title: Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination.
abstract: BACKGROUND
  Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum.
  
  
  METHODS
  Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR.
  
  
  RESULTS
  Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 x 10(3) copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of >99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion.
  
  
  CONCLUSIONS
  Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination.
authors.affiliation: Macha Research Trust, Choma, Zambia. natasha.laban@macharesearch.org.
authors.forename: Natasha M
authors.initials: NM
authors.lastname: Laban
authors.affiliation: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. tkobaya2@jhu.edu.
authors.forename: Tamaki
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Macha Research Trust, Choma, Zambia. harry.hamapumbu@macharesearch.org.
authors.forename: Harry
authors.initials: H
authors.lastname: Hamapumbu
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. dsulliv7@jhmi.edu.
authors.forename: David
authors.initials: D
authors.lastname: Sullivan
authors.affiliation: Macha Research Trust, Choma, Zambia. sungano.mharakurwa@macharesearch.org.|W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. sungano.mharakurwa@macharesearch.org.
authors.forename: Sungano
authors.initials: S
authors.lastname: Mharakurwa
authors.affiliation: Macha Research Trust, Choma, Zambia. phil.thuma@macharesearch.org.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Thuma
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. cshiff1@jhu.edu.
authors.forename: Clive J
authors.initials: CJ
authors.lastname: Shiff
authors.affiliation: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. wmoss1@jhu.edu.
authors.forename: William J
authors.initials: WJ
authors.lastname: Moss
mesh_terms: D000293:Adolescent; D000328:Adult; D000953:Antigens, Protozoan / Q000097:blood / Q000235:genetics*; D002648:Child; D003430:Cross-Sectional Studies; D006801:Humans; D057230:Limit of Detection; D016778:Malaria, Falciparum / Q000175:diagnosis*; D010963:Plasmodium falciparum / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D015800:Protozoan Proteins / Q000097:blood / Q000235:genetics*; D011933:Reagent Kits, Diagnostic / Q000469:parasitology*; D055815:Young Adult; D015024:Zambia
chemical_list: D000953:Antigens, Protozoan; C514457:HRP-2 antigen, Plasmodium falciparum; D015800:Protozoan Proteins; D011933:Reagent Kits, Diagnostic