RESUMEN
BACKGROUND: Low-density asymptomatic Plasmodium infections are prevalent in endemic areas, but little is known about their natural history. The trajectories of these infections and their propensity to fluctuate to undetectable densities can affect detection in clinical trials and field studies. We aimed to classify the natural history of these infections in a high transmission area over 29 days. METHODS: In this longitudinal cohort study, we enrolled healthy, malaria-asymptomatic, afebrile, adults (age 18-59 years) and older children (age 8-17 years) in Katakwi District, Uganda, who were negative for Plasmodium infection on rapid diagnostic tests. Participants were instructed to self-collect one dried blood spot (DBS) per day for a maximum of 29 days. We excluded people if they were pregnant or taking antimalarials. During weekly clinic visits, staff collected a DBS and a 4 mL sample of venous blood. We analysed DBSs by Plasmodium 18S rRNA quantitative RT-PCR (qRT-PCR). We classified DBS by infection type as negative, P falciparum, non-P falciparum, or mixed. We plotted infection type over time for each participant and categorised trajectories as negative, new, cleared, chronic, or indeterminate infections. To estimate the effect of single timepoint sampling, we calculated the daily prevalence for each study day and estimated the number of infections that would have been detected in our population if sampling frequency was reduced. FINDINGS: Between April 9 and May 20, 2021, 3577 DBSs were collected by 128 (40 male adults, 60 female adults, 12 male children, and 16 female children) study participants. 2287 (64%) DBSs were categorised as negative, 751 (21%) as positive for P falciparum, 507 (14%) as positive for non-P falciparum, and 32 (1%) as mixed infections. Daily Plasmodium prevalence in the population ranged from 45·3% (95% CI 36·6-54·1) at baseline to 30·3% (21·9-38·6) on day 24. 37 (95%) of 39 P falciparum and 35 (85%) of 41 non-P falciparum infections would have been detected with every other day sampling, whereas, with weekly sampling, 35 (90%) P falciparum infections and 31 (76%) non-P falciparum infections would have been detected. INTERPRETATION: Parasite dynamics and species are highly variable among low-density asymptomatic Plasmodium infections. Sampling every other day or every 3 days detected a similar proportion of infections as daily sampling, whereas testing once per week or even less frequently could misclassify up to a third of the infections. Even using highly sensitive diagnostics, single timepoint testing might misclassify the true infection status of an individual. FUNDING: US National Institutes of Health and Bill and Melinda Gates Foundation.
Asunto(s)
Malaria Falciparum , Malaria , Plasmodium , Estados Unidos , Adulto , Niño , Embarazo , Humanos , Masculino , Femenino , Adolescente , Adulto Joven , Persona de Mediana Edad , Estudios Longitudinales , Uganda/epidemiología , Plasmodium falciparum/genética , Malaria/diagnóstico , Malaria/epidemiología , Plasmodium/genética , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Estudios de Cohortes , Infecciones Asintomáticas/epidemiologíaRESUMEN
BACKGROUND: Many Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia. Self-collected dried blood spots (DBS) analysed using pooled and individual quantitative reverse transcription polymerase chain reaction (qRT-PCR) provide such a solution. Here, the feasibility and acceptability of daily at-home DBS collections for qRT-PCR was studied to better understand low-density infections. METHODS: Rapid diagnostic test (RDT)-negative individuals in Katakwi District, northeastern Uganda, were recruited between April and May 2021. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits. Participants were trained in DBS collection and asked to collect six DBS weekly between clinic visits. Opinions about the collection process were solicited using daily Diary Cards and a Likert scale survey at the final study visit. Venous blood and DBS were analysed by Plasmodium 18S rRNA qRT-PCR. The number of participants completing the study, total DBS collected, and opinions of the process were analysed to determine compliance and acceptability. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for at-home vs. clinic-collected DBS and venous blood to assess quality and accuracy of at-home collected samples. RESULTS: One-hundred two adults and 29 children were enrolled, and 95 and 26 completed the study, respectively. Three individuals withdrew due to pain or inconvenience of procedures. Overall, 96% of participants collected ≥ 16 of 24 at-home DBS, and 87% of DBS contained ≥ 40 µL of blood. The procedure was well tolerated and viewed favourably by participants. At-home collected DBS were acceptable for qRT-PCR and showed less than a one qRT-PCR cycle threshold shift in the human control mRNA compared to clinic-collected DBS. Correlation between Plasmodium falciparum 18S rRNA from paired whole blood and DBS was high (R = 0.93). CONCLUSIONS: At-home DBS collection is a feasible, acceptable, and robust method to obtain blood to evaluate the natural history of low-density Plasmodium infections by qRT-PCR.
Asunto(s)
Malaria Falciparum , Malaria , Adulto , Niño , Estudios de Factibilidad , Humanos , Malaria/diagnóstico , Malaria/epidemiología , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero , ARN Ribosómico 18S/genética , Transcripción ReversaRESUMEN
The ultrasensitive Alere Plasmodium falciparum Malaria Ag histidine-rich protein 2 rapid diagnostic test (Alere uRDT, Suwon City, South Korea) is a new diagnostic tool which is more expensive than other malaria rapid diagnostic tests (RDTs) routinely used in Ugandan clinics. The manufacturer recommends testing samples within 2 days and scoring results after 20 minutes, which may be impractical in high-volume resource-poor clinics. We compared testing by the Alere Ag rapid diagnostic test (uRDT), CareStart RDT, microscopy, and an ultrasensitive I8S rRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) using survey and clinical samples. For the Alere uRDT, we used survey blood samples stored at 4°C for 44 days and for some clinical samples deliberately scored results beyond 20 minutes. The Alere uRDT and qRT-PCR identified asymptomatic parasitemia cases in 56% and 72%, respectively, of survey samples originally scored as negative by the CareStart RDT. Using qRT-PCR as a gold standard, the Alere uRDT was superior to the CareStart RDT in estimating asymptomatic parasite prevalence in a cross-sectional survey (P = 0.007) and in detection of clinically significant malaria; both RDTs were comparable in detecting asymptomatic parasitemia in the clinic (P = 0.599). Scoring Alere uRDT results at 20 minutes produced valid results confirmed by the CareStart RDT, but there was a consistent background; scoring the Alere uRDT beyond 20 minutes produced false-positive results. The Alere uRDT outperformed the CareStart RDT (ACCESSBIO, Somerset, NJ) in a field survey in estimating malaria prevalence and in the clinic for symptomatic malarial illness. It produced reliable results using samples stored at 4°C for 44 days, but test results read beyond 20 minutes were invalid.