A Four-Center Retrospective Study of the Efficacy and Toxicity of Low-Dose Trimethoprim-Sulfamethoxazole for the Treatment of Pneumocystis Pneumonia in Patients without HIV Infection.

The dose of trimethoprim-sulfamethoxazole (TMP-SMX) for the treatment of Pneumocystis pneumonia (PCP) in patients without human immunodeficiency virus (HIV) infection has not been verified. The aim of this study was to investigate the efficacy and toxicity of a low-dose TMP-SMX regimen in such patients. A retrospective study was conducted in four hospitals. We reviewed the medical records of patients with PCP but not HIV (non-HIV-PCP) who were treated with TMP-SMX between 2003 and 2016. The patients were divided into conventional-dose (TMP, 15 to 20 mg/kg/day) and low-dose (TMP, <15 mg/kg/day) groups after patients who received high-dose (TMP, >20 mg/kg/day) treatment were excluded. Grouping was done according to a correction dose, which was based on renal function. Eighty-two patients had non-HIV-PCP. The numbers of patients who received high-, conventional-, and low-dose treatments were 5, 36, and 41, respectively. Kaplan-Meier analysis for death associated with PCP showed no statistically significant difference in survival rates between the conventional- and low-dose groups. Ninety-day cause-specific mortality rates were 25.0% and 19.5% in the conventional-dose and low-dose groups (P = 0.76), respectively. Adverse events that were graded as ≥3 according to the Common Terminology Criteria for Adverse Events (version 4.0) (National Cancer Institute, 2010) were 41.7% and 17.1% in the conventional-dose and low-dose groups (P = 0.02), respectively. Moreover, vomiting (P = 0.03) and a decrease in platelet count (P = 0.03) occurred more frequently in the conventional-dose group. Treatment of non-HIV-PCP with low-dose or conventional-dose TMP-SMX produces comparable survival rates; however, the low-dose regimen is better tolerated and associated with fewer adverse effects.

The ecology of pneumocystis: perspectives, personal recollections, and future research opportunities.

I am honored to receive the second Lifetime Achievement Award by International Workshops on Opportunistic Protists 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 < 5 µm 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 health care 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.

[Observation on the ultrastructure of Pneumocystis carinii].

OBJECTIVE: To study the life cycle and morphology of Pneumocystis carinii by ultrastructural observation. METHODS: Wistar rat model of P. carinii infection was established by subcutaneous injection with dexamethasone. Lung tissue of the infected rats was used for the transmission electron microscopical study. RESULTS: The organisms were mainly present in the lung alveolar cavity, and also in the alveolar septum, pulmonary macrophages and neutrophils. More trophozoites of P. carinii attached to the type I alveolar epithelial cells, and rarely to the type II alveolar epithelial cells. Most of these trophozoites showed pseudopodial evaginations on their pellicles. The nucleus-associated organelle and spindle microtubules were observed in some trophozoites. The precyst phase was in three forms: early, intermediate and late form. Synaptonemal complexes indicating meiotic nuclear divisions and a clump of mitochondria were also observed in the precyst. The pellicle of the cyst has a thickened portion with a pore. There were nucleus with nucleolus, mitochondrion, vesicles, endoplasmic reticulum and numerous ribosomes in the organisms, and tubular expansions on its surface. CONCLUSION: The life cycle of P. carinii consists of trophozoite, precyst and cyst stages. The presence of a single pore in the cyst wall reveals that pore formation may be a mode of excystation for intracystic bodies of P. carinii.

Pneumocystis carinii pneumonitis. The nature and diagnostic significance of the methenamine silver-positive "intracystic bodies".

The cyst forms of Pneumocystis carinii in specimens stained with methenamine silver contain single or paired discrete foci of enhanced staining that measure 1-2 micron in maximum size. The nature of these foci and their location within the cysts have been disputed. We demonstrate by electron microscopy of silver-stained sections that the darkly stained foci correspond to a focal thickening of the cyst wall and are unrelated to sporozoites and other intracystic organelles. The morphology of these structures by light microscopy is characteristic, and their recognition is helpful in identifying P. carinii cysts and in differentiating them from yeast-form fungi and argyrophilic tissue elements in histologic sections and cytology specimens.

Fluorescence of Pneumocystis carinii in Papanicolaou smears.

Pneumocystis carinii organisms can be identified in routine cytologic smears stained by the Papanicolaou method and viewed under ultraviolet light in a fluorescence microscope. The organisms can be identified easily in thin, well-preserved cytologic smears. No false-positive results were obtained when 55 Papanicolaou smears of bronchial washings were observed by this method. Twenty-three of these smears were from patients with Pneumocystic carinii pneumonia in whom the acquired immunodeficiency syndrome (AIDS) had been well documented with Gomori's methenamine silver stain. With this method no additional special stains are required, no time delay for special stains is involved, and no additional material for the preparation of extra smears for special stains is needed. An additional advantage is the possibility of the retrospective analysis of smears already stained by the Papanicolaou method.

Disseminated Pneumocystis carinii infection in a patient with acquired immunodeficiency syndrome.

A case of disseminated pneumocystosis occurring in a patient with the acquired immunodeficiency syndrome is described. Postmortem examination of this patient, who had three episodes of Pneumocystis carinii pneumonia during his 3-year clinical course, revealed clinically unsuspected infiltration of lymph nodes, spleen, adrenal glands, and bone marrow, in addition to persistent pulmonary infection by the organism.

Increased Pneumocystis carinii recovery from the upper lobes in Pneumocystis pneumonia. The effect of aerosol pentamidine prophylaxis.

STUDY OBJECTIVE: To determine the relative distribution of Pneumocystis carinii in the lungs of patients with P carinii pneumonia and to see the effect of aerosol pentamidine prophylaxis on this distribution. DESIGN: A prospective study of all human immunodeficiency virus-infected patients with pulmonary symptoms over a nine-month period. Patients were followed up for at least six weeks after bronchoscopy. SETTING: Inpatient and outpatient service at one referral center. PATIENTS: Human immunodeficiency virus-infected patients with pulmonary symptoms were referred for evaluation. Those patients subsequently found to have P carinii pneumonia were studied. INTERVENTION: Bronchoalveolar lavage was performed in the middle lobe (or lingula) and the apical segment of the same lung. MEASUREMENTS AND RESULTS: The aspirated fluids were kept separate and modified Wright-Giemsa-stained cytocentrifuge-prepared slides were made from each area, and the number of P carinii clusters per 500 nucleated cells was counted. Fifty patients were studied: 27 receiving pentamidine prophylaxis and 23 receiving no aerosol therapy. There was no significant difference in the amount of fluid retrieved by lavage from the middle or upper lobe for either group. Both groups had significantly lower numbers of P carinii clusters per 500 cells in the middle lobe (receiving pentamidine: 10 +/- 15.8 [SD]; not receiving pentamidine: 15 +/- 12.3) than in the upper lobe (receiving pentamidine: 22 +/- 19.8; not receiving pentamidine: 24 +/- 21.5; p < 0.02). In six patients, there were no P carinii organisms seen in the middle lobe lavage specimen. CONCLUSION: Pneumocystis carinii has a preference for the upper lobes which may be apparent even in patients not receiving aerosol pentamidine. In addition, yield for P carinii may be increased by performing lavage in the apical segment.

Bronchoalveolar lavage as the exclusive diagnostic modality for Pneumocystis carinii pneumonia. A prospective study among patients with acquired immunodeficiency syndrome.

Pneumocystis carinii pneumonia (PCP) is the most common life-threatening opportunistic infection among patients with the acquired immunodeficiency syndrome (AIDS). Because retrospective studies suggested that bronchoalveolar lavage (BAL) compared favorably to lung biopsy in the diagnosis of PCP, we prospectively evaluated the utility of BAL in 40 consecutive patients with AIDS or risk of AIDS who presented with respiratory complaints. The BAL revealed P carinii in 36 of 42 episodes of pneumonia (86 percent) among 40 patients. Clinical follow-up of the six patients whose BAL was negative for PCP suggested only one possible false negative BAL for PCP. Therefore, BAL detected PCP in 36 of 37 patients for a sensitivity of 97 percent. BAL detected cytomegalovirus in 15 of 38 patients, as well as Mycobacterium avium-intracellulare and Cryptococcus (each in one patient). By virtue of accuracy and lack of morbidity demonstrated in our study, BAL should supplant lung biopsy techniques in the evaluation of AIDS patients with pulmonary symptoms.

Use of the transbronchial biopsy for diagnosis of opportunistic pulmonary infections in acquired immunodeficiency syndrome (AIDS).

The authors used a method that includes Giemsa-stained touch preparations of a lung biopsy and culture of the same biopsy for viruses and microorganisms. The yield of etiologic agent diagnoses by this cytologic method was compared with yield from a simultaneous biopsy processed by usual histologic methods and stained by hematoxylin and eosin, silver methenamine, and other stains as necessary. Fifty-nine transbronchial biopsies and eight open lung biopsies were processed on 38 male patients with the clinical and laboratory features of acquired immunodeficiency syndrome (AIDS) to determine the etiology of their pulmonary disease. Pneumocystis carinii pneumonia was diagnosed in 16 patients. When sufficient material was available, the Giemsa-stained touch preparation agreed with the histologic examination. The touch preparation specimens were cultured, and seven of the patients with Pneumocystis carinii also had cytomegalovirus (CMV). Six additional patients had pulmonary CMV infection without evidence of Pneumocystis carinii pneumonia. Mycobacterial and fungal infections also were identified in these six patients. Sixteen patients had no specific diagnosis. With adequate material, their method provides reliable results and diagnosis within two to three hours of Pneumocystis carinii. In addition, it conserves the amount of specimen required, since the same material can be used for culture.

Giemsa staining for cysts and trophozoites of Pneumocystis carinii.

Although Giemsa staining has been routinely used for the detection of trophozoites and intracystic bodies in smears of bronchoalveolar lavage fluid (BAL) from patients with Pneumocystis carinii pneumonia, it does not normally stain the cyst wall. For detection of the cysts other stains such as toluidine Blue 'O' and methenamine silver must be used as well. Sulphation of smears before staining with Giemsa allows cysts to be visualised, thus enabling a single stain to be used to show all the stages of BAL or sputum, which is particularly useful, considering the increase in the prevalence of P carinii pneumonia in conjunction with the spread of AIDS.

Pneumocystis carinii pneumonia therapy with 9-deazainosine in rats.

An inosine analog, 9-deazainosine, has previously been demonstrated to inhibit Pneumocystis carinii in culture with WI-38 cells. The present study shows that it is also effective against Pneumocystis carinii in immunosuppressed Sprague-Dawley rats with Pneumocystis carinii pneumonia. After 8 wk of immunosuppression, rats that developed severe Pneumocystis carinii pneumonia were treated with either 9-deazainosine or served as controls. After 15 days of therapy, animals were sacrificed and severity of infection determined by morphologic examination of lungs for numbers of Pneumocystis carinii. Treated animals had greatly reduced numbers of Pneumocystis carinii trophozoites and cysts, compared with controls. This drug shows promise for therapy of Pneumocystis carinii pneumonia and should be studied further.

Silver staining of Pneumocystis carinii in the rat's lung.

The backscattered electron imaging mode of the scanning electron microscope was used to study the ontogenetic acquisition of argyrophilia in Pneumocystis carinii in rats. Silver staining continually increased from the late trophozoite to the mature cyst stage. The silver uptake began with a fine outline at the surface of the bodies of the late trophozoites; their cellular extensions, however, did not stain. The oblate precyst forms acquired the silver in heterogeneous patches. On spherical cysts the silver staining became more uniform and intense with at least one dense spot. The spherical and collapsed cysts also had short silver staining projections that may represent microvilli. Collapsed forms were paler than spherical ones and appear to be cysts that have undergone partial or complete release of sporozoites. These cell surface observations confirm and amplify previous transmission electron microscopical and histochemical studies indicating that silver staining correlates with the acquisition of the cell pellicle.

The use of an indirect fluorescent antibody test for detecting Pneumocystis carinii.

Pneumocystis carinii pneumonia is a major cause of morbidity and mortality in immunocompromised patients. An indirect fluorescent antibody (IFA) test has been developed using monoclonal antibodies specific for antigens on the surface of P carinii. We tested the sensitivity and specificity of this IFA test for detecting P carinii in respiratory specimens of immunocompromised patients with pulmonary symptoms undergoing bronchoscopy. Both the bronchial wash and bronchoalveolar lavage specimens of patients with and without P carinii pneumonia were studied. The bronchoalveolar lavage and bronchial wash specimens were examined using modified Wright-Giemsa and methenamine silver stains. In addition, aliquots of the specimen were fixed and stained with IFA and read with a fluorescent microscope. Fifty-nine patients were found to have P carinii organisms. The bronchial wash specimen has been shown to be less sensitive than the bronchoalveolar lavage specimen for detecting the presence of P carinii. In the bronchial wash specimen from these 59 patients, only 60% had positive modified Wright-Giemsa stains, and 70% had positive methenamine silver stains. The IFA stain was positive in 93% of the specimens tested (significantly higher than the other two stains). There was only one false-positive IFA test result among the 54 patients tested with negative results. We found the IFA stain to be superior to conventional stains when examining less-than-adequate specimens, such as those from bronchial washes.

Morphological evaluation of Pneumocystis carinii after extraction from infected lung.

Changes in Pneumocystis carinii induced by the extraction of the parasite from rabbit lung have been investigated. Samples obtained using 4 extraction methods were evaluated by light and transmission electron microscopy. Light microscopic evaluation was insufficient to give a measure of the P. carinii viability or to detect parasitic cellular alterations. In contrast, ultrastructural evaluation provided information on host and P. carinii cell integrity, which is a critical condition for viability. None of the tested methods was ideal. How thoroughly and in what shape P. carinii need to be extracted from tissues will determine which extraction technique is of best use.

Ultrastructural demonstration of a pore in the cyst wall of Pneumocystis carinii.

The proposed life cycle of the opportunistic organism Pneumocystis carinii (PC) includes the formation of a thick-walled cyst containing haploid progeny. The progeny cells mature within the cyst and are somehow released, leaving a collapsed empty cyst behind. Pneumocystis carinii cysts containing 8 intracystic bodies (ICB) as well as empty collapsed cysts have been commonly observed. In this study a single pore in the cyst walls of PC was observed by transmission electron microscopy in infected rat-lung preparations. Presence of a single pore in the cyst wall of an empty but noncollapsed cyst and in collapsed, empty cysts suggests that pore formation may be a mode of excystation for ICB of P. carinii.

An improved method of isolating Pneumocystis carinii from infected rat lungs.

A new method of isolating Pneumocystis carinii from infected lungs of cortisonized rats is described. Clumping of parasites and host lung material was diminished by suspension of macerated Pneumocystis-laden rat lung in a modified calcium, magnesium-free Hanks' balanced salts solution at physiologic pH and osmolality, containing the wetting agent G-acid. After washing, this material was suspended in a second buffer system for digestion. The digestion step was done in the same buffer but with the addition of calcium, magnesium, collagenase, hyaluronidase and deoxyribonuclease. These innovations allowed enumeration of trophozoites as well as cysts. Following digestion, the parasites were separated from particulate host lung debris by Percoll density gradients designed to pellet the debris, leaving parasites in the gradient. Density studies done prior to this step revealed that trophozoites and non-nucleated cysts had similar densities, 1.028 g/ml, whereas nucleated cysts were heavier at 1.030 g/ml. Particulate host lung debris could be removed due to its heavier density, 1.040 g/ml. The significance of this study includes: relatively clump-free suspensions of infected rat lung, enumeration of trophozoites as well as cysts, and characterization of nucleated cysts.

Pneumocystis carinii infection in corticosteroid-treated cats.

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage.

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.

Cytologic detection of Pneumocystis carinii: a comparison of Papanicolaou and other histochemical stains.

Pneumocystis carinii, a common pathogen among immunocompromised patients, has been investigated in cytologic specimens using both the histochemical stains and ultraviolet (UV) fluorescence following Papanicolaou staining. We reviewed 57 pulmonary cytologic specimens obtained from 23 patients and compared the results of specific histochemical stains and Papanicolaou-stained preparations under UV excitation. Specific Pneumocystis fluorescence was observed in 26 of 49 Papanicolaou-stained specimens. Thirty-seven specimens were examined using both histochemical staining and after Papanicolaou UV staining. A comparison of the two techniques showed the Papanicolaou UV technique to have 89% sensitivity and 95% specificity. Cellulosic filters were the most valuable Papanicolaou-stained preparation for Pneumocystis diagnosis. The authors conclude that UV fluorescence of Papanicolaou-stained specimens obtained by noninvasive procedures is a rapid, accurate, and economical method for diagnosing pulmonary Pneumocystis carinii infections.

Red blood cell "ghosts" as look-alikes for infectious organisms. A report of two cases.

Two cases are reported in which degenerated red blood cells were initially interpreted as representing infectious organisms. The morphology and staining characteristics of the altered red blood cells are discussed.