Comparison of plasma PCR and bronchoalveolar lavage fluid culture for detection of cytomegalovirus infection in adult bone marrow transplant recipients

Aspin, M M; Gallez-Hawkins, Ghislaine; Giugni, Terrence D.; Tegtmeier, Bernard; Lang, David J.; Schmidt, G.; Forman, Stephen J.; Zaia, John A.

doi:10.1128/jcm.32.9.2266-2269.1994

Cited by 41 publications

(12 citation statements)

References 26 publications

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“…16s rRNA gene sequencing (University of Washington) was used to confirm identification of top ranking bacterial pathogens at the genus level. Finally, alpha diversity of the respiratory microbiome in each subject was assessed using the Simpson Diversity Index (SDI) (35).…”

Section: Pathogen Detection Bioinformaticsmentioning

confidence: 99%

Metagenomic Sequencing Detects Respiratory Pathogens in Hematopoietic Cellular Transplant Patients

Langelier

Zinter

Kalantar

et al. 2017

Preprint

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Commentary: Lower respiratory tract infections are a leading cause of death in hematopoietic cellular transplant recipients, but the microbiologic etiology of these infections is frequently unknown. The limits of current diagnostics preclude targeted antimicrobial treatments, resulting in excess morbidity and mortality. Metagenomic next-generation sequencing may enable precision diagnosis of respiratory infections in hematopoietic cellular transplant patients by simultaneously detecting microbial pathogens, transcriptional biomarkers of the host response, and the pulmonary microbiome. This approach may facilitate targeted antimicrobial prescription and provide a new method for distinguishing infectious and non-infectious post-transplant respiratory illness. ABSTRACT RATIONALE: Current microbiologic diagnostics often fail to identify the etiology of lower respiratory tract infections (LRTI) in hematopoietic cellular transplant recipients (HCT), which precludes the implementation of targeted therapies. OBJECTIVES: To address the need for improved LRTI diagnostics, we evaluated the utility of metagenomic next generation sequencing (mNGS) of bronchoalveolar lavage (BAL) to detect microbial pathogens in HCT patients with acute respiratory illnesses. METHODS: We enrolled 22 post-HCT adults ages 19-69 years with acute respiratory illnesses who underwent BAL at the University of Michigan between January 2012 and May 2013. mNGS was performed on BAL fluid to detect microbes and simultaneously assess the host transcriptional response. Results were compared against conventional microbiologic assays. MEASUREMENTS & MAIN RESULTS: mNGS demonstrated 100% sensitivity for detecting respiratory microbes (human metapneumovirus, respiratory syncytial virus, Stenotrophomonas maltophilia, human herpesvirus 6 and cytomegalovirus) when compared to standard testing.Previously unrecognized LRTI pathogens were identified in six patients for whom standard testing was negative (human coronavirus 229E, human rhinovirus A, Corynebacterium propinquum and Streptococcus mitis); findings were confirmed by independent PCR and 16S rRNA sequencing. Relative to patients without infection, patients with infection had increased expression of immunity related genes (p=0.022) and significantly lower diversity of their respiratory microbiome (p=0.017). CONCLUSIONS:Compared to conventional diagnostics, mNGS enhanced detection of pathogens in BAL fluid from HCT patients. Furthermore, our results suggest that combining unbiased microbial pathogen detection with assessment of host gene biomarkers of immune response may hold promise for enhancing the diagnosis of post-HCT respiratory infections. Abstract Word Count: 249

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Section: Pathogen Detection Bioinformaticsmentioning

confidence: 99%

Metagenomic Sequencing Detects Respiratory Pathogens in Hematopoietic Cellular Transplant Patients

Langelier

Zinter

Kalantar

et al. 2017

Preprint

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“…Approaches to reduce this overtreatment have relied on the development of rapid, sensitive and reliable surveillance methods to diagnose CMV replication at the earliest opportunity, allowing therapy to be targeted to those at greatest risk of subsequent CMV disease (pre-emptive therapy) (Einsele et al, 1995;Boeckh et al, 1996). These methods include the CMV pp65 antigenaemia assay (van der Bij et al, 1988) and detection of CMV DNA by polymerase chain reaction (PCR) in peripheral blood leucocytes, plasma or serum (Jiwa et al, 1989;Einsele et al, 1991;Wolf & Spector, 1993;Aspin et al, 1994). Antigenaemia-guided pre-emptive ganciclovir therapy has been shown to reduce the use of ganciclovir and the number of invasive fungal infections but has also been shown to be associated with an increase in CMV disease before day 1100 compared with routine prophylactic usage (Boeckh et al, 1996).…”

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confidence: 99%

Extended routine polymerase chain reaction surveillance and pre-emptive antiviral therapy for cytomegalovirus after allogeneic transplantation

Peggs¹,

Preiser²,

Kottaridis³

et al. 2000

Br J Haematol

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Pre-emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)-guided pre-emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty-three of the 52 high-risk patients (CMV-seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5.7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high-risk patients (27%) required pre-emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow-up of 402 d (range 117-952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR-guided pre-emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.

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“…Since the first detection of CMV in serum and plasma (7,23,36), many reports on the qualitative (diagnostic) detection of CMV DNA in plasma or serum have appeared. In bone marrow transplant recipients (3,21,23,28,39), renal transplant recipients (7,11,16,39), and liver transplant recipients (16,29,30,33), the presence of CMV DNA in plasma or serum has been shown to be an early marker for CMV infection and CMV disease. In congenitally infected newborns (26) and in children with AIDS (27), CMV can readily be detected in serum.…”

mentioning

confidence: 99%

“…Another problem in the detection of CMV is the preparation of CMV DNA from serum and plasma. Although simple pretreatments (e.g., proteinase digestion and heat treatment or treatment with alkali) will detect high viral loads, low loads may remain undetected (3,23). In previous studies in which CMV DNA was purified before PCR, controls that monitored DNA extraction efficiency were not included and controls that monitored PCR inhibition were usually absent.…”

mentioning

confidence: 99%

A Highly Sensitive Assay for Detection and Quantitation of Human Cytomegalovirus DNA in Serum and Plasma by PCR and Electrochemiluminescence

et al. 1999

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We describe a diagnostic PCR assay (D-PCR) and a quantitative PCR assay (Q-PCR) for the detection of human cytomegalovirus (CMV) in plasma and serum. In the D-PCR, DNA was purified from plasma or serum together with internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR efficiency. DNA was subjected to PCR with a single primer pair, and the amount of PCR products was determined by electrochemiluminescence (ECL) in the QPCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2′-bipyridine) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity of the D-PCR was reached at about 25 CMV particles/ml. Even with extremely low DNA inputs (four molecules of IC DNA/200 μl of plasma), very high yields (near 100%) were reached. DNA extracted from specimens that were CMV positive by the D-PCR was subsequently used in the Q-PCR, which was similar to the D-PCR. The viral load was calculated directly from the ratio of CMV and IC signals obtained by ECL. The Q-PCR assay is quantitative in the range of 100 to 150,000 copies of CMV/ml, independent of the anticoagulant. Interassay variation, intra-assay variation, and interspecimen variation were about 25%, suggesting that the Q-PCR will reliably detect fourfold differences in viral load. Comparison of paired serum and plasma specimens from CMV-infected individuals showed that serum CMV loads were frequently more than 10-fold lower than plasma CMV loads.

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Comparison of plasma PCR and bronchoalveolar lavage fluid culture for detection of cytomegalovirus infection in adult bone marrow transplant recipients

Cited by 41 publications

References 26 publications

Metagenomic Sequencing Detects Respiratory Pathogens in Hematopoietic Cellular Transplant Patients

Metagenomic Sequencing Detects Respiratory Pathogens in Hematopoietic Cellular Transplant Patients

Extended routine polymerase chain reaction surveillance and pre-emptive antiviral therapy for cytomegalovirus after allogeneic transplantation

A Highly Sensitive Assay for Detection and Quantitation of Human Cytomegalovirus DNA in Serum and Plasma by PCR and Electrochemiluminescence

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