Pneumocystis Carinii Organisms Research Articles

Binding of Pneumocystis carinii to the lung epithelial cell receptor HSPA5 (GRP78).

The importance of lung macrophages in Pneumocystis-host interaction is well known, but little is known about the initial binding/colonization of the airway epithelium. Our prior studies have documented cell-signalling events that occur following binding of the organisms to lung epithelial cells; however, the receptors that mediate Pneumocystis attachment to lung surfaces have not yet been fully defined. Using affinity chromatography, we identified heat shock protein 5 (HSPA5), also known as GRP78, as a potential host receptor that may have relevance in Pneumocystis lung colonization. Pneumocystis carinii (Pc) organisms not only bound HSPA5 on a rat lung epithelial cell line, but also on primary rat airway epithelial cells (AECs). Furthermore, Pc bound CHO1 cells overexpressing HSPA5 more than the CHO1 parent line alone, supporting a role for Pc-HSPA5 protein interaction in mediating organism attachment. These results provide new insights into the interactions of Pneumocystis with host lung epithelium.

Uptake and degradation of Pneumocystis carinii by macrophages in vitro.

Pneumocystis carinii organisms, isolated from lungs of rats with glucocorticoid-induced pneumocystosis, were ingested and degraded in vitro by alveolar and peritoneal macrophages obtained from healthy rats and mice. Uptake of the organisms was inhibited at 4 degrees C and by treatment of macrophages with iodoacetate. Neither uptake nor degradation was inhibited by pretreatment of macrophages with hydrocortisone sodium phosphate in vitro. Although peritoneal macrophages of any age in vitro were capable of ingesting Pneumocystis, alveolar macrophages that had been cultivated in vitro for less than 2 days were incapable of phagocytosis of the organism, a functional defect that corrected itself spontaneously during cultivation in vitro.

Polymerase chain reaction assay for the diagnosis of pneumocystis carinii and cytomegalovirus pneumonia

We evaluated the utility of the polymerase chain reaction (PCR) method in detecting Pneumocystis carinii (PC) and cytomegalovirus (CMV) in bronchoalveolar lavage (BAL) specimens from patients suspected of having opportunistic infection of the lung. We evaluated BAL samples obtained from 41 immunosuppressed patients who had signs and symptoms of acute lower respiratory tract infection. In addition, we studied 16 immunocompetent patients. PC-DNA was detected in 19 cases and CMV-DNA was detected in three cases, in the immunosuppressed group. The diagnosis could not be established by conventional techniques in nine of these positive cases. PC-DNA was detected in all cases in which PC organisms had been demonstrated by silver or Giemsa staining. Neither PC nor CMV-DNA was detected in immunocompetent patients. We conclude that the PCR assay has greater sensitivity for the detection of PC and CMV-DNA in BAL specimens than conventional approaches and therefore enhances our diagnostic capability for PC and CMV pneumonia.

Inhibition of surfactant activity byPneumocystis cariniiorganisms and components in vitro

This study examines the direct inhibitory effects of Pneumocystis carinii (Pc) organisms and chemical components on the surface activity and composition of whole calf lung surfactant (WLS) and calf lung surfactant extract (CLSE) in vitro. Incubation of WLS suspensions with intact Pc organisms (10(7) per milligram of surfactant phospholipid) did not significantly alter total phospholipid levels or surfactant protein A content. Incubation with intact Pc organisms also did not impair dynamic surface tension lowering in suspensions of WLS or centrifuged large surfactant aggregates on a bubble surfactometer (37 degrees C, 20 cycles/min, 0.5 and 2.5 mg phospholipid/ml). However, exposure of WLS or CLSE to disrupted (sonicated) Pc organisms led to severe detriments in activity, with minimum surface tensions of 17-19 mN/m vs. <1 mN/m for surfactants alone. Extracted hydrophobic chemical components from Pc (98.8% lipids, 0.1 mM) reduced the surface activity of WLS and CLSE similarly to sonicated Pc organisms, whereas extracted hydrophilic chemical components from Pc (primarily proteins) had only minor effects on surface tension lowering. These results indicate that in addition to surfactant dysfunction induced by inflammatory lung injury and edema-derived inhibitors in Pc pneumonia, disrupted Pc organisms in the alveolar lumen also have the potential to directly inhibit endogenous and exogenous lung surfactants in affected patients.

Complementation and characterization of the Pneumocystis carinii MAPK, PCM

Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Δ kss1Δ mutants with α-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 μM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl 2, pH 6.5, temperature 30–35°C, and 10 μM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.

Pneumocystis carinii pneumonia alters expression and distribution of lung collectins SP-A and SP-D

Surfactant proteins SP-A and SP-D, members of the collectin family, have been shown to play a significant role in lung host defense. Both proteins selectively bind Pneumocystis carinii (PC) organisms and modulate the interaction of this pathogen with alveolar macrophages. We hypothesized that the expression and distribution of lung collectins SP-A and SP-D is altered by PC lung infection. PC organisms (2 × 105) were inoculated intratracheally into C.B–17 scid/scid mice that do not require steroids for immunosuppression. Four weeks after inoculation, bronchoalveolar lavage (BAL) fluid was fractionated into three fractions—cell pellet, large aggregate (LA), and small aggregate (SA) surfactant—and each fraction was analyzed for the expression of surfactant components. In uninfected mice, the majority of SP-A (62% ± 10%) was found in association with lipids in the LA fraction, while 55% ± 14% of SP-D was distributed in the SA fraction. In contrast, both hydrophobic proteins SP-B and SP-C were associated exclusively with LA. PC infection resulted in major changes in the expression of all surfactant components. Total protein content of LA was unchanged by PC infection (115% ± 18% of control), whereas SA protein content markedly increased (240% ± 18% of control level, P < .001). In contrast, the phospholipid content of LA was significantly decreased (53% ± 5% of control level, P < .001), whereas the SA phospholipid content of infected mice was increased (172% ± 16% of control level, P < .001). By Western blotting, PC pneumonia (PCP) induced a 3-fold increase in the total alveolar SP-D protein that was reflected mainly in increases in SA SP-D (454% ± 135% of control, P < .05). The total alveolar SP-A protein content was also increased in PCP because of a large increase in SP-A in SA (720% ± 115% of control, P < .05); SP-A levels in LA were unchanged. The increases in lung collectin expression were selective, because PCP resulted in the down-regulation of both SP-B and SP-C in LA (5% ± 2% and 13% ± 2% of control, respectively, P < .001). We conclude that PCP induces marked elevations in alveolar collectin levels because of increased expression and accumulation of SP-A and SP-D protein in SA surfactant. (J Lab Clin Med 2001;137:429-39)

This month in J Lab Clin Med: Issue highlights for June 2001

This month in J Lab Clin Med: Issue highlights for June 2001

Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts.

Pneumocystis carinii organisms constitute a large group of heterogeneous atypical microscopic fungi that are able to infect immunocompromised mammals by an airborne route and to proliferate in their lungs, inducing Pneumocystis carinii pneumonia. This pneumonia remains a crucial epidemiological challenge, since neither the source of Pneumocystis carinii infection in humans nor the process by which humans become infected has been clearly established. Polymerase chain reaction (PCR) assays have shown that profoundly immunosuppressed patients without pneumocystosis can be subclinically infected with Pneumocystis. Other PCR-based studies have suggested that healthy immunocompetent hosts are not latent carriers of the parasite. However, recent reports have indicated that Pneumocystis carinii can persist for limited periods in the lungs of convalescent rats after recovery from corticosteroid-induced pneumocystosis, and also that immunocompetent mammals can be transiently parasitized by Pneumocystis carinii after close contact with hosts with Pneumocystis carinii pneumonia. Can transiently parasitized hosts be a source of infection for immunosuppressed hosts? In order to investigate this important clinical question, the ability of immunocompetent BALB/c mice, which were carrying subclinical levels of Pneumocystis carinii, to transmit the infection by the airborne route to highly susceptible, uninfected mice with severe combined immunodeficiency was studied. The results indicated that the immunocompetent mice, transiently parasitized by Pneumocystis carinii organisms after close contact with Pneumocystis carinii-infected mice, were able to transmit the infection to Pneumocystis carinii-free mice with severe combined immunodeficiency.

Effects of atovaquone and diospyrin-based drugs on ubiquinone biosynthesis in Pneumocystis carinii organisms.

The naphthoquinone atovaquone is effective against Plasmodium and Pneumocystis carinii carinii. In Plasmodium, the primary mechanism of drug action is an irreversible binding to the mitochondrial cytochrome bc(1) complex as an analog of ubiquinone. Blockage of the electron transport chain ultimately inhibits de novo pyrimidine biosynthesis since dihydroorotate dehydrogenase, a key enzyme in pyrimidine biosynthesis, is unable to transfer electrons to ubiquinone. In the present study, the effect of atovaquone was examined on Pneumocystis carinii carinii coenzyme Q biosynthesis (rather than electron transport and respiration) by measuring its effect on the incorporation of radiolabeled p-hydroxybenzoate into ubiquinone in vitro. A triphasic dose-response was observed, with inhibition at 10 nM and then stimulation up to 0.2 microM, followed by inhibition at 1 microM. Since other naphthoquinone drugs may also act as analogs of ubiquinone, diospyrin and two of its derivatives were also tested for their effects on ubiquinone biosynthesis in P. carinii carinii. In contrast to atovaquone, these drugs did not inhibit the incorporation of p-hydroxybenzoate into P. carinii carinii ubiquinone.

Pulmonary colonization with Pneumocystis carinii in human immunodeficiency virus-negative patients: assessing risk with blood CD4+ T cell counts.

Use of PCR analysis has led to detection of low numbers of Pneumocystis carinii organisms, which were undetectable by microscopy, in bronchoalveolar lavage (BAL) fluid specimens from immunosuppressed patients who showed no evidence of acute P. carinii pneumonia (PCP) (1). These low levels of par- asites were usually considered to reflect pulmonary coloniza- tion, but their significance was a subject of controversy. Cases of colonization were described mainly in patients positive for HIV who had blood CD4 1 T cell counts of /L (2). 6 ! 400 3 10 There were little data concerning HIV-negative patients (3). This study investigates the presence of P. carinii DNA in BAL fluid specimens from HIV-negative patients with no evidence of PCP and examines the link between colonization and decreases in blood CD4 1 T cell counts in this patient population.

Pneumocystis carinii induces interleukin 6 production by an alveolar epithelial cell line.

Pneumocystis carinii (PC) infection often results in severe pneumonia in immunocompromised patients. Attachment of PC organisms to alveolar epithelial cells is a hallmark of PC pneumonia; however, few studies have investigated the response of alveolar epithelial cells to PC infection. Interleukin 6 (IL-6) is a multifunctional cytokine that is produced by alveolar epithelial cells in response to a variety of stimuli. Our investigation was undertaken to determine whether PC organisms induce production of IL-6 by alveolar epithelial cells and to determine the effect of IL-6 on PC attachment. Incubation of the human alveolar epithelial cell line, A549, with PC organisms resulted in a significant increase in IL-6 secreted into the cell culture media. Time-course studies showed that IL-6 production was detected as soon as 2 h after addition of PC and continued up to 48 h of exposure. Further studies demonstrated that preincubation of A549 cells with IL-6 resulted in a concentration-dependent increase in both A549 cell production of fibronectin and PC attachment. Thus, PC attachment to an alveolar epithelial cell line results in epithelial cell production of IL-6, which can act to further increase PC attachment. This may provide an important mechanism whereby PC organisms directly affect the host response to PC infection.

Characterization of natural occurring Pneumocystis carinii pneumonia in pigs by histopathology, electron microscopy, in situ hybridization and PCR amplification.

Macroscopic, histologic, ultrastructural, microbiologic, in situ hybridization (ISH) and PCR detection results in three 8-week-old pigs naturally infected with Pneumocystis carinii (PC) are described. All animals had a nonsuppurative interstitial pneumonia and intra-alveolar Pneumocystis organisms with foamy eosinophilic and PAS positive appearance. Ultrastructurally, PC trophozoites and cysts were observed in pigs No. 2 and No. 3, with the former being much more numerous. PC organisms were located on the alveolar surface or within the alveolar septa. Trophozoites had numerous filopodia and were thick-walled. Cysts had no or few filopodia, were thick-walled and contained intracystic bodies. Using non-isotopic ISH on formalin-fixed, paraffin-embedded lung tissue sections, PC DNA from pigs No. 2 and No. 3 hybridized with a probe specific for PC ribosomal RNA (rRNA). Using primers specific for mitochondrial rRNA gene (pAZ102-E/pAZ102-H), and for the internal transcriber spacers of ribosomal gene of PC, PCR methods amplified a product in the lung of pigs No. 2 and No. 3 using either frozen or formalin-fixed and paraffin-embedded lung tissue. DNA from Pig No. 1 samples did not amplify with any primer. This is the first time that molecular biology techniques (in situ hybridization and PCR) have been applied to the study of porcine pneumocystosis.

Inhibitory Effect of Human Natural Yeast Killer Toxin-like Candidacidal Antibodies on Pneumocystis carinii

Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT). Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms. Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P. carinii. Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P. carinii attachment to epithelial lung cells as well as infectivity to nude rats. Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P. carinii organisms. Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P. carinii organisms that are susceptible to PaKT itself. The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4. Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P. carinii organisms. The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.

Synthesis and dihydrofolate reductase inhibitory activities of 2,4-diamino-5-deaza and 2,4-diamino-5,10-dideaza lipophilic antifolates.

Two series of nonclassical antifolates (2,4-diamino-5-deaza compounds 2-5 and 5,10-dideaza compounds 6-13) were synthesized as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) organisms that are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity. Syntheses of the target 5-deaza compounds were achieved by initial construction of the pivaloyl-protected 2,4-diamino-6-bromopyrido[2,3-d]-pyrimidine 17 via a cyclocondensation of 2,4,6-triaminopyrimidine with bromomalonaldehyde. Sequential Heck coupling of 17 with styrene followed by ozonolysis afforded the 6-formyl derivative 19. Reductive amination of 19 with 3,4,5-trimethoxyaniline afforded the N10-H analog. The N10-Me and N10-Et analogs were synthesized by nucleophilic displacement of the 6-bromomethyl derivative 22 (obtained from the 6-formyl derivative 19 by reduction and bromination) with the appropriate N-alkylaniline. The trans-5,10-dideaza analogs 6-8 were synthesized via a Heck coupling of the appropriate methoxystyrene with 17, and selective reduction of the resulting 9,10-double bond afforded target compounds 9-11. Further reduction to the tetrahydro derivatives afforded analogs 12 and 13. The 5-deaza N10-Me 3,4,5-trimethoxy analog 3 maintained the best balance of potency and selectivity against both tgDHFR and pcDHFR. Compared to trimethoprim, compound 3 was only slightly less selective but was 300-fold more potent against tgDHFR. The 5,10-dideaza analogs were generally less potent and selective than the 5-deaza compounds.

Assessment of in vivo attachment/phagocytosis by alveolar macrophages

Alveolar macrophages (AMs) are recognized as an important first line of celular host defense within the lung. Although mechanisms underlying AM response to microorganisms or particulates are well characterized in vitro, experimental approaches to the study of AMs in vivo are limited. To circumvent these limitations, a new assay was developed using fluorescently labelled liposomes or Pneumocystis carinii (PC) organisms which were administered intratracheally into mechanically ventilated rats. After 30 min, the lungs were lavaged and the percentage of administered liposomes or PC bound to AMs was determined by quantifying fluorescence. Factors known to enhance attachment/phagocytosis by AMs in vitro were assayed to determine their effect in vivo. For example, vitronectin (VN)-coated liposomes increased attachment from 25.2 ± 2.4% to 47.2 ± 3.0% ( p < 0.001), while addition of VN increased the binding of PC to AMs from 16.5 ± 1.7% to 24.5 ± 2.2% ( p < 0.05). Confocal laser microscopy of cells obtained by lavage provided morphologic evidence of attachment/phagocytosis by AMs. This model will permit the quantitative assessment of the interaction of fluorescently labelled liposomes or microorganisms with AMs in the lower respiratory tract of living animals.

DifferentiatingPneumocystisCysts fromCandidaSP. Yeasts in Pulmonary Specimens Using Methenamine Silver

Pneumocystis carinii (PC) pneumonia in the immunocopromised patient requires prompt therapeutic intervention; therefore, rapid identification of PC organisms in cytopathologic specimen is essential. Conclusive diagnoses of PC are achievable using Grocott's methenamine silver (GMS), the "gold-standard" stain for PC cyst visualization. However, non-budding Candida sp. yeasts can simulate PC cysts with GMS and thus pose significant diagnostic challenges. After qualitative and semi-quantitative analysis of 49 cytopulmonary cases, this study aimed to establish morphologic criteria that differentiate these organisms using GMS. The results showed that spherical/demilune PC cysts (4 to 7 microns in diameter) are monomorphic and mainly transparent, with intracyst densities being commonly evident. Demilune cysts typically display wall wrinkling with longitudinal clefts. Relative to cysts, Candida sp. yeasts reveal increased argyrophilia, range 4 to 10 microns in diameter, are mainly oval and budding, polymorphic, and exhibit wall deformation with variable internal structure. Differentiating criteria are (a) budding; (b) cyst transparency, demilune shape; (c) longitudinal cyst clefts; (d) paired comma-like intracyst densities; (e) cyst monomorphism ; (f) alveolar cast formations; (g) overall cytomorphologic presentation; and (h) relative argyrophilia.

Attachment of Pneumocystis carinii to Primary Cultures of Rat Alveolar Epithelial Cells

Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type H alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 ± 1.2% on Day 2 to 18.4 ± 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 ± 2.5% to 9.4 ± 1.9% ( P < 0.01). Addition of antibodies to the α v and α 5 integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of α v and α 5 integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both α v and α 5 integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells.

Small Volume Bronchoalveolar Lavage Used in Diagnosing Pneumocystis carinii Pneumonia in HIV-Infected Patients

To determine the volume of bronchoalveolar lavage (BAL) fluid necessary to diagnose Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, specimens from 25 patients were evaluated. Twenty-one patients were HIV infected. BAL was performed using three to four 60-mL aliquots of room temperature, sterile, saline solution. Each syringe of BAL effluent was numbered and its volume was measured. Immunofluorescent stains were performed on about 8-mL aliquots of the initial, final, and aggregate BAL specimens, and a modified Giemsa stain was also performed on a 0.4-mL aliquot of the aggregate specimen. Of 25 patients, Pneumocystis carinii organisms were identified in 9 with HIV infection, in whom all BAL specimens were positive with both immunofluorescence and Giemsa stains. In 16 patients, BAL specimens were negative for P carinii on both immunofluorescent and modified Giemsa testing. Both staining methods were 100% specific (95% confidence interval [CI], 83 to 100%) and 100% sensitive (95% CI, 72 to 100%). The volume of BAL effluent in the initial specimens positive for P carinii ranged from 15 to 25 mL. We conclude that in this small group of patients, PCP was accurately diagnosed from a single 60-mL BAL specimen stained with immunofluorescence methods.

Pneumocystitis carinii organisms from in vitro culture are highly infectious to the nude rat.

Many in vitro systems have been used to cultivate Pneumocystis, but only limited parasite growth has been obtained by different authors. A reliable in vitro system enabling a sustained propagation of Pneumocystis appears to be an important condition for a better definition of the transmission of P. carinii pneumonia. In this work, Pneumocystis in vitro culture was performed on monolayers of L2 rat lung epithelial-like cells. Ultrastructural assessment revealed that culture parasites were structurally intact. Pneumocystis culture samples were intratracheally inoculated into corticosteroid-treated nude rats (nonlatently infected by P. carinii), which developed P. carinii pneumonia at 40 days postinoculation. The infectious power of parasites obtained in vitro was 7-10 times higher than that of parasites freshly extracted from parasitized rat lung. In summary, the present results show that it is possible to obtain in vitro highly infectious Pneumocystis forms, and this study provides a promising infectivity test for use by investigators working on Pneumocystis in vitro systems.

Persistence of Pneumocystis carinii in patients with AIDS receiving chemoprophylaxis.

Pneumocystis carinii organisms have been shown to persist throughout therapy in the majority of patients with Pneumocystis carinii pneumonia (PCP). This study evaluated the relationship between persistence of organisms and recurrence of disease, and the effect of chemoprophylaxis on bronchoalveolar lavage specimens. Seven patients receiving PCP chemoprophylaxis underwent serial bronchoalveolar lavage (BAL) examinations at 1, 4, and 7 mo after recovery from a first episode of PCP. Specimens were examined for persistent organisms with Gomori's methenamine silver stain and immunofluorescent antibody staining. There were no persistent organisms 1 mo after completion of antimicrobial treatment in six of the seven patients. The one patient with persistent organisms demonstrated clearance of organisms by 4 mo and had no recurrence of PCP. One patient had a recurrence of PCP at 4 mo, after a negative 1-mo BAL. We conclude that a positive BAL result by silver stain or immunofluorescent antibody staining more than a month after ending treatment may indicate clinical recurrence of PCP and not just persistence of nonpathologic cysts. These findings suggest that recurrences of PCP are more likely due to new infection than to relapse of prior disease.