Polymerase chain reaction-based tests for pan-species and species-specific detection of human Plasmodium parasites.
BACKGROUND
There is still a need to improve the sensitivity of polymerase chain reaction (PCR) tests for malaria to detect submicroscopic asexual stage Plasmodium infections during the early phase and chronic, asymptomatic phase of infection when the parasite burden is very low.


METHODS
The inhibitory effect of hemoglobin (Hb) on PCR limits the volume of blood that can be used in the PCR-based detection of intraerythrocytic Plasmodium parasites. We lysed red blood cells with saponin to reduce the Hb concentration in extracted nucleic acid and, as a result, significantly increased the volume of blood that can be tested by PCR. The analytical sensitivity of the PCR was determined using whole blood spiked with ring-stage Plasmodium falciparum parasites, and its clinical sensitivity by testing blood film-positive and blood film-negative samples from individuals living in an endemic area in Ghana.


RESULTS
We have developed a pan-Plasmodium PCR that detects all five human Plasmodium species with the highest analytical sensitivity of two P. falciparum parasites/mL of whole blood and species-specific PCR tests that distinguished between the five human Plasmodium species. Pan-Plasmodium PCR detected 78 of 78 (100%) blood film-positive and 19 of 101 (18.81%) blood film-negative samples from asymptomatic individuals living in Ghana. Pan-Plasmodium PCR was equally sensitive with samples collected as anticoagulated whole blood and clotted blood and in blood collected by finger stick into capillaries.


CONCLUSIONS
We have developed PCR tests with the highest reported sensitivity to date for pan-Plasmodium diagnosis and species-specific diagnosis and detected blood film-negative asymptomatic infections in individuals living in malaria-endemic countries.
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Rockville, Maryland 20852, USA.
Babita
B
Mahajan
Hong
H
Zheng
Phuong Thao
PT
Pham
Mary Y
MY
Sedegah
Victoria F
VF
Majam
Namita
N
Akolkar
Maria
M
Rios
Isaac
I
Ankrah
Parnor
P
Madjitey
George
G
Amoah
Ebenezer
E
Addison
Isabella A
IA
Quakyi
Sanjai
S
Kumar
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001483:Base Sequence; D001774:Blood Chemical Analysis / Q000379:methods; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D005869:Ghana / Q000453:epidemiology; D006801:Humans; D007223:Infant; D008288:Malaria / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D010961:Plasmodium / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D016053:RNA, Protozoan / Q000032:analysis / Q000097:blood; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000097:blood; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013379:Substrate Specificity / Q000235:genetics; D055815:Young Adult
D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S