key: cord-331413-fejho1of
authors: Nakayama, Eiichi; Hasegawa, Keiko; Morozumi, Miyuki; Kobayashi, Reiko; Chiba, Naoko; Ubukata, Kimiko; Iitsuka, Taketoshi; Tajima, Takeshi; Sunakawa, Keisuke
title: Rapid optimization of antimicrobial chemotherapy given to pediatric patients with community-acquired pneumonia using PCR techniques with serology and standard culture
date: 2007-12-31
journal: Journal of Infection and Chemotherapy
DOI: 10.1007/s10156-007-0535-6
sha: 
doc_id: 331413
cord_uid: fejho1of

Abstract Children (n =117; mean age 2.4 ± 2.9 years) were diagnosed as having community-acquired pneumonia (CAP) using clinical symptoms, chest X-rays, and hematological data. The causative pathogen was determined using real-time polymerase chain reaction (PCR) (6 bacteria), multiple reverse transcription-PCR (MPCR; 11 viruses), bacterial culture, and serology. The initial chemotherapy was evaluated based on the pathogens identified using PCR. We found 27 viral cases (23.1%), 25 bacterial cases (21.4%), 45 mixed infections with virus and bacteria (38.5%), 10 Mycoplasma pneumoniae (8.5%), 7 mixed infections with M. pneumoniae and another pathogen (6.0%), 1 Chlamydophila pneumoniae (0.9%), and 2 unknown pathogens (1.7%). Streptococcus pneumoniae and Haemophilus influenzae accounted for 58 (49.5%) and 27 (23.0%) of the cases, respectively. The median values (50%) of the white blood cell count (WBC) and C-reactive protein (CRP) using the box-and-whisker and plot method, respectively, were 11.7 × 103 mm−3 and 1.4mg/dl in viral infections, 15.6 × 103 mm−3 and 4.8mg/dl in mixed infections with virus and bacteria, 17.8 × 103 mm−3 and 6.3mg/dl in bacterial infections, 6.7 × 103 mm−3 and 1.4mg/dl in M. pneumoniae infections, and 21.5 × 103 mm−3 and 6.4mg/dl in mixed infections with M. pneumoniae and other bacterial infections. Sulbactam/ampicillin (n =61), carbapenems (n =12), and ceftriaxone (n =7) were selected for the patients suspected of having bacterial infections alone or mixed infections with bacterial and viruses in accordance with our criteria defined tentatively. For those with M. pneumoniae and C. pneumoniae infections, azithromycin or minocycline was initially used. Treatments averaged 3–5 days. The empirical chemotherapy was improper in 9.4% of cases in relation to the etiologic agents finally identified. We conclude that rapid and comprehensive identification using PCR can provide optimal antimicrobial chemotherapy for CAP patients.

Community-acquired pneumonia (CAP) is one of the most common infections occurring in children. [1] [2] [3] CAP is caused by multiple etiologic agents including viruses, Streptococcus pneumoniae, Haemophilus infl uenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, and other agents. [4] [5] [6] The rates of these causative microorganisms are quite different depending on many factors including the detection methods, seasonal epidemics, and the antibiotics predominantly used. [7] [8] [9] In Japan, antimicrobial chemotherapy for patients with CAP is begun empirically based on (1) chest X-rays, (2) clinical fi ndings including respiratory status, (3) age, and (4) laboratory tests such as white blood cell count (WBC) and C-reactive protein concentration (CRP). Recently, the guidelines to optimize empirical chemotherapy for CAP patients were published. 10 However, we believe the goal of chemotherapy is to select the most appropriate antibiotic for every patient within a short time after admission, based on laboratory results and immediately determining the causative agent.

Recently, the detection of viruses and bacteria using polymerase chain reaction (PCR) in addition to serological diagnosis has determined etiologic agents with high precision. [11] [12] [13] [14] [15] [16] In children with CAP, the determination of the causative pathogen is diffi cult because of the diffi culty in collecting direct clinical samples from alveoli, unlike in adults. Because the pathogens must be identifi ed using indirect nasopharyngeal samples that have low invasiveness, the physician has to determine whether the isolated microorganism is the etiologic agent or not. Therefore, a system to estimate the causative pathogens using obtainable clinical samples on the day of hospitalization is needed to quickly select the appropriate chemotherapy.

Our aim here was to use a multiplex PCR (MPCR) for viruses in parallel with bacterial detection using real-time PCR 17 in nasopharyngeal samples that were obtained from patients with pediatric CAP. Conventional bacterial cultures using the same samples and serological diagnosis with paired sera from the patient were performed to verify the results of the PCR. The clinical fi ndings and laboratory test results from the patient were compared for every causative pathogen, and the appropriateness of the antimicrobial agents selected empirically according to the clinical fi ndings was evaluated.

Pediatric patients with CAP (male: n = 59; female: n = 58) were admitted to the Pediatric Department of Hakujikai Memorial Hospital, Tokyo, from May 2004 to April 2005. The criteria for hospitalization were: (1) the presence of pulmonary infi ltrates found in the chest X-ray, (2) acute respiratory symptoms (e.g., tachypnea), and (3) deterioration of the general clinical state. We excluded patients requiring intensive therapy including artifi cial ventilation, those with chronic respiratory disease, those with congenital heart disease, those hospitalized with the same disease within a 1-month period, and patients with congenital or acquired immunosuppressive conditions. Identifi cation of the causative pathogens After informed consent obtained from the child's parents or guardians, blood samples were taken to determine WBC, CRP, and serum antibody titers for several pathogens. Nasopharyngeal samples were also collected to determine the causative pathogens.

Each sample was used for: (1) real-time PCR screening for six bacterial pathogens, (2) multiple-reverse transcription PCR (MPCR) screening for 11 viral pathogens, and (3) conventional bacterial culture. These techniques were performed immediately after collection of the samples at the Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences.

Streptococcus pneumoniae, Haemophilus infl uenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Streptococcus pyogenes, and Legionella pneumophila were identifi ed within 1.5 h after using the real-time PCR with the RTI kit 17 (Takara Bio, Kyoto, Japan) and Stratagene Mx3000P (Stratagene, La Jolla, CA, USA). The sensitivity and specifi city were high at 95% and 98%, respectively, as compared with standard culture as previously described. 17, 18 

For the identifi cation of viruses, an MPCR kit (Maximbio, San Francisco, CA, USA) was used according to the manufacturer's instructions. The MPCR kit identifi es seven viruses: respiratory syncytial virus (RSV), adenovirus (Adeno), infl uenza virus A (FluA), infl uenza virus B (FluB), and parainfl uenza virus-1, -2, and -3 (PIV-1, PIV-2, PIV-3). In addition, four primer sets to identify rhinovirus (Rhino), 19 human metapneumovirus (hMPV), 20 human bocavirus (hBoV), 21 and coronavirus 19 were prepared and the PCRs were performed using the same conditions as used for the MPCR kit. MPCR required 5 h.

Bacterial culture was performed according to the Manual of Clinical Microbiology. 22 Serotyping of S. pneumoniae was performed using antiserum purchased from Statens Serum Institut (Denmark).

Antibody titers against M. pneumonia, C. pneumonia, RSV, Adeno, FluA and FluB, and PIV-1, -2, and -3 were determined in paired sera from the acute and convalescent phase using the complement fi xation (CF) test, hemagglutination inhibition (HI) test, or enzyme-linked immunosorbent assay (ELISA). When a signifi cant rise in antibody titer was noted in the convalescent phase, the corresponding microorganism was considered to be the causative pathogen. A fourfold rise in titer for M. pneumoniae and Adeno; for RSV using the CF assay; and for PIV-1, -2, and -3, and FluA and FluB using the HI assay were used as indicators. Chlamydophila pneumonia was diagnosed using ELISA and the identifi ed patient had an index value (ID) of 1.35 for IgG in the paired sera.

The decision to begin antimicrobial chemotherapy was based on four conditions described previously; 23 clinical course, chest X-ray fi ndings, age, and the laboratory fi ndings. For clinical observations we used: (1) the presence or absence and timing of fever and respiratory symptoms such as tachypnea, wheezing, and retractive breathing; (2) presence or absence of nasal discharge and its properties; and (3) recurrent fever during the period of recovery of common cold-like symptoms. The diagnosis of tachypnea used World Health Organization (WHO) criteria. 24 Chest X-rays were divided into typical pneumonia (segmental or bronchial pneumonia), atypical pneumonia (ground-glass appear-ance, skip lesion, pleurisy, and segmental), and viral pneumonia (bronchial pneumonia and interstitial shadow).

With regard to age, patients aged 5 years or older were usually thought to have M. pneumoniae infection and those aged 4 years or younger to have viral or mixed viral and bacterial infections. Bacterial infection and mixed infection were suspected in patients aged 4 years or younger with a WBC count of 13 × 10 3 mm −3 or greater, or a CRP value of 3.8 mg/dl or greater. These values may be low in some cases in the early stage after onset and the WBC may readily fl uctuate for various reasons in infants. However, ultimately the estimation of infection to be bacterial, viral, mycoplasmal, or mixed was determined using a composite of the clinical course, chest X-rays, age, and the laboratory data.

Of the four parenteral antibiotics of ampicillin-sulbactam (SBT/ABPC), panipenem (PAPM), meropenem (MEPM), and ceftriaxone (CTRX), a single agent was selected for the patients suspected of having a bacterial infection alone or a viral-bacterial mixed infection from four conditions described above. The respective doses were as follows: SBT/ABPC 100-120 mg kg −1 day −1 (q.i.d.), PAPM and MEPM 60 mg kg −1 day −1 (t.i.d.), and CTRX 40-60 mg kg −1 day −1 (b.i.d.). The antibiotics were administered for 3 days after defervescence (37.5°C).

The febrile period varied considerably in patients with viral and bacterial mixed infections, so the antibiotic was withdrawn 1-2 days after defervescence where we judged the antibiotic had been effective against the bacterial pathogen. For patients suspected of having M. pneumoniae or C. pneumoniae infection from the four conditions, azithromy-cin (AZM) or minocycline (MINO) was used. We did not use antimicrobial agents for patients where viral infection alone was strongly suggested from the four conditions described above.

Criteria for etiological classifi cation Table 1 shows tentative criteria for the etiological classifi cation in CAP patients. Seven categories are as follows: (1) bacterial, (2) mixed infection of viral and bacterial, (3) viral, (4) mycoplasmal, (5) mixed infection of mycoplasmal (chlamydial) and bacterial, (6) mixed infection of mycoplasmal (chlamydial) and viral, and (7) unknown that could not identify any etiological agent.

Statistical analysis of the difference in clinical fi ndings in relation to the causative pathogens was performed using the Fisher's exact test. WBC and CRP values on the day of admission were analyzed using the box-and-whisker plot method. The lower hinge, median, and upper hinge of the box corresponded to the 25%, 50%, and 75% percentiles, respectively; half of the cases were included in the box. The dotted line in each box is 1.5 times the quartile deviation.

One hundred and seventeen CAP cases were 59 male and 58 female patients during May 2004 to April 2005. The As described previously, the decision to admit was determined using chest X-rays (i.e., segmental pneumonia, bronchial pneumonia, atypical pneumonia, or interstitial shadows), acute respiratory symptoms, and deterioration in their general state. Fifty-eight cases (49.6%) had previously received oral antimicrobial agents within the 2 weeks prior to their hospitalization. Viral pathogens MPCR with 11 viruses were correlated with serological test results ( Table 2 ). All PCR positive cases for RSV, Adeno, FluA, FluB, PIV-1, and PIV-3 showed signifi cantly high antibody titers to the corresponding virus. The specifi city of PCR for these agents was 100%; however, the sensitivity of the PCR was 63.6%-100% for viral infections alone and only 21.1%-75.0% for mixed infections. Simultaneous infections with two viruses with Adeno and RSV or hMPV and PIV-3 were identifi ed in two patients each.

The cumulative positive cases determined by serology and PCR were 23 RSV (19.7%) cases, 23 PIV1-3 (19.7%), 13 Adeno (11.1%), 9 FluA (7.7%), 9 FluB (7.7%), 6 Rhino (5.1%), and 3 hMPV (2.6%) cases. No cases having Corona and hBoV infection were identifi ed.

The bacteria suspected to be the causative pathogens was determined by standard culture and real-time PCR for six pathogens: Streptococcus pneumoniae, Haemophilus infl uenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Streptococcus pyogenes, and Legionella pneumophila (Table 3) In the patients suspected of having an infection caused by S. pneumoniae and H. infl uenzae, the real-time PCR results were all positive with early threshold cycles of 15-25 that indicated more than 1000 CFU per sample. Direct bacterial count showed more than 10 4 CFU per sample using standard culture and was found 83.3% and 84.6% of the time, respectively, for S. pneumoniae and H. infl uenzae.

Seventeen cases identifi ed as M. pneumoniae infection showed single (ten cases; 8.5%) and mixed (seven cases; 6.0%) infections with other microorganisms. Fourteen (82.3%) of these cases were identifi ed rapidly by real-time PCR. The rate of individual pathogens in the patients distributed by age is shown in Fig. 2 . Viral infection and mixed infection with viruses and bacteria were the most frequent among children 6 months to 3 years of age, bacterial infection alone occurred in those aged 1-6 years, and M. pneumoniae occurred in those aged 3 years or older.

Clinical characteristics according to etiologic agents Table 4 The following clinical fi ndings on admission were classifi ed according to the respective criteria: (1) chest X-ray fi ndings, (2) with/without asthma, (3) respiration rate, (4) WBC, and (5) CRP. They were analyzed statistically using the Fisher's exact test. As expected, signifi cant differences were noted in chest X-rays among the fi ve groups with signs of interstitial pneumonia being frequently found in patients with viral infection, segmental pneumonia in those with bacterial infections, bronchial or segmental pneumonia in those with mixed infections, and with atypical pneumonia in patients having M. pneumoniae infections.

Viral infection and viral and bacterial mixed infections were more frequent in children with asthma compared with other infections. With regard to respiratory rate, tachypnea was not observed in M. pneumoniae infections. WBC and CRP differed signifi cantly between viral and bacterial infection, and between bacterial or mixed and M. pneumoniae groups.

WBC and CRP values compared with causative pathogens WBC and CRP values of 112 patients on the day of admission were plotted according to the respective causative pathogens (Figs. 3 and 4) . Patients with an unclear causative pathogen (n = 2), C. pneumoniae (n = 1), and mixed infections with M. pneumoniae and virus (n = 2) were excluded. The data were analyzed using the box-and-whisker plot method where each box encompasses 50% of the cases.

As shown in Fig. 3 , the median WBC values in the patients with viral, viral and bacterial, bacterial, M. pneumoniae, and M. pneumoniae and bacterial infections were 11.7 × 10 3 , 15.6 × 10 3 , 17.8 × 10 3 , 6.7 × 10 3 , and 21.5 × 10 3 mm −3 , respectively. In patients with defi ned viral and bacterial mixed infections, the 50% box of WBC values was located between those of the viral and bacterial cases.

As shown in Fig. 4 

The relation between the empirically selected antibiotic for 117 cases and the causative pathogens is shown in Table 5 . The decision to use antimicrobials and the selective criterion for the initial treatment were as outlined in Patients and methods. Antibiotics were used in 97 patients (82.9%), namely SBT/ABPC in 61 (52.1%); CTRX in 8 (6.8%); a carbapenem, either MEPM or PAPM, in 12 (10.3%); AZM in 10 (8.5%); MINO in 3 (2.6%); and SBT/ABPC and AZM in 2 (1.7%) patients. No antibiotic was used in 20 (17.0%) patients.

The initially selected antibiotic was retrospectively considered inappropriate in 9 patients with viral infection (Adeno, 4; RSV, 3; InfA, 1; PIV-3, 1), 1 with M. pneumonia and viral mixed infection, and 1 patient with an undetermined causative pathogen. Out of the 117 cases, antimicrobials were inappropriately used in 11 (9.4%) cases.

Clinically, defervescence was achieved within 24 h after admission in all the patients with bacterial infections. The average duration of antimicrobial treatment was 3-5 days. The duration of antibiotic therapy was also 4 days in four patients in whom S. pneumoniae or H. infl uenzae was isolated from blood cultures taken at admission.

After the termination of treatment, no patient experienced a relapse or was refractory to treatment. However, we experienced four cases having recurrent fever whose hospitalization was slightly prolonged, but this was not due to relapse of pneumonia and all of them recovered spontaneously without further antimicrobial use.

Identifi cation of the causative pathogens in children with CAP is not always easy because sputum or bronchoalveolar lavage (BAL) samples cannot be obtained as routinely performed in adults. To diagnose children, the causative pathogen is suspected from the history, chest X-ray, and blood examination data while taking into account the patient's age, and then the antibiotic is selected empirically. Recently in Japan, 10 the guidelines for the treatment and management of respiratory tract infection in pediatric patients have been proposed to promote optimal chemotherapy, and similar guidelines are found in other countries. 25, 26 However, the incidence rate of pathogenic microorganisms in pediatric CAP in other countries likely differs due to many factors such as the health insurance system, vaccination programs, kinds of antibiotics predominantly used, and population density. There are published studies of the use of PCR methods to determine the pathogens in CAP, [11] [12] [13] [14] [15] [16] and the simultaneous detection of both bacteria and viruses is expected to enhance the accuracy of CAP diagnosis. Here our aim was to identify bacteria and viruses using PCR within a short time frame and to evaluate the PCR results in relation to clinical fi ndings. As previously described, 17 DNA/RNA samples were extracted from clini- In the near future, it is anticipated we will also be able to accomplish viral identifi cation with real-time PCR. We used nasopharyngeal samples as a source to identify the causative pathogens. These materials are appropriate to identify viruses, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, but this sample type is questionable for Streptococcus pneumoniae and Haemophilus infl uenzae because they can be present in normal individuals. A positive result for S. pneumoniae and H. infl uenzae by real-time PCR should be carefully considered as to whether it indicates their causal relation with the infection or not where the physician should consider the chest X-rays, clinical symptoms, clinical laboratory fi ndings such as WBC and CRP, bacterial amounts, and infl ammation fi ndings of leucocytes in nasopharyngeal samples.

The rate of viruses and bacteria identifi ed in this study as the causative pathogens was similar to the data reported by Michelow et al. 12 and Juvèn et al., 5 although the rate of H. infl uenzae differed. This was presumably because Hib vaccination has not been approved in Japan 27 where there are cases of pneumonia due to Hib and nontypable H. infl uenzae. Three CAP cases with Hib having positive blood cultures and positive real-time PCR were found among our 117 cases. Chlamydophila pneumoniae was detected in only one patient possibly because there were few children older than 6 years in this study.

As for the relation between the diagnosis of CAP and blood examination test, although some studies have only concluded that CRP and WBC can provide useful informa-tion for pneumococcal pneumonia, [28] [29] [30] [31] these values are used by Japanese pediatricians as useful references in addition to routine chest X-ray to diagnose pneumonia. In clinical practice, we have observed these values do not fl uctuate for about half a day after the onset of bacterial infection. In addition, in the cases of adenovirus infection, and virus or M. pneumoniae infection in school-age children, the values of WBC and CRP are relatively high. The physician must be mindful of these kinds of exceptional cases; however, as shown in our data, signifi cant differences in these values are found and are correlated to the causative pathogens. At present, we believe the time from onset to presentation in hospital is relatively uniform in Japan as compared with other countries because of our universal health insurance system.

In this study, the antimicrobial agent that was empirically selected was found to have been inappropriately administered to 9.4% of the 117 cases. Using our techniques, we found the treatment for most cases was completed within 3-5 days. In general, the dosing period recommended in the guidelines is 7-10 days; however, we consider this is somewhat long because the antibiotics acted against the causative bacteria within a shorter period. Of the 58 strains of S. pneumoniae, 25 were Penicillin resistant streptococcus pneumonia (PRSP), and none of the patients experienced a relapse after treatment with SBT/ABPC and a carbapenem antibiotic (data not shown).

Viral and bacterial mixed infections were identifi ed in 40.2% of the cases in addition to viral infection in 23.1%. Thus, involvement with the viral-related cases was 74 (63.2%) cases in total. Furthermore, cases relating to virus infections may be present, which could not be demonstrated when 5 days or more had elapsed after onset. Timing of acquiring the clinical samples is also very important to demonstrate the causative viruses.

In the future, we expect comprehensive and rapid identifi cation of the causative pathogens outlined here will become routine in clinical practice.

Community-acquired pneumonia

Etiology and treatment of pneumonia

Diagnosis and management of pneumonia in children

Community-acquired pneumonia in children

Etiology of community acquired pneumonia in 254 hospitalized children

Management of community-acquired pediatric pneumonia in an era of increasing antibiotic resistance and conjugate vaccines

Mycoplasma pneumoniae and Chlamidia pneumoniae in pediatric community-acquired pneumonia: comparative effi cacy and safety of clarithromycin vs erythromycin ethylsuccinate

Etiology of childhood pneumonia: serologic results of a prospective, population-based study

Rationalised prescribing for community-acquired pneumonia: a closed loop audit

Practice guidelines for respiratory tract infections in childhood

Etiology and treatment of community-acquired pneumonia in ambulatory children

Epidemiology and clinical characteristics of communityacquired pneumonia in hospitalized children

Rapid identifi cation of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study

National disease burden of respiratory viruses detected in children by polymerase chain reaction

Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction

Population-based incidence of severe pneumonia in children in Kiel

Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specifi c molecular beacon probes

Assessment of real-time PCR for diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays

Human metapneumovirus infection in Japanese children

Detection of human bocavirus in Japanese children with lower respiratory tract infections

Manual of clinical microbiology

Etiology and clinical study of community-aquired pneumonia in 157 hospitalized children

World Health Organization

British Thoracic Society of Standards of Care Committee. BTS guidelines for the management of community acquired pneumonia in childhood

A practical guide for the diagnosis and treatment of pediatric pneumonia

High prevalence of type b β-lactamase-non-producing ampicillin-resistant Haemophilus infl uenzae in meningitis: the situation in Japan where Hib vaccine has not been introduced

Differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital

White blood cells, C-reactive protein and erythrocyte sedimentation rate in pneumococcal pneumonia in children

C-Reactive protein in viral and bacterial respiratory infection in children

Bacteremic pneumococcal pneumonia in children

The authors are grateful to Akiko Ono and Matsumoto Masato of Meiji Seika Kaisha for their assistance.