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Use of different primer directed sequence amplification by polymerase chain reaction for identification of
Rashmi Gupta1, Bijay Ranjan Mirdha, Randeep Guleria
1Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
Background:
Pneumocystis carinii pneumonia (PCP), caused by opportunistic agent Pneumocystis jirovecii (formerly, Pneumocystis carinii is one of the most serious respiratory infection in immunocompromised patients.
Aim:
The present study was conducted to compare polymerase chain reaction (PCR) assays targetting three different genes of Pneumocystis to study their application in its diagnosis.
Methods:
One hundred and eighty (n = 180) clinical samples from 145 immunocompromised patients with clinical suspicion of PCP and 35 samples from control group of 30 immunocompetent individuals with respiratory infections other than PCP were prospectively examined for the presence of Pneumocystis jirovecii (P. jirovecii). All the samples were subjected to microscopic examination, one single [major surface glycoprotein, (MSG)] and two nested [mitochondrial large subunit ribosomal ribonucelic acid, (mtLSU rRNA) and internal transcribed spacer (ITS) region], polymerase chain reaction assays.
Results:
Microscopic examination was positive in only six (n = 6) patients, whereas single round MSG PCR detected P. jirovecii deoxyribonucleic acid (DNA) in 16 cases. When the clinical samples were tested by mtLSU rRNA and ITS nested PCR assays, it was possible to detect seven additional cases of PCP, making it to a total of 23 cases. None of the clinical specimens in control group (n = 30) were positive by any of the above-mentioned techniques. Amongst the 81 bronchoalveolar lavage (BAL) samples tested, 16 were positive by MSG PCR, while 20 were positive by both nested, i.e., mtLSU rRNA and ITS PCR assays. Similarly, out of 50 sputum samples, only three were positive by MSG, seven by mtLSU rRNA and six by ITS nested PCR assays.
Conclusion:
It has been observed that MSG is relatively more sensitive when single round PCR assay is used for detection of human Pneumocystosis compared to the first (single) rounds of either ITS or mtLSU rRNA nested PCRs. However, the two nested PCRs using ITS and mtLSU rRNA have been found to be more sensitive. On comparison of two nested PCR assays, the results have been more or less comparable.
Insights
Nested PCR assays targeting ITS and mtLSU rRNA genes are more sensitive for diagnosing Pneumocystis pneumonia (PCP) in immunocompromised patients than single-round MSG PCR. These molecular methods improve detection rates for this serious respiratory infection.
Area of Science:
- Medical Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Pneumocystis pneumonia (PCP) is a severe respiratory infection in immunocompromised individuals.
- Pneumocystis jirovecii (formerly Pneumocystis carinii) is the causative agent of PCP.
- Accurate diagnosis is crucial for effective treatment and management of PCP.
Purpose of the Study:
- To compare the diagnostic performance of three distinct Polymerase Chain Reaction (PCR) assays targeting different Pneumocystis jirovecii genes.
- To evaluate the utility of single-round major surface glycoprotein (MSG) PCR versus nested PCR assays for mitochondrial large subunit ribosomal ribonucelic acid (mtLSU rRNA) and internal transcribed spacer (ITS) regions.
Main Methods:
- Prospective examination of 180 clinical samples from 145 immunocompromised patients and 35 controls.
- Samples were analyzed using microscopy, single-round MSG PCR, and nested PCR assays for mtLSU rRNA and ITS regions.
- Comparison of detection rates across different diagnostic techniques.
Main Results:
- Microscopy detected PCP in only 6 patients.
- Single-round MSG PCR identified P. jirovecii DNA in 16 cases.
- Nested PCR assays (mtLSU rRNA and ITS) increased detection to 23 cases, identifying seven additional PCP cases. Nested PCRs showed higher sensitivity than single-round MSG PCR.
Conclusions:
- Single-round MSG PCR shows some sensitivity for detecting Pneumocystis jirovecii.
- Nested PCR assays targeting ITS and mtLSU rRNA regions are significantly more sensitive for diagnosing human Pneumocystosis.
- The ITS and mtLSU rRNA nested PCR assays offer comparable and superior sensitivity for PCP diagnosis.
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