scholarly journals Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens

2021 ◽  
Vol 8 ◽  
Author(s):  
Jie Yi ◽  
Nan Wang ◽  
Jie Wu ◽  
Yueming Tang ◽  
Jingjia Zhang ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii ◽  
Immunocompromised Patients ◽  
Balf Sample ◽  
Bronchoalveolar Fluid ◽  
Life Threatening ◽  
Digital Polymerase Chain Reaction

Background:Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

scholarly journals Pneumocystis jirovecii detection in asymptomatic patients: what does its natural history tell us?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 739 ◽  
Author(s):  
Alexandre Alanio ◽  
Stéphane Bretagne
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii ◽  
Immunocompromised Patients ◽  
Healthy Individuals ◽  
Real Time Quantitative Pcr ◽  
Asymptomatic Patients ◽  
Fungal Load ◽  
Invasive Infections ◽  
Life Threatening

Pneumocystis jiroveciiis an unusual ascomycetous fungus that can be detected in the lungs of healthy individuals. Transmission from human to human is one of its main characteristics in comparison with other fungi responsible for invasive infections.P. jiroveciiis transmitted through the air between healthy individuals, who are considered to be the natural reservoir, at least transiently. In immunocompromised patients,P. jiroveciimultiplies, leading to subacute infections and acute life-threatening pneumonia, called Pneumocystis pneumonia [PCP]. PCP is caused by genotypically distinct mixtures of organisms in more than 90% of cases, reinforcing the hypothesis that there is constant inhalation ofP. jiroveciifrom different contacts over time, although reactivation of latent organisms from previous exposures may be possible. Detection ofP. jiroveciiDNA without any symptoms or related radiological signs has been called “colonization”. This situation could be considered as the result of recent exposure toP. jiroveciithat could evolve towards PCP, raising the issue of cotrimoxazole prophylaxis for at-risk quantitative polymerase chain reaction (qPCR)-positive immunocompromised patients. The more accurate way to diagnose PCP is the use of real-time quantitative PCR, which prevents amplicon contamination and allows determination of the fungal load that is mandatory to interpret the qPCR results and manage the patient appropriately. The detection ofP. jiroveciiin respiratory samples of immunocompromised patients should be considered for potential risk of developing PCP. Many challenges still need to be addressed, including a better description of transmission, characterization of organisms present at low level, and prevention of environmental exposure during immunodepression.

scholarly journals Clarity Plus™ digital PCR: A novel platform for absolute quantification of SARS-CoV-2

2021 ◽  
Author(s):  
Shawn Yi Han Tan ◽  
Milton Sheng Yi Kwek ◽  
Huiyu Low ◽  
Yan Ling Joy Pang
Keyword(s):  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Viral Loads ◽  
Nucleic Acid Analysis ◽  
Polymerase Chain ◽  
The Absolute ◽  
Assay Precision ◽  
Digital Polymerase Chain Reaction ◽  
Higher Sensitivity

In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. Considering the growing demand for improved dPCR technology, the Clarity Plus™ dPCR system which features enhanced multiplexing capability and a wider dynamic range for nucleic acid analysis was recently launched. In this study, a dPCR assay optimized for use on Clarity Plus™ was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intra- assay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARS-CoV-2 and can potentially be adopted as a molecular tool in applications such as detecting low viral loads in patients as well as in wastewater surveillance of COVID-19.

scholarly journals Invasive multifocal cryptococcal airway disease in a teenager with hypogammaglobulinaemia

2021 ◽  
Author(s):  
Michael-John Fay ◽  
Catherine Byrnes ◽  
Naveen Pillarisetti ◽  
Andrew Fox-Lewis ◽  
Brent McSharry ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Respiratory Tract ◽  
Cellular Immunity ◽  
Airway Disease ◽  
Paediatric Population ◽  
Immunocompromised Patients ◽  
Life Threatening ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Cryptococcosis is an invasive, opportunistic, fungal infection that predominantly effects the respiratory tract and central nervous system in immunocompromised patients. It is classically associated with defects in cellular immunity such as acquired immunodeficiency syndrome. Here we describe a case of life-threatening laryngitis, endobronchitis and pneumonia due to Cryptococcus neoformans in a teenager with hypogammaglobulinaemia. To the best of our knowledge, no previous cases of laryngeal cryptococcosis have been reported in the paediatric population.

scholarly journals Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients

2016 ◽  
Vol 54 (6) ◽  
pp. 1487-1495 ◽  
Author(s):  
T. Fauchier ◽  
L. Hasseine ◽  
M. Gari-Toussaint ◽  
V. Casanova ◽  
P. M. Marty ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Quantitative Pcr ◽  
Pneumocystis Jirovecii ◽  
Hiv Positive ◽  
Immunocompromised Patients ◽  
Infection Status ◽  
Content Type ◽  
Fungal Burden ◽  
Life Threatening ◽  
Hiv Negative

Pneumocystisjiroveciipneumonia (PCP) is an acute and life-threatening lung disease caused by the fungusPneumocystis jirovecii. The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O2desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (CT) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate theCTvalues to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a meanCTvalue for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a meanCTvalue for colonized patients of 35 (95% CI, 34 to 36) (P< 10−3). For the subgroup of HIV-positive patients, we demonstrated that aCTvalue below 27 excluded colonization and aCTvalue above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that aCTvalue below 31 excluded colonization and aCTvalue above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia inP. jirovecii-infected immunocompromised patients and patients stratified on the basis of HIV infection status with differentCTvalues.

scholarly journals Pneumocystis jirovecii Pneumonia Prophylaxis for Cancer Patients during Chemotherapy

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 237
Author(s):  
Kazuto Takeuchi ◽  
Yoshihiro Yakushijin
Keyword(s):  
Cancer Treatment ◽  
Treatment Outcomes ◽  
Cancer Patients ◽  
Potential Problem ◽  
Corticosteroid Treatment ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Immunocompromised Patients ◽  
Healthy Individuals ◽  
Life Threatening

Pneumocystis jirovecii pneumonia (PJP) is one type of life-threatening pneumonia in immunocompromised patients. PJP development should be considered in not only immunocompromised individuals, but also patients undergoing intensive chemotherapies and immunotherapies, organ transplantation, or corticosteroid treatment. Past studies have described the clinical manifestation of PJP in patients during chemotherapy and reported that PJP affects cancer treatment outcomes. Therefore, PJP could be a potential problem for the management of cancer patients during chemotherapy, and PJP prophylaxis would be important during cancer treatment. This review discusses PJ colonization in outpatients during cancer chemotherapy, as well as in healthy individuals, and provides an update on PJP prophylaxis for cancer patients during chemotherapy.

A rapid nucleic acid concentration measurement system with large-field-of-view for droplet digital PCR microfluidic chip

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Jinrong Shen ◽  
Jihong Zheng ◽  
Zhenqing Li ◽  
Yourong Liu ◽  
Fengxiang Jing ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Field Of View ◽  
Large Field ◽  
Chain Reaction ◽  
Effective Technique ◽  
Nucleic Acid Concentration ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction(ddPCR) is an effective technique for the absolute quantification of target mucleic acid unparalleled sensitivity. However, current commerical ddPCR device for the detection of the gene...

scholarly journals Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii

2016 ◽  
Vol 85 (4) ◽  
Author(s):  
Brenda L. Tesini ◽  
Terry W. Wright ◽  
Jane E. Malone ◽  
Constantine G. Haidaris ◽  
Martha Harber ◽  
...  
Keyword(s):  
Pneumocystis Carinii ◽  
Surface Protein ◽  
Pneumocystis Pneumonia ◽  
Negative Control ◽  
Cross Reactivity ◽  
Pneumocystis Jirovecii ◽  
Content Type ◽  
Life Threatening ◽  
Potential Vaccine ◽  
Preventive Methods

ABSTRACT Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina. Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii. Potential orthologs of Pca1 have been identified in P. jirovecii. Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

scholarly journals An overview of Pneumocystis jirovecii pneumonia for the African generalist practitioner

1970 ◽  
Vol 19 (4) ◽  
pp. 3200-3207
Author(s):  
I Govender ◽  
O M Maphasha ◽  
S Rangiah ◽  
C Steyn
Keyword(s):  
Pneumocystis Pneumonia ◽  
Diagnostic Methods ◽  
Pneumocystis Jirovecii ◽  
Causative Organism ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Immunocompromised Patients ◽  
Productive Cough ◽  
First Line ◽  
Duration Of Symptoms ◽  
Uninfected Patient

Introduction: Pneumocystis jirovecii is the causative organism of Pneumocystis pneumonia (PCP) in humans, which is more common among immunocompromised patients. Classically patients present with fever, non-productive cough, and dyspnoea. In the HIV-infected individuals the symptoms may be subtle at first, but gradually progress over several weeks. In the HIV-uninfected patient, however, the duration of symptoms is shorter and more severe, mainly due to the increased inflammatory response of the HIV-uninfected patient.Methods: This article focuses on the diagnostic methods and then the management and prophylaxis principles of PCP by reviewing the best current practices and guidelines in Africa.Conclusion: This overview is presented by clinicians who have experience with PCP and is directed mainly at first-line healthcare providers.Keywords: Pneumocystis jirovecii pneumonia, african generalist practitioner.

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