Search

Previous serologic surveys show >80% of infants in Chile have anti-Pneumocystis antibodies by 2 years of age, but the seroepidemiology of Pneumocystis infection beyond infancy is unknown. We describe the sero-epidemiology in infants, children, and adults at different locations in Chile. Serum samples were prospectively obtained from 681 healthy adults (age ≥ 17 years) and 690 non-immunocompromised infants/children attending eight blood banks or outpatient clinics (2 in Santiago) in Chile. ELISA was used to measure serum IgM and IgG antibodies to Pneumocystis jirovecii major surface antigen (Msg) constructs MsgA and MsgC1. Serologic responses to Pneumocystis Msg showed a high frequency of reactivity, inferring infection. Among infants/children increasing age and the proportion with detectable IgM responses to MsgA, and IgG responses to MsgA, and MsgC1 were positively associated. Among adults there was almost universal seropositivity to one or more Pneumocystis Msg constructs. In infants and children rates of detectable IgM responses to MsgC1 and MsgA were greater than IgG responses. In Santiago, rates of seropositivity among infants/children were greater in clinics located in a more socio-economically deprived part of the city. In Chile, a serological response to Pneumocystis Msg constructs was common across ages regardless of geographical location and climatic conditions. Observed higher rates of IgM responses than IgG responses is consistent with concept of recent/ongoing exposure to Pneumocystis in children and adults. Higher rates of seropositivity in infants/children residing in more densely populated areas of Santiago infers crowding poses an increased risk of transmission.

Previous serologic surveys show80% of infants in Chile have anti

Over the past decade, there has been rising interest in the interaction ofWe undertook a systematic review in order to identify environmental factors associated withThe systematic review found evidence of associations betweenThis systematic review provides evidence of associations between

In at risk populations, Pneumocystis pneumonia (PCP) may occur as a solitary event as well as in a cluster- or outbreak setting due to interpatient transmission of Pneumocystis jirovecii. Despite the data and insights obtained from studies of outbreaks of PCP, the development and implementation of comprehensive recommendations for the prevention of healthcare related transmission of P. jirovecii have been delayed. Both optimization of chemoprophylaxis strategies as well as combination with prudent use of isolation precautions are warranted to achieve this goal. The rationale of the available measures for the prevention of PCP should be viewed in the context of what is currently known about the complex biology and epidemiology of P. jirovecii. From there, phased but practical prevention strategies can be deducted to balance the efforts, costs and negative consequences of chemoprophylaxis and isolation precautions with the beneficial effect of preventing healthcare related transmission of P. jirovecii and development of PCP.

Background: Over the past decade, there has been rising interest in the interaction of Pneumocystis with the environment. This interest has arisen in part from the demonstration that environmental factors have important effects on the viability and transmission of microbes, including Pneumocystis. Environmental factors include climatological factors such as temperature, humidity, and precipitation, and air pollution factors including carbon monoxide, nitrogen dioxide, sulfur dioxide, and particulate matter. Methods: We undertook a systematic review in order to identify environmental factors associated with Pneumocystis infection or PCP, and their effects on human and animal hosts. Results: The systematic review found evidence of associations between Pneumocystis infection in animal and human hosts, and climatological and air pollution factors. Data from human studies infers that rather than a seasonal association, presentation with PCP appears to be highest when the average temperature is between 10 and 20°C. There was evidence of an association with hospitalization with PCP and ambient air pollution factors, as well as evidence of an effect of air pollution on both systemic and bronchoscopic lavage fluid humoral responses to Pneumocystis. Interpretation of human studies was confounded by possible genetically-determined predisposition to, or protection from infection. Conclusions: This systematic review provides evidence of associations between Pneumocystis infection in both animal and human hosts, and climatological and environmental air pollution factors. This information may lead to an improved understanding of the conditions involved in transmission of Pneumocystis in both animal and human hosts. Such knowledge is critical to efforts aimed at prevention.

Pneumocystis pneumonia (PCP) is caused by the yeastlike fungus Pneumocystis. Despite the widespread availability of specific anti-Pneumocystis prophylaxis and of combination antiretroviral therapy (ART), PCP remains a common AIDS-defining presentation. PCP is increasingly recognized among persons living in Africa. Pneumocystis cannot be cultured and bronchoalveolar lavage is the gold standard diagnostic test to diagnose PCP. Use of adjunctive biomarkers for diagnosis requires further evaluation. Trimethoprim-sulfamethoxazole remains the preferred first-line treatment regimen. In the era of ART, mortality from PCP is approximately 10% to 12%. The optimal time to start ART in a patient with PCP remains uncertain.

Background: Immune responses to Pneumocystis jirovecii are not well understood in HIV infection, but antibody responses to proteins may be useful as a marker of Pneumocystis risk or presence of Pneumocystis pneumonia (PcP). Design: Retrospective analysis of a prospective cohort. Methods: Enzyme-linked immunosorbent assays of antibodies to recombinant Pneumocystis proteins of major surface glycoprotein fragments (MsgCl, C3, C8, and C9) and of antibody titers to recombinant kexin protein (KEX1) were performed on 3 sequential serum samples up to 18 months before and 3 samples after first AIDS-defining illness from Multicenter AIDS Cohort Study participants and compared between those who had PcP or a non-PcP AIDS-defining illness. Results: Fifty-four participants had PcP and 47 had a non-PcP AIDS-defining illness. IgG levels to MsgC fragments were similar between groups before first AIDS-defining illness, but the PcP group had higher levels of IgG to MsgC9 (median units/mL 50.2 vs. 22.2, P = 0.047) post-illness. Participants with PcP were more likely to have an increase in MsgC3 [odds ratio (OR): 3.9, P = 0.02], MsgC8 (OR: 5.5, P = 0.001), and MsgC9 (OR: 4.0, P = 0.007). The PcP group was more likely to have low KEX1 IgG before development of PcP (OR: 3.6, P = 0.048) independent of CD4 cell count and to have an increase in high IgG titers to KEX1 after PcP. Conclusions: HIV-infected individuals develop immune responses to both Msg and kexin proteins after PcP. Low KEX1 IgG titers may be a novel marker of future PcP risk before CD4 cell count has declined below 200 cells per microliter.

Summary Objectives To characterize the seroepidemiological features of Pneumocystis jirovecii infection in healthy Chilean children using overlapping fragments (A, B, C) of the P. jirovecii major surface glycoprotein (Msg). Methods Serum antibodies to MsgA, MsgB, and MsgC were measured every 2 months by enzyme-linked immunosorbent assay (ELISA) in 45 Chilean infants from about age 2 months to 2 years. Results Peak antibody levels (usually reached at age 6 months) and the force (or rate) of infection were somewhat greater for MsgC than for MsgA. Significant seasonal variation in antibody levels was only found with MsgA. Respiratory infections occurred in most children, but nasopharyngeal aspirates were of limited value in detecting the organism. In contrast, serological responses commonly occurred, and higher levels only to MsgC were significantly related to the number of infections. Conclusions Serological responses to recombinant Msg fragments provide new insights into the epidemiological and clinical features of P. jirovecii infection of early childhood. MsgA, the amino terminus fragment, is more sensitive in detecting seasonal influences on antibody levels, whereas MsgC is better able to detect changes in antibody levels in response to clinical infection.

A seasonal variation in the presentation of Pneumocystis jirovecii pneumonia (PCP) has been reported and a previous study from this centre noted a seasonal variation in mortality rates. This study examined seasonal influences (including climatic factors) within-host factors (clinical and laboratory-derived variables), the infectious burden of P. jirovecii in bronchoalveolar lavage (BAL) fluid, the presence of dihydropteroate synthase (DHPS) mutations in P. jirovecii, variations in knowledge and skills of junior medical staff, and mortality in 547 episodes of PCP occurring in 494 HIV-infected patients. The overall mortality rate was 13.5%. There was a seasonal variation in mortality: highest in autumn (21.2%) and lowest in spring (9.7%), P = 0.047. After adjustment was made for prognostic factors previously identified as being associated with mortality (increasing patient age, second/third episode of PCP, low haemoglobin, low PaO2, presence of medical co-morbidity and pulmonary Kaposi sarcoma), there was no seasonal association with mortality, P = 0.249. The quantity of P. jirovecii DNA in BAL fluid showed no evidence of seasonal variation, P = 0.67; DHPS mutations were identified with equal frequency in each season and the mortality rate for February and August (when junior medical staff arrive in new posts) was 16.7%, only slightly greater than for other months (13.0%).

The use of recombinant fragments of the major surface glycoprotein (Msg) of Pneumocystis jirovecii has proven useful for studying serological immune responses of blood donors and human immunodeficiency virus (HIV)-positive (HIV + ) patients. Here, we have used ELISA to measure antibody titres to Msg fragments (MsgA, MsgB, MsgC1, MsgC3, MsgC8 and MsgC9 in sera isolated in the USA (n=200 and Spain (n=326), to determine whether geographical location affects serological responses to these antigens. Blood donors from Seville exhibited a significantly greater antibody titre to MsgC8, and significantly lower responses to MsgC3 and MsgC9, than did Cincinnati (USA) donors. Spanish blood donors (n=162 also exhibited elevated responses to MsgC1, MsgC8 and MsgC9 as compared with Spanish HIV + (n=patients. HIV + patients who had Pneumocystis pneumonia (PcP + ) exhibited a higher response to MsgC8 than did HIV + PcP - patients. These data show that geographical location plays a role in responsiveness to Msg fragments. Additionally, these fragments have utility in differentiating HIV + PcP and HIV + PcP + among patient populations.

Humans may be a reservoir for this pathogen and transmit it from person to person., The reservoir and mode of transmission of Pneumocystis jirovecii remain uncertain. We conducted a cross-sectional study of 126 San Francisco General Hospital staff in clinical (n = 103) and nonclinical (n = 23) occupations to assess whether occupational exposure was associated with immune responses to P. jirovecii. We examined antibody levels by ELISA for 3 overlapping fragments that span the P. jirovecii major surface glycoprotein (Msg): MsgA, MsgB, and MsgC1. Clinical occupation participants had higher geometric mean antibody levels to MsgC1 than did nonclinical occupation participants (21.1 vs. 8.2, p = 0.004); clinical occupation was an independent predictor of higher MsgC1 antibody levels (parameter estimate = 0.89, 95% confidence interval 0.29–1.48, p = 0.003). In contrast, occupation was not significantly associated with antibody responses to either MsgA or MsgB. Healthcare workers may have occupational exposure to P. jirovecii. Humans may be a reservoir for P. jirovecii and may transmit it from person to person.

Despite medical advances, Pneumocystis jirovecii remains a major cause of lethal pneumonia (Pneumocystis pneumonia [PCP]) in patients with HIV infection. A recent study of >500 episodes of PCP in patients with HIV infection at a single medical center revealed no statistically significant changes in the mortality rate over the past 2 decades [1]. The development of sensitive molecular techniques has permitted detection of P. jirovecii in the respiratory tract of different patient populations. The highest frequency of P. jirovecii colonization or subclinical infection occurs among patients with HIV infection; specific risk factors, such as low CD4+ cell counts and smoking, have been identified [2]. P. jirovecii colonization induces local and systemic inflammatory responses and has been associated with more-severe airway obstruction [3, 4]. Thus, the potential problems resulting from the interaction of P. jirovecii with its human host extend not only to immunocompromised patients but also to immunocompetent patients. Analysis of immune responses to P. jirovecii has long been hampered by the lack of suitable reagents [5–14]. The development of recombinant P. jirovecii antigens has begun to change this situation [15–20]. We selected the major surface glycoprotein (Msg) as our target antigen, because it is highly immunogenic, contains protective B and T cell epitopes, and plays a central role in the interaction of the organism with the host [21–25]. We developed 3 over-lapping recombinant fragments (MsgA, MsgB, and MsgC) that span the entire length of a single Msg isoform. Our studies have shown that MsgC, the carboxyl terminus, is the fragment that is most strongly recognized by serum antibodies of patients with HIV infection who have recovered from PCP [19, 20, 26]. To better delineate this antibody reactivity, we developed 3 variants of our original MsgC (termed MsgC1)—MsgC3, MsgC8, and MsgC9—which exhibited 70%–99% homology at the amino acid level [27]. So far, our studies have shown that these variants react with serum specimens independently, but they also seem to have unique epitopes. The Multicenter AIDS Cohort Study (MACS) is an ongoing, prospective cohort study of HIV infection in men who have sex with men that began in 1984 and involves 4 cities: Baltimore, Maryland; Chicago, Illinois; Pittsburgh, Pennsylvania; and Los Angeles, California. The careful patient follow-up and extensive database of the MACS have made it an invaluable resource for numerous studies of HIV infection and its complications. From 1992 through 2000, 106 patients from this cohort died and had lung tissue samples obtained at autopsy. Fifteen patients died from PCP. Ninety-one patients died from other causes, and of these, 42 (46%) were colonized with P. jirovecii, as determined by polymerase chain reaction (PCR) [28]. Multivariate analysis showed that cigarette smoking was associated with an increased risk for colonization, whereas residence in Los Angeles was associated with a decreased risk. These data raised intriguing questions about the relationship between the clinical and epidemiologic factors of P. jirovecii colonization, the occurrence of PCP, and serologic responses to Msg. In the present study, we have examined the serologic responses to the MsgC constructs over the last 5.5 years of life in these MACS patients according to their status at death (PCP, P. jirovecii colonization, or neither), the specific host and environmental factors for all patients associated with high antibody levels, and whether the first AIDS-defining illness was PCP.

We examined the effects of surfactant protein A (SP-A), a collectin, on the interaction of Pneumocystis murina with its host at the beginning, early to middle, and late stages of infection. Pneumocystis murina from SP-A wild-type (WT) mice inoculated intractracheally into WT mice (WT(S)-WT(R)) adhered well to alveolar macrophages, whereas organisms from SP-A knockout (KO) mice inoculated into KO mice (KO(S)-KO(R)) did not. Substitution of WT mice as the source of organisms (WT(S)-KO(R)) or recipient host macrophages (KO(S)-WT(R)) restored adherence to that found with WT(S)-WT(R) mice. In contrast, when immunosuppressed KO and WT mice were inoculated with P. murina from a homologous source (KO(S)-KO(R), WT(S)-WT(R)) or heterologous source (WT(S)-KO(R), KO(S)-WT(R)) and followed sequentially, WT(S)-KO(R) mice had the highest levels of infection at weeks 3 and 4; these mice also had the highest levels of the chemokine macrophage inflammatory protein-2 and neutrophils in lavage fluid at week 3. Surfactant protein-A administered to immunosuppressed KO(S)-KO(R) mice with Pneumocystis pneumonia for 8 wk as a therapeutic agent failed to lower the organism burden. We conclude that SP-A can correct the host immune defect in the beginning of P. murina infection, but not in the middle or late stages of the infection.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) remains the leading cause of opportunistic infection among human immunodeficiency virus (HIV)-infected persons. Previous studies of PCP that identified case-fatality risk factors involved small numbers of patients, were performed over few years, and often focused on patients who were admitted to the intensive care unit. OBJECTIVE: The objective of this study was to identify case-fatality risk factors present at or soon after hospitalization among adult HIV-infected patients admitted to University College London Hospitals (London, United Kingdom) from June 1985 through June 2006. PATIENTS AND METHODS: We performed a review of case notes for 494 consecutive patients with 547 episodes of laboratory-confirmed PCP. RESULTS: Overall mortality was 13.5%. Mortality was 10.1% for the period from 1985 through 1989, 16.9% for the period from 1990 through June 1996, and 9.7% for the period from July 1996 through 2006 (P = .142). Multivariate analysis identified factors associated with risk of death, including increasing patient age (adjusted odds ratio [AOR], 1.54; 95% confidence interval [CI], 1.11-2.23; P = .011), subsequent episode of PCP (AOR, 2.27; 95% CI, 1.14-4.52; P = .019), low hemoglobin level at hospital admission (AOR, 0.70; 95% CI, 0.60-0.83; P < .001), low partial pressure of oxygen breathing room air at hospital admission (AOR, 0.70; 95% CI, 0.60-0.81; P < .001), presence of medical comorbidity (AOR, 3.93; 95% CI, 1.77-8.72; P = .001), and pulmonary Kaposi sarcoma (AOR, 6.95; 95% CI, 2.26-21.37; P = .001). Patients with a first episode of PCP were sicker (mean partial pressure of oxygen at admission +/- standard deviation, 9.3+/-2.0 kPa) than those with a second or third episode of PCP (mean partial pressure of oxygen at admission +/- standard deviation, 9.9+/-1.9 kPa; P = .008), but mortality among patients with a first episode of PCP (12.5%) was lower than mortality among patients with subsequent episodes of PCP (22.5%) (P = .019). No patient was receiving highly active antiretroviral therapy before presentation with PCP, and none began highly active antiretroviral therapy during treatment of PCP. CONCLUSIONS: Mortality risk factors for PCP were identifiable at or soon after hospitalization. The trend towards improved outcome after June 1996 occurred in the absence of highly active antiretroviral therapy.

Recombinant clones of the carboxyl terminus of the major surface glycoprotein (MsgC) of Pneumocystis jirovecii are useful for analyzing serologic responses in humans. However, there is no standardized set of antigens in general use, which could lead to conflicting results. We have previously shown that human immunodeficiency virus type 1 (HIV-1)-infected patients with prior Pneumocystis pneumonia (PcP + ) responded more frequently and more strongly to a clone of MsgC than did HIV-1-infected patients without PcP (PcP − ). Here we test three new clones of MsgC to determine the effect of antigenic sequence variation on immune reactivity in blood donors and HIV-infected patients previously analyzed for reactivity to our original MsgC clone. In Western blot analyses, PcP + patients exhibited the highest frequency of reactivity to each MsgC clone, and the frequency of reactivity with all four MsgC clones together was significantly higher in sera from PcP + patients than in sera from the other patient groups. Furthermore, in an enzyme-linked immunosorbent assay we found that the PcP + population had the highest level of reactivity to two of the four clones tested. One of the new clones could distinguish between PcP + and PcP − populations, and two MsgC clones could distinguish blood donors from the other patient populations. The results show that inherent differences in MsgC amino acid sequence can affect recognition by antibodies independently of variations in protein length or patient population, and the utility of a clone depends on its sequence and on the populations tested.

We conducted a prospective pilot study of the serologic responses to overlapping recombinant fragments of the Pneumocystis jirovecii major surface glycoprotein (Msg) in HIV-infected patients with pneumonia due to P. jirovecii and other causes. Similar baseline geometric mean antibody levels to the fragments measured by an ELISA were found in both groups. Serum antibodies to MsgC in P. jirovecii patients rose to a peak level 3–4 weeks (p 50 cells/µL and first episode of pneumocystosis were the principal host factors associated with this rise (both p

Bisbenzamidines, such as pentamidine isethionate, are aromatic dicationic compounds that are active against Pneumocystis and other microbes but are oftentimes toxic to the host. To identify potential anti- Pneumocystis agents, we synthesized bisbenzamidine derivatives in which the parent compound pentamidine was modified by a 1,4-piperazinediyl, alkanediamide, or 1,3-phenylenediamide moiety as the central linker. Several of the compounds were more active against P. carinii and less toxic than pentamidine in cytotoxicity assays. For this study, we evaluated nine bisbenzamidine derivatives representing a range of in vitro activities, from highly active to inactive, for the treatment of pneumocystosis in an immunosuppressed mouse model. Six of these in vitro-active compounds, 01, 02, 04, 06, 100, and 101, exhibited marked efficacies against infection at a dose of 10 mg/kg of body weight, and four compounds, 01, 04, 100, and 101, showed significant increases in survival versus that of untreated infected control mice. Compound 100 was highly efficacious against the infection at 20 mg/kg and 40 mg/kg, with >1,000-fold reductions in burden, and resulted in improved survival curves versus those for pentamidine-treated mice (at the same doses). All six bisbenzamidine compounds that exhibited high in vitro activity significantly decreased the infection in vivo; two compounds, 12 and 102, with marked to moderate in vitro activities had slight or no activity in vivo, while compound 31 was inactive in vitro and was also inactive in vivo. Thus, the selection of highly active compounds from in vitro cytotoxicity assays was predictive of activity in the mouse model of Pneumocystis pneumonia. We conclude that a number of these bisbenzamidine compounds, especially compound 100, may show promise as new anti- Pneumocystis drugs.

Among a library of 17 bisbenzamidines connected with various linkers, compounds with a flexible pentanediamide (10) or hexanediamide (12) linker were the most potent derivatives against rat Pneumocystis carinii (IC50 values of 3 and 2 nM, respectively) and had the highest selectivity index ratios (GI50 of human tumor cells/IC50 of rat P. carinii cells) of >104. Seven compounds caused 50% growth inhibition (GI50) of tumor cells at concentrations of

Changes in incidence of PCP, groups at risk for PCP, and possible trends in the disease are discussed., Pneumocystis pneumonia (PCP) has historically been one of the leading causes of disease among persons with AIDS. The introduction of highly active antiretroviral therapy in industrialized nations has brought about dramatic declines in the incidence of AIDS-associated complications, including PCP. In the adult population, the incidence of PCP has significantly decreased, but it remains among the most common AIDS-defining infections. Similar declines have been documented in the pediatric population. In much of the developing world, PCP remains a significant health problem, although its incidence among adults in sub-Saharan Africa has been debated. This review discusses the epidemiology of PCP during the current era of the AIDS epidemic. Although fewer cases of PCP occur in industrialized countries, increasing drug-resistant HIV infections, possible drug-resistant PCP, and the tremendous number of AIDS cases in developing countries make this disease of continued public health importance.

A series of pentamidine congeners has been synthesized and screened for their in vitro activity against Pneumocystis carinii. Among the tested compounds, bisbenzamidines linked by a flexible pentanediamide or hexanediamide chain (7 and 9) emerged as exceptionally potent agents that were more effective and less toxic than pentamidine in the assays described in this study.

Seroepidemiologic studies of Pneumocystis pneumonia (PCP) in humans have been limited by inadequate reagents. We have developed an enzyme-linked immunosorbent assay (ELISA) using three overlapping recombinant fragments of the human Pneumocystis major surface glycoprotein (MsgA, MsgB, and MsgC) for analysis of antibody responses in HIV-positive patients and healthy blood donors. HIV-positive patients had significantly higher antibody levels to all Msg fragments. Furthermore, HIV-positive patients who experienced a previous episode of PCP (PCP-positive) had higher levels of antibodies to MsgC than patients who never had PCP. A significant association was found between ELISA antibody level and reactivity by Western blot in HIV-positive patients, especially those who were PCP-positive. Thus, this ELISA will be useful in studying serum antibody responses to Pneumocystis in different human populations.

The immune response to the opportunistic pulmonary pathogenPneumocystiscan have beneficial and harmful effects on the host despite the presence of corticosteroids. We hypothesized that this deleterious hyperinflammatory response is associated with exaggerated cytokine production. The adoptive transfer of at least 107immune splenocytes reduced the cyst count in rats with corticosteroid-induced pneumocystosis. About 18% of these rats developed clinical illness, an increased lung weight/body weight (LW/BW) ratio, and elevated levels of interleukin 1α (IL-1α), IL-1β, IL-6, tumor necrosis factor alpha, IL-5, IL-10, and gamma interferon in the lungs. This hyperinflammatory reaction was not observed in rats that remained clinically well or in control rats. Thus, in this model, corticosteroids have little effect on the cytokine cascade or other adverse effects of the host immune response toPneumocystis.

The CD4+T lymphocyte plays a central role in host defense againstPneumocystispneumonia but has received only limited attention in rats. CD4+T-cell-depleting (OX-38) and nondepleting (W3/25) monoclonal antibodies, which recognize an identical or adjacent epitope, were administered for up to 14 weeks to Lewis rats that had been exposed toPneumocystis. While OX-38 produced a greater decrease in circulating CD4+cells than W3/25, both antibody treatments resulted in similar effects on the health of the rats and the levels ofPneumocystispneumonia, which were milder than those found with corticosteroids. W3/25 also did not enhance the severity ofPneumocystispneumonia achieved with corticosteroids alone. We conclude that CD4+cell function is more important than CD4+cell number in host defense againstPneumocystisin the rat and that this new model permits study of opportunistic infections in the rat without the confounding effects of corticosteroids.

The major surface glycoprotein (Msg) of Pneumocystis jiroveci (P. jiroveci) is important in the immunopathogenesis of Pneumocystis pneumonia (PcP), but is difficult to study in humans. We generated 3 overlapping recombinant Msg fragments (MsgA, MsgB and MsgC), and analyzed their reactivity with serum samples from 95 healthy blood donors and 94 human immunodeficiency virus (HIV)-infected persons. Reactivity to the Msg fragments varied with HIV infection and prior episodes of PcP but not with geographic origin. Recognition of MsgA was lower-and recognition of MsgB was significantly lower-in HIV(+) serum compared with donor serum. Serum samples from HIV-positive patients with prior PcP recognized MsgC more frequently than did serum samples from those without PcP. None of the serum samples drawn from 9 patients before they had developed PcP recognized MsgC. These data suggest that these novel recombinant proteins are useful for the analysis of antibody responses to Msg.

BACKGROUND: Humoral immunity plays an important role against Pneumocystis jirovecii infection, yet clinical and environmental factors that impact bronchoalveolar antibody responses to P. jirovecii remain uncertain. METHODS: From October 2008-December 2011 we enrolled consecutive HIV-infected adults admitted to San Francisco General Hospital (SFGH) who underwent bronchoscopy for suspected Pneumocystis pneumonia (PCP). We used local air quality monitoring data to assign ozone, nitrogen dioxide, and fine particulate matter exposures within 14 days prior to hospital admission. We quantified serum and bronchoalveolar lavage fluid (BALF) antibody responses to P. jirovecii major surface glycoprotein (Msg) recombinant constructs using ELISA. We then fit linear regression models to determine whether PCP and ambient air pollutants were associated with bronchoalveolar antibody responses to Msg. RESULTS: Of 81 HIV-infected patients enrolled, 47 (58%) were diagnosed with current PCP and 9 (11%) had a prior history of PCP. The median CD4+ count was 51 cells/μl (IQR 15-129) and 44% were current smokers. Serum antibody responses to Msg were statistically significantly predictive of BALF antibody responses, with the exception of IgG responses to MsgC8 and MsgC9. Prior PCP was associated with increased BALF IgA responses to Msg and current PCP was associated with decreased IgA responses. For instance, among patients without current PCP, those with prior PCP had a median 73.2 U (IQR 19.2-169) IgA response to MsgC1 compared to a 5.00 U (3.52-12.6) response among those without prior PCP. Additionally, current PCP predicted a 22.5 U (95%CI -39.2, -5.82) lower IgA response to MsgC1. Ambient ozone within the two weeks prior to hospital admission was associated with decreased BALF IgA responses to Msg while nitrogen dioxide was associated with increased IgA responses. CONCLUSIONS: PCP and ambient air pollutants were associated with BALF IgA responses to P. jirovecii in HIV-infected patients evaluated for suspected PCP.

Quinupristin-dalfopristin (Q-D), which is active against bacteria and Toxoplasma gondii , was examined for its activity against Pneumocystis carinii . After 72 h of incubation with rat P. carinii in an ATP cytotoxicity assay, the 50% inhibitory concentration of Q-D was 10.6 μg/ml, a level that can be achieved in serum with high-dose administration. Q-D administered intraperitoneally at doses of 50 to 200 mg per kg of body weight per day in the treatment and 100 mg/kg/day three times per week in the prophylaxis of pneumocystosis in immunosuppressed mice reduced the organism burden up to 15- and 302-fold, respectively. We conclude that Q-D has activity against P. carinii in vitro and in vivo.

Immunosuppressed Swiss Black mice deficient in surfactant protein A (SP-A(-/-)) and wild-type control mice (SP-A(+/+)) were exposed to Pneumocystis carinii by environmental exposure, intratracheal inoculation, and direct exposure to other infected animals. The frequency and intensity of P. carinii infection were significantly greater in the SP-A(-/-) mice by all 3 methods of exposure. P. carinii free of SP-A and alveolar macrophages were isolated from SP-A(-/-) mice and were tested in an in vitro attachment assay. Pretreatment of P. carinii with human SP-A resulted in a significant dose-dependent increase of the adherence of P. carinii to the macrophages. Thus, SP-A plays a role in host defense against P. carinii in vivo, perhaps by functioning as a nonimmune opsonin.

In a previous cross-sectional study, we showed that clinical staff working in a hospital had significantly higher antibody levels than nonclinical staff to Pneumocystis jirovecii. We conducted a longitudinal study, described here, to determine whether occupation and self-reported exposure to a patient with P. jirovecii pneumonia were associated with antibody levels to P. jirovecii over time. Baseline and quarterly serum specimens were collected and analyzed by using an ELISA that targeted different variants of the Pneumocystis major surface glycoprotein (MsgA, MsgB, MsgC1, MsgC3, MsgC8, and MsgC9). Clinical staff had significantly higher estimated geometric mean antibody levels against MsgC1 and MsgC8 than did nonclinical staff over time. Significant differences were observed when we compared the change in antibody levels to the different MsgC variants for staff who were and were not exposed to P. jirovecii pneumonia- infected patients. MsgC variants may serve as indicators of exposure to P. jirovecii in immunocompetent persons.

Pneumocystis carinii is an important opportunistic pulmonary pathogen in human immunodeficiency virus (HIV) patients and other immunocompromised hosts. Basic research on P. carinii has been hampered by the lack of a reliable in vitro culture system; nevertheless, through the use of molecular

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose, and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanvalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host.

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5′ end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein–MSG-B fusion protein ( MBP MSG-B 41–1065 ) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

Pneumocystis carinii is a common cause of life-threatening pneumonia in immunodeficient patients. Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate recognition domains (CRD), binds P. carinii and enhances adherence to alveolar macrophages. In this study, we examined the structural basis of the interaction between SP-A and the major surface glycoprotein of P. carinii (MSG). Rat SP-A bound to purified rat P. carinii-derived MSG in a saturable and calcium-dependent manner, which was partially reversible by coincubation with excess monosaccharides, or pretreatment of MSG with N-glycanase. Mutant recombinant SP-As with neutral amino acid substitutions for the predicted calcium- and carbohydrate-coordinating residues of the CRD were synthesized in insect cells using baculovirus vectors and tested for binding to MSG. Substitutions of negatively charged (Glu195, Glu202, and Asp215) and polar residues (Asn214) of the CRD with alanine but not substitution of the Arg197 with glycine reduced the binding of SP-A to mannose-Sepharose beads and to MSG. Deletion of the N-linked oligosaccharides from SP-A by mutagenesis of the consensus sequences for glycosylation had no effect on binding. We conclude that the CRD mediates the binding of SP-A to oligosaccharides attached to MSG.

Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii-exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-gamma, although in differing amounts, implicating a Th1 response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii, MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.

Background: Ambient air pollution (AAP) may be associated with increased risk for Pneumocystis pneumonia (PCP). The mechanisms underlying this association remain uncertain. Objectives: To determine if real-life exposures to AAP are associated with suppressed IgM antibody responses to P. jirovecii in HIV-infected (HIV+) patients with active PCP, and to determine if AAP, mediated by suppressed serologic responses to Pneumocystis, is associated with adverse clinical outcomes. Methods: We conducted a prospective cohort study in HIV+ patients residing in San Francisco and admitted to San Francisco General Hospital with microscopically confirmed PCP. Our AAP predictors were ambient air concentrations of particulate matter of < 10 μm in diameter (PM 10) and < 2.5 μm in diameter (PM2.5), nitrogen dioxide (NO2), ozone (O3), and sulfur dioxide (SO 2) measured immediately prior to hospital admission and 2 weeks prior to admission. Our primary outcomes were the IgM serologic responses to four recombinant P. jirovecii major surface glycoprotein (Msg) constructs: MsgC1, MsgC3, MsgC8, and MsgC9. Results: Elevated PM10 and NO2 exposures immediately prior to and two weeks prior to hospital admission were associated with decreased IgM antibody responses to P. jirovecii Msg. For exposures immediately prior to admission, every 10 μg/m3 increase in PM10 was associated with a 25 to 35% decrease in IgM responses to Msg (statistically significant for all the Msg constructs), and every 10 ppb increase in NO2 was associated with a 19-45% decrease in IgM responses to Msg (statistically significant for MsgC8 and MsgC9). Similar findings were seen with exposures two weeks prior to admission, but for fewer of the Msg constructs. Conclusions: Real life exposures to PM10 and NO2 were associated with suppressed IgM responses to P. jirovecii Msg in HIV+ patients admitted with PCP, suggesting a mechanism of immunotoxicity by which AAP increases host susceptibility to pulmonary infection. © 2013 Blount et al.

BackgroundHumoral immune responses in human immunodeficiency virus (HIV)-infected and uninfected children with Pneumocystis pneumonia (PcP) are poorly understood.MethodsConsecutive children hospitalized with acute pneumonia, tachypnea, and hypoxia in South Africa were investigated for PcP, which was diagnosed by real-time polymerase chain reaction on lower respiratory tract specimens. Serum antibody responses to recombinant fragments of the carboxyl terminus of Pneumocystis jirovecii major surface glycoprotein (MsgC) were analyzed.Results149 children were enrolled of whom 96 (64%) were HIV-infected. PcP occurred in 69 (72%) of HIV-infected and 14 (26%) of HIV-uninfected children. HIV-infected children with PcP had significantly decreased IgG antibodies to MsgC compared to HIV-infected patients without PcP, but had similar IgM antibodies. In contrast, HIV-uninfected children with PcP showed no change in IgG antibodies to MsgC, but had significantly increased IgM antibodies compared to HIV-uninfected children without PCP. Age was an independent predictor of high IgG antibodies, whereas PcP was a predictor of low IgG antibodies and high IgM antibodies. IgG and IgM antibody levels to the most closely related MsgC fragments were predictors of survival from PcP.ConclusionsYoung HIV-infected children with PcP have significantly impaired humoral immune responses to MsgC, whereas HIV-uninfected children with PcP can develop active humoral immune responses. The children also exhibit a complex relationship between specific host factors and antibody levels to MsgC fragments that may be related to survival from PcP.

I am honored to receive the second Lifetime Achievement Award by International Workshops on Opportunistic Protists (IWOP) and to give this lecture. My research involves Pneumocystis, an opportunistic pulmonary fungus that is a major cause of pneumonia (“PcP”) in the immunocompromised host. I decided to focus on Pneumocystis ecology here because it has not attracted much interest. Pneumocystis infection is acquired by inhalation, and the cyst stage appears to be the infective form. Several fungal lung infections, such as coccidiomycosis, are not communicable, but occur by inhaling < 5μ spores from environmental sources (buildings, parks), and can be affected by environmental factors. PcP risk factors include environmental constituents (temperature, humidity, SO2, CO) and outdoor activities (camping). Clusters of PcP have occurred, but no environmental source has been found. Pneumocystis is communicable and outbreaks of PcP, especially in renal transplant patients, are an ongoing problem. Recent evidence suggests that most viable Pneumocystis organisms detected in the air are confined to a patient’s room. Further efforts are needed to define the risk of Pneumocystis transmission in healthcare facilities; to develop more robust preventive measures; and to characterize the effects of climatological and air pollutant factors on Pneumocystis transmission in animal models similar to those used for respiratory viruses.

Background. Pneumocystis pneumonia (PcP) is the second leading cause of morbidity and mortality in human immunodeficiency virus (HIV)–infected patients in the United States. Although the host risk factors for the development of PcP are well established, the environmental (climatological, air pollution) risk factors are poorly understood. The major goal of this study was to determine the environmental risk factors for admissions of HIV-positive patients with PcP to a single medical center. Methods. Between 1997 and 2008, 457 HIV-positive patients with microscopically confirmed PcP were admitted to the San Francisco General Hospital. A case-crossover design was applied to identify environmental risk factors for PcP hospitalizations. Climatological and air pollution data were collected from the Environmental Protection Agency and Weather Warehouse databases. Conditional logistic regression was used to evaluate the association of each environmental factor and PcP hospital admission. Results. Hospital admissions were significantly more common in the summer than in the other seasons. Increases in temperature and sulfur dioxide levels were independently associated with hospital admissions for PcP, but the effects of sulfur dioxide were modified by increasing carbon monoxide levels. Conclusions. This study identifies both climatological and air pollution constituents as independent risk factors for hospitalization of HIV-positive patients with PcP in San Francisco. Thus, the environmental effects on PcP are more likely complex than previously thought. Further studies are needed to understand how these factors exert their effects and to determine if these factors are associated with PcP in other geographic locations.

Background: Little is known about the serologic responses to Pneumocystis jirovecii major surface glycoprotein (Msg) antigen in African cohorts, or the IgM responses to Msg in HIV-positive and HIV-negative persons with respiratory symptoms. Methods: We conducted a prospective study of 550 patients, both HIV-positive (n=467) and HIV-negative (n=83), hospitalized with cough $2 weeks in Kampala, Uganda, to evaluate the association between HIV status, CD4 cell count, and other clinical predictors and antibody responses to P. jirovecii. We utilized ELISA to measure the IgM and IgG serologic responses to three overlapping recombinant fragments that span the P. jirovecii major surface glycoprotein: MsgA (amino terminus), MsgB (middle portion) and MsgC1 (carboxyl terminus), and to three variations of MsgC1 (MsgC3, MsgC8 and MsgC9). Results: HIV-positive patients demonstrated significantly lower IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 compared to HIV-negative patients. We found the same pattern of low IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 among HIV-positive patients with a CD4 cell count ,200 cells/ml compared to those with a CD4 cell count $200 cells/ml. HIV-positive patients on PCP prophylaxis had significantly lower IgM responses to MsgC3 and MsgC9, and lower IgG responses to MsgA, MsgC1, MsgC3, and MsgC8. In contrast, cigarette smoking was associated with increased IgM antibody responses to MsgC1 and MsgC3 but was not associated with IgG responses. We evaluated IgM and IgG as predictors of mortality. Lower IgM responses to MsgC3 and MsgC8 were both associated with increased in-hospital mortality. Conclusions: HIV infection and degree of immunosuppression are associated with reduced IgM responses to Msg. In addition, low IgM responses to MsgC3 and MsgC8 are associated with increased mortality.

OBJECTIVES We examined 96 bronchoalveolar lavage fluid (BALF) specimens from AIDS patients with proven Pneumocystis carinii pneumonia (PCP) in order to compare the relationship of organism burden, viability and antigen expression with disease severity at the time of clinical presentation. METHODS Tinctorial analysis of BALF specimens with proven PCP using Diff-Quik, cresyl echt violet and erythrosin B stains to evaluate organism burden and viability. P. carinii antigen examination was performed by Western blot analysis. RESULTS P. carinii cluster ratios were more sensitive than cyst counts as an indicator of organism burden, and correlated well with the alveolar-arterial oxygen gradient as a measure of disease severity. Erythrosin B, the vital stain used to measure P. carinii viability, displayed a wide range of values and provided little useful information. Antigens of 35-45 and 95kD, which were specific for P. carinii, were found by immunoblot analysis in BALF cellular fraction of most patients with pneumocystosis. By contrast, antigens of 52 and 66 kD, which were found in both BALF supernatant and cellular fractions of P. carinii patients and controls, most likely represented albumin and immunoglobulin G heavy chain, respectively, of host origin. The 35-45 kD antigen was found in 88% of the BALF specimens and appeared to represent an important marker of P. carinii infection. The 95 kD antigen was detected in 49% of the specimens. CONCLUSIONS We conclude that analysis of P. carinii characteristics in BALF specimens of patients with pneumocystis may provide additional information. These data will also be helpful in developing more sensitive assays and in targeting specific P. carinii factors for future investigation.

The major surface glycoprotein (MSG) of rat-derived Pneumocystis carinii represents a group of related molecules that are encoded by multiple genes. We isolated seven unique MSG cDNAs from a library prepared from a single infected rat lung. The cDNAs displayed both conserved and variant regions to previously described cDNAs. These clones contained inserts that ranged in size from 0.4 to 1.8 kb and all contained a poly(A) tail. The largest clone, Pc1410, hybridized to all 15 chromosomes resolved by pulsed-field gel electrophoresis (PFGE). Protein produced by in vitro translation from Pc1410 was immunoprecipitated with affinity-purified MSG antibodies. The clones were characterized by DNA sequencing of their 3' and 5' ends. Analysis of the untranslated and coding regions demonstrated that the clones contained unique and conserved regions of sequence, but none of the clones were identical. Isolation of seven additional unique clones picked from a single screening of a cDNA library suggests that numerous MSG transcripts exist within a population of P. carinii.

A prognostic scoring tool (PST) was created to aid prediction of outcome from HIV-associated Pneumocystis jirovecii pneumonia (PCP) using data obtained from 577 episodes of PCP among 540 patients presenting to a specialist HIV treatment centre in London, UK. It used risk factors identifiable at/soon after hospitalization, previously identified as being associated with mortality: repeat episode of PCP, patient's age, haemoglobin (Hb) and oxygen partial pressure (PaO2) on admission, presence of medical co-morbidity (Comorb) and of pulmonary Kaposi sarcoma (PKS). The derived PST was 25.5+(age in years/10) + 2 (if a repeat episode of PCP) + 3 (if Comorb present) + 4 (if PKS detected) – PaO2 (kPa) – Hb (g/dL), and produced scores that ranged between 0 and 19. Patients were divided into five groups according to their prognostic score: 0-3.9 = group 1 (0% mortality), 4-7.9 = group 2 (3% mortality), 8-10.9 = group 3 (9% mortality), 11-14.9 = group 4 (29% mortality) and ≥15 = group 5 (52% mortality). This PST facilitates rapid identification of patients early in their hospitalization who have mild or severe HIV-associated PCP and who are at high and low risk of in-hospital death from PCP. The PST may aid assessment of severity of illness and in directing treatment strategies, but requires validation in patient cohorts from other healthcare institutions.

During the past 30 years, major advances have been made in our understanding of HIV/AIDS and Pneumocystis pneumonia (PCP), but significant gaps remain. Pneumocystis is classified as a fungus and is host-species specific, but an understanding of its reservoir, mode of transmission, and pathogenesis is incomplete. PCP remains a frequent AIDS-defining diagnosis and is a frequent opportunistic pneumonia in the United States and in Europe, but comparable epidemiologic data from other areas of the world that are burdened with HIV/AIDS are limited. Pneumocystis cannot be cultured, and bronchoscopy with bronchoalveolar lavage is the gold standard procedure to diagnose PCP, but noninvasive diagnostic tests and biomarkers show promise that must be validated. Trimethoprim-sulfamethoxazole is the recommended first-line treatment and prophylaxis regimen, but putative trimethoprim-sulfamethoxazole drug resistance is an emerging concern. The International HIV-associated Opportunistic Pneumonias (IHOP) study was established to address these knowledge gaps. This review describes recent advances in the pathogenesis, epidemiology, diagnosis, and management of HIV-associated PCP and ongoing areas of clinical and translational research that are part of the IHOP study and the Longitudinal Studies of HIV-associated Lung Infections and Complications (Lung HIV).