APPLICATION OF INNOVATE MOLECULAR BIOLOGICAL TECHNOLOGIES FOR THE DETECTION OF ANAEROBIC MICROORGANISMS IN ODONTODENIC INFLAMMATORY PROCESSES

ABSTRACT

Background: Odontogenic infections often originate from oral microbiota, particularly anaerobic bacteria, which play a critical role in the development and progression of inflammatory lesions in the maxillofacial region. Methods: Clinical specimens (n=80) including purulent exudates and tissue biopsies. Species-specific primers were used to detect major anaerobic pathogens such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. Statistical analysis was conducted to evaluate detection sensitivity and pathogen prevalence. Results: Molecular diagnostics successfully identified anaerobic bacteria in 92.5% of samples, significantly outperforming conventional culture methods (63.7%). P. gingivalis and F. nucleatum were the most prevalent, detected in 58.7% and 49.3% of cases, respectively. A significant correlation (p < 0.01) was found between bacterial load and the severity of clinical symptoms. Conclusion: The implementation of innovative molecular biological methods, particularly PCR-based diagnostics, offers a rapid, sensitive, and specific approach to identifying anaerobic microorganisms in odontogenic infections.

АННОТАЦИЯ

Введение: Одонтогенные инфекции часто возникают из-за микробиоты полости рта, особенно анаэробных бактерий, которые играют ключевую роль в развитии и прогрессировании воспалительных очагов в челюстно-лицевой области. Методы: Были проанализированы клинические образцы (n = 80), включая гнойные выделения и биоптаты тканей. Для выявления основных анаэробных патогенов, таких как Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum и Tannerella forsythia, использовались видоспецифичные праймеры. Проведен статистический анализ для оценки чувствительности методов обнаружения и распространенности патогенов. Результаты: Молекулярная диагностика успешно выявила анаэробные бактерии в 92,5% образцов, что значительно превышает эффективность традиционных методов культивирования (63,7%). P. gingivalis и F. nucleatum были самыми распространенными, обнаруженными в 58,7% и 49,3% случаев соответственно. Обнаружена значительная корреляция (p < 0,01) между бактериальной нагрузкой и тяжестью клинических симптомов. Заключение: Внедрение инновационных молекулярно-биологических методов, в частности ПЦР-диагностики, представляет собой быстрый, чувствительный и специфичный подход к идентификации анаэробных микроорганизмов при одонтогенных инфекциях.

Keywords: Odontogenic infections, microbiological detection, Dental abscess, Inflammation.

Ключевые слова: одонтогенные инфекции, микробиологическое выявление, зубной абсцесс, воспаление.

I. Introduction

Odontogenic infections are commonly associated with a diverse array of anaerobic microorganisms that reside within the oral cavity. These infections, including periapical abscesses, cellulitis, and osteomyelitis, often progress rapidly and require prompt diagnosis and treatment. Anaerobes such as P. gingivalis and F. nucleatum are frequently implicated in these conditions and contribute to the destruction of soft and hard tissues through the release of proteolytic enzymes and inflammatory mediators.

Conventional culture-based diagnostic methods are limited by their inability to cultivate fastidious anaerobic species and their long processing times. In contrast, molecular diagnostic technologies—especially polymerase chain reaction (PCR)—provide accurate, rapid, and species-specific identification of bacterial DNA directly from clinical specimens.

II. Materials and Methods

A. Materials

This study involved 80 patients with clinically diagnosed odontogenic infections treated at the Department of Oral and Maxillofacial Surgery, Tashkent State Dental Institute (2023–2025). Pus aspirates and inflamed tissue samples were obtained under sterile conditions and stored at −20°C prior to DNA extraction.

B. Methods

Genomic DNA was extracted using the "DNA-Express Blood and Tissue Kit" (Litech, Moscow). Species-specific primers were selected based on 16S rRNA sequences of key anaerobic pathogens. Conventional PCR and real-time qPCR assays were performed using a DTlite PCR instrument. Amplification products were verified by agarose gel electrophoresis. Quantitative data from qPCR were analyzed to determine bacterial load. Statistical Analysis: Sensitivity, specificity, and detection rates were calculated. Chi-square (χ²) tests and logistic regression were used to assess correlations between bacterial presence and clinical parameters (e.g., lesion size, systemic involvement).

III. Results and Discussion

A) Results

This study involved the collection of 80 clinical samples from patients diagnosed with odontogenic infections at the Tashkent State Dental Institute. Molecular diagnostic methods, including PCR and real-time qPCR, identified anaerobic microorganisms in 74 of the 80 samples, which equates to a detection rate of 92.5%. In comparison, traditional culture methods, which rely on bacterial growth in anaerobic conditions, identified pathogens in only 51 samples (63.7%).

The most prevalent anaerobic pathogens identified were:

Porphyromonas gingivalis – 47 cases (58.7%)

Fusobacterium nucleatum – 39 cases (49.3%)

Prevotella intermedia – 30 cases (37.5%)

Tannerella forsythia – 21 cases (26.2%)

Figure 1. The most prevalent anaerobic pathogens identified in  %

Co-infection with two or more anaerobic species was detected in 42% of cases (34 patients), highlighting the polymicrobial nature of odontogenic infections. Furthermore, real-time qPCR allowed for the quantification of microbial load, revealing a strong positive correlation (r = 0.72, p < 0.01) between bacterial load and the severity of clinical symptoms, such as swelling, fever, and systemic involvement. This correlation suggests that the microbial load could serve as an important indicator of infection severity.

For example, among patients with a higher bacterial load (above 10⁶ copies of bacterial DNA), 75% presented with extensive swelling and systemic symptoms, whereas only 25% of patients with a lower bacterial load (below 10⁴ copies) exhibited such severe symptoms. These findings support the potential role of microbial load as a prognostic marker for the clinical severity of odontogenic infections.

Real-time PCR also demonstrated its ability to detect multiple pathogens simultaneously, making it an essential tool for identifying polymicrobial infections, which are common in odontogenic abscesses and cellulitis. This was especially beneficial in identifying cases where pathogens like P. gingivalis and F. nucleatum were present together, which often resulted in more aggressive infections.

B) Discussion

The results of this study underline the superiority of molecular diagnostics, particularly PCR and real-time qPCR, over conventional culture-based methods for detecting anaerobic bacteria in odontogenic infections. Anaerobes, such as P. gingivalis and F. nucleatum, are difficult to culture due to their growth requirements and sensitivity to oxygen. Traditional culture methods have long been the gold standard, but their limitations, such as long incubation times (often 48-72 hours) and the need for specialized anaerobic conditions, make them inefficient and prone to false negatives, especially when bacterial loads are low.

In this study, the molecular techniques provided results much more rapidly—within hours, as opposed to several days needed for culture—and with significantly higher sensitivity. The ability of PCR to detect even small amounts of bacterial DNA allows for the identification of pathogens that might not grow well or at all in culture. This is particularly important for pathogens like P. gingivalis and F. nucleatum, which are known to be involved in the progression of odontogenic infections, especially in chronic cases. These bacteria are potent virulence factors, producing enzymes such as proteases and lipopolysaccharides, which degrade tissues and promote inflammation.

Moreover, the detection of multiple anaerobic pathogens in a single sample through multiplex qPCR is crucial, as polymicrobial infections are common in odontogenic abscesses. For example, P. gingivalis and F. nucleatum are often found together, as they synergistically exacerbate tissue destruction. Studies have shown that the presence of both pathogens increases the severity of infection and its resistance to treatment (Slots, 2002). In this study, co-infections were identified in 42% of cases, underlining the complexity of these infections and the need for a broad-spectrum diagnostic approach.

The correlation between bacterial load and clinical severity further strengthens the clinical relevance of molecular diagnostics. Higher bacterial loads were associated with more severe symptoms, such as significant facial swelling, fever, and systemic infection. These findings are consistent with those of other studies, which have demonstrated that the quantity of bacteria in odontogenic infections correlates with the severity of clinical manifestations (Siqueira & Rôças, 2013).

IV. Conclusion

The findings of this study highlight the considerable advantages of using molecular biological techniques, particularly PCR and real-time qPCR, for the detection of anaerobic microorganisms in odontogenic infections. These methods not only offer faster results compared to traditional culture-based techniques but also provide a higher detection rate, which is crucial for effective diagnosis and treatment. In this study, molecular diagnostics identified anaerobic pathogens in 92.5% of clinical samples, significantly outperforming culture-based methods (63.7%).

The study also demonstrates the clinical relevance of microbial load as a prognostic factor, with higher bacterial loads correlating with more severe clinical symptoms. The ability to identify multiple pathogens simultaneously using multiplex qPCR adds another layer of accuracy, enabling a more comprehensive understanding of polymicrobial infections, which are common in odontogenic abscesses.

Given these advantages, the implementation of PCR-based diagnostic methods in routine clinical practice should be prioritized. These techniques can help ensure that odontogenic infections are accurately diagnosed, thus guiding clinicians to provide targeted, effective treatment. Furthermore, the ability to monitor bacterial load through qPCR could assist in assessing infection progression and response to treatment, ultimately leading to better patient outcomes.

In conclusion, molecular biological technologies represent a significant advancement in the diagnosis and management of odontogenic infections. As these methods become more accessible and integrated into dental practice, they will likely improve the quality of care and enhance patient outcomes by enabling earlier and more precise interventions.

References:

1. Ashimoto A, Chen C, Bakker I, Slots J. (1996). Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiology and Immunology [7,p. 266–273].
2. Li X, Kolltveit KM, Tronstad L, Olsen I. (2000). Systemic diseases caused by oral infection. Clinical Microbiology Reviews [11, p.  547–558].
3. Siqueira JF Jr, Rôças IN. (2013). Microbiology and treatment of acute apical abscesses. Clinical Microbiology Reviews [18,p. 255–273].
4. Slots J. (2002). Periodontal microbiology: current concepts in oral bacterial infections. Oral Diseases[3,p. 37–40].
5. Tang H., Pei H., Xia Q., Tang Y., Huang J., Huang J., Pei F. Role of gene polymorphisms/haplotypes and serum levels of interleukin-17A in susceptibility to viral myocarditis. Mol. Biol. Rep. 2017;44. [15, p. 420–445].
6. Yang HW, Huang YF, Chang SW, Chen YW. (2010). Detection of Porphyromonas gingivalis in non-periodontal endodontic lesions. Journal of Endodontics [5,p.  985–990].