Blood samples
This study was conducted in accordance with institutional review board guidelines and requirements of the University of Florida and the ethical review board of the Haitian MSPP after obtaining all permits and approvals (UF IRB 201400202; UF IRB201400224; MSPP Ref. 1314-12; MSPP Ref. 1314-62). Subjects were recruited through a cross-sectional sampling of reproductive age women across Haiti in gynecology and maternity clinics. Study team collaborated with NGOs and government dispensaries to enrol women for free malaria screening. Each participant during the waiting time for enrolment was asked to answer a demographic survey in Haitian Creole. The questionnaire included general demographic and behaviour questions such as gender, age, geographic residency, history of travel, sleeping pattern (outdoor and/or indoor), presence of window screens, use of insecticide-treated bed nets, presence of bed nets whether treated or not, and history of malaria transmission for the participant and household members. Each questionnaire was labelled with a unique identifying number that matched the consent form and sample to provide de-identified data for analysis.
Study sites shown in Fig. 1 were selected to cover diverse settings across the entire country of Haiti, including areas of high and low transmission. Subjects living in rural sites located in six of the ten departments, (Artibonite, Central Plateau, Grand Anse, Nord, Ouest, Sud-Est) were screened. Historically, malaria transmission has been reported in the Nord, Artibonite and Sud-Est Departments, and Grand Anse experienced an elevated level of transmission beginning in July 2014. In contrast, the Ouest and Central Plateau were known to be areas of low transmission. Between August 2014 and March 2015 a total of 563 venous blood samples were collected in an EDTA-coated, purple-top Vacutainer tube from attendants of maternity and gynecology clinics. Blood was mixed gently within the collection tube and 400 µL were transferred in a cryotube pre-filled with 1200 µL of DNA/RNA Shield™ (DRS, ZymoResearch, Inc.) for preservation of RNA and DNA. Approximately 25–50 µL of blood were used to make two microscopic slides (thick and thin smear) and run a CareStart Malaria HRP2 RDT. RDT results were recorded immediately in a study logbook. Microscopic slides were left to air-dry then Giemsa stained, following CDC protocol [23]. Whole blood in DRS was maintained at 4 °C or on wet ice continuously until placed at −20 °C upon return to the University of Florida laboratory in Gressier. The average travel time for the study team for sample collection, storage and sample freezing ranged from five to 15 days. Samples were shipped frozen on blue ice to the University of Florida, Gainesville, FL, USA.
qRT-PCR assay design
A set of primers and a probe were designed to develop a highly sensitive molecular test to detect the presence of P. falciparum small sub-unit ribosomal RNA type A [24, 25]. These reagents were developed by aligning the small sub-unit rRNA gene sequences from the four most common Plasmodium spp. infecting humans (Plasmodium
ovale, P. falciparum, Plasmodium malariae, Plasmodium
vivax) along with human small sub-unit rRNA using ClustalW2 software, and selecting specific primer pairs and probe using Integrated DNA Technologies, Inc. software. Care was taken to assure that the primers and probe would detect the small sub-unit rRNA, which is present predominantly in P. falciparum blood stage parasites. The following primers were selected for use in the qRT-PCR assay employed here: forward (5-GATACCGTCGTAATCTTAACCATAAAC-3), reverse (5-AAGGTACTGAAGGAAGCAATCT-3) and probe (6-FAM-ACACTTTCATCCAACACCTAGTCGGC-BHQ-1). The specificity of the primers was confirmed experimentally with RNA from P. falciparum clone 3D7 and RNA from uninfected human blood.
Sample processing and qRT-PCR assay
Total RNA was extracted from 800 µL of the mixture of blood preserved in DRS using the Quick-RNA™ MiniPrep Kit following the RNA isolation from red blood cells protocol (Zymo Research) in a PCR workstation. The RNA was eluted in a volume of 50 µL of which 5 µL was used in the qRT-PCR reaction. Total nucleic acid extraction was also performed from blood samples in DRS using the same kit, but following a modification recommended by the manufacturer for obtaining total nucleic acids. Total nucleic acids were also eluted in 50 µL of which 5 µL was used in subsequent qPCR and qRT-PCR reactions.
The qRT-PCR was conducted using the Express One-Step SuperScript qRT-PCR Kit (Invitrogen) following manufacturer’s instructions with a final concentration of 0.4 µM of each primer and 0.2 µM for the probe in a 20 µL reaction. The PCR master mix preparation was conducted in a separate PCR work station. Amplification and real-time measurements were performed on a CFX96 Touch ™ Real-Time PCR Detection system (Bio-Rad). Reaction conditions were as follows: 20 min at 58 °C for reverse transcription step, one cycle of 2 min at 95 °C for Taq DNA Polymerase activation and 40 cycles of 95 °C for 15 s, 60 °C for 1 min for annealing and extension of amplified product. Every PCR run was conducted with two negative controls and one positive control. Samples were tested in separate batches where those with positive RDT results were extracted and tested separate from the ones with negative RDTs to reduce the possibility of cross contamination.
In vitro limit of detection
A synchronous culture of P. falciparum 3D7 free of mature parasites and most culture debris was prepared as follows to obtain parasitized cells containing ring stage parasites, the developmental stage expected in the peripheral blood of infected patients. A fresh, semi-synchronous culture was prepared by the sorbitol method [26] and mature schizonts collected on a magnetic column [27]. The mature schizonts were released from the column and incubated with fresh red blood cells for 12–15 h to allow release of merozoites and re-invasion. The mature parasites that had not released merozoites were removed using a second magnetic column. The parasitaemia of the ring stage parasites in culture which passed through the column was determined using both microscopy and fluorescence activated cell sorting, and the number of parasites per volume of culture calculated based upon the number of red blood cells per volume of culture determined using a Coulter counter. Counted parasitized red cells from culture were mixed with whole human blood to prepare an initial sample containing 105 parasites/ml.
A standard curve was prepared from RNA extracted from the initial sample containing 105 parasites/ml and serially diluted in RNase/DNase free water in intervals of tenfold dilution with the final dilution equivalent to 0.1 parasites/ml of blood. All dilutions were treated with RNase inhibitor using RNase Out (Life Technologies) with a final concentration of 1 unit per µL. The same blood sample having 105 parasites/ml was serially diluted as well in un-infected blood at intervals of ten-fold to reach a concentration of 10 parasites/ml of blood, then at intervals of two-fold dilution until reaching a concentration of 0.3 parasites/ml of blood. Infected blood (200 µL) was then mixed with 600 µL of DRS and RNA was extracted from all 800 µL as described above. RNA was eluted in a volume of 50 µL, of which 5 µL was used as a template in each qRT-PCR reaction.
Statistical analysis
Demographic and survey items were used to assess differences in the likelihood of being an asymptomatic carrier of P. falciparum parasites. After stratification for demographic location by department, simple logistic regression models were used to determine the likelihood of carrying P. falciparum parasites using survey questions in Stata V.11 (Statacorp, College Station, TX, USA). Questions included items relating to participant knowledge of malaria, risk factors for malaria infection, demographic characteristics, and human mobility characteristics. Statistical significance was determined at an alpha level of 0.05 in all logistic regression models.