Ethical Approval

The protocols for these studies were reviewed and approved by the Institutional Review Boards of Tulane University and the Hôpital Albert Schweitzer (Deschapelles, Haiti). All samples were collected after obtaining informed consent.

Study Site

Studies were performed in the low-lying Artibonite Valley. The valley has abundant rainfall and is heavily farmed; 80% is irrigated for the cultivation of rice and other crops. The major peak in malaria cases (>99% caused by P. falciparum) (11,14–16) is during November–January (11,12,17). The population of the Artibonite Valley relies primarily on subsistence farming and informal trade (barter) for income. This population is poor; only 18% of households have electricity and just 12% have piped water (12). As a result, members of the population rarely travel outside the study area, and international travel to other malaria-endemic countries is uncommon. The primary malaria control activities currently being implemented include improvement of microscopy at Hôpital Albert Schweitzer, a facility that is supported by the Global Fund (www.theglobalfund.org/en/worldmalariaday/2007) and vector control (10).

Hôpital Albert Schweitzer was the base of operations for the household surveys, passive case detection, and laboratory studies (i.e., thick and thin blood smears, antigen testing by using rapid diagnostic tests [RDTs], clinical examinations, and clinical and laboratory follow-up of patients). This hospital provides comprehensive inpatient care at its 100-bed facility and delivers preventive and primary health services to a population of 300,000 through a network of health centers, dispensaries, and workers in the community. Data from Hôpital Albert Schweitzer indicate that malaria transmission in this area of Haiti varies annually according to rainfall. For example, 157 of 2,739 suspected cases were confirmed by microscopy and treated with CQ in 2005 (smear positivity rate 5.7%), and only 29 of 1,307 suspected cases were confirmed and treated in 2006 (smear positivity rate 2.2%). The prevalence of P. falciparum infection in this area of Haiti is estimated to be 3.1% (13).

Household Survey in 2006 (Active Case Detection)

A 2-stage cluster design, in which probability was proportional to cluster size, was used to generate a sample of 200 households within the study area, as described elsewhere (11,12). Thick and thin blood films and 4 blots of blood on filter paper for PCR were collected from 714 persons >1 month of age within selected households. All smear-positive case-patients were treated with CQ.

Passive Case Detection in 2006 and 2007

Diagnosis of Malaria by Blood Smear or RDT and Species-Specific PCR for P. falciparum Small Subunit rRNA Gene

Thick and thin Giemsa-stained blood smears were examined for malaria parasites at Hôpital Albert Schweitzer by trained laboratory technologists by using standard methods (18,19). Filter paper blots were transported from Haiti to New Orleans where parasite DNA was extracted (20,21), and microscopy results were confirmed by using a PCR for the P. falciparum small subunit (SSU) rRNA gene (22). DNA was extracted from filter paper blots by using the Charge Switch Forensic DNA Purification Kit (catalog no. CS 11200; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. This extraction yielded 150 μL of DNA in buffer (10 mmol/L Tris, pH 8.5, 1 mmol/L EDTA) from each specimen.

PCR for the P. falciparum SSU rRNA gene used a P. falciparum–specific forward primer (which hybridizes only with P. falciparum DNA) and a genus-specific reverse primer (which hybridizes with DNA from all 4 Plasmodium spp. that infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae) (22) (Table 1). To perform this PCR, 4 μL of DNA extracted from filter paper blots was added to 19 μL of PCR master mixture (Promega, Madison, WI, USA) and 1 μL of each primer. Parasite DNA was amplified after an initial denaturation at 95°C for 15 min; 43 cycles of denaturation at 95°C for 45 s and annealing at 60°C for 90 sec; and a final extension at 72°C for 5 min in an i-Q thermocycler (Bio-Rad, Hercules, CA, USA). Positive controls for these assays contained DNA from in vitro culture of the Haiti I/CDC strain of P. falciparum (26). Resulting amplicons were visualized by electrophoresis on 1% agarose gels stained with ethidium bromide (27,28). Amplicon sizes were estimated by using a 100–600-bp DNA ladder (catalog no. 15628–019; Invitrogen).

Amplification of P. falciparum pfcrt Gene from Specimens Positive for P. falciparum SSU DNA

Two protocols were used to amplify the P. falciparum CQ resistance transporter (pfcrt) gene responsible for CQ resistance (23–25). The first protocol (single-step PCR) was used to screen 79 smear-positive and RDT-positive specimens that were positive for P. falciparum SSU DNA (23). In this assay, 4 μL of DNA extracted from filter paper blots was mixed with 21 μL of PCR master mixture (Promega) plus 2 μL of primers (Table 1) and amplified by an initial denaturation at 95°C for 7 min; 40 cycles of denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, and extension at 72°C for 30 sec; and a final extension at 72°C for 10 min in an i-Cycler thermocycler (Bio-Rad).

The second protocol (nested PCR) (24,25) was used to retest 58 specimens positive for SSU DNA that were negative in the single-step PCR for pfcrt. The nested PCR protocol used primers CRTP1 and CRTP2 for the first round of amplification and primers CRTD1 and CRTD2 for the second round (24,25). Samples in the first round were amplified by an initial denaturation at 94°C for 3 min; 45 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 60°C for 1 min; and a final extension at 60°C for 3 min. Samples in the second round were amplified by an initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 92°C for 30 sec, annealing at 48°C for 30 sec, and extension at 65°C for sec; and a final extension at 65°C for 3 min (Table 1).

Digestion of Amplicons from pfcrt with ApoI

For each sample positive for SSU DNA, an aliquot (10 μL) of the pfcrt gene PCR product was digested with 10 U of ApoI (New England Biolabs, Beverly, MA, USA) according to the manufacturer’s instructions. Briefly, 10 U of ApoI in 1× NE buffer 3 (100 mol/L NaCl, 50 mmol/L Tris-HCl, 10 mmol/L MgCl₂, 1 mmol/L dithiothreitol) and bovine serum albumin (100 μg/μL) were incubated overnight with 10 μL of the PCR product at 50°C (23–25). DNA fragments from samples and positive and negative controls were resolved by electrophoresis on 3% agarose gels stained with ethidium bromide.

ApoI digests most wild-type pfcrt genes (with CVMNK haplotype sequences at positions 72–76) but not the CQ-resistant mutant gene (i.e., K76, not T76) (23–25). On the basis of a single-step PCR for pfcrt, which yields an amplicon of 170 bp, amplicons with a lysine at position 76 (K76) are digested into 2 fragments (98 bp and 72 bp). Amplicons from CQ-resistant parasites (i.e., parasites with CVIET and CVMNT sequences at positions 72–76) are not digested by ApoI, resulting in an unchanged amplicon of 170 bp. The nested PCR product is slightly smaller (134 bp vs. 170 bp). As with the single-step PCR, most amplicons from CQ-susceptible parasites are digested by ApoI (in this instance to 30-bp and 104-bp fragments); amplicons from CQ-resistant parasites are not digested (unchanged amplicons of 134 bp; 24,25).

Amplification, Cloning, and Sequencing of pfcrt Genes Not Digested by ApoI

Samples not digested by ApoI for which DNA was available (9 of 10) were reamplified under the conditions described above for nested pfcrt PCR, cloned into the pCRII-TOPO vector, and transfected into the TOP10 strain of Escherichia coli by using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s instructions (29,30). Cloned pfcrt amplicons were sequenced in both directions by using CRTD1 and CRTD2 primers at an automated DNA sequencing facility (Davis Sequencing, Davis, CA, USA). Data for >3 clones sequenced in both directions were compared by using the multiple sequence alignment function in Lasergene version 7.2 software (DNASTAR, Madison, WI, USA) (31,32).