Klebsiella pneumoniae subsp. rhinoscleromatis : causal agent of rhinoscleroma – Molecular diagnosis (PCR); Molecular identification (PCR and sequencing)
The genus Klebsiella is composed of opportunistic pathogens that cause a wide range of infections in man. This bacterial genus is currently classified into the species: Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena and Klebsiella ornithinolytica. In turn, K. pneumoniae is divided into three subspecies: Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, and Klebsiella pneumoniae subsp. rhinoscleromatis. The first one, K.pneumoniae subsp. pneumoniae plays an important role as a cause of opportunistic infections in humans, mainly pneumonia and urinary tract infection. In contrast, the other two subspecies that make up the species, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis are associated with chronic diseases of the upper airways uncommon nowadays in the developed countries, denominated respectively ocena and rhinoscleroma. Initially, the category of species was assigned to the causative agents of these diseases because they cause defined clinical syndromes. However, the taxonomic study of the genus Klebsiella, based on 16S ribosomal DNA and DNA-DNA hybridization, reclassified K. ozaenae and K. rhinoscleromatis as subspecies of K. pneumoniae due to the high similarity of their sequences.
K. pneumoniae subsp. rhinoscleromatis is the causative agent of rhinoscleroma, a chronic and progressive granulomatous infection of the upper respiratory tract. Rhinoscleroma is acquired by direct or indirect contact with the nasal discharge of an infected person. The infection begins in the nasal passages and extends to the upper respiratory tract, and less frequently to the lower tract, causing an infiltrative granuloma with marked tendency to sclerosis and subsequent obstruction. Consequently, lesions associated with rhinoscleroma generally affect the nasal cavity, but they can also affect the larynx, nasopharynx, oral cavity, paranasal sinuses, and soft tissues of the lips, nose, trachea, and bronchi. The disease progresses through three stages: a catarrhal or rhinitic phase (with nonspecific inflammation and symptoms of the common cold), a proliferative phase (with the formation of large granulomatous masses), and a scar or fibrotic phase (with the formation of a tissue repair). The clinical picture of rhinoscleroma includes: dysphonia, dyspnea, stridor, bronchial involvement, and productive cough with laryngotracheal involvement. The main effect noticed is the obstruction of the air passage. Although rarely fatal, rhinoscleroma is a progressive disease, prone to recurrence and extremely difficult to cure. The relative rarity of the disease and the low specificity of the early symptoms make the clinical diagnosis of rhinoscleroma difficult. As a result, some patients progress to advanced stages that can lead to severe respiratory failure. Histologically, the affected tissues show infiltrates of granulomatous tissue in the submucosa, characterized by the presence of plasma cells, lymphocytes and eosinophils. The most characteristic feature of the disease is the observation in biopsies of vacuolated macrophages of foamy appearance, called Mikulicz cells, in which bacilli are observed after staining with hematoxylin-eosin.
Rhinoscleroma is rare today in developed countries, although endemic in tropical and subtropical areas such as North and Central Africa, Southeast Asia, Central and South America, and some areas of Eastern Europe. This disease is associated with population of low socioeconomic status, ruralism, poor hygiene, and malnutrition. Also, some authors have associated rhinoscleroma with abnormalities of the immune system, and due to the greater frequency of the disease in certain families, it has been proposed that the susceptibility to this infection may have a genetic factor. In addition, it affects women, and young adults more frequently. Despite being rare in developed countries, K. pneumoniae subsp. rhinoscleromatis is ubiquitous, and sporadic cases of this disease have been diagnosed all over the world. Some authors suggest that its incidence in developed countries could be on the rise due to the current migration rate from endemic regions. In addition to rhinoscleroma, K. pneumoniae subsp. rhinoscleromatis has been related sporadically in other types of infections, such as pneumonia or bacteremia.
In clinical microbiology laboratories, strains of Klebsiella spp. they are usually identified by the use of automated instruments based on classical biochemical tests such as the Vitek and API systems. However, identification at the species level through these tests is often difficult, because some of the species share similar biochemical profiles. Therefore, methods of DNA detection of pathogenic Klebsiella species have been developed that allow the identification of Klebsiella strains. However, due to the high sequence homology that exists between the three subspecies of Kebsiella pneumoniae, the genetic determination of K. pneumoniae subsp. rhinoscleromatis is complicated. The detection and identification of this subspecies can be done from the analysis of the localized variations in the sequences of internal transcribed spacer regions (ITS), ribosomal DNA 16-23S that show high interspecies variations and low number of intraspecies polymorphisms. Another strategy used for the detection and identification of K. pneumoniae subsp. rhinoscleromatis is the detection of the K3 capsular serotype, which all the strains of this subspecies possess, although also some strains of K. pneumoniae subsp. pneumoniae. Finally, the study of single-nucleotide polymorphisms (or SNPs) of K.pneumoniae, has allowed the design of specific primers for this subspecies based on the identification of two SNPs located in the phoE gene of phospho-phosphine E, existing only in the subspecies K.pneumoniae subsp. rhinoscleromatis.
Tests carried out in IVAMI:
- Molecular identification of the subspecies Klebsiella pneumoniae subsp. rhinoscleromatis through the amplification of internal spacer regions of 16-23S ribosomal DNA (PCR).
- For molecular diagnosis due to clinical suspicion: sample of exudate of the suspicious lesion.
- For identification of an isolate in culture: cultivation of strain of Klebsiella spp.
Conservation and shipment of the sample:
- Clinical sample for molecular diagnosis: refrigerated (less than 24 hours) or frozen (more than 24 hours).
- Bacterial cultures can be stored and sent at room temperature or refrigerated.
Delivery of results:
- Molecular diagnosis (PCR): 24 hours.
- Molecular identification of the subspecies Klebsiella pneumoniae subsp. rhinoscleromatis through the amplification of internal spacer regions of 16-23S ribosomal DNA (PCR): 24 hours.
Cost of the test:
- Molecular diagnosis or molecular identification of the subspecies Klebsiella pneumoniae subsp. rhinoscleromatis through the amplification of internal spacer regions of ribosomal DNA 16-23S (PCR): consult firstname.lastname@example.org.