Investigation Of Clinical Relevance Of Bacterial Colonization In ...

Dis Mol Med 2013;1(1):2-7

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ORIGINAL ARTICLE

Investigation Of Clinical Relevance Of Bacterial Colonization In Patients With Suspected Viral Respiratory Tract Infection By Using Multiplex PCR Method Vedat Turhan1,2, Abdullah Kilic2,3 , Gurkan Mert4, Haijing Li2, Susan E. Sefers2, Charles W. Stratton2, Yi-Wei Tang2 Received: December 15, 2012 Accepted: December 24, 2012 Dis Mol Med 2013;1(1):2-7 DOI:10.5455/dmm.20130122062607 Key words: respiratory pathogens, acute respiratory tract infection, multiplex PCR.

Abstract Numerous viral and bacterial pathogens have been reported causing acute respiratory tract infection (ARTI). Nasopharyngeal swab (NPS) specimens from 351 patients (278 children, 73 adults) with suspected upper and lower ARTI were submitted during the study period from Jan. 2005 to Dec. 2006. Organism-specific nucleic acids were detected using TemPlex technology (ResPlex I and II, Genaco Biomedical Products, Huntsville, AL). Amplified products were identified using a suspension array for multiplex detection performed on a Luminex 100 instrument (Luminex, Austin, TX). A total of 221 viral and bacterial respiratory agents were detected in 148 patients (135 [48.5%] of the 278 children and 13 [17.8%] of the 73 adults) with suspected ARTI. A single respiratory pathogen was detected in 89 patients [25.35%], whereas mixed infection with two or three pathogens was found in 59 [16.8%] of 351 suspected patients. S. pneumonia was the most frequently isolated strain (54 [15.3%] of 351 patients), followed by H. influenzae (37 [10.5%]), rhinoviruses (35 [9.9%]), influenza A virus (23 [6.5%]), enteroviruses (19 [5.4%]), hMPV (14 [3.9%]), PIV-1 (12 [3.4%]), PIV-3 (11 [3.1%]), RSV (10 [2.8%]), and influenza B virus (6 [1.7%]). Mixed infections were more frequent in children (56 [20.1%] of 278) than adult patients (3 [4.1%] of 73 patients). The detection rate of the bacteria peaked in the spring season (37 [40.6%] of 91 bacteria), followed by winter (24 infections), autumn (18 infections) and summer (12 infections). The prevalence of co-infection is ~40%, finding a much higher incidence of co-infection with more than one agent than that reported previously.

Introduction Acute respiratory tract infections (ARTIs) are common among children and adults. Many bacterial and viral pathogens can cause ARTIs. Since bacterial and viral ARTIs usually present with similar signs and symptoms, it is difficult to distinguish them from each other by their clinical findings (1). Mycoplasma pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Acinetobacter baumannii as bacterial respiratory pathogens and influenza A virus (Flu-A) and Flu-B, respiratory syncytial virus (RSV) A and B, parainfluenza virus 1 (PIV-1), PIV-2, PIV-3, and PIV-4, human metapneumovirus (hMPV), rhinoviruses (RhVs), entero-

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viruses (EnVs), and severe acute respiratory syndrome (SARS) coronavirus (CoV) as viral respiratory pathogens are well recognized (2,3). Rapid and accurate diagnosis of these pathogens is very important for initiating antiviral and antibacterial therapy, preventing nosocomial spread, decreasing the duration of hospital stay, and Author affiliations: 1Department of Infectious Diseases, Gulhane Military Medical Academy and School of Medicine, Haydarpasa Training Hospital, Istanbul, Turkey.3Department of Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara, Turkey; 4Department of Infectious Diseases, Gulhane Military Medical Academy and School of Medicine, 06018, Ankara, Turkey,2Departments of Pathology and Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-USA. Correspondence to: [email protected] (V.Turhan) This study was presented in part at the 107th General Meeting of the American Society for Microbiology, Toronto, Canada, 21-25 May, 2007

©2012 Disease and Molecular Medicine. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Turhan V et al.

reducing healthcare costs (3). One of the important problems in the diagnosis of ARTIs is to make a distinction between causative infectious agents clinically. Conventional diagnosis of respiratory tract pathogens is usually based on the microbial culture, cell culture and/or detection of antigens. Culture techniques are very difficult for some pathogens, such as M. pneumoniae, L. pneumophila, C. pneumoniae, and viruses, and culturing pathogens is time-consuming (2,4). Molecular techniques have been established for detection of respiratory pathogens and they have some advantages over conventional methods. Therefore, multiplex RT-PCR-based techniques have been developed for simultaneous detection of a panel of respiratory pathogens in a single reaction (1). We reported here the use of ResPlex Kits (Genaco Biomedical Products, Huntsville, AL), which use a target-enriched multiplex PCR to amplify and detect a panel of gene targets within one reaction, to detect 20 bacterial and viral pathogens commonly encountered in the respiratory tract.

Materials and Methods Clinical samples Nasopharyngeal swab (NPS) specimens were collected over the study period from January 2005 to December 2006. The specimens were submitted to the Clinical Virology Laboratory at Vanderbilt University Medical Center for detection of respiratory viruses and bacterial pathogens. Specimens with sufficient residual volume after routine diagnostic testing were stored at –80oC for further study.

Total nucleic acid extraction A NucliSens easyMAG platform (bioMerieux, Durham, NC) was used to extract total nucleic acid from the frozen NPS specimens. The starting specimen volume was 0.2 ml, and total nucleic acid was eluted in 55 μl of RNase-free water.

Genaco ResPlex I and II The ResPlex I and II assays entail the simultaneous amplification of a total of 20 bacterial and viral pathogens. ResPlex Panel 1 covers DNA pathogens including M. pneumoniae, L. pneumophila, C. pneumoniae, N. meningitidis, S. pneumoniae, H. influenzae, and A. baumannii. ResPlex Panel 2 covers RNA pathogens includ3

Dis Mol Med 2013;1(1):2-7

ing Flu-A, Flu-B, RSV-A, RSV-B, PIV-1, PIV-2, PIV-3, PIV-4, hMPV, RhVs, EnVs, and SARS CoV. Organism-specific nucleic acids were detected using TemPlex technology (6), followed by product detection and identification using a Luminex suspension microarray (3). In brief, 6µl ResPlex II and ResPlex I SuperPrimers were added to 25 µl of Qiagen HotStarTaq master Mix (Qiagen Inc, Valencia, CA), followed by the addition of 5 µl of extracted nucleic acid and 14 µl of water for a final volume of 50 µl. Amplification was initiated with reverse transcription at 50°C for 30 min, followed by the five-stage Templex cycling program, as previously described. Amplified products were identified using a suspension array for multiplex detection performed on a Luminex 100 instrument (Luminex, Austin, TX), as previously described. Results for each channel are expressed as the median fluorescence intensity (MFI) value. The cutoff value for each target was determined as the sum of the mean plus 4 times the standard deviations of the negative controls.

Patients Review In total, 351 patients with suspected viral ARTI were retrospectively reviewed. A standardized set of 45 demographical, clinical and laboratory items was extracted from the patients’ files, entered into Excel files, controlled, and reviewed by 2 infectious disease specialists blindly. All patients were categorized as upper, lower, and non-ARTI. Subjects were grouped as pediatric (age 18) patients. This study protocol was approved by the Vanderbilt University institutional local study review board and ethics committee.

Results A total of 351 subsequently clinical samples collected from ARTI-suspected patients were tested. Of the patients included, 190 (54.1%) were male and 161 (45.9%) were female. There were 278 (79.2%) pediatric and 73 (20.8%) adult patients. The average age of the patients was 10 ± 18.24 years. A total of 293 patients (248 children and 45 adults) was diagnosed as lower ARTI (39 children and 9 adults) and upper ARTI (209 children and 36 adults), whereas 58 patients (30 children and 28 adults) were not to fulfill ARTI entry criteria according to their clinical and laboratory findings (7). In 150 of 293 (51.1%) ARTI patients, no infectious agent was detected in NPS samples. On the other hand, microorganisms (four bacteria and one virus) were detected in 5 of 58 (8.6%) non-ARTI patients Disease and Molecular Medicine - www.dismolmed.org

DOI:10.5455/dmm.20130122062607

Microorganisms in respiratory tract infections

(Table 1). A total of 221 viral and bacterial respiratory agents were detected in 148 patients suspected with ARTI. A single respiratory pathogen was detected in 89 (25.35%) patients, whereas mixed infection with two or three pathogens was found in 59 (16.8%) of 351 patients sus-

pected with RTI. Two different organisms were detected in 45 patients and three different organisms were detected in 14 patients. The most frequent combinations were S. pneumoniae plus H. influenzae or RhVs (Table 2). Among single and mixed infections, S. pneumoniae was the most frequently isolated pathogen (54 [15.3%]

Table 1. Diagnostic category of the 351 patients suspected with respiratory tract infection according to their clinical and laboratory evidence by two infectious diseases specialists blindly Diagnostic Category, No. (%) Children (n=278)

Pathogens

Bacterial pathogens

Adult (n=73)

Upper RTI

Lower RTI

Non-RTI

Upper RTI

Lower RTI

Non-RTI

(n=209)

(n=39)

(n=30)

(n=36)

(n=9)

(n=28)

65 (31.1)

14 (35.8)

3 (10.0)

5 (13.8)

3 (33.3)

1 (3.5)

Streptococcus pneumoniae

40 (19.1)

8 (20.5)

2 (6.6)

2 (5.5)

1 (11.1)

1 (3.5)

Haemophilus influenzae

25 (11.9)

6 (15.3)

1 (3.3)

3 (8.3)

2 (22.2)

-

103 (49.2)

19 (48.7)

1 (3.3)

7 (19.4)

-

-

Influenza A virus

15 (7.1)

3 (7.6)

1 (3.3)

4 (11.1)

-

-

Influenza B virus

4 (1.9)

2 (5.1)

-

-

-

-

Respiratory syncytial virus

8 (3.8)

2 (5.1)

-

-

-

-

Parainfluenza virus 1

10 (4.7)

2 (5.1)

-

-

-

-


10 (4.7)

1 (2.5)

-

-

-

-

Human metapneumovirus

12 (5.7)

2 (5.1)

-

-

-

-

Rhinovirus

28 (13.3)

5 (12.8)

-

2 (5.5)

-

-

Enterovirus

16 (7.6)

2 (5.1)

-

1 (2.7)

-

-

88 (42.1)

29 (74.3)

26 (86.6)

26 (72.2)

7 (77.7)

27 (96.4)

Viral pathogens

Pathogen not detected (n=203)

S. pneumonia

-

H. influenza

15

-

Influenza A virus

3

2

Influenza B virus

4


9

2


1

2

Enteroviruses

Rhinoviruses





Influenza B virus

Influenza A virus

H. influenzae

S. pneumoniae

Table 2. Combination of pathogens in the 59 patients with more than one pathogen

-


3

1


5

4

Rhinovirus

10

5

Enterovirus

5

2

Disease and Molecular Medicine - www.dismolmed.org

1

-

1

1

3

1

7

-

4

Turhan V et al.

Dis Mol Med 2013;1(1):2-7

of 351 patients), followed by Haemophilus influenzae (37 [10.5%]), RhVs (35 [9.9%]), Flu-A (23 [6.5%]), EnVs (19 [5.4%]), hMPV (14 [3.9%]), PIV-1 (12 [3.4%]), PIV-3 (11 [3.1%]), RSV (10 [2.8%]), and Flu-B (6 [1.7%]). The infectious pathogen detection rate was higher in pediatric patients (135 [48.5%] of 278) than adult patients (13 [17.8%] of 73 patients). Mixed infections were also more frequent in pediatric patients (56 [20.1%] of 278 patients) than adult patients (3 [4.1%] of 73 patients) (Table 3). The detection rate of the bacteria peaked in the spring season (37 [40.6%] of 91 bacteria), followed by winter (24 infections), autumn (18 infections) and summer (12 infections).

Discussion Acute respiratory tract infections are the most common causes of childhood morbidity and mortality worldwide, and account for about 30% of all childhood deaths in the developing world. On the other hand, ARTIs cause loss of time, labor and huge healthcare costs directly or indirectly, especially in developed countries (8). Detection and classification of the common respiratory pathogens are impressively important because of

the fact that pathogens have potentially high morbidity and mortality rates. Culture-based, antigen-based (FA or direct antigen detection), and molecular techniques are currently used as diagnostic methods. Among them, molecular methods have the potential for the highest sensitivity, with assay turn-around times on the order of a few hours and foreseeable capability to be run in a high-throughput batch process (3). Several multiplex techniques have been developed so far which have the capacity to identify the majority of common respiratory viruses and bacteria in clinical samples (9). The ResPlex I and II assays provide a rapid and easy test for semi-quantitation of viral and bacterial material present in respiratory samples (1,9). Both assays can be performed daily with acceptable sensitivities and specificities. Another advantage of simultaneous detection of a panel of pathogens is the recognition of co-infection. When monoplex RT-PCR is used for pathogen detection, the clinician often does not consider the possible presence of other possible pathogens (5). However, the ResPlex I/II assays do not detect any DNA from the other bacteria and viruses associated with the respiratory tract infections, not including in the kits, suggesting a

Table 3. No (%) of pathogens detected in patients studied according to age and sex. No. (%) of patients, by age and sex group Total (%)

Children (age 18 years)

(n=351)

(n=278)

(n=73)

91 (25.9)

82 (29.4)

9 (12.3)

0.003

Streptococcus pneumoniae

54 (15.3)

50 (17.9)

4 (5.4)

Haemophilus influenzae

37 (10.5)

32 (11.5)

5 (6.8)

130 (37.1)

123 (44.2)

23 (6.5)

19 (6.8)

Pathogens

Bacterial pathogens

Viral pathogens Influenza A virus

5

Male

Female

(n=190)

(n=161)

53 (27.8)

38 (23.6)

0.361

0.008

29 (15.2)

25 (15.5)

0.945

0.248

24 (12.6)

13 (8.1)

0.166

7 (9.5)