Carolina A. Ponce

Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants.

To determine whether Pneumocystis carinii is associated with clinical illness in the competent host, 107 normal, healthy infants were enrolled in a 2-year prospective cohort study in Chile. P. carinii was identified by specific stains and nested--deoxyribonucleic acid (DNA) amplification of the large subunit mitochondrial ribosomal ribonucleic acid gene of P. carinii f. sp. hominis, and seroconversion was assessed by enzyme-linked immunosorbent assay of serum samples drawn every 2 months. P. carinii DNA was identified in nasopharyngeal aspirates obtained during episodes of mild respiratory infection in 24 (32%) of 74 infants from whom specimens were available for testing. Three (12.5%) of those 24 infants versus 0 of 50 infants who tested negative for P. carinii had apnea episodes. Seroconversion developed in 67 (85%) of 79 infants who remained in the study by 20 months of age and occurred in the absence of any symptoms of disease in 14 (20.8%). The study indicates that P. carinii DNA can be frequently detected in healthy infants, and it raises the hypothesis that they may be an infectious reservoir of P. carinii in the community. Further investigation is needed to identify whether P. carinii causes overt respiratory disease in infants.

Pneumocystis Colonization Is Highly Prevalent in the Autopsied Lungs of the General Population

BACKGROUND Increasing reports of Pneumocystis DNA in noninvasive respiratory specimens from immunocompetent asymptomatic adults and the characteristic lung tropism of Pneumocystis suggest that asymptomatic pulmonary infections with Pneumocystis occur after primary infection. However, studies searching for Pneumocystis in the autopsied lungs of healthy immunocompetent adults have not met with success. METHODS Lungs of people who died of violent causes (accidents, homicide, and suicide) and of nonviolent causes (diseases causing a rapid demise in the street) in Santiago, Chile-for whom an autopsy was legally required-were examined for Pneumocystis by nested polymerase chain reaction (PCR) DNA amplification of the mitochondrial large subunit ribosomal RNA-specific P. jirovecii gene and immunofluorescent microscopic analysis. Lung tissue concentration methods and analysis of approximately 3% of the weight of the right upper lobe (RUL) were needed to reach the sensitivity threshold of the assays. Individuals determined to be P. jirovecii negative after analysis of 3% of the RUL weight in the violent death group were confirmed to be negative by analyzing additional tissue, totaling 6%-7% of the RUL weight. RESULTS P. jirovecii was identified by nested PCR in 50 (64.9%) of 77 individuals (34 [61.8%] of 55 in the violent death group and 15 [78.9%] of 19 in the nonviolent death group; P > .05) and additionally by microscopic analysis in all individuals who tested positive for P. jirovecii DNA in the violent death group. Analysis of tissue beyond 3.0% of the RUL weight for the individuals who tested negative yielded consistently negative results. CONCLUSIONS A mild P. jirovecii pulmonary infection is prevalent in more than half of the general adult population. Our results strengthen the concept that immunocompetent adults develop frequent self-limited reinfections throughout life and participate in the circulation of P. jirovecii as an infective reservoir for susceptible individuals.

Association of Primary Pneumocystis carinii Infection and Sudden Infant Death Syndrome

To delineate clinical and histological features of the first Pneumocystis carinii infection affecting the immunocompetent host, P. carinii-specific histological stains were performed on autopsy lung specimens from 534 consecutive pediatric patients (those with AIDS and malignancies were excluded) in Santiago, Chile. P. carinii clusters were found in 4 (25%) of 16 infants who died of no apparent cause at arrival to the emergency department, and in 10 (2.9%) of 342 infants who died of multiple conditions at the hospital (P=.002, Fishers exact test). This prompted us to analyze additional series of infants with sudden infant death syndrome (SIDS). In 161 additional SIDS cases, 47 (35.1%) of 134 infants from Chile and 4 (14.8%) of 27 infants from Oxford, United Kingdom, were found to have P. carinii clusters in the lungs. The quantity of P. carinii cysts was small compared with the numbers seen in immunocompromised hosts with P. carinii pneumonitis. This study provides histological evidence that primary P. carinii infection is associated with SIDS.

Near-Universal Prevalence of Pneumocystis and Associated Increase in Mucus in the Lungs of Infants With Sudden Unexpected Death

We demonstrate that Pneumocystis reaches a >90% prevalence peak at 3–5 months of age and associates with increased mucus (MUC5AC), suggesting airway epithelium stimulation in infants during this age range. Host ability to clear mucus would determine pathogenic expression.

Pneumocystis colonization in older adults and diagnostic yield of single versus paired noninvasive respiratory sampling.

The presence of Pneumocystis was assessed in oropharyngeal wash specimens from 110 adults (median age, 76 years; age range, 69-95 years), 66 of whom had a paired nasal swab specimen. Pneumocystis jirovecii DNA was detected in 12.8% of oropharyngeal wash specimens, and the frequency increased to 21.5% in paired specimens. Pneumocystis colonization is prevalent in older adults. Double noninvasive sampling increases the diagnostic yield.

Detection of Pneumocystis carinii f. sp. hominis and Viruses in Presumably Immunocompetent Infants Who Died in the Hospital or in the Community

Fresh-frozen lung and tracheal-aspirate specimens obtained from 112 infants who died in Santiago, Chile, during 1998-2000 were analyzed for the presence of Pneumocystis DNA, by use of nested DNA amplification of the large subunit mitochondrial rRNA, and for the presence of viruses, by use of culture and immunofluorescence. Pneumocystis DNA was detected in specimens from 45 (51.7%) of 87 infants who died in the community and from 5 (20%) of 25 infants who died in the hospital (P=.006). Primary infection with Pneumocystis was highly frequent among infants who die unexpectedly in the community. Infection with viruses was more common in infants who died in the hospital.

Distribution of Pneumocystis carinii f. sp. hominis types in the lung of a child dying of Pneumocystis pneumonia

Pneumocystis f. sp. hominis causes pneumonia in immunocompromised persons. In order to determine the types and distribution of P. carinii organisms within a single human lung, multiple samples were obtained from the lung of a child who died of P. carinii pneumonia. P. carinii DNA was detected in all of the samples and 2 different genotypes of P. carinii were identified, with uneven distribution in the lung, demonstrating that infection of the human lung is not necessarily clonal, and that different P. carinii genotypes may predominate in different areas of the lung.

Seroepidemiological study of Pneumocystis jirovecii infection in healthy infants in Chile using recombinant fragments of the P. jirovecii major surface glycoprotein

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.

Fungal colonization with Pneumocystis correlates to increasing chloride channel accessory 1 (hCLCA1) suggesting a pathway for up-regulation of airway mucus responses, in infant lungs

Fungal colonization with Pneumocystis is associated with increased airway mucus in infants during their primary Pneumocystis infection, and to severity of COPD in adults. The pathogenic mechanisms are under investigation. Interestingly, increased levels of hCLCA1 – a member of the calcium-sensitive chloride conductance family of proteins that drives mucus hypersecretion – have been associated with increased mucus production in patients diagnosed with COPD and in immunocompetent rodents with Pneumocystis infection. Pneumocystis is highly prevalent in infants; therefore, the contribution of Pneumocystis to hCLCA1 expression was examined in autopsied infant lungs. Respiratory viruses that may potentially increase mucus, were also examined. hCLCA1 expression was measured using actin-normalized Western-blot, and the burden of Pneumocystis organisms was quantified by qPCR in 55 autopsied lungs from apparently healthy infants who died in the community. Respiratory viruses were diagnosed using RT-PCR for RSV, metapneumovirus, influenza, and parainfluenza viruses; and by PCR for adenovirus. hCLCA1 levels in virus positive samples were comparable to those in virus-negative samples. An association between Pneumocystis and increased hCLCA1 expression was documented (P=0.028). Additionally, increasing Pneumocystis burden correlated with increasing hCLCA1 protein expression levels (P=0.017). Results strengthen the evidence of Pneumocystis-associated up-regulation of mucus-related airway responses in infant lungs. Further characterization of this immunocompetent host-Pneumocystis-interaction, including assessment of potential clinical significance, is warranted.

High prevalence of Pneumocystis jirovecii dihydropteroate synthase gene mutations in patients with first episode of Pneumocystis pneumonia in Santiago, Chile, and their clinical response to trimethoprim-sulfamethoxazole therapy.

ABSTRACT Mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis jirovecii are associated with the failure of sulfa prophylaxis. They can develop by selection in patients receiving sulfa drugs or be acquired via person-to-person transmission. DHPS mutations raise concern about the decreasing efficacy of sulfa drugs, the main available therapeutic tool for Pneumocystis pneumonia (PCP). The prevalence of Pneumocystis DHPS mutations was examined in Pneumocystis isolates from 56 sulfa-prophylaxis-naive adults with a first episode of PCP from 2002 to 2010 in Santiago, Chile. Their clinical history was reviewed to analyze the effect of these mutations on response to trimethoprim-sulfamethoxazole (TMP-SMX) therapy and outcome. Mutant genotypes occurred in 22 (48%) of 46 HIV-infected patients and in 5 (50%) of 10 HIV-uninfected patients. Compared to patients with a wild-type genotype, those with mutant genotypes were more likely to experience sulfa treatment-limiting adverse reactions and to have a twice-longer duration of mechanical ventilation if mechanically ventilated. Specific genotypes did not associate with death, which occurred in none of the HIV-infected patients and in 50% of the non-HIV-infected patients. Chile has a high prevalence of DHPS mutations, which were presumably acquired through interhuman transmission because patients were not on sulfa prophylaxis. These results contrast with the low prevalence observed in other Latin American countries with similar usage of sulfa drugs, suggesting that additional sources of resistant genotypes may be possible. The twice-longer duration of mechanical ventilation in patients with mutant DHPS genotypes suggests a decreased efficacy of TMP-SMX and warrants collaborative studies to assess the relevance of DHPS mutations and further research to increase therapeutic options for PCP.