Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin

Wakefield, Ann E.; Pixley, Fiona J.; Banerji, Suneale; Sinclair, Kathryn; Miller, R F; Moxon, E. Richard; Hopkin, Julian M.

doi:10.1016/0166-6851(90)90131-5

Cited by 176 publications

(104 citation statements)

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“…The mtLSU rRNA gene, which is involved in basic metabolic mechanisms during translation, providing peptidyl transferase activity to the mitochondrial ribosome, is considered to be a highly informative marker for discerning associations between specific genotypes and geographical or clinical data, and it has been used most widely for PCR-based studies of P. jirovecii (8,24,31,35,37). The mtLSU rRNA sequencing results were similar to those reported in recent studies on Portuguese subjects (7)(8)(9).…”

Section: Discussionsupporting

confidence: 73%

“…The simultaneous amplification of mtLSU rRNA, SOD, and DHFR was achieved by using three pairs of primers (MWG Biotech), pAZ102-X and pAZ102-E (0.6 M), MnSODFw and MnSODRw2 (1 M), and FR 208 and FR 1018 (1.2 M), respectively. Except for MnSODRw2 (5Ј-CACTTCCTTGAAT CCCAGACAA-3Ј), all primers were described previously (8,21,35,37).…”

Section: Methodsmentioning

confidence: 99%

“…One major problem in P. jirovecii research is the absence of an in vitro culture system. Molecular typing, particularly PCR-based approaches, have been used to describe differences between organisms (1,24,37). The advances in the understanding of the genetic diversity of P. jirovecii raised important questions, such as whether some organisms are more pathogenic than others and whether resistant or more virulent genotypes spread between hosts (5, 7, 8, 13-15, 24, 30).…”

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confidence: 99%

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Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

et al. 2011

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Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

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Section: Discussionsupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

et al. 2011

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“…P. jirovecii was detected using toluidine blue O stain and immunofluorescence assay (IFA). The results for the samples were confirmed by a PCR specific for each organism (17,19,20). Each sample was recorded as a code number and kept at Ϫ20°C in order to use in a blind study of the multiplex nested PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Amplification of the mitochondrial large-subunit (mtLSU) rRNA gene of P. jirovecii was performed using the oligonucleotide primers published by Wakefield et al (19,20), as shown in Table 1. The first-round amplification was performed using primers pAZ102-E and pAZ102-H as the forward and reverse outer primers, respectively, and yielded a 346-bp fragment.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

et al. 2010

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A multiplex PCR assay for the simultaneous detection of Mycobacterium tuberculosis and Pneumocystis jirovecii was developed using IS6110-based detection for M. tuberculosis and mitochondrial large-subunit (mtLSU) rRNA gene detection for P. jirovecii. Ninety-five pulmonary blinded samples were examined using the developed multiplex PCR assay, and the results were compared with those obtained by the single nested PCRs targeting IS6110 for M. tuberculosis and mtLSU rRNA for P. jirovecii. Of the 95 pulmonary samples tested, the multiplex nested PCR developed here could detect 36 cases of M. tuberculosis infection, 35 cases of P. jirovecii infection, and 17 cases of M. tuberculosis and P. jirovecii coinfections. The sensitivities of the multiplex nested PCR in detecting M. tuberculosis and P. jirovecii were 92.1% and 81.4%, respectively, whereas the specificities in detecting M. tuberculosis and P. jirovecii were 98.2% and 100%, respectively.

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New Insights Into Transmission, Diagnosis, and Drug Treatment of <EMPH TYPE="ITAL">Pneumocystis carinii</EMPH> Pneumonia

Kovacs

Gill²,

Meshnick

et al. 2001

JAMA

201

106

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Pneumocystis carinii has been recognized as a human pathogen for nearly 50 years. We present a case of P carinii infection that typifies clinical presentation in the era of the acquired immunodeficiency syndrome epidemic. The high incidence of P carinii pneumonia in persons infected with human immunodeficiency virus (HIV) has served to focus laboratory and clinical research efforts on better understanding the biology of the organism and on improving diagnosis, treatment, and prevention of this disease. Although inability to culture P carinii has hampered research efforts, molecular and immunologic approaches have led to the recognition that the organism represents a family of fungi with a very restricted host range and have allowed characterization of clinically relevant antigens and enzymes. Molecular epidemiologic studies have identified more than 50 strains of human-derived P carinii and have suggested that recently acquired infection, as opposed to reactivation of latent infection, may account for many cases of clinical disease. Diagnosis has been improved by the development of organism-specific monoclonal antibodies and, more recently, by polymerase chain reaction using multicopy gene targets, together with induced sputum or oral wash samples. Chemotherapeutic prophylaxis is very effective in preventing P carinii pneumonia; the combination of trimethoprim-sulfamethoxazole remains the first-line agent for both therapy and prophylaxis. Prophylaxis needs to be administered only during periods of high risk; in HIV-infected patients responding to effective antiretroviral therapies, prophylaxis no longer needs to be lifelong. Molecular studies have identified mutations in the target of sulfa drugs that appear to represent emerging resistance in P carinii. Resistance to atovaquone, a second-line agent, may also be developing.

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Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin

Cited by 176 publications

References 9 publications

Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

New Insights Into Transmission, Diagnosis, and Drug Treatment of <EMPH TYPE="ITAL">Pneumocystis carinii</EMPH> Pneumonia

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