Pneumococcus testing service

Pneumococcus Testing Service – Comprehensive Microbiological and Serological Analysis for Vaccine Development, Clinical Diagnostics and Epidemiological Surveillance

As an ISO/IEC 17025 accredited contract research and diagnostic laboratory, we provide specialised testing services for Streptococcus pneumoniae (pneumococcus) – a leading cause of pneumonia, meningitis, otitis media, and sepsis – serving Bulgarian and international pharmaceutical companies, vaccine manufacturers, clinical research organisations, public health laboratories, and hospital infection control units. Our test portfolio covers pneumococcal serotyping, antibiotic susceptibility testing, vaccine antigen quantification, opsonophagocytic killing assays (OPKA), and molecular detection of serotype‑specific genes, all designed to support vaccine efficacy studies, epidemiological surveillance, and patient management. All methods are aligned with CLSI (Clinical and Laboratory Standards Institute), WHO recommendations, ISO 20776, and BDS (Bulgarian Institute for Standardisation) standards. Our reports are recognised by the Bulgarian Food Safety Agency (BFSA), the National Centre of Infectious and Parasitic Diseases (NCIPD) – the national reference laboratory for pneumococcal diseases – and the European Centre for Disease Prevention and Control (ECDC).

Clinical and Environmental Specimens We Test

Our laboratory accepts a wide variety of clinical specimens and isolates for pneumococcal detection and characterisation. Typical samples include:

• Clinical specimens – sputum, bronchoalveolar lavage (BAL), nasopharyngeal swabs, cerebrospinal fluid (CSF), pleural fluid, blood cultures, and middle ear fluid (for otitis media studies)
• Bacterial isolates – pure cultures of Streptococcus pneumoniae from clinical or environmental sources, submitted as agar stabs, glycerol stocks, or lyophilised cultures
• Vaccine antigens – purified pneumococcal polysaccharides (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, etc.) and protein-conjugate vaccines
• Serum samples – from vaccinated individuals or animal models, for opsonophagocytic and antibody titre measurement
• Environmental swabs – from surfaces, air, and water sources where pneumococcal contamination is suspected
• Culture media and transport media – for validation of sample collection and handling procedures

Serotyping and Molecular Identification – Determining Vaccine Coverage

• Quellung reaction (capsular swelling test) – the gold standard for serotyping, using type‑specific antisera to identify the capsular serotype. We have a comprehensive panel of antisera (Statens Serum Institut) covering all clinically relevant serotypes. This method is essential for epidemiological surveillance and for predicting vaccine coverage (pneumococcal conjugate vaccines – PCV10, PCV13, PPSV23).
• Multiplex PCR (mPCR) for serotype detection – we use validated multiplex PCR assays targeting serotype‑specific genes (e.g., cps loci) to identify up to 30 serotypes in a single reaction. This is particularly useful for non‑viable or slow‑growing specimens, and for confirmation of Quellung results.
• Whole‑genome sequencing (WGS) – serotype prediction – for research and outbreak investigation, we perform WGS on pneumococcal isolates and use bioinformatic pipelines (e.g., PneumoCaT, SeroBA) to predict the serotype and identify sequence types (STs) for epidemiological typing.
• MLST (Multi‑Locus Sequence Typing) – we determine the sequence type (ST) of isolates using the standard pneumococcal MLST scheme (7 housekeeping genes), providing global epidemiological context and tracking of virulent clones (e.g., ST305, ST320).

Antimicrobial Susceptibility Testing (AST) – Guiding Therapy

• Minimum Inhibitory Concentration (MIC) – broth microdilution – ISO 20776‑1 / CLSI M07 – we test a panel of antibiotics relevant to pneumococcal infections: penicillin, amoxicillin, cefotaxime, ceftriaxone, erythromycin, azithromycin, clarithromycin, levofloxacin, moxifloxacin, vancomycin, linezolid, and tetracycline. MICs are determined in cation‑adjusted Mueller‑Hinton broth with 2‑5 % lysed horse blood. Results are interpreted according to CLSI (M100) and EUCAST breakpoints.
• Disk diffusion (Kirby‑Bauer) – CLSI M02 – for routine screening, we perform disk diffusion on Mueller‑Hinton agar with 5 % sheep blood and measure inhibition zone diameters. This is a fast and cost‑effective method for detecting resistance to penicillin, macrolides, and fluoroquinolones.
• Etest (gradient strip) – for penicillin and ceftriaxone – for precise MIC determination of beta‑lactams, we use Etest strips applied directly to agar plates, providing a quantitative result that can identify penicillin non‑susceptible isolates (intermediate or resistant).
• Resistance mechanism detection – for penicillin‑resistant isolates, we screen for altered pbp genes by PCR or sequencing. For macrolide resistance, we detect ermB (methylase) and mefA (efflux pump) genes. For fluoroquinolone resistance, we sequence the gyrA and parC quinolone resistance‑determining regions (QRDR).

Opsonophagocytic Killing Assay (OPKA) – Vaccine Functional Antibody Testing

• Standard OPKA protocol (based on WHO reference method) – we measure the ability of serum antibodies to opsonise pneumococci for killing by phagocytic cells (HL‑60 human myeloid cells). The assay uses a serotype‑specific pneumococcal strain and a complement source (baby rabbit complement). After incubation, viable bacteria are enumerated by plating on agar; the opsonisation titre (OPKA titre) is defined as the reciprocal serum dilution that results in 50 % killing.
• Multiplex OPKA – for vaccine trials, we have developed a multiplex format that allows testing of up to 8 serotypes in a single assay, reducing sample volume and cost.
• Quality control – we include a reference serum (WHO standard) and a negative control (serum from non‑vaccinated subjects) in each run. The assay is validated for precision (intra‑assay CV ≤ 20 %, inter‑assay CV ≤ 25 %).
• Correlation with antibody titres – we can also measure serotype‑specific IgG concentrations by ELISA (using 22F adsorption) and correlate them with OPKA titres to assess functional immune responses.

Antigen Quantification – Vaccine Formulation and Potency Testing

• Polysaccharide quantification – colorimetric and HPLC methods – we quantify capsular polysaccharide content in vaccine intermediates using the anthrone‑sulfuric acid method (total sugars) and high‑performance anion‑exchange chromatography with pulsed amperometric detection (HPAEC‑PAD) for individual serotypes. This is essential for lot‑release and stability testing.
• Protein content – Lowry or Bradford assay – for conjugate vaccines, we measure carrier protein (e.g., CRM197, tetanus toxoid, diphtheria toxoid) content using colorimetric protein assays, ensuring consistent conjugation ratios.
• Molecular size distribution – SEC‑HPLC – we use size‑exclusion chromatography (SEC‑HPLC) to assess the molecular weight distribution of conjugated polysaccharides, which correlates with immunogenicity.
• Residual impurities – host cell proteins, endotoxins – we perform ELISA for residual host cell proteins (HCP) and LAL (Limulus Amebocyte Lysate) for endotoxin detection, per ICH guidelines and pharmacopoeial standards.

Molecular Detection – Direct from Clinical Specimens

• Real‑time PCR (qPCR) – targeting the lytA or pneumolysin gene – we offer highly sensitive and specific detection of pneumococcal DNA directly from clinical specimens (sputum, CSF, blood, pleural fluid) using validated qPCR protocols. The limit of detection is typically ≤ 10 CFU/reaction. This is particularly useful for culture‑negative cases (e.g., patients on prior antibiotics).
• Serotype‑specific qPCR – for rapid serotype identification without culture, we use serotype‑specific qPCR assays targeting the cps locus of individual serotypes, enabling early determination of vaccine coverage.
• Multiplex PCR panels – we also offer commercial and in‑house multiplex PCR panels that can detect and serotype up to 20 serotypes in a single reaction, providing a cost‑effective option for outbreak investigation and surveillance.
• Quantitative bacterial load assessment (by qPCR) – we can estimate the bacterial burden in clinical specimens by interpolation from a standard curve of known CFU equivalents, which is useful for monitoring response to therapy.

Surveillance and Epidemiological Typing

• Antimicrobial resistance (AMR) surveillance – we aggregate AST data from clinical isolates to generate local and national AMR reports, aiding the Bulgarian health authorities in updating treatment guidelines and detecting emerging resistance.
• Serotype distribution analysis – for epidemiological studies, we compile serotype data and compare them to historical data and to PCV10/PCV13/PPSV23 vaccine compositions, helping to assess the impact of vaccination programmes.
• Outbreak investigation – using WGS and MLST, we can link cases in suspected outbreaks (e.g., in nursing homes, hospital wards) and determine the relatedness of isolates.
• Population structure analysis – we perform phylogenetic analysis (e.g., eBURST, goeBURST) to identify global and regional clonal complexes, such as the Spain23F‑1, Netherlands3‑31, and Taiwan19F‑14 clones.

Quality Control and Reference Strains

To ensure the reliability and reproducibility of our tests, we strictly adhere to quality control protocols, including:

• Daily and weekly QC checks – using ATCC reference strains (e.g., S. pneumoniae ATCC 49619) for AST and serotyping, we verify the performance of media, reagents, and instruments.
• Proficiency testing – we participate in external quality assessment (EQA) schemes such as those organised by the European Antibiotic Resistance Surveillance System (EARS‑Net) and the World Health Organization (WHO) reference laboratories.
• Internal validation of new assays – before introducing a new method, we validate it against current gold‑standard methods and document sensitivity, specificity, positive and negative predictive values.
• Traceability – all reference materials (antisera, antibiotics, control DNA) are sourced from certified suppliers and are traceable to international standards where available.

Report Acceptance & Compliance with Bulgarian and European Public Health Standards

All pneumococcus testing is performed under our ISO/IEC 17025 accreditation, and for clinical samples we adhere to the Bulgarian Health Act and ECDC surveillance guidelines. Our final test reports include: a complete description of the specimen (type, source, date, collection method), the test performed (e.g., serotyping, AST, OPKA, molecular detection), a summary of results (serotype, MIC values, susceptibility interpretation, OPKA titre, bacterial load, etc.), interpretation of the results (resistance, vaccine coverage, immune response), and a clear conclusion with clinical or epidemiological recommendations where applicable. Reports are prepared in accordance with the requirements of the National Centre of Infectious and Parasitic Diseases (NCIPD), the Bulgarian Food Safety Agency (BFSA), and international pharmaceutical and biotech companies. Bilingual (Bulgarian/English) versions are available for ease of submission to national and international regulatory and public health bodies.