Abstract
Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.
Highlights
ĞƉĂƌƚŵĞŶƚ ŽĨ DŝĐƌŽďŝŽůŽŐLJ
&Žƌ ĂƐƐĞƐƐŵĞŶƚƐ ŽĨ ƚŚĞ ůŽǁĞƌ ůŝŵŝƚ ŽĨ ĚĞƚĞĐƟŽŶ ;>>Ϳ ƐĞƌŝĂů ϭϬͲĨŽůĚ ĚŝůƵƟŽŶƐ ĞƋƵŝǀĂůĞŶƚ ƚŽ ĨƌŽŵ κϮε п ϭϬ5 to Ϭκ ŐĞŶŽŵĞ ĐŽƉŝĞƐͬђů ;ϭ ŶŐͬђů ƚŽ ϭ ĨŐͬђůͿ ŽĨ ƉƵƌŝĮĞĚ E ǁĞƌĞ ƉƌĞƉĂƌĞĚ ĂŶĚ ƚŚĞ ĂůŝƋƵŽƚƐ ǁĞƌĞ ƚĞƐƚĞĚ ďLJ ƚŚĞ plylytA, psaA-͕ ĂŶĚ ^ƉŶεΘϬϮͲƐƉĞĐŝĮĐ ƉƌŝŵĞƌͲƉƌŽďĞ ƐĞƚƐ ŝŶ ŵƵůƟƉůĞdž ƌĞĂĐƟŽŶ Ϯϳ͘
& Ğƚ Ăů ŝĂŐŶŽƐŝƐ ŽĨ Streptococcus pneumoniae ůŽǁĞƌ ƌĞƐƉŝƌĂƚŽƌLJ ŝŶĨĞĐƟŽŶ ŝŶ ŚŽƐƉŝƚĂůŝnjĞĚ ĐŚŝůĚƌĞŶ ďLJ ĐƵůƚƵƌĞ ƉŽůLJŵĞƌĂƐĞ ĐŚĂŝŶ ƌĞĂĐƟŽŶ ƐĞƌŽůŽŐŝĐĂů ƚĞƐƟŶŐ ĂŶĚ ƵƌŝŶĂƌLJ ĂŶƟŐĞŶ ĚĞƚĞĐƟŽŶ ůŝŶ /ŶĨĞĐƚ ŝƐ ϮϬϬϮϯκϭϭϭŚƩƉͬͬ ĚdžĚŽŝŽƌŐͬϭϬϭϬΘςͬϯϮκϯρΘ WD/͗ϭϭϳϯϭεςρ Ϯε ǀĂŶ ,ĂĞŌĞŶ Z WĂůůĂĚŝŶŽ ^͕
Summary
^ƚĂŶĚĂƌĚŝƐĂƟŽŶ ĂŶĚ ĞǀĂůƵĂƟŽŶ ŽĨ Ă ƋƵĂŶƟƚĂƟǀĞ ŵƵůƟƉůĞdž ƌĞĂůͲƟŵĞ WZ ĂƐƐĂLJ ĨŽƌ ƚŚĞ ƌĂƉŝĚ ŝĚĞŶƟĮĐĂƟŽŶ ŽĨ Streptococcus pneumoniae. ŴŽŶŐƐƚ ƉŶĞƵŵŽĐŽĐĐƵƐͲůŝŬĞ ǀŝƌŝĚĂŶƐ ŐƌŽƵƉ ƐƚƌĞƉƚŽĐŽĐĐŝ Ă ŶĞǁůLJ ƌĞĐŽŐŶŝƐĞĚ ƐƉĞĐŝĞƐ ĐůĂƐƐŝĮĞĚ ĂƐ S. pseudopneumoniae ǁŚŝĐŚ ŝƐ ƉŽƐŝƟǀĞ ďLJ ĐĐƵWƌŽďĞ ĂƐƐĂLJ ŚĂƐ ďĞĞŶ ĚĞƐĐƌŝďĞĚ ĂŶĚ ĐŚĂƌĂĐƚĞƌŝƐĞĚ Ϯ͘ ^ƚƵĚŝĞƐ ŚĂǀĞ ƐŚŽǁŶ ƚŚĂƚ lytA and psaA ŐĞŶĞ ƐĞƋƵĞŶĐĞƐ ĐĂŶ ƌĞůŝĂďůLJ ĚŝƐƟŶŐƵŝƐŚ S. pneumoniae from S. pseudopneumoniae ϭς͘ &ƵƌƚŚĞƌ ^ƵnjƵŬŝ Ğƚ Ăů ϭϳ ĚĞǀĞůŽƉĞĚ Ă ŚŝŐŚůLJ ƐƉĞĐŝĮĐ ^ƉŶεΘϬϮ ƉƌŝŵĞƌ ƐĞƚ ĨŽƌ S. pneumoniae ŝŶ ŽƌĚĞƌ ƚŽ ĚŝƐĐƌŝŵŝŶĂƚĞ S. pneumoniae ĨƌŽŵ ƉŶĞƵŵŽĐŽĐĐƵƐͲůŝŬĞ ŽƌĂů ƐƚƌĞƉƚŽĐŽĐĐŝ ŚĂƌďŽƵƌŝŶŐ ƚŚĞ ply and lytA ŐĞŶĞƐ ŚŽǁĞǀĞƌƚŚĞƐĞ ^ƉŶεΘϬϮ ƉƌŝŵĞƌƐ ŵĂLJ ŶŽƚ ĚŝƐĐƌŝŵŝŶĂƚĞ S. pseudopneumoniae ϭρ͘ &Žƌ ƚŚĞ ŝĚĞŶƟĮĐĂƟŽŶ ŽĨ ƉŶĞƵŵŽĐŽĐĐŝ WZ ƚĞƐƚƐ ŚĂǀĞ ďĞĞŶ ĂƉƉůŝĞĚ ďLJ ǀĂƌŝŽƵƐ ƌĞƐĞĂƌĐŚĞƌƐ ŚŽǁĞǀĞƌƚŚĞƌĞ ĂƌĞ ƌĞƉŽƌƚƐ ŽĨ ĂŵďŝŐƵŽƵƐ ƌĞƐƵůƚƐ ρ͘ ^ƵĐŚ ĂŵďŝŐƵŝƟĞƐ ĂƌĞ ƉĞƌŚĂƉƐ ŶŽƚ ƐƵƌƉƌŝƐŝŶŐ ŐŝǀĞŶ ƚŚĞ ĚŝǀĞƌƐŝƚLJ ƚŚĂƚ ĞdžŝƐƚƐ ǁŝƚŚŝŶ ƚŚĞ ŽƌŐĂŶŝƐŵƐ ƚŚĂƚ ĂƌĞ ĐůŽƐĞůLJ ƌĞůĂƚĞĚ ƚŽ ƉŶĞƵŵŽĐŽĐĐŝ /ƚ ŝƐ ĞǀŝĚĞŶƚ ƚŚĂƚ ƚŚĞƐĞ ŽƌŐĂŶŝƐŵƐ ĐĂŶ ŚĂƌďŽƵƌ ƉŶĞƵŵŽĐŽĐĐĂů ǀŝƌƵůĞŶĐĞ ĚĞƚĞƌŵŝŶĂŶƚƐ ƐƵĐŚ ĂƐ ply and lytA ŐĞŶĞƐ ƚŚĂƚ ĂƌĞ ŶŽƟŽŶĂůůLJ ƵƐĞĚ ĨŽƌ ĚŝĂŐŶŽƐŝƐ. 2.2 Serum specimens dǁĞŶƚLJ ƐĞƌƵŵ ƐĂŵƉůĞƐ ĨƌŽŵ ƉĂƟĞŶƚƐ ǁŚŽ ǁĞƌĞ ƉŽƐŝƟǀĞ ĨŽƌ ƉŶĞƵŵŽĐŽĐĐĂů ďůŽŽĚ ĐƵůƚƵƌĞ Ϯς ĨƌŽŵ ƉĂƟĞŶƚƐ ǁŚŽ ǁĞƌĞ ŶĞŐĂƟǀĞ ĨŽƌ ƉŶĞƵŵŽĐŽĐĐĂů ďůŽŽĚ ĐƵůƚƵƌĞ ĂŶĚ ϯϬ ĨƌŽŵ ŚĞĂůƚŚLJ ƐƵďũĞĐƚƐ ǁĞƌĞ ŝŶĐůƵĚĞĚ ŝŶ ƚŚĞ ƐƚƵĚLJdŚĞ E ĞdžƚƌĂĐƚĞĚ ĨƌŽŵ ƚŚĞ ƐĞƌƵŵ ƐĂŵƉůĞƐ ŽĨ ŚĞĂůƚŚLJ ƐƵďũĞĐƚƐ ǁĂƐ ƐƵďƐĞƋƵĞŶƚůLJ ƐƉŝŬĞĚ ǁŝƚŚ κϮε п ϭϬ2 ĐŽƉŝĞƐͬђů Žƌ ϭ ƉŐͬђů ŽĨ S. pneumoniae d κεςϭε ŐĞŶŽŵŝĐ E ^ĞƌƵŵ ƐĂŵƉůĞƐ ǁĞƌĞ ƐŽƵƌĐĞĚ ĨƌŽŵ ƚŚĞ WŶĞƵŵŽĐŽĐĐĂů ZĞƐĞĂƌĐŚ ŝǀŝƐŝŽŶ ĞŶƚƌĂů ZĞƐĞĂƌĐŚ >ĂďŽƌĂƚŽƌLJ
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