Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

ABSTRACT 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.

[1]  S. Blacksell,et al.  Commercial Dengue Rapid Diagnostic Tests for Point-of-Care Application: Recent Evaluations and Future Needs? , 2012, Journal of biomedicine & biotechnology.

[2]  Luzia Gonçalves,et al.  Bayesian Latent Class Models in Malaria Diagnosis , 2012, PloS one.

[3]  M. Leeflang,et al.  Prospective Evaluation of Three Rapid Diagnostic Tests for Diagnosis of Human Leptospirosis , 2013, PLoS neglected tropical diseases.

[4]  Jonathan J Deeks,et al.  Rapid diagnostic tests for diagnosing uncomplicated P. falciparum malaria in endemic countries. , 2011, The Cochrane database of systematic reviews.

[5]  Haruo Watanabe,et al.  flaB‐Polymerase Chain Reaction (flaB‐PCR) and Its Restriction Fragment Length Polymorphism (RFLP) Analysis Are an Efficient Tool for Detection and Identification of Leptospira Spp. , 2001, Microbiology and immunology.

[6]  E. Harris,et al.  Early Clinical Features of Dengue Virus Infection in Nicaraguan Children: A Longitudinal Analysis , 2012, PLoS neglected tropical diseases.

[7]  E. Bertherat,et al.  Rapid tests for diagnosis of leptospirosis: current tools and emerging technologies. , 2014, Diagnostic microbiology and infectious disease.

[8]  R. Peeling,et al.  Point-of-care tests for diagnosing infections in the developing world. , 2010, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[9]  D. Mckay,et al.  A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp , 2002, BMC infectious diseases.

[10]  N. Ahmed,et al.  Annals of Clinical Microbiology and , 2005 .

[11]  P. Fournier,et al.  Leptospirosis in a French traveler returning from Mauritius. , 2012, Journal of travel medicine.

[12]  G. Baranton,et al.  Comparison of polymerase chain reaction with microagglutination test and culture for diagnosis of leptospirosis. , 1995, The Journal of infectious diseases.

[13]  Jeremy D. DeBarry,et al.  Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA , 2011, Journal of Clinical Microbiology.

[14]  Jian Ye,et al.  Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction , 2012, BMC Bioinformatics.

[15]  Rosanna W. Peeling,et al.  Evaluation of diagnostic tests: dengue , 2010, Nature Reviews Microbiology.

[16]  P. Alonso,et al.  A direct comparison of real time PCR on plasma and blood to detect Plasmodium falciparum infection in children , 2012, Malaria Journal.

[17]  G. Chang,et al.  Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses , 2006, Journal of Clinical Microbiology.

[18]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[19]  M. M. Pereira,et al.  Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic surveillance. , 2012, Diagnostic microbiology and infectious disease.

[20]  E. Harris,et al.  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 , 2013, Journal of Clinical Microbiology.

[21]  K. Leder,et al.  Acute and potentially life-threatening tropical diseases in western travelers--a GeoSentinel multicenter study, 1996-2011. , 2013, The American journal of tropical medicine and hygiene.

[22]  Herbert L DuPont,et al.  Patterns of illness in travelers visiting Mexico and Central America: the GeoSentinel experience. , 2011, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[23]  J. Jacobs,et al.  Malaria rapid diagnostic tests in travel medicine. , 2013, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[24]  A. M. de Oliveira,et al.  Comparison of molecular tests for the diagnosis of malaria in Honduras , 2012, Malaria Journal.

[25]  R. Gilman,et al.  Polymerase chain reaction detection of Plasmodium vivax and Plasmodium falciparum DNA from stored serum samples: implications for retrospective diagnosis of malaria. , 2007, The American journal of tropical medicine and hygiene.

[26]  J. Rosenblatt,et al.  Diagnostic performance of rapid diagnostic tests versus blood smears for malaria in US clinical practice. , 2009, Clinical Infectious Diseases.

[27]  R. Steele,et al.  Fever in a Returning Traveler , 2016, Clinical pediatrics.

[28]  B. Cooper,et al.  Fool's Gold: Why Imperfect Reference Tests Are Undermining the Evaluation of Novel Diagnostics: A Reevaluation of 5 Diagnostic Tests for Leptospirosis , 2012, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[29]  N. Lennon,et al.  Dynamics of Dengue Disease Severity Determined by the Interplay Between Viral Genetics and Serotype-Specific Immunity , 2011, Science Translational Medicine.

[30]  A. Hoffmaster,et al.  Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study , 2011, PloS one.

[31]  E. Harris,et al.  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 , 2013, Journal of Clinical Microbiology.

[32]  E. Harris,et al.  Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses , 2013, PLoS neglected tropical diseases.

[33]  G. Baranton,et al.  Oligonucleotides specific for pathogenic and saprophytic leptospira occurring in water. , 1997, FEMS microbiology letters.

[34]  M. Cetron,et al.  Spectrum of disease and relation to place of exposure among ill returned travelers. , 2006, The New England journal of medicine.

[35]  Eileen M. Burd,et al.  Validation of Laboratory-Developed Molecular Assays for Infectious Diseases , 2010, Clinical Microbiology Reviews.

[36]  C. Dolea,et al.  World Health Organization , 1949, International Organization.

[37]  L. Smythe,et al.  Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene , 2006, BMC Microbiology.

[38]  H. Margolis,et al.  Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus , 2013, PLoS neglected tropical diseases.

[39]  R. Price,et al.  Considerations on the use of nucleic acid-based amplification for malaria parasite detection , 2011, Malaria Journal.

[40]  E. Caumes,et al.  Travel-related leptospirosis: a series of 15 imported cases. , 2013, Journal of travel medicine.

[41]  Weltgesundheitsorganisation.  World malaria report , 2005 .

[42]  J. Jacobs,et al.  Malaria rapid diagnostic tests in endemic settings. , 2013, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[43]  R. Lanciotti,et al.  Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction , 1992, Journal of clinical microbiology.