Evolving Health Effects of Pneumocystis: Title and subTitle BreakOne Hundred Years of Progress in Diagnosis and Treatment

A man in his early 50s with chronic lymphocytic leukemia was admitted with 3 weeks of progressive cough, shortness of breath, and fever. His chronic lymphocytic leukemia, which was associated with a deletion in chromosome 11q22-23, had been diagnosed 2 years previously and was in partial remission following 6 cycles of fludarabine and rituximab therapy administered as part of a clinical trial at the National Institutes of Health. Because of a Coombs-positive hemolytic anemia, he was also receiving prednisone, 1 mg/kg per day. He was receiving aerosol pentamidine rather than trimethoprim-sulfamethoxazole for Pneumocystis prophylaxis because of a history of a severe sulfa allergy.

On physical examination, the patient had a temperature of 41.5°C and diffuse crackles throughout his lung fields. His oxygen saturation was 86% on room air. Chest radiographs and computed tomographic examination of the chest showed diffuse ground glass–appearing infiltrates symmetrically in all lobes (Figure 1). Bronchoalveolar lavage performed on admission showed many clusters of Pneumocystis jirovecii on direct fluorescent antibody staining. No other pathogens were identified.

The patient was treated with intravenous pentamidine, and corticosteroids were continued. He had disease progression (Figure 1), necessitating intubation on hospital day 13. Two additional bronchoscopy samples continued to show moderate numbers of Pneumocystis organisms as well as Candida , Aspergillus , and herpes simplex. Sequencing of the dihydropteroate synthase (DHPS) gene of Pneumocystis from 3 separate bronchoalveolar lavage samples showed only wild-type sequence. The patient died of respiratory failure on day 31 of hospitalization.

The autopsy showed extensive involvement of his bone marrow, adrenal glands, lymph nodes, and lung with leukemic cells. Histologic examination of the lungs also showed many Pneumocystis organisms (Figure 2) and 1 small cluster of Aspergillus but no evidence of herpes simplex or Candida .

2009 marks the 100th anniversary of the identification of Pneumocystis by Carlos Chagas, who initially reported this organism to be part of the life cycle of Trypanosoma cruzi.¹ Two years later, Chagas identified the organisms in the lungs of a patient who died of trypanosomiasis.² It was not until 1912 that Pneumocystis was recognized as a unique organism by the Delanoes,³ who named it Pneumocystis carinii in honor of Antonio Carini (the second person to study the organism). Studies of this apparently clinically irrelevant organism languished over the next 40 years, until the 1940s and 1950s, when Pneumocystis was ultimately identified as the cause of a perplexing pneumonia, characterized by prominent plasma cell infiltration, that was causing epidemics in premature and malnourished infants.^{4 - 5} Over the next 30 years, as immunosuppressive therapy was expanding in use, clinicians began to recognize the potential for this pathogen to cause life-threatening pulmonary disease in immunosuppressed hosts, especially in children receiving intensive chemotherapy for treatment of leukemia.^{6 - 7} Its clinical relevance became strikingly clear as an epidemic of Pneumocystis cases in previously healthy adults served to announce the arrival of the AIDS epidemic and to highlight that AIDS was a disorder of profound immunodeficiency.^{8 - 9} The past 30 years has seen impressive advances in the diagnosis and management of Pneumocystis pneumonia (PCP), yet, as illustrated by the case presentation, PCP remains a potentially fatal disease that continues to be diagnosed at most major medical centers that manage large numbers of immunosuppressed patients.

Quiz Ref IDPneumocystis pneumonia is a disease that occurs exclusively in patients who have substantial immunosuppression. Immunologically normal patients do not develop clinically important disease due to this pathogen. Although some studies suggested that sudden infant death syndrome (SIDS) was associated with Pneumocystis infection, well-designed studies have in fact found no difference in the frequency of Pneumocystis infection in infants who died of SIDS vs other causes.^{10 - 11} Pneumocystis has been identified in patients with chronic pulmonary obstructive disease, although what role, if any, it plays in modulating clinical manifestations remains undefined.¹² Among immunosuppressed populations, the number of cases of PCP in patients infected with human immunodeficiency virus (HIV) has decreased over the past 20 years, initially because of the broad use of prophylaxis in susceptible patients and more recently because of the immune recovery associated with antiretroviral therapy.¹³ However, the number of patients receiving blood and solid organ transplants has increased, and oncologists and rheumatologists have treated patients with progressively more potent immunosuppressive drugs, including high-dose corticosteroids and immunomodulating monoclonal antibodies, so the number of cases of PCP in these populations has grown.

Pneumocystis, which was long thought to be a protozoan, is now clearly recognized as a fungus based on an expanding range of information, including ribosomal RNA and other gene sequence homologies, the composition of its cell wall, and the structure of key enzymes, among other factors.¹⁴ Based on molecular and antigenic studies, the organism that infects humans has been recognized as unique and distinct from that infecting animals,¹⁵ and this has led to a change in the species name from Pneumocystis carinii to Pneumocystis jirovecii (pronounced “yee row vet zee”) in honor of Otto Jirovec, one of the investigators who recognized the association of Pneumocystis with plasma cell pneumonia, although this name has not been universally accepted.^{16 - 17} In fact, each mammalian species appears to be infected with a unique strain of Pneumocystis. Thus, humans acquire Pneumocystis only from other humans. Rat, mouse, ferret, or horse species infect only their specific host and are not sources of human infection.

Pneumocystis jirovecii is now recognized as a worldwide pathogen, with cases reported from all continents except Antarctica. Although it was initially thought to be a rare cause of pneumonia in Africa, a prospective study in Uganda using bronchoscopy and immunofluorescent staining identified Pneumocystis in nearly 40% of HIV-infected patients with fever and respiratory symptoms who had negative results from acid-fast bacilli smears.¹⁸

It is well documented that transmission of Pneumocystis occurs by the respiratory route among rodents, and it presumably spreads among humans in a similar manner.¹⁹ However, it is unlikely that the organism is transmitted only by humans with overt PCP, given the widespread nature of this infection: serologic data indicate that most humans are infected with Pneumocystis, usually within the first 2 to 4 years of life.²⁰ Data from infants offer dramatic support for the suggestion that exposure to the organism is ubiquitous. An autopsy study of otherwise healthy infants younger than 1 year who died of a variety of causes identified Pneumocystis DNA by polymerase chain reaction (PCR) in 100%.²¹ In addition, infants with HIV infection have a peak incidence of PCP between the ages of 2 and 6 months²² ; given that in animal models it takes 2 to 3 months after exposure to develop a heavy infection,²³ this suggests exposure to the organism soon after birth. Thus, Pneumocystis must presumably be transmitted to these infants by apparently healthy humans in their environment, and inapparent infection must be extremely prevalent for so many infants to become infected so quickly.

In the past, it was thought that most cases of PCP were due to reactivation of a strain acquired many years previously (eg, during infection as an infant). However, recent data support the concept that, at least in some patients, disease is caused by a recently acquired strain. The strongest evidence for this comes from a study of mutations in the DHPS gene of Pneumocystis. (GenBank AY628435 has the entire sequence for a trifunctional gene of Pneumocystis jirovecii, part of which is DHPS.) DHPS is the microbial target of sulfa and sulfone therapy.²⁴ These mutations, which are strongly associated with prior sulfa exposure,^{25 - 26} were identified in more than 50% of patients in whom HIV infection had not been previously diagnosed and who had not been exposed to sulfa drugs, suggesting that they had recently acquired the strain from others with such exposure. Reports of clusters of cases of PCP with a restricted Pneumocystis genotype also support recent transmission.²⁷

There is substantial evidence that many adults with PCP are infected with more than 1 strain of human Pneumocystis. Genotyping of isolates from individual patients with PCP using a variety of techniques have identified 2 or more strains concomitantly in more than 50% of patients (Figure 3).^{28 - 29} There are also data to show that distinct strains are responsible for each episode in patients who developed multiple episodes of PCP.³⁰ Multiple episodes of PCP are common only among patients with HIV infection (relapse occurred in 75%-90% of patients in the AIDS era prior to PCP prophylaxis and antiretroviral therapy, depending on how long patients survived), but when they occur, they are in some cases due to reinfection rather than relapse.

Pneumocystis pneumonia can largely be prevented by administration of chemoprophylaxis to susceptible individuals. For patients with HIV infection, peripheral blood CD4 counts provide a simple and effective method for evaluating PCP susceptibility.³¹ As the CD4 count decreases to less than 200 cells/μL, the likelihood that a patient will develop PCP increases substantially. Quiz Ref IDThus, in this population, a CD4 count below 200 cells/μL is an extremely useful biomarker for initiating prophylaxis. Approximately 10% to 15% of cases of PCP have been reported at CD4 counts above 200 cells/μL, but the incidence of such cases is very low given the large number of HIV-infected patients who belong to this category; patients with CD4 counts greater than 200 cells/μL who have a CD4 percentage of less than 14% may merit prophylaxis.³¹ Prophylaxis recommendations for HIV-infected children are age-based. Prophylaxis should be provided for children 6 years or older based on adult guidelines, for children aged 1 to 5 years if CD4 counts are less than 500 cells/μL or CD4 percentage is less than 15%, and for all HIV-infected infants younger than 12 months.³²

For patients immunosuppressed by processes other than HIV, there is no reliable laboratory marker for susceptibility. Non–HIV-infected patients with CD4 counts of less than 200 cells/μL have a higher likelihood of PCP than patients with higher CD4 counts,³³ but this test is not nearly as sensitive or specific as it is in HIV-infected patients. Thus, clinicians estimate susceptibility to PCP based on clinical parameters, such as the dose and duration of corticosteroid administration, exposure to other immunosuppressive drugs, or time since transplantation.

For the case presented here, the patient's prednisone therapy and his recent treatment with both rituximab and fludarabine exposed him to 3 immunosuppressive regimens associated with the occurrence of PCP, although the duration of susceptibility has not been clearly delineated.^{34 - 35} Management of prophylaxis in patients receiving corticosteroids has also been debated. One reasonable approach has been to administer prophylaxis to patients who are receiving at least 20 mg of prednisone per day for at least 1 month.³⁶

The most studied and most effective chemotherapeutic agent for prevention of PCP is trimethoprim-sulfamethoxazole. This combination therapy is effective when given as a single-strength tablet per day, a double-strength tablet per day, or either regimen twice daily.³⁷ Many hematologists and oncologists worry about the effects of trimethoprim-sulfamethoxazole on bone marrow function and either prescribe the drug 2 to 3 days per week or prescribe an alternative agent, even though the alternatives are less effective.³⁸

The patient presented here was provided alternative therapy (aerosolized pentamidine) because of his prior sulfa allergy. Although some patients with HIV have been given escalating doses of trimethoprim-sulfamethoxazole with apparent success in inducing tolerance,³⁹ such an approach has not been assessed in non–HIV-infected patients. Moreover, rechallenge with any dose would be inappropriate in patients if the history documented an immediate hypersensitivity reaction. Patients with a history of immediate hypersensitivity reactions can be desensitized in the intensive care unit if there is an urgent reason to do so, but such patients must then maintain consistent serum levels to avoid recurrent anaphylaxis. Dapsone, aerosol pentamidine, and atovaquone are alternative agents that are safe and effective, although not as effective as trimethoprim-sulfamethoxazole, based on randomized controlled trials in patients with HIV infection.^{40 - 41} No adequately powered studies have compared these agents for prophylactic efficacy in other immunosuppressed populations.

In the current case, dapsone was avoided because a substantial number of patients (probably ~50%) who developed adverse reactions to trimethoprim-sulfamethoxazole have similar reactions to dapsone. Atovaquone is a reasonable alternative if a patient can tolerate a good diet, especially one rich in fat to increase absorption, but is less reliable in patients with chemotherapy-induced nausea or gut graft-vs-host disease. Aerosol pentamidine was a good option in this patient, but many centers no longer support staff and facilities for delivering aerosol pentamidine.

Quiz Ref IDWhy are cases of PCP still seen at a time when susceptible populations and effective preventive regimens have been identified? For many immunosuppressed patients, the major reason is that appropriate chemoprophylaxis is not administered. For patients with HIV infection, the Adult and Adolescent Spectrum of HIV Disease Study found that 40% of patients developing PCP had never started prophylaxis. In the United States, 25% of HIV-infected patients are unaware of their infection and thus would not be candidates for prophylaxis.⁴² Moreover, 39% of patients with a new diagnosis of HIV infection are “late testers”; ie, they develop AIDS within 1 year of their HIV diagnosis. These patients, as well as patients with poor access to care, account for a large fraction of the patients who develop HIV-related PCP. Additional patients fail to take their prophylaxis or have clinicians who forget to prescribe prophylaxis when their CD4 count decreases to less than 200 cells/μL. A small proportion of HIV-infected patients will develop PCP at CD4 counts above the threshold recommended for chemoprophylaxis, as noted previously, and some will break through chemoprophylaxis, especially when receiving regimens other than trimethoprim-sulfamethoxazole.

In many reports of PCP among cancer patients and transplant recipients, PCP occurs during periods of recognized susceptibility, not because the risk was overlooked, but because the patient either was not prescribed prophylaxis or did not take the prescribed drug.

Quiz Ref IDPatients with PCP, like the one presented here, come to medical attention with fever, shortness of breath, cough, hypoxemia, and/or diffuse pulmonary infiltrates.⁴³ Clinicians need to recognize that many infectious and noninfectious processes can present identically and should also recognize that hallmarks such as fever, shortness of breath, and diffuse infiltrates do not invariably occur, especially early in the course while the disease is mild.

One interesting feature of PCP is that in patients with cancer or transplants, compared with patients with HIV infection, PCP is much more likely to be an acute illness causing severe respiratory distress within the first several days.⁴³ Subacute presentations, such as what the current patient showed, are much more common among patients with HIV infection. However, there is considerable variation in the presentation with each underlying condition. Patients can be hypoxemic but can also have normal alveolar-arterial gradients and normal chest radiographs if identified early in the natural history of their disease. In such patients, a computed tomographic scan is almost always abnormal.⁴⁴

However, most patients, like this one, present radiologically with diffuse, symmetrical interstitial or reticulogranular infiltrates. As the disease progresses, the infiltrates become more alveolar. Of note, almost every conceivable radiographic presentation has been linked to PCP, including asymmetrical infiltrates, nodules, cavities, pleural effusions, and pneumothorax.

Extrapulmonary manifestations of PCP—including retinitis; sepsis; digital necrosis; and space occupying lesions of the liver, spleen, kidney, and brain—are distinctly unusual and have been reported primarily among HIV-infected patients, especially those who received aerosol prophylaxis for PCP.⁴⁵ Aerosol pentamidine does not achieve systemic levels of drug, and thus extrapulmonary disease plausibly occurs more commonly when this agent is used.

Quiz Ref IDPneumocystis pneumonia is currently diagnosed by detection of organisms in respiratory secretions or tissue. The organisms can be stained by a variety of techniques: methenamine silver and toluidine blue O are most commonly used to visualize cysts in tissue samples, although hematoxylin-eosin show a characteristic foamy, eosinophilic material filling the alveoli that is difficult to confuse with other pulmonary processes (Figure 2). For respiratory secretions, including both induced sputum and bronchoalveolar lavage, immunofluorescent staining is the most sensitive and specific diagnostic reagent, although colorimetric stains such as methenamine silver, Giemsa, or toluidine blue O can also be used.⁴⁶

Humans are probably infected multiple times during their lives with Pneumocystis, but there is no evidence that healthy humans have organisms that are detectable by light microscopy in their respiratory secretions. Thus, while healthy humans without pulmonary dysfunction may have organisms detectable by molecular techniques such as PCR, clinicians should presume that whenever patients with fever or pulmonary dysfunction have organisms identified in tissue or secretions by light microscopy, their symptoms are due to Pneumocystis. Polymerase chain reaction testing of induced sputum or bronchoalveolar lavage can also identify and quantitate Pneumocystis, and several studies have suggested that PCR can be a sensitive and specific diagnostic test on these samples, as well as on oral gargles, but no commercial PCR test is broadly available at this time.⁴⁷

A major advance in the diagnosis of PCP would be the availability of an accurate serologic test. Antibody testing is not useful: there are many different IgG antibody tests, all of which are positive in most asymptomatic adults.^{48 - 49} Neither IgG nor IgM tests have been useful for diagnosis of acute disease. One group of investigators has reported that Pneumocystis cannot synthesize S-adenosylmethionine, an essential growth factor, and that the presence of the organism in the lung can be correlated with depletion of S-adenosylmethionine from the serum.⁵⁰ These results have not yet been confirmed by other laboratories,⁵¹ and a Pneumocystis gene encoding a functional S-adenosylmethionine synthetase has been identified.⁵² Tests measuring lactate dehydrogenase, C-reactive protein, and β-D-glucan⁵³ are biologically interesting, but results to date have not demonstrated that such tests are useful clinically for establishing a diagnosis of PCP or for assessing prognosis.

Once a patient has been diagnosed with PCP and is receiving treatment, the demonstration of organisms by light microscopy or PCR has less relevance. For HIV-infected patients, even in patients who are rapidly improving clinically, organisms can persist in tissue and secretions for many weeks.⁵⁴ When patients with PCP do not improve and they undergo repeat bronchoalveolar lavage or lung biopsy, the persistent presence of Pneumocystis organisms is hard to interpret.

In the patient presented here, Pneumocystis cysts continued to be present in the second and third bronchoscopy samples. Two other potential pathogens, Aspergillus and herpes simplex, were also present. There is no ideal way to determine which of these potential pathogens was causing the pulmonary dysfunction. Finding angioinvasive Aspergillus in a lung biopsy should be an obvious indication for treatment, but even if such a finding had been present in this case, it would have been uncertain whether the invasive Aspergillus was causing the diffuse lung injury or was a second process. Both herpes simplex and Candida are uncommon causes of diffuse pulmonary dysfunction. Most clinicians would treat both the herpes simplex and Aspergillus in this case, because effective therapy is easy to administer and relatively nontoxic, but would do so with no conviction that these pathogens were likely to be the cause of pulmonary dysfunction.

Trimethoprim-sulfamethoxazole, which can be administered orally or intravenously, is the first-line agent for the treatment of any form or severity of PCP.^{55 - 56} It is highly effective and generally well tolerated; adverse reactions, which can include leukopenia, rash, and hepatitis, are more common in HIV-infected patients than other populations.

This patient was treated with intravenous pentamidine because of history of a severe hypersensitivity to trimethoprim-sulfamethoxazole. Although desensitization could potentially allow the use of trimethoprim-sulfamethoxazole, there is no published regimen for doing this that has been shown to be safe in patients with severe reactions.

Intravenous pentamidine (there is no oral formulation) has been shown to have equivalent efficacy to trimethoprim-sulfamethoxazole in trials of patients with HIV infection or with cancer.^{57 - 58} However, pentamidine is not the preferred therapy because of its substantial toxicity profile.^{40 ,59} The drug is both nephrotoxic and toxic for pancreatic islet cells. It is not uncommon for patients to have hypoglycemia at some point during the course of receiving pentamidine due to a surge in insulin as islet cells are destroyed; this can occur even weeks or months after the course is completed. Some patients subsequently develop insulin-dependent diabetes mellitus. Pentamidine can also cause leukopenia.

Other drugs, including clindamycin-primaquine, dapsone-trimethoprim, and atovaquone, are also highly effective, although probably not as effective as either trimethoprim-sulfamethoxazole or intravenous pentamidine.^{55 - 56 ,60} For this patient, clindamycin-primaquine would have been an alternative choice for initial therapy, even though primaquine is available only in oral form and gastrointestinal absorption might be unreliable. Atovaquone is also effective but would be a poor choice for this patient because only an oral preparation is available; absorption is enhanced by fatty meals, which this patient was unable to take; and steady state is not attained for 4 days. Dapsone is also available only as an oral drug and cross-reacts with sulfonamides. Thus, dapsone-trimethoprim would have been a poor choice for this patient.

It is unclear how this patient's corticosteroids should have been managed to optimize his response to PCP therapy. For HIV-infected patients who are not receiving corticosteroids at the time PCP develops, there are data clearly demonstrating that prednisone therapy can accelerate symptomatic and physiologic improvement and improve survival in patients with moderate or severe infection, ie, those with a room-air PaO₂ of less than 70 mm Hg at onset of therapy.^{61 - 62} Corticosteroids likely modulate an inflammatory response that is elicited in the lungs following initiation of anti-Pneumocystis therapy.

There are no randomized data proving that corticosteroid therapy is effective in cancer or transplant patients, but most clinicians presume efficacy based on data from cohort studies. It is especially difficult to determine how to manage corticosteroids in patients who were receiving these drugs when PCP developed. It is plausible that corticosteroid therapy should not be reduced as PCP therapy is instituted. However, whether doses should be increased is unknown.

When disease progresses in a patient despite an anti-Pneumocystis regimen, optimal management is not well defined. There are no tests currently available for assessing clinically relevant drug resistance. There have been multiple studies looking for mutations in the DHPS gene (that confer potential sulfonamide or sulfone resistance), and these mutations are clearly more common in recent years and more common among patients who have been exposed to trimethoprim-sulfamethoxazole or dapsone in the past.^{26 ,63} Similar mutations do encode measurable resistance when present in Plasmodia and pneumococci. However, the studies linking these mutations to therapeutic failure in patients with PCP have been inconclusive, and many patients in whom these mutations were retrospectively identified were successfully treated with trimethoprim-sulfamethoxazole or dapsone.^{64 - 66} Mutations conferring potential atovaquone resistance have also been identified, but again their clinical relevance is uncertain.⁶⁷

There are no randomized trial data indicating when specific anti-Pneumocystis therapy should be modified because of inadequate response. Published literature would suggest that in the pre–corticosteroid era, patients generally improved after 4 to 8 days of therapy.⁴³ Thus, it is logical to maintain the initial regimen for at least 4 days before considering another strategy. Many clinicians would treat progressively deteriorating PCP with the following sequence of drugs: trimethoprim-sulfamethoxazole, pentamidine, and clindamycin-primaquine. Whether there is synergy or antagonism by adding drugs, rather than switching drugs, has not been elucidated by animal studies or human trials; physicians will often switch rather than add to minimize cumulative toxicities.

While adjusting PCP therapy, physicians must always evaluate patients for other treatable processes. In a few patients, depending on the underlying disease, another treatable infection can be identified by repeat bronchoalveolar lavage or lung biopsy, but finding such treatable causative organisms is unusual. Clinicians also need to carefully assess patients to determine whether congestive heart failure, pulmonary emboli, mechanical problems such as pneumothorax or large effusion, or progressive neoplastic disease are part of the process causing lung dysfunction.

There are 2 major challenges to confront to reduce the impact of PCP on immunosuppressed patients. First, systems need to be developed so that susceptible patients are reliably offered chemoprophylaxis and then continued with that prophylaxis until their immunity improves. Second, as patients are exposed to more trimethoprim-sulfamethoxazole, clinically important drug resistance is likely to continue to develop. Potent and well-tolerated drugs will need to be developed, but currently there are few incentives for new drugs to be identified and developed.