Reliable and Sensitive Nested PCR for the Detection of Chlamydia in Sputum

Abstract

Chlamydia are Gram-negative, intracellular pathogens colonizing epithelial mucosa. They cause primarily atypical pneumonia and have recently been associated with chronic diseases. Diagnostics relies almost exclusively on serological methods; PCR tests are used rarely because in patients with positive ELISA, it is nearly impossible to identify chlamydial DNA. This paradox is associated with DNA degradation in sputum samples, low abundance, and low sensitivity of PCR systems. In a newly designed and validated “nested” PCR (NPCR) assay, it was possible to amplify DNA of Chlamydia known to infect humans in 31% samples. The reliability of the assay was confirmed by DNA sequencing, and all PCR products belonged exclusively to the Chlamydiales, mainly recognized as Chlamydia pneumoniae. Three samples were related to Ca. Rhabdochlamydia porcellionis and Ca. Renichlamydia lutjani, which infect arthropods. In one case, samples were taken from sick individual, indicating the potential as a human pathogen.

Are ELISA and PCR Discrepancies in the Identification of Chlamydia pneumoniae Caused by the Presence of “Chlamydia-Related Bacteria”?

“…Microbiological Laboratory, Bratislava, Slovakia for routine plating tests (four males and six females; mean age: 42; range, 24 to 68 years). Another 156 samples were provided by healthy volunteers from the Faculty of Natural Sciences and the Faculty of Physical Education and Sports [ 23 ]. We also examined 95 clotted blood samples (33 men, 62 women, average age 49 years, range 5–90 years) from patients with pneumonia and unclotted blood and sputum samples from one patient with chronic pneumonia, followed for 4 years.…”

“…The samples were collected in 2018 and 2019, transported to the laboratory, and stored in a freezer at −20 °C. Standards are described in detail elsewhere [ 23 ].…”

“…Sputa. The DNA was obtained from sputa according to Freise et al [ 27 ], with minor modifications as described previously [ 23 ].…”

“…There are several PCR variants, such as real-time PCR, digital droplet PCR, or nested PCR, that help to increase the sensitivity and specificity of detection [ 19 , 20 , 21 , 22 ]. However, analysis in silico of the previously published methods revealed their limited use due to the primer design and the extremely high polymorphism of favored target genes such as ompA [ 23 ]. From the sensitivity and specificity point of view, the target of choice appears to be 16S rRNA, which contains hypervariable regions that are highly specific for a biological species or genus [ 24 , 25 , 26 ].…”

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

“…Particularly, molecular biology technologies have attracted much more attention for the merits of accurate, fast and easy-operation [17,18]. With advantages of high sensitivity, specificity and the nucleic acid-based molecular diagnosis, molecular biologybased methods have become the preferred option for the accurate detection of pathogens gradually [19][20][21]. As well-established nucleic acid analysis approaches, polymerase chain reaction (PCR) and isothermal nucleic acid amplification have become the mainstream for the development of pathogen detection methods [22][23][24].…”