Specimens Prior to Flu- orescence Staining section. Following fixation, the slides are stained with the MONOFLUO™ Pneumocystis jirovecii Staining Reagent. The Staining Reagent binds to any Pneumocystis jir- ovecii organisms present in the specimen. A subsequent rinse step removes unbound Staining Reagent from the specimen.

2 The slides are then mounted with the MONOFLUO™ Mounting Medium included in the kit. The Mounting Medium contains an anti-quencher which increases the length of time slides may be examined before the fluorescence fades. When viewed with a fluorescence microscope, infected speci- mens fluoresce bright apple-green. Stained cysts appear as large, round-to-elliptical structures found both individually and in clusters. Sporozoites and trophozoites may also be stained, appearing as relatively small, crescent-shaped or pleomorphic structures. In addition, the amorphous, extracellular matrix should also fluoresce brightly. Other organisms and cellular material from respiratory specimens are counterstained red to orange-red or gold without characteristic fluorescence of Pneu- mocystis jirovecii cysts. PRODUCT DESCRIPTION Table 1: Materials Provided Store reagents at 2-8°C. Slides should be stored at 2-28°C. Component Contents Preparation R1 • Pneumocystis • FITC-labeled monoclonal Mix gently jirovecii Staining antibodies (murine) before use. Reagent* • Counterstain (Evans Blue) 1 Dropper bottle • Sodium azide** (2.2 mL) • Protein-stabilized buffer R2 • Mounting • Buffered glycerol Ready to use as Medium • Formaldehyde*** supplied. 1 Dropper bottle • Anti-quencher (3.5 mL) R3 • Fluorescence • Fluorescence microscopy Wipe with lint- Microscopy Slides slides free tissue 24 (2 wells each) before use. * 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.

3 • 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 for reading FITC are available from different manufacturers. Wide band (420-490 nm) or narrow band (470-490 nm) excitation wavelengths with a 515 nm dichromatic mirror and a 515 nm barrier filter is advisable. The narrow band excitation wavelength will diminish background fluores- cence. Variations in the wattage, intensity and alignment of the bulb and differences in type of illuminator and filters may 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 available from Caldon Biotech; Use as directed. • Phosphate Buffered Saline (PBS). PRECAUTIONS For in vitro diagnostic use only. For professional use only.

1. This test should only be performed by personnel who are properly trained in the handling of clinical specimens that may contain HIV-1. The slides should be examined by per- sonnel who have experience in reading immunofluorescence assays. 2. Handle all clinical specimens as potentially infectious material. 3. Procedures that create aerosols should be conducted in a biological safety cabinet. All materials should be disinfected after use or prior to disposal.

4 4. Use good laboratory technique when handling specimens or the MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immu- nofluorescence Test Kit reagents. Do not eat, drink or smoke in the laboratory. Do not mouth pipette. Employ Standard and Universal Precautions; handle these reagents, all human blood and specimens as if capable of transmitting infectious disease, in a Biosafety Level 2 laboratory, applying the guidelines from the current CDC/NIH Biosafety in Microbio- logical and Biomedical Laboratories21 or the WHO Laboratory Biosafety Manual20 or equivalent local, regional and national regulations. Avoid contaminating the reagents with microbes or other substances. 5. The Staining Reagent contains Evans Blue, which is an irri- tant. Avoid spilling or splashing the Staining Reagent on skin or clothing. Flush any areas which may have come in contact with the Staining Reagent thoroughly with water. 6. The Mounting Medium has ≤ 2% formalin that contains ≤ 0.8% formaldehyde, which, according to the United States National Toxicity Program (NTP), may be reasonably antici- pated to be carcinogenic. However, existing data do not conclusively establish the carcinogenicity of formaldehyde at the concentrations used in this kit (< 1%). Avoid spilling or splashing the Mounting Medium on skin or clothing. Flush any areas which may have come in contact with the Mount- ing Medium thoroughly with water. ≤ 2% Formalin (≤ 0.8% Formaldehyde [HCHO]): H317: May cause an allergic skin reaction. P280: Wear protective gloves/protective clothing/ WARNING eye protection/face protection. H317* P302 + P352: IF ON SKIN: Wash with plenty of soap and water. P333 + P313: If skin irritation or rash occurs: Get medical advice/attention. 7. The Staining Reagent contains sodium azide. Sodium azide may react with lead and copper plumbing to form metal azides that are highly explosive. If the Staining Reagent is

5 disposed of in the sink, flush plumbing with a large volume of water to prevent azide buildup. 0.1% Sodium Azide [NaN3]: WARNING H303: 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. The Safety Data Sheet is available at bio-rad.com and upon request. 8. Dispose of product packaging and waste in accordance with all 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 prevent any cross-contamination of Pneumocystis jirovecii organ- isms. It is also recommended that each slide be washed separately. 12.Slides should be prepared using acetone fixation. Formalin fixed slides or ethanol fixed slides are not recommended for this test. REAGENT STABILITY AND STORAGE Do 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. If stored at the specified temperatures, opened or unopened reagents are stable for the printed shelf life. PNEUMOCYSTIS jirovecii TEST PROCEDURE

Specimen Collection Clinical specimens should be freshly collected using standard methods for obtaining induced sputum, bronchial wash or bronchoalveolar lavage.17,18 The specimen should be processed immediately. Reserve a portion of the specimen for culture, if needed.

6 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 an equal volume of dithiothreitol reagent. Vortex the tube and incubate for 5-10 minutes at 37°C or 15 minutes at room temperature (RT=15-30°C). - OR -

1b.If the sample is too viscous to easily transfer to a centrifuge tube, add an estimated equal volume of dithiothreitol reagent direct to the collection vessel, stir vigorously to mix, and incubate for 5-10 minutes at 37°C or 15 minutes at room temperature (RT=15-30°C). Transfer to a centrifuge tube as soon as possible during the incubation and vortex before proceeding. 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 tube and thoroughly vortex. 3. Centrifuge at 1500 x G for 5 minutes. 4. Carefully decant or aspirate the supernatant, leaving 0.5 mL or less of the pellet and supernatant in the tube. Steps 2, 3 and 4 may be repeated, if necessary, to remove all traces of mucus. 5. Resuspend the pellet thoroughly. Using a pipettor, apply one drop (approximately 25-50 μL) onto a microscope slide pro- vided with the kit. Spread the drop evenly over the well with the 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 be processed immediately or frozen at -20°C for up to one month 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-

7 dure. For these samples, the protocol can begin with centrifugation, step 3 above, in a 15 mL centrifuge tube or 1.5 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, controls should be processed in the same manner. Slides prepared with a cytospin centrifuge are fixed for ten minutes in acetone and stained as described below in the Fluorescence Staining Proce- dure section. PREPARATION OF CONTROL SLIDES A positive control slide should be included each time the test is performed. Bronchial wash and bronchoalveolar lavage speci- mens are the most suitable for use as a positive control. The specimens chosen should previously have been confirmed as positive for Pneumocystis jirovecii by histological stain. Follow the 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 stain appropriately and be interpreted as described in the Evaluation of Test Results section. FLUORESCENCE STAINING PROCEDURE 1. 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 Staining Reagent to each well containing a specimen (2-3 drops) to cover the specimen, keeping within the boundary of the well. 4. Incubate for 30-35 minutes at 37±1°C. Do not allow the Pneumocystis jirovecii Staining Reagent to dry on the slide during the procedure. 5. Remove excess Pneumocystis jirovecii Staining Reagent by draining 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-

8 tent agitation. Remove excess water by draining the slide onto clean paper toweling or by aspiration. 7. Dry the slide thoroughly by air drying or with a slide warmer at 37±1°C. 8. Apply one to two drops of Mounting Medium to the slide and apply a coverslip, being careful to avoid the formation of air bubbles. 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. Slides stored in the cold must be allowed to reach room tempera- ture before reading to prevent condensation from obscuring the sample. 10.For negative results to be valid, confirm by light microscopy that specimen is present on the slide. EXAMINATION OF STAINED SLIDES Scan each well at 200X or greater magnification. If fluorescence is observed, use 400X or 630X (with immersion oil) magnifica- tion to confirm the characteristic staining patterns described in the Evaluation of Test Results section. Confirm that specimen is present on each slide after the completion of the assay. EVALUATION OF TEST RESULTS 1. Specimens infected with Pneumocystis jirovecii contain large round-to-elliptical shaped cysts, found both individually and in clusters, which will be stained bright apple-green. Two or more well-defined, fluorescing cysts must be observed for the specimen to be considered positive. 2. Sporozoites and trophozoites may also be stained. They appear as small, crescent-shaped or pleomorphic struc- tures. Brightly staining, amorphous, extracellular matrix may also be present. Specimens containing only brightly-stain- ing, amorphous material or typical sporozoite and trophozo- ite forms should prompt active searching for cysts before a definite diagnosis can be made. In the absence of cysts, the specimen should be suspected as positive for Pneumocys- tis jirovecii, and another specimen should be obtained for staining.

9 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 verified on a subsequent specimen collected by bronchial wash or bronchoalveolar lavage. LIMITATIONS OF THE PROCEDURE A negative result does not exclude the possibility of Pneumo- cystis jirovecii infection in the patient. Sample collection and preparation 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 of the specimen, or misusing the reagents provided can result in failure to detect Pneumocystis jirovecii. Diagnosis should be made in conjunction with clinical symptoms. Run only one specimen on each slide. Care must be taken to process, 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 Reagent may 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 light microscopy. If the bronchial wash or bronchoalveolar lavage specimen is negative, but the patient continues to exhibit symptoms associ- ated with respiratory illness, other etiologic agents should be suspected and appropriate diagnostic procedures (including culture) should be performed. PERFORMANCE CHARACTERISTICS Three investigators participated in a clinical trial to evaluate the MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunofluo- rescence test. Clinical specimens were collected from patients who were suspected of having Pneumocystis jirovecii pneumo-

10 nia. An induced sputum specimen was collected from each patient and was tested in parallel with the MONOFLUO™ (MF) test and by histologic stain (reference method). Two histologic stains were used: a Wright-Giemsa stain [Diff-Quik™ (DQ)] and toluidine 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 the induced sputum specimen, a bronchoalveolar lavage (BAL) specimen was obtained and tested. If the BAL was positive the patient was positive for P. jirovecii. If the BAL was negative the patient was considered negative for P. j ir o v e ci i . A total of 100 patients were included in the analysis. The results obtained are shown in Table 2. Table 2: Patient Results Sites 1&2 Site 3 Specimen DQ MF TBO MF Patients Positive for P. 62 63 14 14 jirovecii • by Induced Sputum 53 53 8 14 • by BAL 91066**

**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 with the MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunoflu- orescence Test Kit in comparison to the reference test is pre- sented in Table 3. 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 +– + 76 1 Monofluo™ – 023

11 Of the 100 patients tested, 76 were positive for P. jirovecii and 24 were negative for P. jirovecii as determined by histologic staining. The MONOFLUO™ test correctly identified all 76 of the positive patients and was negative on 23 patients who were negative for P. jirovecii. One induced sputum specimen was negative by both the MONOFLUO™ test and the reference test, but the BAL from this patient was negative by the reference and positive with the MONOFLUO™ test. Based on these clinical data, the performance characteristics of the MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunoflu- orescence Test Kit were: Sensitivity = 100% Specificity = 95.8%

12 CROSS-REACTIVITY The following bacterial and fungal cultures from culture isolates and from specimens were tested for cross-reactivity with the MONOFLUO™ Pneumocystis jirovecii (P. carinii) Immunofluo- rescence Test Kit and were found to be nonreactive: Bacteria Acinetobacter calcoaceticus Mycobacterium tuberculosis Actinomyces israelii Mycobacterium kansasii Bacteroides spp. Mycobacterium avium-intracellu- Bifidobacterium dentium lare Campylobacter sputorum Neisseria spp. Cardiobacterium hominis Nocardia asteroides Corynebacterium ulcerans Pepto streptococcus Eggerthella lenta Proteus mirabilis Enterobacter cloacae Pseudomonas spp. Escherichia coli Rothia dentocariosa Fusobacterium nucleatum Selenomonas sputigena Haemophilus spp. Staphylococcus aureus Klebsiella pneumoniae Staphylococcus spp. Lactobacillus catenaformis Streptococcus spp. Legionella pneumophila Streptococcus pneumoniae Leptotrichia buccalis Streptococcus, Groups A,B,C,F,G Micrococcus luteus Treponema denticola Moraxella lacunata Veillonella parvula Moraxella (Branhamella) catarrhalis Fungi Aspergillus niger Candida parapsilosis Aspergillus flavus Candida pseudotropicalis Aspergillus fumigatus Candida tropicalis Aspergillus terreus Cryptococcus neoformans Aspergillus versicolor Histoplasma capsulatum Candida albicans Saccharomyces cerevisiae Candida krusei Torulopsis glabrata Candida guillermondii Trichosporon spp.

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

1. Stringer JR, Beard CB, Miller RF, Wakefield AE: A New Name (Pneumocystis jiroveci) for Pneumocystis from Humans. Emerg Infect Dis 8(9):891-896, 2002. 2. Mills J: Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS. Rev Infect Dis 8(6):1001- 1011, 1986. 3. Kovacs JA, Hiemenz JW, Macher AM, et al: Pneumocystis carinii pneumonia: A comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med 100:663-671, 1984. 4. Posner D, Khan FA: Respiratory infections in AIDS: A comprehensive care approach. J Respir Dis pp 83-94, June 1987. 5. Simone JW, Holland E, Johnson W: Fatalities during remission of childhood leukemia. Blood 39:759-770, 1972. 6. Gajdusek DC: Pneumocystis carinii-etiologic agent of interstitial plasma cell pneumonia of premature and young infants. Pediatrics 19:543-565, 1957. 7. Suffredini AF, Masur H: Pneumocystis carinii infection in AIDS, in Wormser GP, Stahl RE, Bottone EJ (eds): AIDS, Aquired Immune Deficiency Syndrome, and Other Manifestations of HIV Infection. Park Ridge, NJ, Noyes Publications, 1987, chap 23. 8. Walzer PD, Perl DP, et al: Pneumocystis carinii pneumonia in the United States. Epidemiologic, diagnostic, and clinical features. Ann Intern Med 80:83-93, 1974. 9. Brenner M, Ognibene FP, et al: Prognostic factors and life expectancy of patients with acquired immunodeficiency syndrome and Pneumocystis carinii pneumonia. Am Rev Respir Dis 136:1199-1206, 1987. 10.Chaisson RE, Hopewell PC: Empiric diagnosis of pneumocystis pneumonia. JAMA 258(23):3385, 1987.

111 11.Chandra P, Delaney MD, Tuazon CU: Role of special stains in the diagnosis of Pneumocystis carinii infection from bronchial washing specimens in patients with the acquired immune deficiency syndrome. Acta Cytol 32(1):105-108, 1988. 12.Nieberg RK, Gong Jr: Diagnosis of Pneumocystis carinii pneumonia by bronchoalveolar lavage in patients with the acquired immunodeficiency syndrome. Amer Clin Prod Rev 6:28-31, 1987. 13.Kovacs JA, Ng VL, et al: Diagnosis of Pneumocystis carinii pneumonia: Improved detection in sputum with use of monoclonal antibodies. NEngl J Med 318(10):589-593, 1988. 14.Lim SK, Eveland WC, Porter RJ: Direct fluorescent-antibody method for the diagnosis of Pneumocystis carinii pneumonitis from sputa or tracheal aspirates from humans. Appl Microbiol 27(1):144-149, 1974. 15.Kovacs JA, Gill V, et al: Prospective evaluation of a monoclonal antibody in diagnosis of Pneumocystis carinii pneumonia. Lancet 2(8497):1-3, July 5, 1986. 16.Gill VJ, Evans G, et al: Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J Clin Microbiol 25(10):1837-1840, 1987. 17.Bigby TD, Margolskee D, Curtis JL, Michael PF, Sheppard D, Hadley WK, Hopewell PC: The usefulness of induced sputum in the diagnosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Am Rev Respir Dis 133:515-518, 1986. 18.Broaddus C, Dake MD, Stulberg MS, Blumenfeld W, Hadley WK, Golden JA, Hopewell PC: Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern 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.

112 20.World Health Organization. Laboratory Biosafety Manual. 3rd ed. Geneva: World Health Organization, 2004. 21.US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, DC: US Government Printing Office, HHS Publication No. (CDC) 21-1112. December 2009.

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Also in: Gladesville, Australia, Ph. 61-2-9914-2800, Fx. 61-2-9914- 2888 Vienna, Austria, Ph. 43-1-877-8901, Fx. 43-1-876-5629 Nazareth, Belgium, Ph. 32-9-385-5511, Fx. 32-9-385-6554 Rio de Janeiro, Brazil, Ph. 5521-3237-9400, Fx. 5521-2527-3099 Montréal, Canada, Ph.1-514-334-4372, Fx. 1-514-334-4415 Shanghai, China, Ph. 86-21-64260808, Fx. 86-21-64264988 Prague, Czech Republic, Ph. 420-241-430-532, Fx. 420-241-431-642 Symbion Science Park, Denmark, Ph. +45-4452-1000, Fx. +45-4452-1001 Espoo, Finland, Ph. 358-9-804-22-00, Fx. 358-9-7597-5010 Marnes-la-Coquette, France, Ph. 33-1-47-95-60-00 Fx. 33-1-47-41-91-33 Munich, Germany, Ph. +49-(0)89-318-840, Fx. +49-(0)89-318-84100 Athens, Greece, Ph. 30- 210-7774396, Fx. 30-210-7774376 Quarry Bay, Hong Kong, Ph. 852- 2789-3300, Fx. 852-2789-1290 Budapest, Hungary, Ph. +36-1-459- 6100, Fx. +36-1-459-6101 Haryana, India, Ph. 91-124-4029300, Fx. 91- 124-2398115 Rishon Le Zion, Israel, Ph. 972-3-9636050, Fx. 972-3- 9514129 Milan, Italy, Ph. +39-02-216091, Fx. +39-02-21609-398 Tokyo, Japan, Ph. 81-3-6361-7070, Fx. 81-3-5463-8483 Seoul, Korea, Ph. 82-2-3473-4460, Fx. 82-2-3472-7003 Mexico D.F., Mexico, Ph. 52(55)5200-0520, Fx. 52(55)1107-7246 Veenendaal, The Netherlands, Ph. +31-318-540666, Fx. +31-318-542216 Auckland, New Zealand, Ph. 64-9-415-2280, Fx. 64-9-415-2284 Oslo, Norway, Ph. 47-23-38-41- 30, Fx. 47-23-38-41-39 Warsaw, Poland, Ph. 48-22-3319999, Fx. 48- 22-3319988 Amadora, Portugal, Ph. 351-21-472-7700, Fx. 351-21- 472-7777 Moscow, Russia, Ph. 7-495-721-14-04, Fx. 7-495-721-14-12 Singapore, Ph. 65-6415-3188, Fx. 65-6415-3189 Johannesburg, South Africa, Ph. 27-11-442-85-08, Fx. 27-11-442-85-25 Madrid, Spain, Ph. 34-91-590-5200, Fx. 34-91-590-5211 Sundbyberg, Sweden, Ph. 46-8-555-127-00, Fx. 46-8-555-127-80 Reinach BL, Switzerland, Ph. 41-61-717-95-55, Fx. 41-61-717-95-50 Bangkok, Thailand, Ph. 662-651-8311, Fx. 662-651-8312 Hemel Hempstead, United Kingdom, Ph. +44-(0)20-8328-2000, Fx. +44-(0)20-8328-2550

Revised July 2012 506396