Design and development of primers for detection of Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis

dgkh000545 10.3205/dgkh000545 urn:nbn:de:0183-dgkh0005454 Research Article Design and development of primers for detection of Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis Design und Entwicklung von Primern für den Nachweis von Streptococcus pneumoniae, Haemophilus influenzae und Neisseria meningitidis Azimi Azimi Leila L MD

Pediatric Infections Research Center, Research Institute of children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran. P.O. Box 15468-15514, Iran; phone: +98 21 22227004Pediatric Infections Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran

leilaazimi1982@gmail.com author Shirkavand Shirkavand Fatemeh F

Pediatric Infections Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran

author Armin Armin Shahnaz S

Pediatric Infections Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran

author Karbasian Karbasian Fereshteh F

Department of pediatric gastroenterology and hepatology, Ali-Asghar children’s hospital, Iran University of Medical Sciences, Tehran, Iran

author Khodaei Khodaei Hannan H

Pediatric Infections Research Center, Research Institute for Children’s Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran

author German Medical Science GMS Publishing House

Düsseldorf

610 Streptococcus pneumoniae Haemophilus influenzae Neisseria meningitidis primer sensitivity Streptococcus pneumoniae Haemophilus influenzae Neisseria meningitidis primer sensitivity 20250430 engl This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). 2196-5226 20 GMS Hygiene and Infection Control GMS Hyg Infect Control 16 Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Researcher Grant Committee Hintergrund: Die Sterblichkeitsrate bei Meningitis ist in bestimmten Regionen der Welt immer noch alarmierend hoch. Ziel der Arbeit ist es, die wirksamsten Primer für den Nachweis von Streptococcus (S.) pneumoniae, Haemophilus (H.) influenzae und Neisseria (N.) meningitidis mithilfe der Real-Time-PCR-Technologie zu ermitteln.Material und Methoden: Es wurden zwei Primer-Sets zum Nachweis von S. pneumoniae, H. influenzae und N. meningitidis unter Verwendung der Primer Biosoft Allele ID 7.6 entwickelt. Untersucht wurden die minimalen bakteriellen Kopienzahlen, die von jedem Primer nachgewiesen werden können, sowie ihre Spezifität.Ergebnisse: CtrA und hpd2 konnten 400 Kopienzahlen/ml von H. influenzae und N. meningitidis und LytA2 40 Kopienzahlen/ml von S. pneumoniae nachweisen. Die Sensitivität und Spezifität aller Primer betrug 100 % (CI: 95 %). Schlussfolgerung: Die Verwendung empfindlicherer Primer zum Nachweis des für die bakterielle Meningitis verantwortlichen Erregers erhöht die Chance, die verursachenden Bakterien zu identifizieren. Die entwickelten Primer konnten die ausgewählten Bakterien mit mindestens zehnmal höherer Empfindlichkeit identifizieren als kommerzielle Diagnosekits im Iran. Background: The mortality rate of meningitis is still alarmingly high in certain regions across the globe. The objective of this research is to identify the most effective primers for detecting Streptococcus (S.) pneumoniae, Haemophilus (H.) influenzae, and Neisseria (N.) meningitidis using Real-Time PCR technology.Materials and methods: Two sets of primers were developed for detecting S. pneumoniae, H. influenzae, and N. meningitidis using the Primer Biosoft Allele ID 7.6 application. The study examined the minimum bacterial copy numbers detectable by each primer, as well as their specificity.Results: CtrA and hpd2 could detect the 400 copy numbers/ml of H. influenzae, and N. meningitidis and LytA2 could detect the 40 copy numbers/ml of S. pneumoniae. The sensitivity and specificity of all primers was 100% (CI: 95%). Conclusion: Using more sensitive primers to detect the bacterial agent responsible for causing bacterial meningitis increases the chance of identifying the causative bacteria. The primers designed in this study could identify the selected bacteria with at least 10 times more sensitivity than the currently available commercial diagnostic kits in Iran. IntroductionH. influenzae, N. meningitidis, and S. pneumoniae can cause infection in important tissues such as blood and cerebrospinal fluid (CSF), requiring rapid intervention and healing , , , , . Fast and early diagnosis of these bacteria is necessary for quick treatment and appropriate antibiotic therapy. Blood and CSF cultures (conventional culture or BACTEC) are traditional methods used to identify bacteria . However, these methods are time-consuming and, in some cases, bacteria cannot grow in the culture medium due to the use of antibiotics and despite the clinical symptoms, bacterial growth is not observed. Molecular methods can be helpful in overcoming these disadvantages. Real-time PCR, a molecular method, can be used for early diagnosis and fast treatment of the patient. Many commercial diagnosis kits with different levels of sensitivity are used worldwide to identify the cause of infections. The sensitivity and minimum detectable copy numbers of bacteria are related to the primers of the kits. As such, the primers play a key role in these diagnosis kits. In this regard, the aim of this study was to design a diagnostic panel using real-time PCR to identify H. influenzae, N. meningitidis, and S. pneumoniae. Materials and methodsSamples and setting H. influenzae, N. meningitidis, and S. pneumoniae, which were isolated from bacterial meningitides and had their identity confirmed by real-time PCR in the last study by our group , were selected. These strains were used to evaluate the designed primers. Primer design Two different pairs of primers were designed by Allele ID 7.6 for different genes or different parts of genes to detect H. influenzae (hpd gene), N. meningitidis (CtrA and SodC), and S. pneumoniae (lytA) (Table 1 ). DNA extractionBacterial genome extraction was performed using a DNA extraction kit (Qiagen Cat No./ID: 51304). The DNA concentration was read by Qubit. 10 dilutions were prepared by a factor of 101 for each bacterium, and the DNA concentration of all dilutions was determined by the Qbit instrument to determine the sensitivity and the cut-off point of these primers.Real-time PCR assay Real time-PCR assay was performed for the identification of bacteria using differently designed primers. Cyber green master mix and ABI step-one, in addition to the real-time PCR instrument, were used in this setup. The DNA copy number calculator works according to the following equation: where Amount (ng) is the amount of DNA in nanograms (ng) in the tube, 6.022x1.023 is Avogadro’s constant and represents the number of molecules per mole, Length (bp) is the length of DNA, in base pairs (bp), in the template (H. influenzae 1.830.137 bp, N. meningitidis 2.184.406 bp and S. pneumoniae 2.160.837 bp), 1x109 is the factor used to convert to ng, Mass of DNA bp stands for the average mass of a DNA bp, which is either 660 (dsDNA) or 330 (ssDNA) g/mole. This value depends on what is selected as the type of DNA in the calculator (https://toptipbio.com/dna-copy-number-calculator/).The genomes of Acinetobacter baumannii, Pseudomonsa (P.) aeruginosa, Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Enterobacter spp., Staphylococcus (S.) aureus and Enterococcus spp. were used as a negative control to evaluate the specificity of these primers. ResultsThe results of real-time PCR showing the sensitivity of these primers to detect the included bacteria are presented in Table 2 .None of the duplication observed with the genomes of A. baumannii, P. aeruginosa, E. coli, K. pneumoniae, Enterobacter spp., S. aureus and Enterococcus spp. confirmed the 100% (CI: 95%) specificity of these primers. DiscussionBacterial meningitis occurs most often in childhood and the etiological pathogens can be diverse in different age groups of children , , . Based on previous studies, the incidence of bacterial meningitis can vary depending on factors such as time, geographical location, and patient age , , . A systematic review and meta-analysis on the worldwide etiology of bacterial meningitis showed that the most prevalent causative pathogens were N. meningitidis and S. pneumoniae in all age groups, while S. pneumoniae was most prevalent in children . An accurate method to identify these bacteria can be helpful in saving human lives and prevent them from becoming disabled in the future due to meningitis. The results of this study showed higher sensitivity of CtrA primer than SodC for the detection of N. meningitidis. On the other hand, different genome regions of hpd and LytA genes in H. influenzae and S. pneumoniae, respectively, were selected to design primers. The different results when various genes are selected to identify the bacteria, showing that the primer selected for nucleotide sequence between 777–903 has greater sensitivity for detecting H. influenzae. Also, primer design for nucleotide sequences between 350–424 is more sensitive for detecting S. pneumoniae.In the study by Haddad-Boubaker et al. the results showed that Real-time PCR could detect up to 67.10–4 ng/µL DNA for S. pneumoniae, 38.10–6 ng/µL and 38.10–3 ng/µL for N. meningitidis ctrA gene and sodC gene, respectively, and 97.10–4 ng/µL for H. influenzae . In the current study, the primers used were ctrA, hpd.2, and LytA.2. These results are near to ours.Cyber green was used in the current study and is more affordable in comparison to using the probe of the Haddad-Boubaker et al. study . One of the commercial molecular-diagnosis kits for the detection of these three bacteria is being used (Sacace™ NHS Meningitidis Real-TM) in Iran. The analytical sensitivity, genome equivalents/ml of this kit is 1*103 (genome of bacteria/ml) , but the primers we designed have a minimum detectable bacterial genome 4*102 for N. meningitidis and H. influenzae and 4*10 for S. pneumoniae. These results showed that the primers designed in the current study detected bacteria with at least 10 times greater sensitivity. This is an important advantage of molecular diagnosis kits, especially when patients used antibiotics before the test. ConclusionsThe detection of the causative bacteria of meningitis can be helpful to choose the best therapeutic process as soon as possible. In this regard, selecting the most accurate and rapid method with the greatest possible sensitivity to identify relevant agents is necessary. It is important to know the exact cause of meningitis, because the choice of treatment depends on it. NotesCompeting interestsThe authors declare that they have no competing interests.Ethical approval Ethical approval No. IR.SBMU.RICH.REC.1399.061 was granted by the Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.FundingThe research reported in this publication was supported by the Researcher Grant Committee under grant number [20326] from the Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.Authors’ ORCID Azimi L: https://orcid.org/0000-0002-7216-2530Shirkavand F: https://orcid.org/0009-0001-5207-4883Armin S: https://orcid.org/0000-0002-4993-482X Karbasian F: https://orcid.org/0000-0003-2494-6231Khodaei H: https://orcid.org/0000-0002-2297-4733 Pouladfar G Sanaei Dashti A Kadivar MR Jafari M Pourabbas B Jamalidoust M Evaluation of multiplex real-time PCR and WHO criteria for diagnosing childhood bacterial meningitis in a tertiary referral hospital in Iran 2022 Arch Pediatr Infect Dis e101822 Pouladfar G, Sanaei Dashti A, Kadivar MR, Jafari M, Pourabbas B, Jamalidoust M. 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