Pneumocystis Jiroveci in HIV/AIDS Patients:Detection by FTA Filter Paper Together with

PCR in Noninvasive Induced Sputum Specimens

Siraya Jaijakul MD*,Wilai Saksirisampant MSc**, Juraratt Prownebon BSc* ,Sutin Yenthakam BSc*, MathirutMungthin MD, PhD***,

Saovanee Leelayoova PhD***, Surang Nuchprayoon MD, MPH, PhD*,**

* Department of Parasitology, ** Lymphatic Filariasis Research Unit, Faculty of Medicine, Chulalongkorn University.*** Department of Parasitology, Phramongkutklao College of Medicine

Objectives: To detect P.jiroveci (previously named P. carinii) by PCR using FTA filter paper to extract theDNAJrom noninvasive induced sputum samples of HI V/AIDSpatients.Material and Method: Fifty two HIV/AIDS patients suspected of Pneumocystis jiroveci pneumonia (PJP) inKing Chulalongkorn Memorial Hospital were recruited. Both cytological method and PCR with FTAfilterpaper technique wereperformed to detect P.jiroveci from each specimen. ,

Results: The detectability rate ofP.jiroveci infection was 21%. The PCR with FTAfilter paper method was 4folds much more sensitive than Giemsa staining technique. P.jiroveci was detected in 18% of the HIV/AIDSpatients in spite of receiving standard PJP prophylaxis.Conclusion: Detection of P.jiroveci by using FTA filter paper together with PCR in induced sputu!'" samplescould detect more cases of P. jiroveci infection than by using cytological method. DNA extraction using the

FTA filter paper was more rapid and convenient than other extraction methods.. The causes of failure of PJPprophylaxis should be evaluated.

Keywords: Pneumocystis jiroveci pneumonia, PJP, Polymerase chain reaction, FTA filter paper

J Med Assoc Thai 2005; 88(SuppI4): S294-9Full text. e-Journal: http://www.medassocthai.org/journal

Pneumocystis pneumonia (PJP) is caused byPneumocystis jiroveci, formerly named Pneumocystiscarinii(l). Although an incidence of this disease hasdeclined as a result of highly active antiretroviraltherapy (HAART)(2), PJP remains one ofthe most com-mon opportunistic infection in patients infected withhuman immunodeficiency virus (HlV)(3).The incidenceof pneumocystis pneumonia in developing countries,including Thailand, has continuously increased(4).

Diagnosis ofPJP is based on history, physi-cal examination, and laboratory investigation. Therisk of pneumocystis pneumonia among HIV /AIDSpatients is greatly increased when the T-helper cellcount (CD4+) is less than 200 cells per millimeter5)

Correspondence to : Nuchprayoon S, Lymphatic FilariasisResearch Unit, Faculty of Medicine, Chulalongkorn Univer-sity, Bangkok 10330, Thailand. Phone: 0-2256-4387, Fax:0-2252-4963, E-mail: fmedstt@md2.md.chula.ac.th

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because the host defence against P. jiroveci dependsmainly on the cellular immunity. The dominant presen-tations of the disease are low-grade fever, progressivedyspnea, nonproductive cough, bilateral perihilarinterstitial infiltrates, and hypoxemia(6). Because theseclassic symptoms do not always appear(71,the diagno-sis of P. jiroveci infection needs to be confirmed bylaboratory investigation. Nevertheless, Pneumocystisspp. cannot be cultured; therefore, the current diag-nostic method still requires direct microscopic exami-nation after Giemsa, 0- Toluidine, or Gomon methena-

mine silver (GMS) staining of respiratory specimens inorder to identify the Pneumocystis organism(S,91.

Recently, the polymerase chain reaction(PCR) has been used to amplify P.jiroveci-specific DNAsequences from respiratory specimens, so as to increasethe detection of the parasite. A single PCR has greatersensitivity and specificity than staining methods, when

J Med Assoc Thai Vol.88 SlIppl.4 2005

the PCR primers for Pneumocystis mitochondriallarge-subunitribosomalRNA (rRNA)areused(lO,ll).Theinducedsputum samples often contain many endo-genoussubstances that will inhibit PCR, and yieldfalse-negativeresults. In order to achieve appropriateDNA template preparations, the methods currentlyusedfor PCR to detect microorganisms often requiremultiple steps(12).However, the simple filter-basedtechnique(ie. Flinders Technology Associates; FTA@paper) has been developed to obtain DNA for PCRtechnique effectively<12,13).

The appropriate specimens for detection ofP.jeroveci are induced sputum, bronchoalveolar fluid(BALF), or lung tissue. BALF has traditionally beenthe definitive clinical sample. However, the procedureto obtain the specimen is invasive while the inducedsputum sample is not sensitive compared to BALF andlung tissue for detection of P.jiroveci (14,15).The collec-tion methods of sputum specimens are less invasive,and more convenient than those of BALF or lung tis-sue. Therefore, increasing sensitivity and specificityfor PJP detection assay using sputum samples shouldbe collected in the laboratory.

This study is the first report to evaluate theusefulness of FTA filter paper together with PCR todetect P.jiroveci in HIV /AIDS patients.

Material and Method

Study population and clinical specimens

Respiratory specimens were induced sputum'samples from 52 patients with HIV /AIDS and suspectedof Pneumocystis jiroveci pneumonia in King Chula-longkom Memorial hospital, between January 2002 andJune 2004. The study was approved by the Ethics Com-mittee of the Faculty of Medicine, Chulalongkom Uni-versity. Each sputum specimen was treated with muco-lytic agent (6.5 mM dithiothreitol, Gibco, Grand Island,USA) and followed by centrifugation. The pellet wasprocessed for cytological method and DNA extraction.

CytologyThe pellet was directly smeared onto a slide

pellet, dried and fixed with methanol. Giemsa stainingwas performed using methodology as described pre-viously<16).The slides were examined by two indivi-duals independently.

DNA extraction and detection of P.jiroveciiThe DNA was extracted from the sa~ple by

using FTAfilterpaper (FTA,Whatman Inc, Kent, UK)as previously described(l2,13).Each specimen was

J Med Assoc Thai Vol.88 Suppl.4 2005

spotted onto the FTApaper,air cirjedat room tempera-ture,and washed twicewith 200ml ofFTA purificationbufferfor 15min.The repeatedwashingwasperformedtwice with 200 ml ofTE-l buffer (1OmMTris-HCl pH8.0,0.1 mM EDTApH8.0) for 5 min. The product wasused as a DNA templatefor the PCR amplification.

The primers used were rAZ 102E-(5'GATGGCTGTTTCCAAGCCCA-3')andpAZ1O2H-(5'GTGTACGTTGCAAAGTACTC-3') (Invitrogen,Gibco,Tl)kyo, Japan) in order to amplify a 346-bpsegment from a specific region of a gene coding for alarge subunit of mitochondrial rRNA of human P.

jiroveci<lO).

The DNA was amplified in PCR buffer (1OmM

Tris-HC1,pH 8.4, 50 mM KCL 1.5mM MgC~) containing200 flM dNTPs, 1pmol of each primer, and 0.5 units ofTaq DNA polymerase. The PCR was performed by pre-denaturation at 94"C for 3 min and followed by deria-turation at 94 °C for I mill, annealing at 52 °C for 30 sec,and extension at 72"C for 1min, for 35 cycles, by usinga DN A Thermocylcer (GeneAmp@ PCR system 2400,Perkin Elmer, USA). The PCR products were determinedby 0.8% agarose gel electrophoresis (Gib co GrandIsland, USA) with Tris-acetate EDTA buffer (Tris 0.04M,Acetic acid 0.02M, EDTA, disodium O.OOIM).The gelwas stained with ethidium bromide and visualized

hy using ultraviolet light. Negative control ( normalhuman sputum samples) and positive control (sputumof HIV/AIDS patient with PJP) were inCluded in eachexperiment.

Results

Of all 52 HN/AIDS patients, 29 were femaleand 23 were male patients. Cytological staining andPCR were performed for detection of P.jiroveci ininducedsputum samples.The346-bpbandof P.jiroveciDNA from some patientswas showed as a single band(Fig. 1).Eleven(21%)caseswerepositiveforP.jeroveciinfection by PCR, 8 were female and the rest (3) weremale (data not showed). Three HIV/AIDS patientswere under age of 15,and 1among them was positivefor PJP detection.The rest (49)weremore than 15yearsold, while 10of themwere positive forP.jeroveci (datanot showed)

The result of P.jiroveci detection is summa-rized in Fig. 2 and Table 1. Of the total 52 specimenstested, 11 were positive by PCR, giving the detect-ability rate of P.jiroveci infectionto 21%. Only 3 (6%)samples were positive by cytology. Eight samplesnegativeby cytologywerepositiveby PCR,while noneof the cytology-positive specimens were ne-gativeby

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PCR. Therefore, the use of FTA filter paper togetherwith PCR could detect more cases of P jiroveci 4 foldsas much compared to staining method.

1,000-+

Fig. 1

500-+

rill 3 6 84 5 72

BP

Table 1. Comparison ofPCR with cytologicalstainingmethod for detection P jiroveci

A.

Sputum No. of samplesPositive Negative

3 4911 41

621

Detectability (%)

CYTPCR

B.

Microscopicexamination

Positive

Negative

PCR detction

Positive Negative3 08 41

Table 2. Correlation of pneumocystis pneumoniaprophylaxis and the P jiroveci detection

PCR detection for P.jirm"eci DNA from induced

sputum specimens. Ten microliters of PCRproducts were subjected to electrophoresis in0.8% agarose gel. The 346-bp band was confirmeda positive result in both positive control andpatients' specimens containing P.jiroveci. LaneM:100 bp DNA marker; Lane 1:positive control: Lane2: negative control; Lane 3-5: HIV +ve iP.jiroveci

+ve; Lane 6- 7: HIV +ve/ P.jiroveci -ve

PCR-/CYT- 41

Fig. 2

+ = positive result -=negative result

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Venn diagram of Cytology (CYT) and PCR todetect P.jiroveci in 52-induced sputum samplesobtained from HIV/AIDS-infected patients

Among the totai 52 HIVI AIDS patients, 94%(49/52) received prophylactic drug against the P.

jiroveci infection (Table 2). We found that 9 (18%)patients given prophylaxis were still positive tor detec-tion of Pneumocystis. However, one of three HIViAIDSpatients who did not receive the prophylaxis remainednegative for the organism, while the other two becamepositive.

Discussion

Pneumocystisjiroveci is an important oppor-tunistic fungus that causes potentially fatal pneumo-nia in HIV 1AIDS patients. This pathogen cannot becultured. Therefore, the diagnosis of PJP is based ondirect microscopic examination to visualize cysts ortrophozoites by special stains of respiratory specimens(ie. induced sputum, BALF and lung tissue). Identifi-cation ofthe organism is still considered to bethe goldstandard for dianosis of P jiroveci. Giemsa stain isroutinely used to detect trophozoites, while Gomorimethinamine silver (GMS) and 0- Toluidine is used todetect cysts. As Giemsa stain is simple, inexpensiveand familiar to most microbiological laboratories, thisstaining method is widely used for screening samplessuspected of PJPlI7). However, Giemsa cytologicaldetection still presents some disadvantages. Directmicroscopic examination requires enough burden ofthe organisms in picked-up specimens to visualize.

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P1P prophylaxis Total+- 340 + -

P.jiroveci + 9 2 11Detection - 40 1 41Total 49 3 52

Moreover,technical expertise is also needed to deter-mineand differentiatePjiroveci nom artifactsandotherorganisms, such as yeast form of Candida albicanwhichhas approximately the same size as P jirovecicyst.Therefore, the microscopic examination is ratheruncertainof both sensitivity and specificity.

The polymerase chain reaction (PCR) tech-niquehas been widely used as a tool for detection ofPlleumocystisorganisms. PCR has been found moresensitivethan cytological staining with senstitivity of84-100% and specificityof95-1 00%(15,18.19). Our studyalso suggested that PCR was superior to cytologicalmethod in the detection of P jiroveci in induced spu-tumsamples. By using PCR, the detectability rate wasabout4 folds as much as using cytological method.

BALF has been considered to be the defini-tivesample for detection of Pjiroveci. The Pjirovecidetection in BALF is more sensitive than that ininduced sputum samples, with sensitivity of 100%and 84.62% respectively<15).However, the procedureobtaining the BALF specimens is invasive. The moresensitive method to detect Pneumocystis organismsfrom noninvasive induced sputum should be usefulandmore practical for the physicians and patients. TheFTAfilter paper is a filter-based template. It has beenused to extract DNA with more DNA recovery com-pared to phenol-chloroform method which is widelyused as a standard method(:J). When organisms con-tactwith this paper, they will be lysed after solubilizedinFTApurificationbuffer,andtheir DNAwillbe trappedon the matrix. Other debris then will be removed bywashing.UsingFTAfilterpaper,our PCR result showedcomparable detectability rate (4 folds as much as therate of Giemsa staining) to previous PCR studies (2-6times as much)(lS.2O).Moreover, using the FTA filterpaper does not involve several steps as the phenol-chloroform method. Furthermore, the FTA techniqueis a simple and rapid test that can be used with alarge number of samples, as well as does not requireexperienced personnel to handle(l2).

The US Public Health Service Task Force hasrecommended cotrimoxazole, the combination oftrimetroprim (TMP) and sulfamethoxazole (SMZ), tobeused as the first-linedrug for the prophylaxis againstP jiroveci infection. Dose of cotrimoxazole recom-mended are 1double-strength tablet daily or I single-strength tablet daily(21).HIV/AIDS patients shouldreceiveprimaryprophylaxisagainstPJP when theCD4+

. countis less than 200 cellsper millimeter, or if there is ahistory of oropharyngeal candidiasis. Secondary pro-phylaxis should begin in patients who have ever had

J Med Assoc Thai Vot. 88 SlIppl.4 2005

been infected with Pneumocystis organisms. SMZ isan inhibitorof dihydropterate synthase (DHPS), whileTMP inhibits dihydrofolate reductase (DHFR). Ourdata showed 18% (9/49) failure rate in the HIV/AIDSpatients receiving PIP propJJylaxis. Although thepatients provided the information of good drug com-pliance, the possibility of drug allergy and lackingadherencecould not be ruled out.Another explanationfor failureofPJJ:'pw!J;lYldJl.;:'is that the drug resistancemight occur in Thai-HIV/AIDS patients. Both DHPSand DHFR are enzymes involved in the biosynthesisoffolic acidof Pjiroveci. The emergence of Pjirovecidrug resistance has been suggested(22.24).The mecha-nism of drug resistance may result nom the alterationofDHPS or DHFR genes. Further study of drug resis-tance will answer thequestion.There were two ofthreep'ltients,who did not receive PJP prophylaxis, infectedwith P jeroveci. This result suggests that criteria forPJP prophylaxis in mv 1AIDS patients may be neededto be reevaluated.

AcknowledgementsThis study was supported by Rajadapisek

Sompoj Fund, Faculty of Medicine, ChulalongkornUniversity, Bangkok 10330, Thailand. The authors wishto thank Mr. Suradej Siripattanapipong (Department ofMicrobiology, Faculty of Science, Mahidol University),Miss Alisa Junpee, Miss Piyachat Surattanasing,Miss Jatuporn Sutjitkul, and the staffs at Departmentof Parasitology, Faculty of Medicine, ChulalongkornUniversity, for their technical assistance.

References1. James RS, Charles BB, Robert FM, Ann EW A

New Name (Pneumocystis jiroveci) for Pneumo-cystis from Humans. Emerg Infect Dis 2002; 8:891~. .

2. Kaplan JE, Hanson D, Dworkin MS, Frederick T,Bertolli J, Lindegren ML, Holmberg S, Jones JL.Epidemiologyof human immunodeficiency virus-associated opportunistic infections in the UnitedStates in the era of highly active antiretroviraltherapy.Clin InfectDis 2000;30 (Suppll): S5-14.

3. Morris A, Lundgren JD, Masur H, Walzer PO,Hanson DL, Frederick T, Huang L, Beard CB,Kaplan JE. CurrentepidemiologyofPneumocystispneumonia.EmergInfectDis 2004; 10:1713-20.

4. Ruxrungtham K, Phanuphak P. Update on mvIAIDS in Thailand. J Med Assoc Thai 2001; 84(Suppll):SI-17. .

5. Phair J, Munoz A, Detels R, Kaslow R, Rinaldo C,

S297

. SaahA. The risk ofPneumocystis carinii pneumo-nia among men infected with human immunodefi-ciency virus type I. Multicenter AIDS CohortStudy Group. N EnglJ Med 1990; 322: 161-5.

6. Thomas CF Jr, Limper AH. PneiImocystis pneumo-nia. N EnglJ Med 2004; 350: 2487-98.

7. Wilkin A, Feinberg J. Pneumocystis carinii Pneu-monia: A Clinical Review. Am Fam Physician 1999;60: 1699-1708,1713-4.

K el-Nassery SM, Rahmy AB, el Gebaly WM, SadakaHA. Pneumocystis carinii: recognition of theinfection in albino rats using different stains. JEgypt Soc Parasitol1994; 24: 185-94.

9. Savoia D, Caramello P.The microscopic identifica-tion ofPneumocystis carinii. J Protozool1989; 36:72S-4S

10. Wakefield AE, Guiver L, Miller RF, Hopkin JNl.DNA amplification on induced sputum samplesfor diagnosis ofPneumocystis carinii pneumonia.Lancet 1991; 337: 1378-79

11. Ribes JA, Limper AH, Espy MJ, Smith TF. PCRdetection of Pneumocystis caripii in bronchoal-veolar lavage specimens: analysis of sensitiyity;and specificity. J ClinMicrobiol1997; 35: 830-5

12. Orlandi PA, Lampel KA. Extraction-free, filter-based template preparation for rapid and sensitiyePCR detection of pathogenic parasitic protozoa. JClin Microbio12000; 38: 2271-7.

13. Submngruang I, Mungthin M, Chavalitshewin-koon-Petmitr P, Rangsin R, Naaglor T, LeelayoovaS. Evaluation of DNA extraction and PCR methods

for detection of Enterocytozoon bienuesi in stoolspecimens. J Clin Microbiol2004; 42: 3490-4.

14. Mirdha BR, Guleria R. Comparative yield of differentrespiratory samples for diagnosis ofPneumocystiscarinii infections in HIV-seropositive and serone-gative individuals in India. Southeast Asian J TrapMed Public Health 2000; 31: 473-7.

15. Pinlaor S, Mootsikapun P, Pinlaor P, PhunmaneeA, Pipitgool V, Sithithawom P,Chumpia W, Sithi-thawom 1.PCR diagnosis ofPneumocyst;s cariniion sputum and bronchoalveolar lavage samplesin immune-compromised patients. Parasitol Res2004; 94: 213-8.

16. Saksirisampant W, Eampokalap B, Chantharode-

S298

vong R, Changthong R. Comparison of methodsfor identification of Pneumocystis carinii inbronchoalveolar lavage fluid. J Med Assoc Thai2002; 85 (Suppll): S407-14.

17. Holten-Andersen W, Kolmos H1. Comparison ofmethenamine silver nitrate and Giemsa stain fordetection of Pneumocystis carinii in bronchoal-veolar lavage specimens from HIV infectedpatients. APMIS 1989; 97: 745-7.

18. Ishimine T,NakamotoA, HigaF, Koide M, InadomeJ, Kawakami K, Fukuhara H, Kusano N, KitsukawaK, Saito A. Usefulness of polymerase chain reac-tion method in the early diagnosis ofPneumocystiscarinii pneumonia. Kansenshogaku Zasshi 1994;68:751-8.

19. Hua L, Qin S, Wang A, Sheng R, Zhang K. Thediagnostic value of polymerase chain reaction forthe detection ofPneumocystis carinii DNA frominduced sputum samples. Zhonghua Nei Ke ,ZaZhi2002; 41: 610-2.

20. Leibovitz E, Pollack H;Moore T, Papellas J, GalloL, Krasinsld K, Borkowsky W. Comparison ofPCRand standard cytological staining for detl"ction ofPneumocystis carinii from respiratory specimensfrom patients with or at high risk for infection byhuman immunodeficiency virus. J Clin Microbiol1995;33:3004-7.

21. Centers for Disease Control and Prevention (CDC).Recommendations for Prophylaxis Against Pneu-mocystis carinii Pneumonia for Adults and Adoles-cents Infected with Human Immunodeficiency

Virus.MMWR 1992; 4; 41(RR-4); 001.22. Sritangratanakul S, Nuchprayoon S, Nuchprayoon

1. Pneumocystis pneumonia: An update. J MedAssoc Thai 2004; 87 (Supp12): S309-17.

23. NahimanaA, Rabodonirina M, Bille J, Francioli p,Hauser PM. Mutations of Pneumocystis jiroveciidihydrofolate reductase associated with failure ofprophylaxis. Antimicrob Agents Chemother 2004:48:4301-5.

24. Huang L, Crothers K, Atzori C, Benfield T, MillerR, Rabodonirina M, Helweg-Larsen J. Dihydrop-teroate synthase gene mutations in Pneumocystisand sulfa resistance. Emerg Infect Dis 2004; 10:1721-8.

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