Chandrasekar, C

Kumar, J Suria

Madhavan, HN

Revathy, M

Therese, K Lily

Vinayagam, A Kamala

BMC Infectious Diseases

English

PubMed

M Revathy

K Therese

C Chandrasekar

J Kumar

HN Madhavan

A Vinayagam

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Evaluation of PCR based DNA sequencing
targeting mitochondrial larger subunit
(mtLSU) region for the rapid detection of
Pneumocystis jirovecii in sputum from HIV
positive patients
M Revathy1*, K Lily Therese1, C Chandrasekar2, J Suria Kumar2, HN Madhavan1, A Kamala Vinayagam1
From First International Science Symposium on HIV and Infectious Diseases (HIV SCIENCE 2012)
Chennai, India. 20-22 January 2012
Background
To establish rapid and reliable diagnostic techniques for
the detection and identification of Pneumocystis jirovecii
(P. jirovecii) as there are no standard laboratory diagnos-
tic techniques available for Pneumocystis jirovecii Pneu-
monia (PJP) infections.
Materials and methods
A total of 104 sputum specimens from HIV positive and
clinically suspected PJP patients attending Government
Hospital of Thoracic Medicine during Dec 2009-Mar
2011 were included in the study. Direct staining of spu-
tum specimens using KOH Calcoflour and Grocott
methanamine silver staining (GMS) and nested PCR tar-
geting mitochondrial larger subunit region (mtLSU) was
optimized. The optimized PCR was applied on all clini-
cal specimens and amplified products were subjected to
DNA sequencing and the sequencing results were ana-
lyzed using BLAST analysis software to deduce the
sequence homology.
Results
Of the 104 clinical specimens included in the study, P.
jirovecii was detected in 35 sputum specimens (36.4%)
by KOH Calcoflour and GMS staining. Nested PCR
using primers targeting the mtLSU region was specific
and sensitive to detect 500fg of P. jirovecii DNA with
base pair product size of 346 for first round and 120bp
for the second round. The optimized PCR was applied
on all sputum specimens, resulting in 47.84% (46/104)
positivity for detecting the genome of P. jirovecii. The
DNA sequencing of the amplified PCR products showed
98-100% sequence similarity with P. jirovecii deposited
in the genbank.
Conclusions
Nested PCR based DNA sequencing targeting mtLSU
region is a sensitive, specific, rapid and reliable labora-
tory diagnostic tool for the detection of PJP directly
from sputum specimens.
Author details
1L & T Microbiology Research Center, Vision Research Foundation, Sankara
Nethralaya Chennai, India. 2Government Hospital of Thoracic Medicine,
Chennai, India.
Published: 4 May 2012
doi:10.1186/1471-2334-12-S1-P78
Cite this article as: Revathy et al.: Evaluation of PCR based DNA
sequencing targeting mitochondrial larger subunit (mtLSU) region for
the rapid detection of Pneumocystis jirovecii in sputum from HIV
positive patients. BMC Infectious Diseases 2012 12(Suppl 1):P78.
1L & T Microbiology Research Center, Vision Research Foundation, Sankara
Nethralaya Chennai, India
Full list of author information is available at the end of the article
Revathy et al. BMC Infectious Diseases 2012, 12(Suppl 1):P78
http://www.biomedcentral.com/1471-2334/12/S1/P78
© 2012 Revathy et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.


Evaluation of PCR based DNA sequencing targeting mitochondrial larger subunit (mtLSU) region for the rapid detection of Pneumocystis jirovecii in sputum from HIV positive patients

K Lily Therese

J Suria Kumar

A Kamala Vinayagam

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Evaluation of PCR based DNA sequencing targeting mitochondrial larger subunit (mtLSU) region for the rapid detection of Pneumocystis jirovecii in sputum from HIV positive patients

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