"Ae Cll Centr ote Ward se of Fea im GRAND ROUNT New Insights Into Transmission, Diagnosis, and Drug Treatment of Pneumocystis carinii Pneumonia Joseph A. Kovaes, MD Vee J-Gill, PRD Steven Meshnick, MD, PAD. Henry Masur, MD Pneumocystis carinii has been recognized as a human pathogen for nearly 50 years. We present a case of P carinil infection that typifies clinical pre- sentation in the era of the acquired immunodeficiency syndrome epidem Henry Masts MD the high incidence of P carn pneumonia in persons infected with human CASE PRESENTATION immunodeficiency virus (HIV) has served to focus laboratory and clinical e- ‘8 49.yearold Hispanic man was research efforts on better understanding the blology ofthe organism and on ferred to the National Institutes of improving diagnosis, treatment, and prevention of this disease. Although Hs IB) fo herapy of human gait to culture P carn has hampered research efforts, molecular and immanodsticiency vias CHV) gmunologi approaches have ld to the recognition thatthe organism rep- ee eat ea esents a fay offungt with avery restcted host range and have allowed weed uvsiouesnunceounl —eharactetzation of clinically relevant antigens and enzymes. Molecular ep Tenens wth poor wr contrl. in demiologie studies have identified more than 50 stain of human-derved January 2001, he was diagnosed to have P carinif and have suggested that recently acquired infection, as opposed to ‘Pneumocystis carinii pneumonia (PCP) eactivation of latent infection, may account for many cases of clinical di and was treated initially with trimeth- ease, Diagnosis has been improved by the development of organism- ee ee therapy specific monoclonal antibodies and, more recently, by polymerase chain Tee tcateaticcclogedne action using multicopy gene targets, together with induced sputum or uct oral wash samples. Chemotherapeutic prophylaxis is very effective in pre- Tm April 2001, the patient presented venting P carinii pneumonia; the combination of trimethoprim- toanemergency department complain- sulfamethoxazole remains the first-line agent for both therapy and prophy- ing of fever. shortness of breath, and _laxts. Prophylaxis needs to be administered only during periods of high risk; cough that had persisted for 2 weeks. jn HIV-infected patients responding to effective antiretroviral therapies, pro- He was taking an antiretroviral reg men that consisted of lopinavir/ tHtonavir, abacavir, lamivudine, and phylaxis no longer needs to be lifelong. Molecular studies have identified ‘mutations in the target of sulfa drugs that appear to represent emerging re- Jidanosine. He wa also taking ira- 5!8t4NC® in P carinii, Resistance to atovaquone, a second-line agent, may conazole, atovaquone, aerosolized pent- also be developing. amidine, and clarithromycin, al- JAMA 2001,2862450-2460 wu jamacom though adherence to his drug regimens was uncertain. He was empirically Auth Affliatons: ital Care Medicine Depart- appli fr by the US government for use of 2 cn treated witha combination of wugmen metOsKovasandMasu)andlebsogySe-‘eved regu ofthe nar sue Eycpoe gene Mice, Department of Laberatry Medcne (Br Gif, of human P cana Yor ages a P cr pes lun and levofloxacin, but 2 weeks later Cini enfr, Natenalitiuterefesih Gath, mona reported that the shortness of breath Mé-ahd Department of Epidemiology, Universty of Comesponding Autor and Reprints: Joseph A. Ko ported that the sh breath hengan Sehol of Pubic Healtn-Ann Arbor (Or vate MO, Creal Care Medene Departmen. Ch and fever had not improved. A chestra- Msi) (alent Nana tutes of Heath 10, Room liograph showed diffuse interstitial in- Financial isdosue: DreKovacsandMasrarebotn YD43, MSCY66D Bethea, MO 2089-165 (emal. dlograph showed diff rata in eine patti bythe US govern” ova go filtrates and an anterior mediastinal jpntterthe treatment of Peumocytscavin pneu ‘Gand Rounds at he Clincal Center ofthe National mass, His white blood cell count was mena wihtumetisateandatevertrsofagatent Istutes of Heh Secbon Ets ohn Call MO, ; Son © heldby the US government foc moradonal aniood- the Chal Cantera heaton sues of Heath, 4500/pL with 55% neutrophils. The jg, against human P canni. Drs Kovacs, Masur, Bethesda, Md; DavdS. Cooper, MD, Conibuding Ed CD4 T-lymphocyte count was Land and Glare cantentors ofa patent thet has been tr JAMA 2450 JAMA. November 21, 2001—v o, No. 19 Reprints) (©2001 American Medical Association. All rights reserved jamanetwork.com/ by Manuel Mendez Santana on 09/25/2020his viral load was 250000 copies. Room air PaO, was 55 mm Hg, Biopsy of the anterior mediastinal mass re- vealed a large B-cell lymphoma, Bron- choalveolar lavage (BAL) failed to dem- onstrate any pathogen. Cytology was not performed. The patient developed respiratory failure requiring intubation and mechanical ventilation, He was trans- ferred to the NIH for therapy of his lymphoma, where he presented with a lemperature of 39.3°C. Chest exami- nation revealed diffuse rales. Arterial blood gases on 40% oxygen were: pH, 7.48; PO;, 95 mm Hg; and PCO, 40, mm Hg. His white blood cell count was 2300/iL. His chest radiograph showed diffuse infiltrates that were interstitial and alveolar. Over the next 4 days, the patient began EPOCH (etoposide, prednisone, vincristine [Oncovin], cyclophosphamide, and doxorubicin [hydroxydaunorubicin]) chemotherapy for lymphoma, but his respiratory status worsened. A BAL was performed and many P carinii eysis and trophozoites were seen by direct fluorescent antibody (DFA) staining. By quantitative polymerase chain reaction (PCR), 18640 copies of 4 P carinii-specifie major surface gly- coprotein (MSG) gene were detected per 20 pL of BAL fluid, The patient was treated with intrave- nous pentamiine for days. Beeause his condition was not improving, therapy ‘was switched to intravenous trimetho- prim-sullamethoxazole. The DFA stain remained positive and by PCR, 61260 copied20 pL of the MSG gene were de- tected. The patient slowly improved, but alter 9 days of trimethoprimn-sulfa- methoxazole the patient developed a rash and received an additional 14 days of intravenous pentamidine, AU3 weeks and 5 weeks after initiation of therapy, 1190 and 6 copies/20 jtL of the MSG gene, respectively, were detected DFA staining Was positive at 3 weeks but negative at 5 weeks, Analysis ofthe Pca rinti dihydropteroate synthase (DHPS) gene, the target of sulfamethoxazole therapy, revealed a single Thr—>Ala m- lation at position 55, suggesting pos- (©2001 American Medical Association, All rights reserved, sible sulfa resistance. The patient was able to be weaned from mechanical ven- tilation after 6 weeks of ventilatory sup- port and was discharged to the oncol- logy service for additional therapy of his lymphoma, DISCUSSION Pneumocystis carinii (FIGURE 1) isa fun- gus whose importance in the United States as a human pathogen increased dramatically following the onset of the acquired immunodeficiency syn- drome (AIDS) epidemic 20 years ago, Since that time, advances in under standing the biology and in improving the diagnosis, treatment, and preven- ion of PCP have substantially re- duced the morbidity and mortality as- sociated with this organism, Biology and Epidemiology of P earini Historical Perspective. Pncumacystisca- rinii was frst identified 4m 1909 and 1910 in typanosome-infected lungs of animals by Chagas* and Carini? who thought that it was a form of rypano- some. In 1912, Prewnacystis was ree- ognized asa new genus by Delanoe and Delanoe? and was named in honor of Carin In the 1930s and 1940, epidemics of interstitial plasma cell pneumonia were recognized in premature and mal- nourished infants in Europe. Prewno- cystiscarinié was identified asthe eause of this pneumonia by Vanek and Jirovel! in 1952, Pneumocystis carint pneumonia was subsequently recog- nized with increasing frequency as a major cause of pneumonia and death fn immunodeficient and immunosup- pressed paticats. The muimber of ree- cgnized cases exploded in the 1980s, as PCP became one of the hallmark ‘manifestations of AIDS wellas.acom- mon complication of eancer chemo- therapy and organ transplantation.»® Incidence of PCP. Inthe late 1960s and the early 1970s, there were fewer than 100 cases per year of PCP re- ported in the United States (FIGURE 2A), Following the onset of the AIDS ep dlemic in 1982, there was a marked in- PNEUMOCYSTIS PNEUMONIA Figure 1. Prevmocystis cari Infectng a at Lung cton micrograph ilstates both ests white a- rowhead) and Wophozotes (ack sous) crease in the incidence of eases re- ported to the Centers for Disease Control and Prevention that peaked in 1990 at about 20000 cases per year. In the early 1990s, there was decline in incidence that was largely due to the widespread, use of PCP prophylaxis, which fol- lowed a report from a Public Health Se vice task force that recommended that prophylaxis should be universal for HI infected patients who met certain crite Fa, most importantly a CD4 cell count less than 200/L." After 1005, there was a further de- cline due to the widespread use of highly active antiretroviral therapy (HAART), which is very effective in suppressing HIV, augmenting im- rune function, and protecting from op- portunistic infections, as documented by the Adult and Adolescent Spec- trum of Disease study. a prospective co- hort study conducted by the Centers for Disease Control and Prevention (Fig- ure 2B).” Despite this dramatic de- cline in incidence, PCP remains the most common life-threatening oppor lunistic infection diagnosed in HIV- infected patients Clinical Presentation Pneumocystis carinii causes clinically apparent pneumonia virtually exclu sively in immunosuppressed patients The clinical presentation is character (Reprinted) JAMA, November 2, 2001-—Vol 206, No. 19. 2484PNEUMOCYSTIS PNEUMONIA sued for 14 ized by fever, shormmess of breath, sub- will usually demonstrate a character- treatment should be cont eral ghthess,anda nonproductive istic ground glass attenuation” to days for most patents cough. Especially in HIV-infected Diagnosis is dependent on det Patients the symptoms can be rela- tion of the organism in a clinical Advances in Understanding tively mild and slowly progressive, specimen by colorimetric orimmuno- the Biology of P carinii which may delay diognosis,as occurred fluorescent stains (Figure 3B). Hist Substantial advances have been made inthiscase!"Thechesttadiographchar- pathologically, PCP showsa very char- over the past 2 decades in understand- acteristically demonstrates bilateral acteristic intra-alveolar acellular ing the Biology of P carinil To high ‘hich progressto cosinophilicexudate thatean fillthe al- light these advances, itis helpful to an alveolar pattern (FIGURE 3A), veoli (Figure 3C) andis primarily com- compare what was now about the or- although in l0%ormoreofcases,itmay posed of large mumnbers of Pcarnti organism in te pre-AIDS era inthe 1070s be entirely normal: Inthelattersitu- ganisms. The combination of withwhatisknown today. Inthe 1970s, ation, high-resolution (hin section) —trimethoprim-sullamethoxazole isthe P carinii was thought by moat experts computedtomogrphyseanofthechest current first-line therapeutic agent; tobe protozoan. Based largely on mo interstitial infilrat igure 2._ Incidence of Pneumocystis carn Pneumonia (PCP): Impact of the Acquired Immunodeficency Syndrome (AIDS) Epidemic And of Highly Active Antiretroviral Therapy (HAART) [Bror cases reparaaoine coe [Ere nesee| _- 1 Cases of PCP reported othe Centers for Osease Coil and Preven (CDC nthe pre AIDS ea compared wth he ADS cra. Hovzortal basa he op nae ‘he petod dung whch pophynesto prevent PCP (popes) and HAART ware vate. Te row nate theme publeation guidelines bythe US Pub Hesth See fr prevention of PCP inhuman irmnadefeny wus (l-infected patents, Change m meen of CP nthe pr HAART and HAART ea, emonstating the mated decnein incence hat occured elloning the veduction of HAART. ne nme re HAART a (198201995), thee wasa 34% Deryea gece minadence of PCP During the HAAAT ea, te incdnce of PCP decreaced by 215% per year, Oa ae rom the Adu nd Adolescent Spec ot Beco Stuy 7 Ciniel Findings of Patients With Preumocyats carn Pheumona (PCP) |. Chestradiograph ofa patent wth PCP demonstrating fuse iateralinfitats 8 Detection of human derived cari byimmunotorecece ung manana ‘itbodesC Hematowlncosn-staned scion of ug tom an HIV-infected patent wih PCP. An sala esnopic exudate characters of PCP canbe een ing the ave 2452 JAMA November 21 o, No. 19 Reprints) (©2001 American Medical Association. All rights reserved jamanetwork.com/ by Manuel Mendez Santana on 09/25/2020lecular studies, P carinii has been re- classified as a angus.” In the 1970s, P carinit was thought to bea single strain with « broad host range. Molecular as well as imimno- logic studies have shown that in fact there are multiple strains of P carinii, cach of which is restricted to infecting single host species. ©!" Thus, human: derived Pcavinitcannotinfect mice, ras, oF ferrets, or even monkeys, whereas mouse-derived P carini cannot infect rats or humans. These different strains are potentially unique species. In the 1970s, the lifecycle of P cari twas unknown, in large part because the organism could not be cultured. As a consequence, metabolism of the oF sanism wasalso poorly understood. Cur rently thelfeeyele emains unknown, since P carinii still cannot be consis. tently cultured, but there are some en= couraging recent reports of success in this area." However, advances in n- derstanding the metabolism have been made through molecular biologial ap- proaches, Targetenzymes of some avail- able therapeutic regimens have been cloned, sequenced, and characterized. ‘These inchide the genes encoding diby- dropteroate synthase (DHPS) and dihy- drofolate reductase (DHFR), which are the targets of sulfamethoxazole and tri- methoprim, respectively." In the 1970s, protective host tm- mune responses were ill-defined, al- though cell-mediated immunity was be- lieved tobe portant. Studies in animals and humans have documented a criti cal role for CD cellsin controlling Pca- rini infection, while CDS cells appear to have a minor role at best, presi ably because P carni is exclusively an extracellular pathogen." Macro- phages appear to be important ellector cells and certain eytokines, such as tu mor necrosis factor, also appear to be important" Inthe 1970s, the antigens of P arin were essentially uncharacterized. To- day a number of antigens have been identified and cloned, the most impor- tant of which is the MSG. This protein is encoded by a multicopy gene family swith 100 or more genes per genome that (©2001 American Medical Association, All rights reserved, presumably exists to allow P carinii wo lise antigenic variation as a means of avoiding host immune defenses.» Epidemiology in Humans Pheumocystis carinii pneumonia has been diagnosed in patients through- fut the world, The mode of transmis sion in humansis unknown, though the respiratory route is likely important this route has been documented in a smal studies to be the primary mode of ansmission.** The reservoir for hu- rman infection is unknown, but could include environmental sources, other humans, or animals, although the re- stricted host range argues strongly against the latter ‘Serologic studies have shown that healthy humans havea high rate of prior infection with P carinit and that expe sure often occurs by 2 years of age There is no well-defined clinical syn- drome associated with acute infection in the immunocompetent host; pre sumably patients are asymptomatic or experience mild, nonspecific symp- toms. A few studies have demon- strated focal pneumonitis in associa don with P carinii in infants" and there isan intriguing recent report sug gesting association of mild PCP with sudden infant death syndrome, al- though whether this is causal of coin cidental needs to be evaluated fur ther.””°* It has been widely held that latency is established following acute {infection in healthy individuals and that this latent infection can reactivate in the setting of immunosuppression. Molecular typing of isolates has been recently used asa tool to address some of the epidemiologic questions. Typ- {ng with the internal transeribed spacer region of the ribosomal RNA (1RNA) gene has identified more than 50 unique fsolates.» Coinfection with multiple P carinii isolates has been demonstrated {in 20% to 30% of PCP cases.» Type variation has been demonstrated in some recurrent episodes, suggesting that recurrence may be the result of re- infection with a new type rather than reactivation of the type causing the pre- vious episode.” Type variation has also jamanetvrork.com/ by Manuel Mendez Santana on 09/25/2020 PNEUMOCYSTIS PNEUMONIA been associated with place of diagno- sis but not with place of birth, again sug- gesting recently acquired rather than remotely acquired organisms are re- sponsible for clinical disease." In ar cent study, 54% of patients with PCP ‘who had previously unrecognized HIV infection showed mutations in the DHPS gene.” Because such mutations appear to represent resistance in the set- Ling of sulfa drug prophylaxisand these patients were not receiving proph; laxis, this provides compelling evi- dence that the infection was recently ac- quired either directly or indirectly from patients who were receiving prophy axis. Importantly, evaluation of clu ters of PCP have provided no strong evidence to date for single-source out- break. Diagnosis of PCP (Over the past 2 decades, major im- provements have been made in the labo- ratory diagnosis of PCP. Diagnosis in the 1960s and 1970s was primarily made using stains specific for the eyst form of the organism, such as Gomori methenamine silver, Gram-Weigert, or toluidine-blue O. Expe ligators used Giemsa stains to look for ced inves trophozoite forms of P carinii or elus- ters of cysts containing intracystic bod- ies. Because patients with PCP fre- quently raised iule if any sputum and harbored low numbers of organisms, the specimen of choice was open lung biopsy. Expectorated sputum as wel as bronchial washes obtained using rigid bronchoscopes were insensitive for P carinii detection, In the early to mid-1970s, the intro- duction of flexible beroptic bronchos- copy led to transbronchial biopsy and BAL specimens, These specimens have sensitivities for P carinit detection that fare comparable with open lung biop- sies. Bronchoalveolar lavage alone was subsequently used effectively for diag nosing PCP. During the mid-1980s, sputum induction using nebulized sa- line was found to provide an even less invasively obtained specimen with a sensitivity ranging from 60% up 10 95%, though in some centers it was lower.” (Reprinted) JAMA, November 2, 2001-Vol 206, No. 19. 2482PNEUMOCYSTIS PNEUMONIA 1111986, monoclonal antibodies were patient care, the induced sputum re- ora nested PCR procedure requiring 2 developed that reacted with human Pca sults are available prompily, so that a amplification rounds. Allstudiesus- Fini.” Several ofthese anubodies proved BAL.canbe performed within 24 hours. ing these assays have shown PCR to be tobebothscnsitiveandspecificfor ca Although hing biopsies were requested more sensitive than stains for dete rin permitting development of afluo- fequently in the past, these requests ion of P carinii in either BAL or in- rescent antibody stain that allowed de-havedecreased from an average of 2010 duced sputum. This increased sensi- tection ofthe organism within 2hours."* 30submisstons peryearinthelate 1980s tivity may permit the use of PCR to Astain with such specificity has greater to our curtent rate of about 5 per year, detect P carinii in more easily ob- accuraey'somorphologicalysimlaryesst despitean enlarging population of highly tained specimens such as oral washes like Candida will not be misidentified ss immunocompromised patients, Mote- oF gages.” Increased sensitivity may P carni. Moreover, tain that detects over, inthe past 5 years, none of these also allow earlier detection of infec- botheysisand trophozoites, suchas luo- biopsies has been positive for P carinii, tion when there may be very few or rescent stains using monoclonal anli- Experience with PCP diagnosis at ganisms that are difficult to find in body 262, has greater sensitivity than NHLover the past 13 years sshown in stained smears stains that detect cysts only, such assil- FIGURE +. Our data for AIDS patients Several recent studies using PCR to ver stains and other monoclonal anti- are consistent with the national trend, detect P carinié have found individ body stains, because trophozoites greatly witha decrease in both the number of als who are positive by PCR but nega ‘outnumber cysts specimen submissionsand the percent- ive by sain." Although some of thes In addition to specimen type and _ age that were positive. Currently, PCP patients have clinical disease, others do stain, another significant factor alfect- ts diagnosed more often in non-HIV im- not develop PCP, suggesting either ing diagnosis is the patient being ex- munocompromised patients. Thequan- asymptomatic colonization or car- amined. In general, patients with AIDS — lies of P carinii in these patients ate riage, We can therefore expect (0 € who are not receiving P carinii prophy- generally low, making detection more _ counter occasional patients in whom a laxis have a higher organism burden difficult. positive PCR may not represent active than those receiving prophylaxis, and Fortunately, the introduction of mo- infection. As further epidemiological the diagnostic yield of BAL and in- _lecular-based methodshas provided de- studies are undertaken, we should b duced sputum may be decreased in pa- tection assays that are more sensitive gin to understand carriage and trans- lients receiving aerosol pentamidine than tsditional tains and even immu- mission of this organism better, par- prophylaxis." Also, AIDS patients of-nofluorescent tains. The first report us- ticularly in different populations of len have more organismsthan otherim- ing molecular amplification methods immunocompetent and immunocom- munocompromised patients, such as such as PCR for detection of P carinii promised hosts ‘cancer patients." was published by Wakefield et al! in Currently at NIH, we are looking at Thealgorithm used for many yearsat_ 1000. Since thes the NIH Clinical Centerfordiagnosisof PCR methods described that use a va-__ using primers for the mitochondeial PCP hasbeen to obtain an immunofluo- riety of gene targets.**”” Those with the FRNA gene’ and the other using newly rescent-stained smear of induced spu- highest sensitivity use either a multi-. described primers for the major sur- tum, and ifthat smear isnegative,to pro- copy gene target (eg, mitochondrial face glycoprotein gene.” Preliminary r ceed 1o BAL Inordernot to compromise rRNA, majorsurface glycoprotein”) sults with oral washes show a sensitiv therehavebeen many P carinii PCR by 2 different assays, one Figure 4. National Insttutes of Health Experence n the Diagnosis of Pneumocystis carn Preumonia (PCP), 1987-1999 [ise arom anc r [scons a Fv idateshamanimmunodeticene us Both the number of epsode of pananry symptoms eval for CP (A) andthe number of asesoTPCP diagnosed (@) showed a decease over em pans with FV infection (exces) const to paterts without HIV (gay ere). 2454 JAMA. November 21,2001 ol 286, No 19 Reprint) (©2001 American Medical Association. All rights reservedity of up to 80% when compared with, concomitantly obtained BAL or in- duced sputum samples examined by di- rect immunofluorescent staining. By us- ing a sensitive PCR assay, we ean now look atthe possibility of detecting PCP cearlier than by traditional smears and determining if there ate certain PCR- positive but smear-negative patients who will develop P carinit infection later. By following up PCR-positive pa- tients who are without disease, we ean also look at whether carriage oF colo- nization is transitory or long-term and. determine cartier rates among dilfe cent patient populations, ‘Our goal is to develop a new algo- rithm in which a noninvasive speci- men stich as an oral wash is screened. for the presence of P carinii by PCR. AL- though the significance of a positive PCR assay would need to be inter preted in conjunction with the pa tients clinical findings and risk fac tots, a negative PCR would suggest a low likelihood of PCP and the need to look for other etiologies. Our group has developed a quantitative PCR assay that could help predict the likelihood of colonization vs disease and may also provide prognostic information, as sug- gested in the case presentation.» For now, however, pending prospective validation of this approach, diagnosis should follow the traditional algo- rithm (FIGURE 5), namely to examine fan induced sputum sample, ideally by direct immunofluorescence, and ifthis is negative to follow with BAL, reserv- ing bronchoscopic or open lung bi- ‘opsy for patients in whom the BAL is nondiagnostic Prevention of PCP Prevention of PCP isa logical strategy’ to reduce the morbidity and mortality that P carinii can produce in suscep- lible humans. There are 4 baste ap- proaches that could be taken to pre- vent PCP. Because it ishighly likely that ‘human P carinii, like murine is spread * reducing expo- sure of susceptible individuals would be a logical approach to prevention, However, P carinii appears tobe a ubiq- by an aerosol rout (©2001 American Medical Association, All rights reserved, ultous organism, there are no data dem- onstrating transmission of P cari [rom an acutely infected host to a suscep- Uuble patient, and molecular epidemio- logic studies do not suggest single source outbreaks. Thus, respiratory {solation would not be expected to pre- vent most eases of PCP. ‘A second approach to prevention would be to enhance immunocompe- tence so that patients Were not immu nologically susceptible to PCP for extended periods, Management of trans- plant recipients, cancer patients, and patients with HIV seeks to minimize the period and severity of immune defi- ciency by minimizing the intensity of chemotherapy or by controlling HIV replication. Otherstrategies to enhance immunocompetence specifically against Peavini, such asactiveor passive immu- nization, have not been evaluated in humans The most widely used and success- ful strategy for preventing PCP is wouse specific chemoprophylaxis in patients during defined periods of susceptibil- lty. This strategy requires that suscep- tuble patients be identified and that a convenient, nontoxic, effective drug regimen be developed. TABLE 1 shows the estimated risk for PCP in selected patient popula- tions." The risk for developing PCP can be suggested by the degree of ce! lular or humoral deficiency that the pa tienthas by virtue of an underlying dis- cease of an immunostippressive therapy; however, laboratory measures of im- mune competence are imprecise mea- sures of susceptibility. Thus, clinical studies are needed to identify patient populations at high risk, and to define the periods when patients are most sus- ceptible For most patients, the period of sus- cepubilty is discrete, based on the natu ral history of their underlying disease, the extent and duration of immuno- suppression induced by their therapy. and the response to therapy. For pa tients with HIV infection, this period of susceplibility can be sucessfully as- sessed by measuring the peripheral CD4 cell count (ie, patients are most sus- jamanetvrork.com/ by Manuel Mendez Santana on 09/25/2020 PNEUMOCYSTIS PNEUMONIA Figure 5. Agontim for Evaluston of Patients With Suspected Pneumocystis carn Peumonia PCF) v PD nats paid prot ceave, ceptible when their CD4 cell count is lower than 200/jL). For other pa- tients, there are no laboratory mark- cers that are predictive of susceptibil- ity; observational studies have been necessary to define when the high- risk period begins and ends." While such studies have led to the routine use of PCP prophylaxis during high-risk pe- riods for certain patients without HIV Infection (including those listed in Table 1, for other populations the level and period of risk are mote dificult to define *?*** Further, changes in man- agement of underlying diseases, which may increase the intensity of immuno- suppression, require an ongoing reas- sessinent of risk, The recognition that the period of susceptibility is diserete (ie, there isa beginning and end) isan (Reprinted) JAMA, November 2, 2001-Vol 206, No. 19. 2485PNEUMOCYSTIS PNEUMONIA Important concept, especially for pa- tions: rash, fever, transaminase eleva- tients with HIV infection in whom tion, interstitial nephritis, and erystal- guidelines now recommend cessation luria can occur, bul these toxicities are ‘of prophylaxis following an adequate manageable. Patients with HIV infec response to HAART, asdefined byasus- tion have an unusually high frequency tained CD4 T-lymphocyte count greater of toxicity, although several strategies for than 200/pL. gradual dose escalation and for using For patients at high risk, whatare the lower maintenance doses appear to en- chemoprophylactic regimens that are hance tolerability." A variety of dosing preferred based onefficacy,toxicity,con- regimens has been assessed, including, venience, and cost? In 1075, Hughes et 1 single- or double-strength tablet wvice ab demonstrated that daily trimetho- daily or once daily, 1 double-strength prim-sulfamethoxazole was almost tablet 3 times weekly, and 1 doubl 100% effective for preventing PCP in strength tablet twice daily 2 consecu- heavily immunosuppressed children tive days per week. While sufficiently ‘with cancer. That regimen isextremely powered studies to accurately compare well tolerated in most patient popula- _theirefficacy and toxicity have not been Risk of Preumocysts carl Preumonia (PCP) Accorang to Underlying Disease” Disease Period Patients with POP, Se ion Tos 1075 oan daa 067-1070 ‘ete myelocytic aera 067-1070 Feulohmphoctc keri TES Tog Zz ‘Tarug trator heresy = Timproma Tar PROMACE 7 PPROMACE-cyiOW z THE TOO 7 Tar Oa 0R7-1006 7 HTLV Tassocatea bmpnoma TORSO a Five 080-1000 To Regimens for chemoprophyians of Pneumocystis cari Preumanis Dag Dose. Praca, Timethoprin-sutarathoxazole 1 1DS.or1 $8 by mouth daly Tem Tietopin Satara TDS by moun ies we Dapsore 2ormg by Mouth vice daly or TOO ng by Rea cay apse wi Stimg Bj mouth daly Pymetarina wih Sting Bj mouth every Weak iaacovorn ma by math every week mg by math every week 75mg by mouth every week ieacovorn 25 mg bj mouth even week ‘areeotzed ponariane amg every month va Raspigard I rebuRer oT ovaquone 1800 mg by mouth daly done, all of these regimens appear to have a high degree of efficacy and safety. The lower dose regimens (ie, single-strength tablet daily rather than adouble-strength tablet daly) are prob- ably better tolerated. Trimethoprim sulfamethoxazole prophylaxis is quite inexpensive; the wholesale cost for 4 month of prophylaxis can be less than $10. The use of trimethoprim-sulfa- methoxazole is associated with ad- verse effects in addition to toxicity. The widespread use of trimethoprim- sulfamethoxazole for PCP prophy- laxis has an effect on bacterial ecol- ogy, enhancing the prevalence of bacterial resistance to these agents among common enteric and respira- tory flora.” Such use ean also affect the sensitivity of P carinii to sulla drugs TABLE? lists some potes live regimens for patients who cannot tolerate or who fail the trimethoprim- sulfamethoxazole regimen. Most of these regimens have been assessed most completely in patients with HIV infec- tion, but are used in other patient pop lations as well. Dapsone and dapson pyrimethamine appear to be the most elfective alternatives, although a sub- stantial number of patients who ean- not tolerate a sulfonamide also cannot jal alterna tolerate the structurally similar sulfone based regimens. Acrosolized pentami- dine is effective, but not as effective as lrimethoprim-sulfamethoxazole, Aero- solized pentamidine also has the dis- advantage that i is not well distrib- uted toall lobes ofthe lungs, espectally in patients with obstructive hing dis- cease, and it may predispose to extra- pulmonary P carini infection, though such cases are rare (19%) even in pa- lients receiving aerosolized pentami- dine.” Aerosolized pentamidine isalso more expensive to buy and administer than the oral regimens, but it ean be given once monthly, which is an ad- vantage in terms of convenience and adherence. Atovaquone is quite effec- live, but is also expensive, I the period of patient suscepti ity has been defined and effective pro- phylactic regimens are available, why 2456 JAMA. November 21, 2001—Vol 206, No. 19 (Rept) (©2001 American Medical Association. All rights reserved jamanetwork.com/ by Manuel Mendez Santana on 09/25/2020do patients still develop PCP today? Many cases of PCP occur in patients who were not taking prophylaxis be- cause (1) they were unaware of the suscepubility (eg, patients with HIV in- fection and low CD4 T-lymphocyte counts who are unaware of their HIV Infection); (2) they did not adhere 10 prescribed chemoprophylaxis; or (3) their health eare provider did not pre- seribe the appropriate regimen.” A few patients develop PCP when they were not expected to be susceptible (eg, a solid organ transplant recipient with no episode of organ rejection who devel- ‘ops PCP more than 6-12 months post- transplant, or an HIV-infected patient who develops PCP when his or her CD4 T-lymphocyte count is greater than 200/41). Some patients also fail an ap- propriate regimen, regardless of how adherent they ate, because no regi- men is 100% effective, How often these failures occur because of sulfonamide resistance remains to be determined. Chemoprophylaxis of PCP has been fone of the major successes in the field fof infectious diseases in the past 25 years. Careful attention to using the ‘well-established regimens during pe- riods of immunologic susceptibility and proper education of patients about the importance of these regimens are sential (o minimize the impact of PCP ‘on susceptible patient populations. Sulfa Drug-Resistant P cari Sulfa drugs have been the most impor- tant component of therapy for PCP since the 1970s, The most important sulla-containing drug is a combina- lion of 2 antifolates: sulfamethoxazole and trimethoprim.*"° Animal models suggest that trimethoprim may be in- active against P earini.”” Thus, while itis difficult to assess the contribution of trimethoprim to therapeutic effi- cacy in humans, trimethoprim- sulfamethoxazole may functionally be sulfa monotherapy. Dapsone, a sul- fone, is an important second-line agent. Both dapsone and sulfa- methoxazole act by inhibiting the fo- late biosynthetic enzyme DHPS, which catalyzes the condensation of para- (©2001 American Medical Association, All rights reserved, aminobenzoie acid (pABA) and 6-hy- droxymethyl-7,8-dihydropterin pyro- phosphate Sulla and sulfone resistance has been ed in a variety of bac- terial pathogens including Streptococ- dis," Escherichia colt,® Mycobacterium leprae,” and in malaria.” Sulfa re: tance in these organisms results from point mutations in the DHPS gene. In malaria, mutations tend to accumu late over time and impart greater de- agrees of drug resistance.” *" since P carinit has been widely € posed to sulfa, it is reasonable to expect that it might be developing sulla resistance, However, drug resistance in P carinii cannot be confirmed by clas- sic methods beeause itis not possible to culture patient isolates and deter- mine their in vitro drug sensitivities, Thus, the only way to detect resis- tance at present is by an indirect method: looking for mutations in the P carinii DHPS gene. tn 1997, the Pea rinii DHPS gene was sequenced from 6 patient isolates and specific genetic polymorphisms were found." Allof the polymorphisms were nonsynony- ‘mous (ie, all resulted in changes inthe encoded amino acids). This suggested that there was evolutionary selective pressure to induce mutations, possi- bly as a result of drug exposure. Based on homology to the E coli en- zyme whose 3-dimensional structure has been solved. 2 of the mutations area the enzyme active site (FIGURE 6) Threonine 55 and Arg56 are involved inbinding to the pterin substrate; Arg56 isalso involved in binding to pABA and to sulfa, Mutant strains usually re- place the Thrat 55 with an Ala, which lacks the hydroxyl group involved in binding. The Pro at 57 is also replaced with Ser, which may alter the position of the critical Arg56. Interestingly, mu tations at the same 2 sites have re- cently been shown to cause sulfa resis- tance in M leprae.” ‘Mutations at positions 55 (as seen in the ease presentation) and 57 have now been seen in many other studies. TABLE 3 is a summary of 6 studies in well character PNEUMOCYSTIS PNEUMONIA Figure 6. Mutstone atthe Diycropteoste synthase (HPS) Active Site DAP inang| Dinyropterate Synthase "metas ca Diagram ofthe Peumocysts cain DHPS enzyme Sots poston of key amino acs valved ba ing78-dhydroplti- pyrophosphate (OHPPP pas SMinoberzic acd (PABA, and sufonamies ul) based onomatogy fo thengwn cyst structure of {he EScherchia cal enzyme" Two common seen rulaons tat may beasoeated with esstance te ‘baa and Prose which the frequencies of mutations in sulfa-exposed and nonexposed pa- tients were compared.”"®*" The stud= ies range in size from 20 to 152 pa- tients. DHPS mutations occurred in 62% to 100% of patients receiving prophy laxis compared with 11% to 47.5% of patients not receiving prophylaxis. In every study, there were significantly more mutations in the sulla-exposed ‘group than in the nonexposed group. ‘One other important piece of infor mation was added by Ma etal." The study was the first to look for mutac tions in DHER, the target of trimetho- prim, Even though they found many isolates with DHPS mutations, no DHFR mutations were found. This study is consistent with previous animal stud- iessuggesting tha trimethoprim was in- active; it also supports the hypothesis that trimethoprim-sulfamethoxazole may function as sulla monotherapy ‘As one would expect, there is wid spread geographical variation in the prevalence of these mutations." In general, they are more common on the ‘West Coast than the East Coast, with the lowest frequency in Denver and In- dianapolis. (Reprinted) JAMA, November 2, 2001-Vol 206, No. 19. 2487PNEUMOCYSTIS PNEUMONIA Three studies have looked atthe in-patients with mutant DHPS were si cidence of mutations over ime." Mi nificantly less likely to suevive for 3 absent or infrequent be- months than were patients with wild- fore 1993 and then became quite type P carnitinfections" However, in common.**** tna Danish study, the a recent study, there was no effeet on prevalence peared tadeclineagainal- survival, though patients who failed ter 1997, possibly because sulfa drug. sulfa therapy were found to be signi use was declining cantly more likely o be infected with mutant strains than with wild-type strains."' In another recently com- pleted study, no association was seen The occurrence of PCP in patients between mutationsand either response receivingsullaprophylaxissuggeststhat_ wo therapy orsuevval.” Thus, appears the DHPS mutations confer, at mini- thatthe 55 and 57 mutations én the P rum, « low-level resistance that can carinii DHPS gene have, at mos, small overcome the inhibitory ellects of the _ellecton response to therapy. But what low doses of sulla administered dur- wall happen when a strain with addi- ing prophylaxis. What is less clear is onal mutations arises? Ifand when tat how the mutations affect response to oecuts, it could mean a much higher therapeutic doses of trimethoprim level of resistance and loss of the most sullamethoxazole.tn the Danish study, effective drug for treating PCP. ‘Table 3. Associaton of Mutation Frequency in the DIPS Gene of Pheumocystscariii With Prior Sua (Sufamethoxazole or Daprone) Prophylaxis" Proportion With Mutation, Ne Total) Soure,y Prior Sulfa Prophylaxis No Prior Sulfa Prophylaxis pValue aan otal 1908 a Bsa FrawegLaren tal 1000 Tea0 62 area) 0 ieee TH TTS 5) TOTO) a Santos etal 1000 BB 100) aT ar aan ot a> 2000 are) Tae 07 Fang eta 2000, STL, TOUTS) oo Regimens for Weatment of Preumiocysts cara Prearoni™ Da Route De rao Tinebopin suanathorazole youth 20S evay Shows inv Tiahoprin Smog poe clanaroae moto stay Shows aaa Tina opim plus you 220 mg ety Shas psene Syma oom cay Tongans Bye me ie ay Sinan aos By moh ara 200-450 mg eiey OTA primi Syrah Tsamg daly Paar Tavo “img pe oa Tinebele Tras gin ely texovan By mah nara — Aoi? every Shas *oiSeceone foam oP By mouth, int 40 12 hours for 8 cy ro mmbg vith 72 hours The dO mg daly for ae citvatng ha) fren 20 ga or Atovaquone Resistance Pneumocystis carinit may also be devel- ‘oping resistance to atovaquone. Atova- {quone isa second-line therapeutic and prophylactic agent for PCP and is also used against malaria. When used as monotherapy for malaria, almost all pa- lientsdevelop resistance." To slow the development of drug resistance, atova- {quone is now used for malaria only in combination with another antima- larial drug, proguantl The mechanism of action of atova- {quone iswell known. Atovaguone mim- ics ubiquinone and binds to cyto- chrome b.” There is a good molecular explanation for why resistance to atova- {quone develops quickly. Cytochrome bis encoded on the mitochondrial g nome where spontaneous mutation rates are 10-fold higher than in the nucleus." Biophysicists have been map- ping mutations in eytochrome b for many years. Many electron transport in- hibitors resemble atovaquone. Muta- lions conferring resistance to these have bbeen mapped to the Qy box in bact ria, fungi, and protozoa.” The P carinit cytochrome b genes Irom more than 70 patient recently sequenced.” A number of mutations were seen in both ofthe pep- lides involved in the site. There wet 7 different mutations and only 2 were found in more than 1 patient. Five of 15 patients with atovaquone exposure had P carinii cytochrome b mutations, ‘while mutations were seen in only 3 of 45 matched controls with no atova- quone exposure (P=.02). Thus, muta- lions im the P carinii eytochrome b are significantly more common in pa- Lents exposed to atovaquone. The mull- Liplicity of mutations suggests that alovaquone resistance develops de novo in each patient alates wer Current Recommendations Despite the detection of these muta lions, there is no compelling evidence to date that suggests a change is war- ranted in the approach to the treat ment or prevention of PCP. Prophy- {trates sone srerah Gane, OSS Na HSE Ta a eR TG laxisshould continueto be administered ort cetacean a rnep, 3 mata sen & Mr mo summariged in Table 2. Druge of 2458 JAMA. November 21, 2001—Vol 206, No. 19 (Reprnes) (©2001 American Medical Association. All rights reserved jamanetwork.com/ by Manuel Mendez Santana on 09/25/2020choice for treatment of active PCP are summarized in TABLE 4. Since at pre- sent detection of DHPS mutations remains an experimental procedure not broadly available and many patients ‘with DHPS mutations are treated with and do respond to trimethoprim- sulfamethoxazole, this drug remains the first-line agent for therapy as well as for prophylaxis. Folinic acid (leucovorin) should not be coadministered because itdoes not reduce toxicity and may be associated with an increase in failure of therapy and death.” Patients intoler ant of trimethoprim-sulfamethoxa- zole can be treated with one of thealter- es noted in Table 4. Adjunctive prednisone should be administered ‘within 72 hours of starting therapy to patients with an arterial oxygen ten- sion of less than 70 mm Hg.”” ‘CONCLUSION Despite the inability to culture P cari nil, major advances have been made in management of PCP over the past 20 years, much of it based on advances in understanding the basic biology of the ‘organism, Ongoing studies of this per- plexing organism, including a project, lo sequence the genome,” should con- nue to provide clinically relevant insights that will facilitate the care of patients with this potentially lethal infection. Previous Presentation: Presented inp athe Grand Rounds Cnc ttf Conterence othe Nationa In Sites of Heath June 28, 200, detnesda Ma ‘ednowledgment: We hari HarsLase, MD, Steven Facer, Pn lg Ma MO, or ‘oumin he quanlave PCA ay andor deter ing the DHPS sequence or he ave resentation. ERENCE 4. Chagas. 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