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 ~ Introduction
 ~ Methods
 ~ Results
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  Table of Contents  
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 504-507

Validation of pneumococcal iron acquisition (piaA) gene for accurate identification of Streptococcus pneumoniae

1 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
3 Department of Pediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
4 Department of Neonatology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Sujatha Sistla
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_18_274

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 ~ Abstract 

Purpose: The pneumococcal iron acquisition (piaA) gene is found to be highly specific and hence proposed as a diagnostic marker for identification of pneumococci. The objective of the present study was to evaluate the piaA gene as a genetic marker for the identification of pneumococci. Methods: Twenty isolates were initially sequenced for lytA gene using published primers. PiaA-PCR (piaA polymerase chain reaction) was performed using in-house primers and protocol. Based on the sensitivity and specificity results, a final sample of 30 pneumococcal isolates and 11 non-pneumococcal isolates confirmed with lytA- sequencing were selected. Statistical analyses were performed using OpenEpi v3.01 and GraphPad Quickcalc at P < 0.05 as the level of statistical significance. Results: Of the initial 20 samples tested, piaA PCR was positive in only 71.43% (10/14) of the pneumococcal isolates but was 100% specific (0/6 non-pneumococcal isolates) P = 0.011. When the PCR was performed on 41 samples, the sensitivity increased to 73.33% (95% of confidence interval [CI] = 55.55–85.82) and specificity remained the same P < 0.001. The level of agreement between the PCR and lytA-sequencing was found to be moderate (κ = 0.694; 95% CI = 0.432–0.955). Conclusions: PiaA-PCR can be used as a specific marker for the identification of pneumococcus, though it is less sensitive. As the level of agreement was moderate, further analyses on a large number of samples can give conclusive evidence for its use as a diagnostic marker for pneumococcus.

Keywords: LytA gene, pneumococcal iron acquisition gene, polymerase chain reaction, Streptococcus pneumoniae

How to cite this article:
Peela SC, Sistla S, Tamilarasu K, Krishnamurthy S, Adhishivam B. Validation of pneumococcal iron acquisition (piaA) gene for accurate identification of Streptococcus pneumoniae. Indian J Med Microbiol 2018;36:504-7

How to cite this URL:
Peela SC, Sistla S, Tamilarasu K, Krishnamurthy S, Adhishivam B. Validation of pneumococcal iron acquisition (piaA) gene for accurate identification of Streptococcus pneumoniae. Indian J Med Microbiol [serial online] 2018 [cited 2020 Oct 25];36:504-7. Available from:

 ~ Introduction Top

Streptococcus pneumoniae is a major cause of morbidity and mortality in developing countries like India. Clinically, pneumococcus is responsible for a range of infections such as acute pyogenic meningitis and pneumonia. Despite the clinical and epidemiological significance, accurate identification of pneumococcus is a challenge faced by many microbiologists around the world.[1] Previously used protocols such as optochin susceptibility and bile solubility are losing their stand as accurate methods of identification.[2] These tests are less specific in differentiating pneumococcus from other members of viridans streptococci such as Streptococcus pseudopneumoniae and Streptococcus mitis.[3] Furthermore, there are reports of pneumococcus either being optochin-resistant or bile insoluble or both. Such isolates were collectively grouped as 'atypical pneumococci' as they were different from 'typical' pneumococcal strains (optochin sensitive and bile soluble).[3]

The use of molecular assays failed to improve the status of identification in terms of specificity. Of all the molecular targets studied, autolysin (lytA) appears to be highly specific for the identification of pneumococci.[4],[5],[6],[7] This gene has been extensively studied, and its presence in related streptococci has been determined through whole-genome sequencing. Among other molecular targets, piaA has been studied as a diagnostic marker in a few studies.[8],[9],[10] The absence of this gene in related streptococci has raised the hopes of using it as a marker for identification of pneumococcus.[11] The present study was carried out to evaluate the use of piaA as a diagnostic marker for accurate identification of S. pneumoniae.

 ~ Methods Top

Design of piaA-primers and polymerase chain reaction assay

The complete sequences of piaA gene of S. pneumoniae were extracted from NCBI database which were then aligned using CLUSTALW. The consensus region which was conserved in all the sequences was identified, and the primers for piaA polymerase chain reaction (PCR) were designed using NCBI-PRIMER-BLAST tool. After manually assessing the parameters and the amplification sizes, a primer pair piaA_F– 5'-AAGCGGAAAAGCAGGCCTTA-3' and piaA_R– 5'-CGAAGCCAATAGGTCCACCA-3' with an amplification size 365 bp was selected. Furthermore for assessing the specificity of the primers, they were submitted to NCBI-PRIMER BLAST for search in 'nr' database and without specifying the taxon. These primers were found to be 100% specific to pneumococci. There were at least three mismatches with other streptococci there by reducing the chances of any misidentification. Conventional PCR was performed at 25 μL final volume consisting of 12 μL master-mix with Taq polymerase and dNTPs besides the buffers (Origin Biolabs, Kerala, India), 0.6 μM of each primer and DNA template of 5 μL. The following cycling conditions were used: initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15 s annealing at 59.8°C for 15 s and extension at 72°C for 15 s. Final extension was carried out at 72°C for 8 min. The amplified products were loaded onto 2% agarose gels stained with ethidium bromide and visualised under ultraviolet light using GelDoc XR system (Bio-Rad, California, USA).

Pilot study

A pilot sample consisting of 20 isolates of suspected pneumococci from various clinical samples routinely sent to the diagnostic Bacteriology laboratory from January 2017 to June 2017 were tested by both optochin susceptibility and bile solubility assays. DNA was extracted using Mericon Plus Bacterial DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer instructions and the isolates were sequenced for lytA gene using Sanger sequencing method in ABI 3130 electrophoresis instrument (Applied Biosystems, California, USA). The primers for lytA gene were selected based on a previous report.[12] The isolates were also tested for the presence of piaA gene using the primers and protocols mentioned above. Any isolate with the highest similarity to pneumococcal lytA (estimated using NCBI-BLASTN) was considered as pneumococcus. The performance of piaA PCR was compared against the lytA-sequencing results, and the sample size was recalculated for the descriptive study. Later, 41 isolates (according to the sample size) were sequenced for lytA gene similar to the above, and then analysed and submitted to GenBank with accession numbers MH029553-MH029580 and MH029582-MH029594.

Descriptive study

Based on the specificity of the piaA PCR after the pilot study, the sample size was calculated at an estimated specificity of 99.5%, a precision of 2.5% and at 95% confidence level using the nMASTER sample size calculation software.[13] The required sample size was determined to be 30 pneumococcal isolates, and 11 non-pneumococcal isolates were added to the sample size as a control group from the samples sent to the laboratory during the above-specified duration. The piaA PCR was performed on these 41 isolates and 10 isolates of other bacterial species (two each of Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis,  Escherichia More Details coli and Staphylococcus epidermidis), and its performance was assessed using statistical tests.

Statistical analysis

For all the statistical analyses, the results of lytA-sequencing were considered as the gold standard. Sensitivity, specificity and positive- and negative-predictive values were determined, and Cohen's Kappa was used to quantify the agreement between lytA-sequencing results and piaA PCR. Fisher's exact test was used to estimate the level of significance of the results, and P < 0.05 was considered as statistically significant. All the analyses were carried out in OpenEpi v 3.1 (Andrew G. Dean and Kevin M. Sullivan, Atlanta, GA, USA) and GraphPad QuickCalcs (GraphPad Software, San Diego, CA) online software.

Ethical statement

The study has been approved by the Institute Ethics committee for Human studies (JIP/IEC/2015/15/744).

 ~ Results Top

Pilot study

Among the 20 isolates tested initially, 14 isolates were identified as pneumococci based on lytA-sequence similarity (>99%). The piaA PCR was positive in 10 isolates (sensitivity = 71.43%; 95% confidence interval [CI] = 45.35%–88.28%) while negative in all the six non-pneumococcal isolates (specificity = 100%; 95% CI = 60.97%–100%). There was a moderate agreement between results by lytA-sequencing and piaA PCR (κ coefficient = 0.6; 95% CI = 0.198–1.002). The specificity was adjusted to 99.5% and the sample size was recalculated.

Descriptive study

Of the 41 α-haemolytic isolates tested, 28/41 (68.3%) were optochin susceptible, 23/41 (56.1%) were bile soluble and 5/41 (12.2%) were atypical by phenotypic tests. Of these, 22 pneumococcal isolates (sensitivity = 73.33%; 95% CI = 55.55%–85.82%) and none of the pneumococcal isolates (specificity = 100%; 95% CI = 85.54%–100%) were positive by piaA PCR. The results are shown in [Table 1] and [Table 2]. The piaA–PCR was in moderate agreement with the lytA-sequencing results (kappa coefficient = 0.694; 95% CI = 0.432–0.955) and was statistically significant P < 0.001.
Table 1: Results of various tests versus the autolysin - sequencing (the gold standard)

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Table 2: Results of autolysin - sequencing and pneumococcal iron acquisition - polymerase chain reaction in typical and atypical pneumococci

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 ~ Discussion Top

Identification of pneumococcus is a challenge to the microbiologists both clinically and epidemiologically. Misidentification of pneumococcus can lead to over- or under-reporting of antibiotic resistance and serotype patterns in many epidemiological studies.[3] To create a standard modus operandi, the World Health Organisation and the Centres for Disease Control and Prevention released a manual which specifies the assay procedures and interpretations (WHO 2011). This manual is now followed in many centres where epidemiological studies on pneumococcus are being carried out. In spite of the presence of a manual, various investigators have used different concentrations of sodium deoxycholate for bile solubility tests and zone diameters for interpretation of optochin susceptibility.[3]

The shift from phenotypic to molecular assays may have been triggered by the inaccuracy, and many molecular assays such as PCR and its variants, multiplex PCR and real-time PCR have been incorporated into laboratory manuals and other epidemiological studies. Various targets such as pneumolysin, autolysin, pneumococcal surface adhesin, rpo A and penicillin-binding proteins were targeted for species-specific identification.[6],[14],[15] Although these assays were initially found to be successful and accurate, their specificity decreased greatly after multiple evaluation studies. Currently, autolysin-based real-time PCR assay is considered as the gold standard for the identification of pneumococcus in epidemiological studies, even though a few reports found a slight false-positivity among the samples tested.[10] This has been attributed to genetic mosaicism which has led to large-scale recombination of genes among pneumococcus and other closely related streptococci.

piaA is an ABC transporter lipoprotein involved in iron acquisition and is found exclusively in all typical pneumococci, whereas piu A is another member of iron transporters which may be present in both typical and atypical pneumococci.[16] These proteins constitute a part of necessary major virulence systems as determined by murine systemic and pulmonary models.[17] The presence of piaA in all typical pneumococci has opened the possibility to use it as a vaccine candidate and a diagnostic marker. In spite of its specificity, studies employing this gene as a target for identification are very few. It was found that the piaA gene was identified in all 39 of the typical pneumococcal strains tested but absent in only two of the eight acapsular strains tested.[16] In another study, a duplex real-time PCR assay targeting both lytA and piaA was used in determining the pneumococcal carriage in children by trans-nasal and trans-oral nasopharyngeal swabs.[8] By this protocol, the frequency of pneumococcal carriage doubled from 19% to 40%. The presence of the piaA gene in non-typable pneumococcus was shown in another study evaluating multiple targets for identification of pneumococcus, in which the isolates were also positive for cpsA gene.[11] In the present study, four typical isolates were negative by lytA-sequencing, thereby suggesting misidentification by phenotypic tests alone. Interestingly, two typical pneumococcal isolates were negative by piaA–PCR, whereas two 'atypical' pneumococcal isolates and three phenotypically negative pneumococcal isolates (confirmed by lytA-sequencing) were positive for the piaA gene [Table 2].

As per our knowledge, reports on using piaA gene for identification of pneumococcus were only very few, and many studies for testing the utility of other diagnostic markers such as cpsA and lytA usually tested a few isolates closely related to pneumococci. Due to this, the true false-positivity of other diagnostic markers can be under-reported, raising false hopes for highly specific assays. For example, a recent study published from south India assessed the diagnostic value of employing multiplex quantitative PCR by testing various targets such as pneumolysin, autolysin, pneumococcal surface adhesin and SPN9802 (all the primers and probes were designed in-house).[18] They tested only one isolate each in the mitis group of streptococci and found an overall sensitivity and specificity of 100%. Testing a larger number of non-pneumococcal isolates would give a better estimate of specificity. This was further explored in a study where three non-pneumococcal isolates (of the 1562 total isolates tested) were positive by lytA-targeted real-time PCR.[10] In the same study, the presence of piaA gene was also tested by real-time PCR, and these three non-pneumococcal isolates were negative. We used around 11 isolates which were closely related to pneumococci to assess the specificity and found all the isolates were piaA-negative.

Among the eight false-negative isolates, only two isolates revealed typical pneumococcal characteristics (optochin sensitive and bile soluble), two isolates were optochin sensitive and bile insoluble and four isolates were both optochin-resistant and bile insoluble. Six of these strains were similar to atypical pneumococcal strains from a single study,[19] whereas the two other isolates were similar to a single isolate (101/87) from the another study.[20] In the study involving atypical strains, the isolates were closely related to both S. pneumoniae and S. mitis, and the phylogenetic relationship failed to group these isolates either with pneumococcus or other mitis group streptococci (MLST analysis on these isolates showed they belong to diverse clones that were not present in the database at the time of study). The other single isolate (101/87) was identified using a DNA probe based on autolysin. It is interesting to note that identification by autolysin is error-prone, and hence, the actual species of these previous isolates remains questionable. Hence if the eight isolates were not pneumococci, the performance of piaA PCR will be 100% accurate. The limitations of the present study would be the inability to classify the organism based on >1 gene sequences and lytA-sequencing was performed only for 41 isolates.

 ~ Conclusions Top

PiaA gene can be used as an accurate marker for identification of pneumococcus as it is highly specific. Further, testing on a large number of confirmed pneumococcal and non-pneumococcal isolates by advanced techniques like sequencing should be performed to assess its diagnostic value.


The author (SCMP) acknowledges the Council of Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (file no. 09/805 (0010)/2014-EMR-I). The author (SCMP) would like to acknowledge Mr. Jeby Jose (Ph.D. student) for assistance in statistical analyses.

Financial support and sponsorship

The work was supported by JIPMER Intramural Research Fund (JIPMER/01/109/2015/00411) and contingency grant by CSIR (09/805(0010)/2014-EMR-I).

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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Kellogg JA, Bankert DA, Elder CJ, Gibbs JL, Smith MC. Identification of Streptococcus pneumoniae revisited. J Clin Microbiol 2001;39:3373-5.  Back to cited text no. 2
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McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW, et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 2001;39:3446-51.  Back to cited text no. 4
Messmer TO, Sampson JS, Stinson A, Wong B, Carlone GM, Facklam RR, et al. Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae. Diagn Microbiol Infect Dis 2004;49:249-54.  Back to cited text no. 5
Suzuki N, Yuyama M, Maeda S, Ogawa H, Mashiko K, Kiyoura Y, et al. Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae. J Med Microbiol 2006;55:709-14.  Back to cited text no. 6
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Wyllie AL, Rümke LW, Arp K, Bosch AA, Bruin JP, Rots NY, et al. Molecular surveillance on Streptococcus pneumoniae carriage in non-elderly adults; little evidence for pneumococcal circulation independent from the reservoir in children. Sci Rep 2016;6:34888.  Back to cited text no. 9
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Wyllie AL, Pannekoek Y, Bovenkerk S, van Engelsdorp Gastelaars J, Ferwerda B, van de Beek D, et al. Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species. Open Biol 2017;7. pii: 170074.  Back to cited text no. 11
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El Aila NA, Emler S, Kaijalainen T, De Baere T, Saerens B, Alkan E, et al. The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays. BMC Infect Dis 2010;10:104.  Back to cited text no. 14
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Whalan RH, Funnell SG, Bowler LD, Hudson MJ, Robinson A, Dowson CG, et al. Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci. J Bacteriol 2006;188:1031-8.  Back to cited text no. 16
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Ganaie F, Govindan V, Kumar KL. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae. Pneumonia 2015;6:57-66.  Back to cited text no. 18
Obregón V, García P, García E, Fenoll A, López R, García JL, et al. Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble. J Clin Microbiol 2002;40:2545-54.  Back to cited text no. 19
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  [Table 1], [Table 2]


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