Homotypic Dengue Virus Reinfections in Nicaraguan Children.
journal of infectious diseases
2016; 214 (7): 986-993
Infection with one of four related dengue virus serotypes (DENV-1-4) is thought to result in life-long immunity to re-infection with the same serotype (homotypic DENV re-infection). Archived serum samples, collected as part of an ongoing pediatric dengue cohort study in Nicaragua, were tested for DENV by real-time RT-PCR. Samples were collected from 2,892 children who presented with an acute febrile illness clinically attributed to a non-dengue cause ("C" cases). Test results were added to a database of previously-identified symptomatic dengue cases in the cohort to identify repeat infections. Four patients with homotypic DENV re-infections were identified and confirmed among 29 repeat DENV infections with serotype confirmation (13.8% of repeat symptomatic infections). Homotypic re-infections with DENV-1, -2, and -3 occurred 325-621 days after the initial infection. Each patient experienced one symptomatic dengue case and one DENV-positive C case, and two patients presented with symptomatic dengue during their second infection. These DENV-positive C cases did not elicit long-lived humoral immune responses, despite viremia up to 6.44 log10 copies/mL of serum. We describe the first set of virologically confirmed homotypic DENV re-infections. Such cases challenge the current understanding of DENV immunity and have important implications for modeling DENV transmission.
View details for DOI 10.1093/infdis/jiw099
View details for PubMedID 26984144
Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses
EMERGING INFECTIOUS DISEASES
2016; 22 (7): 1295-1297
Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.
View details for DOI 10.3201/eid2207.160326
View details for Web of Science ID 000378563900030
View details for PubMedID 27184629
Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection
JOURNAL OF CLINICAL VIROLOGY
2016; 78: 57-61
Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR).From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/μL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.
View details for DOI 10.1016/j.jcv.2016.01.007
View details for Web of Science ID 000374480000013
View details for PubMedID 26991052
- Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus PLOS ONE 2015; 10 (11)
Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing
JOURNAL OF CLINICAL MICROBIOLOGY
2015; 53 (10): 3226-3233
BK virus (BKV) infection and end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep sequencing methodology and bioinformatics pipeline that identifies BKV variants across the genome and at BKV-specific HLA-A2, HLA-B0702, and HLA-B08 restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets and fragmentation libraries were sequenced on the Ion Torrent PGM. An error model and variant calling algorithm were developed to accurately identify rare variants. 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range 2-37, interquartile range 10), with the majority of variants (77%) detected at a frequency of less than 5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.
View details for DOI 10.1128/JCM.01385-15
View details for Web of Science ID 000365625300017
- Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing PLOS ONE 2015; 10 (7)
Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing.
2015; 10 (7)
Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.
View details for DOI 10.1371/journal.pone.0132988
View details for PubMedID 26177295
- Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis PLOS ONE 2014; 9 (11)
- Commutability of the Epstein-Barr Virus WHO International Standard across Two Quantitative PCR Methods JOURNAL OF CLINICAL MICROBIOLOGY 2014; 52 (10): 3802-3804
Encephalitis Caused by Chikungunya Virus in a Traveler from the Kingdom of Tonga
JOURNAL OF CLINICAL MICROBIOLOGY
2014; 52 (9): 3459-3461
Febrile travelers from countries with unique endemic pathogens pose a significant diagnostic challenge. In this report, we describe the case of a Tongan man presenting with fever, rash, and altered mental status. The diagnosis of Chikungunya encephalitis was made using a laboratory-developed real-time RT-PCR and serologic testing.
View details for DOI 10.1128/JCM.01288-14
View details for Web of Science ID 000341359300055
Improved Detection of Emerging Drug-Resistant Mutant Cytomegalovirus Subpopulations by Deep Sequencing
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
2014; 58 (8): 4697-4702
In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.
View details for DOI 10.1128/AAC.03214-14
View details for Web of Science ID 000339259200053
Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis
JOURNAL OF CLINICAL MICROBIOLOGY
2014; 52 (6): 2011-2018
Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.
View details for DOI 10.1128/JCM.00341-14
View details for Web of Science ID 000337919500028
Human Papilloma Virus is not Prevalent in Nevus Sebaceus
2014; 31 (3): 326-330
Nevus sebaceus (NS) is a common congenital cutaneous hamartoma that typically presents on the scalp and face at birth or in early childhood. Occasionally NS can be associated with the Schimmelpenning-Feuerstein-Mims syndrome, which presents with concomitant severe neurologic, skeletal, cardiovascular, ophthalmic, and genitourologic disorders. In a previous study, maternal transmission of the human papillomavirus (HPV) and infection of ectodermal stem cells by HPV was postulated to result in the development of NS. In this study we aimed to determine the incidence of HPV infection in pediatric NS samples to further clarify the potential link between HPV and the pathogenesis of NS. NS tissue samples (N = 16) were analyzed for HPV DNA using type-specific, real-time polymerase chain reaction (PCR) targeting HPV 6, 11, 16, and 18 and conventional PCR with modified general primers designed for broad-range HPV detection. The tissues were also histologically evaluated for evidence of HPV infection. HPV DNA was not detected in any of the NS tissue samples using PCR and HPV-associated histopathologic changes were absent in all 16 NS tissues. HPV infection is an unlikely etiologic cause of NS.
View details for DOI 10.1111/pde.12249
View details for Web of Science ID 000334884300017
BK Polyomavirus Subtype III in a Pediatric Renal Transplant Patient with Nephropathy
JOURNAL OF CLINICAL MICROBIOLOGY
2013; 51 (12): 4255-4258
BK polyomavirus (BKV) is an emerging pathogen in immunocompromised individuals. BKV subtype III is rarely identified and has not previously been associated with disease. Here we provide the whole-genome sequence of a subtype III BKV from a pediatric kidney transplant patient with polyomavirus-associated nephropathy.
View details for DOI 10.1128/JCM.01801-13
View details for Web of Science ID 000327147100067
View details for PubMedID 24048534
Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing
JOURNAL OF CLINICAL MICROBIOLOGY
2013; 51 (11): 3700-3710
Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations.
View details for DOI 10.1128/JCM.01605-13
View details for Web of Science ID 000325812200035
View details for PubMedID 23985916
Comparison of the FDA-Approved CDC DENV-1-4 Real-Time Reverse Transcription-PCR with a Laboratory-Developed Assay for Dengue Virus Detection and Serotyping
JOURNAL OF CLINICAL MICROBIOLOGY
2013; 51 (10): 3418-3420
Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.
View details for DOI 10.1128/JCM.01359-13
View details for Web of Science ID 000324624300039
View details for PubMedID 23903549
Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays
JOURNAL OF CLINICAL MICROBIOLOGY
2013; 51 (7): 2172-2181
A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic-acid amplification tests (NAATs). However, reports describing the direct comparison of different NAATs are limited. In this study, we report the design of an internally-controlled, real-time reverse-transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two-hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay; a commercially-produced, internally-controlled DENV rRT-PCR (the Altona assay); a widely-used hemi-nested RT-PCR; and a serotype-specific, multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 7.0 to 1.0 log10 complimentary DNA (cDNA) equivalents/μL and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μL depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved more clinically sensitive than either the Altona or hemi-nested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (p≤0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day five of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed, molecular diagnosis of DENV infection can be provided.
View details for DOI 10.1128/JCM.00548-13
View details for Web of Science ID 000320595800024
View details for PubMedID 23637298
- Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses PLOS NEGLECTED TROPICAL DISEASES 2013; 7 (4)
Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B
JOURNAL OF VIROLOGICAL METHODS
2012; 186 (1-2): 137-140
Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B.
View details for DOI 10.1016/j.jviromet.2012.07.023
View details for Web of Science ID 000312763600024
View details for PubMedID 22841669