Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples

Leeuw, Bertie H.C.G.M. de; Voskuil, W. Sebastiaan; Maraha, B.; Zee, Anneke van der; Westenend, Pieter J.; Kusters, Johannes G.

doi:10.1016/j.yexmp.2015.03.021

Cited by 6 publications

(7 citation statements)

References 12 publications

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“…Therefore, it was not unexpected that only 63.4% of the analyzed samples were positive for amplification of the 260 bp fragment of the mt LSU gene. This finding is consistent with reports from other authors who have used this type of material to perform genetic studies [8,9,13,14]. To our knowledge, this study is the first attempt to genotype P. jirovecii DHPS in patients with AIDS-related PcP from FFPE tissue and confirm our previous data on mitochondrial genes using a larger number of samples [14].…”

Section: Discussionsupporting

confidence: 92%

“…Thus, the real possibilities of a successful PCR are limited. Furthermore, the time of exposure to heat and formalin and the temperature for the preservation of tissue samples are important variables to consider [9,14]. Therefore, it was not unexpected that only 63.4% of the analyzed samples were positive for amplification of the 260 bp fragment of the mt LSU gene.…”

Section: Discussionmentioning

confidence: 99%

“…Regarding the use of FFPE samples, some limitations related to ensuring higher PCR efficiency in FFPE samples were previously discussed. Other factors must also be taken into account, for example, the length and temperature of tissue fixation, chemical reagents used for fixation, temperature, and humidity of the place where FFPE samples were stored, as well as the quantity and thickness of cuts to be used in PCR [8][9][10]14,40]. Therefore, all of these variables should be controlled to increase the frequency of detection of pathogens of medical importance in FFPE samples.…”

Section: Discussionmentioning

confidence: 99%

“…However, collecting this type of sample requires invasive medical management, therefore this procedure is not usually performed in many countries to identify the pathogen [1,7]. In view of this limitation, formalin-fixed and paraffin-embedded tissues (FFPE) could be alternative sources of samples for molecular studies [8,9]. However, the number of reports favoring the use of FFPE in P. jirovecii studies is still scarce, and, in addition, they are more directed toward identification than to characterization of the microorganism [10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

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Multilocus Genotyping of Pneumocystis jirovecii from Deceased Cuban AIDS Patients Using Formalin-Fixed and Paraffin-Embedded Tissues

Friaza

Armas

Capó

et al. 2021

JoF

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The results of the genotypic characterization of Pneumocystis jirovecii are described in lung tissue samples from 41 Cubans who died of AIDS with pneumocystosis between 1995 and 2008. Histological sections of the lung preserved as formalin-fixed and paraffin-embedded tissue were examined. PCR amplification and nucleotide sequencing of the two mitochondrial genes (large and small) of the pathogen allowed verification of a predominance of genotype 3 (85T/248C) of the large mitochondrial gene and genotype 3 (160A/196T) of the small mitochondrial gene over a period of 14 years (1995–2008). These results suggest that the 85T/248C//160A/196T genotype circulates with the highest frequency (81.3%) among AIDS patients in Cuba. Multilocus analysis indicates a limited circulation of pathogen genotypes on the island with the existence of a clonal genotype with an epidemic structure. Furthermore, it appears that circulating strains of P. jirovecii have not developed mutations related to sulfonamide resistance. Taken together, the data in this study revealed important elements about pneumocystosis in Cuban patients dying of AIDS and the usefulness of formalin-fixed and paraffin-embedded samples to carry out molecular epidemiology studies of P. jirovecii.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multilocus Genotyping of Pneumocystis jirovecii from Deceased Cuban AIDS Patients Using Formalin-Fixed and Paraffin-Embedded Tissues

Friaza

Armas

Capó

et al. 2021

JoF

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“…57 In a recent report, de Leeuw et al showed that when targeting a small DNA fragment P. jirovecii , real-time PCR can be performed on formalin-fixed paraffin-embedded samples of BAL fluid with sensitivity up to 83.3%. 58 This method will be useful in case the suspicion of PCP arises late after a patient starts to deteriorate rapidly.…”

Section: Presentation and Diagnosismentioning

confidence: 99%

PneumocystisPneumonia in Human Immunodeficiency Virus–infected Adults and Adolescents: Current Concepts and Future Directions

Tasaka

2015

Clin Med Insights Circ Respir Pulm Med

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Pneumocystis jirovecii pneumonia (PCP) is one of the most common opportunistic infections in human immunodeficiency virus-infected adults. Colonization of Pneumocystis is highly prevalent among the general population and could be associated with the transmission and development of PCP in immunocompromised individuals. Although the microscopic demonstration of the organisms in respiratory specimens is still the golden standard of its diagnosis, polymerase chain reaction has been shown to have a high sensitivity, detecting Pneumocystis DNA in induced sputum or oropharyngeal wash. Serum β-d-glucan is useful as an adjunctive tool for the diagnosis of PCP. High-resolution computed tomography, which typically shows diffuse groundglass opacities, is informative for the evaluation of immunocompromised patients with suspected PCP and normal chest radiography. Trimethoprimsulfamethoxazole (TMP-SMX) is the first-line agent for the treatment of mild to severe PCP, although it is often complicated with various side effects. Since TMP-SMX is widely used for the prophylaxis, the putative drug resistance is an emerging concern.

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Identification of infectious organisms in cytopathology: A review of ancillary diagnostic techniques

Allison

Simner

Ali

2018

Cancer Cytopathology

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Cytology samples obtained from exfoliative sources and fine-needle aspiration (FNA) procedures can all be used to detect microorganisms and/or the associated cytopathologic effects (CPE) caused by an infection. There are many advantages to utilizing cytology samples as an adjunct to routine microbiology laboratory methods. For example, cytology samples can be obtained by non-invasive and minimally invasive techniques, and interpretation is affordable, accurate, and fast. Furthermore, routine cytology stains, including the Papanicolaou (Pap) and the Diff-Quik (DQ) stains, can adequately identify a number of microorganisms. Finally, material obtained by these procedures can also be used for cytologic ancillary testing, microbiology culture, and molecular studies. Currently, there are a variety of ancillary diagnostic techniques that are routinely utilized in the cytopathology laboratory. Additionally, the increasing utilization of molecular-based, diagnostic techniques on fluid specimens, as well as FFPE material, is expanding the role of cytopathology for infectious disease diagnostics. In this review, we provide an overview of the most practical ancillary techniques commonly used to identify microorganisms on cytology specimens.

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Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples

Cited by 6 publications

References 12 publications

Multilocus Genotyping of Pneumocystis jirovecii from Deceased Cuban AIDS Patients Using Formalin-Fixed and Paraffin-Embedded Tissues

Multilocus Genotyping of Pneumocystis jirovecii from Deceased Cuban AIDS Patients Using Formalin-Fixed and Paraffin-Embedded Tissues

PneumocystisPneumonia in Human Immunodeficiency Virus–infected Adults and Adolescents: Current Concepts and Future Directions

Identification of infectious organisms in cytopathology: A review of ancillary diagnostic techniques

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