Use and evaluation of molecular diagnostics for pneumonia etiology studies

Comprehensive microbiological testing will be a core function of the Pneumonia Etiology Research for Child Health (PERCH) project. The development stage of PERCH provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. These efforts allowed us to articulate the unique requirements of PERCH, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. This process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. Although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of PERCH, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies.     

The Pneumonia Etiology Research for Child Health Project: a 21st century childhood pneumonia etiology study

The Pneumonia Etiology Research for Child Health (PERCH) project is a 7-country, standardized, comprehensive evaluation of the etiologic agents causing severe pneumonia in children from developing countries. During previous etiology studies, between one-quarter and one-third of patients failed to yield an obvious etiology; PERCH will employ and evaluate previously unavailable innovative, more sensitive diagnostic techniques. Innovative and rigorous epidemiologic and analytic methods will be used to establish the causal association between presence of potential pathogens and pneumonia. By strategic selection of study sites that are broadly representative of regions with the greatest burden of childhood pneumonia, PERCH aims to provide data that reflect the epidemiologic situation in developing countries in 2015, using pneumococcal and Haemophilus influenzae type b vaccines. PERCH will also address differences in host, environmental, and/or geographic factors that might determine pneumonia etiology and, by preserving specimens, will generate a resource for future research and pathogen discovery.     

A literature review and survey of childhood pneumonia etiology studies: 2000-2010

The Pneumonia Etiology Research for Child Health (PERCH) project is the largest multicountry etiology study of childhood pneumonia since the Board on Science and Technology in International Development studies of the 1980s. However, it is not the only recent or ongoing pneumonia etiology study, and even with seven sites, it cannot capture all epidemiologic settings in the developing world. Funding providers, researchers and policymakers rely on the best available evidence to strategically plan programs, new research directions and interventions. We aimed to describe the current landscape of recent pneumonia etiology studies in children under 5 years of age in the developed and developing world, as ascertained by a literature review of relevant studies with data since the year 2000 and a survey of researchers in the field of childhood pneumonia. We collected information on the study population, study design, case definitions, laboratory samples and methods and identified pathogens. A literature review identified 88 studies with child pneumonia etiology results. As of June 2010, our survey of researchers identified an additional 65 ongoing and recently completed child pneumonia etiology studies. This demonstrates the broad existing context into which the PERCH study must be placed. However, the landscape analysis also reveals a multiplicity of case definitions, levels of clinician involvement, facility types, specimen collection, and laboratory techniques. It reinforces the need for the standardization of methods and analyses for present and future pneumonia etiology studies in order to optimize their cumulative potential to accurately describe the microbial causes of childhood pneumonia.     

Disk diffusion bioassays for the detection of antibiotic activity in body fluids: applications for the Pneumonia Etiology Research for Child Health project

To draw inferences about the putative etiologic agents of severe pneumonia, the Pneumonia Etiology Research for Child Health (PERCH) project must be able to objectively assess antibiotic pretreatment in enrolled participants. This review is focused on the disk diffusion bioassay, a simple laboratory method to assess recent antibiotic treatment. In this method, a sensitive indicator organism is used to detect antimicrobial activity in body fluid specimens that have been inoculated on a filter paper disk and placed on agar growth medium. We reviewed and present several variations on the disk diffusion method as applied to serum or urine, including specimen handling, choice of indicator organism and medium, and incubation steps. Although there are limitations to the disk diffusion method, its low cost, ease of use, and ability to broadly detect antibiotic pretreatment make it an appealing method for epidemiologic studies such as PERCH.     

Procedures for collection of induced sputum specimens from children

In most settings, sputum is not routinely collected for microbiological diagnosis from children with lower respiratory disease. To evaluate whether it is feasible and diagnostically useful to collect sputum in the Pneumonia Etiology Research for Child Health (PERCH) study, we reviewed the literature on induced sputum procedures. Protocols for induced sputum in children were collated from published reports and experts on respiratory disease and reviewed by an external advisory group for recommendation in the PERCH study. The advisory group compared 6 protocols: 4 followed a nebulization technique using hypertonic saline, and 2 followed a chest or abdomen massage technique. Grading systems for specimen quality were evaluated. Collecting sputum from children with lower respiratory tract illness is feasible and is performed around the world. An external advisory group recommended that sputum be collected from children hospitalized with severe and very severe pneumonia who participate in the PERCH study provided no contraindications exist. PERCH selected the nebulization technique using hypertonic saline.     

The definition of pneumonia, the assessment of severity, and clinical standardization in the Pneumonia Etiology Research for Child Health study

To develop a case definition for the Pneumonia Etiology Research for Child Health (PERCH) project, we sought a widely acceptable classification that was linked to existing pneumonia research and focused on very severe cases. We began with the World Health Organization's classification of severe/very severe pneumonia and refined it through literature reviews and a 2-stage process of expert consultation. PERCH will study hospitalized children, aged 1-59 months, with pneumonia who present with cough or difficulty breathing and have either severe pneumonia (lower chest wall indrawing) or very severe pneumonia (central cyanosis, difficulty breastfeeding/drinking, vomiting everything, convulsions, lethargy, unconsciousness, or head nodding). It will exclude patients with recent hospitalization and children with wheeze whose indrawing resolves after bronchodilator therapy. The PERCH investigators agreed upon standard interpretations of the symptoms and signs. These will be maintained by a clinical standardization monitor who conducts repeated instruction at each site and by recurrent local training and testing.     

Evaluation of risk factors for severe pneumonia in children: the Pneumonia Etiology Research for Child Health study

As a case-control study of etiology, the Pneumonia Etiology Research for Child Health (PERCH) project also provides an opportunity to assess the risk factors for severe pneumonia in hospitalized children at 7 sites. We identified relevant risk factors by literature review and iterative expert consultation. Decisions for inclusion in PERCH were based on comparability to published data, analytic plans, data collection costs and logistic feasibility, including interviewer time and subject fatigue. We aimed to standardize questions at all sites, but significant variation in the economic, cultural, and geographic characteristics of sites made it difficult to obtain this objective. Despite these challenges, the depth of the evaluation of multiple risk factors across the breadth of the PERCH sites should furnish new and valuable information about the major risk factors for childhood severe and very severe pneumonia, including risk factors for pneumonia caused by specific etiologies, in developing countries.     

The role of postmortem studies in pneumonia etiology research

The diagnosis of etiology in severe pneumonia remains a challenging area. Postmortem lung tissue potentially increases the sensitivity of investigations for identification of causative pathogens in fatal cases of pneumonia and can confirm antemortem microbiological diagnoses. Tissue sampling allows assessment of histological patterns of disease and ancillary immunohistochemical or molecular diagnostic techniques. It may also enhance the recognition of noninfectious conditions that clinically simulate acute pneumonia. Biobanking of lung tissue or postmortem culture isolates offers opportunities for new pathogen discovery and research into host-pathogen interactions. The Pneumonia Etiology Research for Child Health study proposes a percutaneous needle biopsy approach to obtain postmortem samples, rather than a full open autopsy. This has the advantage of greater acceptability to relatives, but risks greater sampling error. Both approaches may be susceptible to microbiological contamination or pathogen degradation. However, previous autopsy studies have confirmed the value of histological examination in revealing unsuspected pathogens and influencing clinical guidelines for the diagnosis and treatment of future pneumonia cases.     

Identification and selection of cases and controls in the Pneumonia Etiology Research for Child Health project

Methods for the identification and selection of patients (cases) with severe or very severe pneumonia and controls for the Pneumonia Etiology Research for Child Health (PERCH) project were needed. Issues considered include eligibility criteria and sampling strategies, whether to enroll hospital or community controls, whether to exclude controls with upper respiratory tract infection (URTI) or nonsevere pneumonia, and matching criteria, among others. PERCH ultimately decided to enroll community controls and an additional human immunodeficiency virus (HIV)-infected control group at high HIV-prevalence sites matched on age and enrollment date of cases; controls with symptoms of URTI or nonsevere pneumonia will not be excluded. Systematic sampling of cases (when necessary) and random sampling of controls will be implemented. For each issue, we present the options that were considered, the advantages and disadvantages of each, the rationale for the methods selected for PERCH, and remaining implications and limitations.     

Bioethical considerations in developing a biorepository for the Pneumonia Etiology Research for Child Health project

The Pneumonia Etiology Research for Child Health (PERCH) project has the potential to provide a highly valuable resource of biospecimens that may be used to inform future studies on the causes of pneumonia. Designing a biorepository for this complex project was done in collaboration with a wide range of experts including bioethicists. In this paper, we describe the most significant ethical issues encountered related to the biorepository, focusing on its structure and informed consent. We also outline the proposed approach to the PERCH biorepository, which was designed to be sensitive to the ethical, practical, and cultural challenges inherent to the study. Through this process, we concluded that biorepository governance plans and strategies for managing informed consent should be implemented in a way to allow for careful study in order to better understand the attitudes of and impact on the stakeholders involved in the study.     

Specimen collection for the diagnosis of pediatric pneumonia

Diagnosing the etiologic agent of pneumonia has an essential role in ensuring the most appropriate and effective therapy for individual patients and is critical to guiding the development of treatment and prevention strategies. However, establishing the etiology of pneumonia remains challenging because of the relative inaccessibility of the infected tissue and the difficulty in obtaining samples without contamination by upper respiratory tract secretions. Here, we review the published and unpublished literature on various specimens available for the diagnosis of pediatric pneumonia. We discuss the advantages and limitations of each specimen, and discuss the rationale for the specimens to be collected for the Pneumonia Etiology Research for Child Health study.     

Diagnosis and management of pneumonia in the emergency department

Pneumonia is a condition that is often treated by emergency physicians. This article reviews the diagnosis and management of pneumonia in the emergency department and highlights dilemmas in diagnostic testing, use of blood and sputum cultures, hospital admission decisions, infection control, quality measures for pneumonia care, and empiric antimicrobial therapy.     

Etiology of community-acquired pneumonia: impact of age, comorbidity, and severity.

The aim of this study was to determine the etiology of community-acquired pneumonia (CAP) and the impact of age, comorbidity, and severity on microbial etiologies of such pneumonia. Overall, 395 consecutive patients with CAP were studied prospectively during a 15-mo period. Regular microbial investigation included examination of sputum, blood culture, and serology. Sampling of pleural fluid, transthoracic puncture, tracheobronchial aspiration, and protected specimen brush (PSB) sampling were performed in selected patients. The microbial etiology was determined in 182 of 395 (46%) cases, and 227 pathogens were detected. The five most frequent pathogens were Streptococcus pneumoniae (65 patients [29%]), Haemophilus influenzae (25 patients [11%]), Influenza virus A and B (23 patients [10%]), Legionella sp. (17 patients [8%]), and Chlamydia pneumoniae (15 patients [7%]). Gram-negative enteric bacilli (GNEB) accounted for 13 cases (6%) and Pseudomonas aeruginosa for 12 cases of pneumonia (5%). Patients aged < 60 yr were at risk for an "atypical" bacterial etiology (odds ratio [OR]: 2.3; 95% confidence interval [CI]: 1.2 to 4.5), especially Mycoplasma pneumoniae (OR: 5.3; 95% CI: 1.7 to 16.8). Comorbid pulmonary, hepatic, and central nervous illnesses, as well as current cigarette smoking and alcohol abuse, were all associated with distinct etiologic patterns. Pneumonia requiring admission to the intensive care unit was independently associated with the pathogens S. pneumoniae (OR: 2.5; 95% CI: 1.3 to 4.7), gram-negative enteric bacilli, and P. aeruginosa (OR: 2.5; 95% CI: 0.99 to 6.5). Clinical and radiographic features of "typical" pneumonia were neither sensitive nor specific for the differentiation of pneumococcal and nonpneumococcal etiologies. These results support a management approach based on the associations between etiology and age, comorbidity, and severity, instead of the traditional syndromic approach to CAP.     

Lower respiratory infections among hospitalized children in New Caledonia: a pilot study for the Pneumonia Etiology Research for Child Health project

We conducted a prospective pilot study over a 1-year period in New Caledonia in preparation for the Pneumonia Research for Child Health (PERCH) project. The pathogens associated with hospitalized lower respiratory infections in children were identified through the use of culture of induced sputum and blood, urinary antigen detection, polymerase chain reaction (PCR) on respiratory specimens, and serology on paired sera. Respiratory viruses were detected on respiratory specimens by immunofluorescence and PCR, and by serology on paired sera. Pathogens were detected in 87.9% of the 108 hospitalized cases. Viruses represented 81.6% of the 152 pathogens detected. Respiratory syncytial virus and rhinovirus were the most frequent, accounting for 32.2% and 24.3% of the pathogens identified, respectively. Only 26.3% of 99 induced sputum specimens collected were determined to be of good quality, which may be a consequence of the collection method used.     

Prospective study of etiologic agents of community-acquired pneumonia in patients with HIV infection.

OBJECTIVES: To study prospectively HIV-positive patients admitted to the hospital because of pneumonia by extensive laboratory tests to determine specific microbiologic diagnoses and to establish the best clinical diagnosis after review of all available data by expert clinicians. METHODS: Patients admitted to one of two hospitals had extensive questionnaires completed and defined diagnostic tests performed on blood, sputum, urine and bronchoalveolar lavage specimens, when available. RESULTS: A total of 230 patients had a diagnosis of pneumonia verified. A definite or probable etiologic diagnosis was made in 155 (67%) of these patients. Pneumocystis carinii caused 35% of all cases of pneumonia. Twenty-seven percent of cases of pneumonia with a single etiology had a definite or probable bacterial etiology. 'Atypical agents' were distinctly uncommon. Few clinical or laboratory parameters could differentiate specific etiologies. CONCLUSIONS: P. carinii continues to be a common cause of pneumonia in these patients. The rarity of 'atypical agents' could simplify the empiric approach to therapy. Despite the use of extensive testing we did not find a definite etiology in a large number of cases.     

Viruspneumonie bei immuninkompetenten Patienten

Pneumonia is a relevant cause of high rates of mortality and morbidity in immunocompromized patients. While most important microbial pathogens of community-acquired pneumonia in immunocompetent adult patients are well characterized according to data from the multicenter study of CAPNETZ, immunocompromized patients often show infections with different, rare and opportunistic microbial pathogens. Especially respiratory viruses should be taken into consideration. Immune suppression can result from HIV infection with depletion of CD4 lymphocytes, in patients with lymphoma, leukemia or solid tumors regarding aggressive cytostatic chemotherapy or transplantation or from immunosuppressive therapy in rheumatic or autoimmune diseases. The diagnosis of viral pneumonia is difficult because of atypical symptoms, unspecific changes in inflammatory blood parameters and several possible types of X-ray changes. This makes it even more important to have a clear proof of viral infection, such as molecular biological diagnostics from respiratory specimens prior to treatment with antiviral therapy, as antiviral therapy is known to possibly cause severe side effects and should thereby be considered carefully.     

Epidemiology of the Rhinovirus (RV) in African and Southeast Asian Children: A Case-Control Pneumonia Etiology Study

Rhinovirus (RV) is commonly detected in asymptomatic children; hence, its pathogenicity during childhood pneumonia remains controversial. We evaluated RV epidemiology in HIV-uninfected children hospitalized with clinical pneumonia and among community controls. PERCH was a case-control study that enrolled children (1–59 months) hospitalized with severe and very severe pneumonia per World Health Organization clinical criteria and age-frequency-matched community controls in seven countries. Nasopharyngeal/oropharyngeal swabs were collected for all participants, combined, and tested for RV and 18 other respiratory viruses using the Fast Track multiplex real-time PCR assay. RV detection was more common among cases (24%) than controls (21%) (aOR = 1.5, 95%CI:1.3–1.6). This association was driven by the children aged 12–59 months, where 28% of cases vs. 18% of controls were RV-positive (aOR = 2.1, 95%CI:1.8–2.5). Wheezing was 1.8-fold (aOR 95%CI:1.4–2.2) more prevalent among pneumonia cases who were RV-positive vs. RV-negative. Of the RV-positive cases, 13% had a higher probability (>75%) that RV was the cause of their pneumonia based on the PERCH integrated etiology analysis; 99% of these cases occurred in children over 12 months in Bangladesh. RV was commonly identified in both cases and controls and was significantly associated with severe pneumonia status among children over 12 months of age, particularly those in Bangladesh. RV-positive pneumonia was associated with wheezing.     

支气管肺泡灌洗在免疫抑制患者肺部感染中的诊断价值

肺部感染是导致免疫抑制患者死亡的重要原因之一。支气管肺泡灌洗是研究肺泡表面液细胞成分和可溶性物质的一种方法,被广泛用于免疫抑制宿主肺部病变的诊断中,是下呼吸道微生物取样的标准方法。现将支气管肺泡灌洗在免疫抑制患者肺部感染中的诊断价值作一综述。