Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity.
BACKGROUND
Quantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared.


METHODS
To quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed.


RESULTS
Intra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (< 1 gametocyte per μL). Logistic models, including log-transformed gametocytaemia estimated by QT-NASBA, explained variability in mosquito feeding experiment results as well as log-transformed gametocytaemia by qRT-PCR (marginal R20.28 and 0.22, respectively). Densities determined by both methods strongly correlated with mosquito infection rates [Spearman's rank correlation coefficient, 0.59 for qRT-PCR and 0.64 for QT-NASBA (P < 0.001 for both)]. Gametocyte densities estimated by qRT-PCR were higher than levels estimated by QT-NASBA or light microscopy at high densities (>100 gametocyte per μL). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze-thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain.


CONCLUSIONS
The experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities.
Department of Medical Microbiology, Radboud University Medical Center, Geert Grooteplein Zuid 26-28, 6500 HB, Nijmegen, The Netherlands.
Helmi
H
Pett
Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, UK.
Bronner P
BP
Gonçalves
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Alassane
A
Dicko
Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso.
Issa
I
Nébié
Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso.
Alfred B
AB
Tiono
Department of Medical Microbiology, Radboud University Medical Center, Geert Grooteplein Zuid 26-28, 6500 HB, Nijmegen, The Netherlands.
Kjerstin
K
Lanke
MRC Tropical Epidemiology Group, Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK.
John
J
Bradley
Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA.
Ingrid
I
Chen
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Halimatou
H
Diawara
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Almahamoudou
A
Mahamar
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Harouna M
HM
Soumare
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Sekou F
SF
Traore
Malaria Research and Training Centre, Faculty of Pharmacy and Faculty of Medicine and Dentistry, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
Ibrahima
I
Baber
Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso.
Sodiomon B
SB
Sirima
Department of Medical Microbiology, Radboud University Medical Center, Geert Grooteplein Zuid 26-28, 6500 HB, Nijmegen, The Netherlands.
Robert
R
Sauerwein
Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA.
Joelle
J
Brown
Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA.
Roly
R
Gosling
Molecular Diagnostics Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland.
Ingrid
I
Felger
Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, UK.
Chris
C
Drakeley
Department of Medical Microbiology, Radboud University Medical Center, Geert Grooteplein Zuid 26-28, 6500 HB, Nijmegen, The Netherlands. teun.bousema@radboudumc.nl.
Teun
T
Bousema
D002050:Burkina Faso; D002648:Child; D002986:Clinical Trials as Topic; D005260:Female; D006801:Humans; D008297:Male; D008302:Mali; D059208:Parasite Load / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D021161:Self-Sustained Sequence Replication / Q000379:methods*
C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins
Anopheles; Gametocytes; Malaria; Mosquito; QT-NASBA; Quantification; Transmission; qRT-PCR