Study design, setting, and participants
This was a cross-sectional study that evaluated febrile patients who attended the Puerto Lempira hospital, in Gracias a Dios, in the Honduran region called “La Moskitia”. Samples were collected during 2021 and from January to August 2022. Gracias a Dios is the easternmost department of the country, bordering Nicaragua, characterized by geographic isolation and lack of land communication with the rest of the territory (Fig. 2). Due to isolation and historical and cultural circumstances, the population of La Moskitia lives in conditions of limited socioeconomic development, without adequate access to health services, and low educational levels. La Moskitia accounts for about 98% of malaria cases in Honduras, and currently, the number of cases due to P. vivax reaches 62% while 36% are due to P. falciparum, with 1.7% of mixed infections (Personal communication by the National Malaria Surveillance Laboratory, Health Ministry, Honduras).
Blood samples were collected on the same day as the medical consultation. The demographic data of the patients (age, sex, and municipality of residence) were recorded together with the clinical history. Most of the patients resided in the municipality of Puerto Lempira (92.88%), and the rest came from four other municipalities in the department (Fig. 2). Febrile patients of both sexes and of all ages were recruited. There were no exclusion criteria. The sample size for a low transmission setting was calculated assuming a sensitivity of at least 55%, a specificity of at least 85%, a malaria prevalence of 33.3%, a relative precision of 12%, and 80% power [17]. These criteria yielded a minimum required sample size of 199 subjects.
Microscopic Diagnosis
After the medical examination of the patients, the clinical laboratory personnel collected blood samples in tubes with EDTA anticoagulant. In accordance with national malaria guidelines, thick and thin blood smears were prepared for parasitological analysis [18]. Slides were examined within hours of sampling. An expert microscopist observed a maximum of 500 microscopic fields at 100X magnification before reporting the slides as negative. Parasite density was estimated using a quantitative approach in those smears positive for P. vivax and/or P. falciparum, reporting the total number of sexual and asexual stages per 200 leukocytes. Parasite density was classified as high, moderate, or low, according to parameters established by Alger et al [19]. Patients with a microscopic diagnosis of malaria were treated with chloroquine and primaquine according to national guidelines. Figure 3 shows the workflow used in this study.
Dna Extraction
Two or three drops of blood from each participant were used to impregnate Whatman No. 3 filter paper to preserve the DNA until its subsequent extraction in the city of Tegucigalpa. The samples were placed in sealed plastic bags with desiccant and stored for up to four months. Three circles of 10 mm2 each were cut from paper impregnated with blood for DNA extraction. Disks were immersed in 200 µL of 1% saponin, vortexed, and incubated at 4°C overnight. The next day, samples were washed four times with PBS and then exposed to a 5% Chelex-100 suspension (Bio-Rad, Hercules, CA, USA). Tubes were incubated at 56°C for 15 min and then at 100°C for 10 min. Tubes were centrifuged for 5 min at 13,000 rpm and the DNA was recovered from the supernatant and stored at 4°C for later analysis.
Nested Pcr
A segment of the 18S ribosomal gene of Plasmodium spp. was amplified using the nested PCR (nPCR) technique described by Singh et al [20]. Both reactions (first and second round) were carried out in a 50 µL volume containing a 2X Taq polymerase master mix (Promega Corp. Madison, WI, USA) and 2 µL of each primer 10 µM (Table 1). The first reaction included 11 µL of nuclease-free water and 10 µL of DNA. The second reaction included 20 µL of nuclease-free water and 1 µL of the PCR product of the first reaction.
Table 1
List of primers used for amplification reactions, nucleotide sequences, annealing temperatures, and amplicon sizes.
Reaction | Primer | Sequence (5´- 3´) | Annealing temperature (ºC) | Product size (bp) |
First PCR for Plasmodium spp. | rPLU1 | TCA AAG ATT AAG CCA TGC AAG TGA | 55 | |
| rPLU5 | CCT GTT GTT GCC TTA AAC TYC | | |
Second PCR for Plasmodium spp. | rPLU3 | TTT YTA TAA GGA TAA CTA CGG AAA AGC TGT | 62 | 240 |
| rPLU4 | TAC CCG TCA TAG CCA TGT TAG GCC AAT ACC | | |
PCR for P. vivax | rVIV1 | CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC | 58 | 107 |
| rVIV2 | ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA | | |
PCR for P. falciparum | rFAL1 | TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT | 58 | 205 |
| rFAL2 | ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC | | |
PET-PCR for Plasmodium spp. | Genus forward | GGC CTA ACA TGG CTA TGA CG | 63 | 91 |
| Labeled - Genus reverse | 6FAM- agg cgc ata gcg cct ggC TGC CTT CCT TAG ATG TGG TAG CT | | |
Negative samples were recorded as negative after the first result. Positive samples were confirmed by a new amplification. If a discordant result was detected between the two amplifications or between the nPCR and the light microscopy (LM), samples were amplified a third time from new DNA extraction. The result was settled by means of two concordant tests. The samples with a final positive result for malaria were analyzed to determine the species of the parasite. Two separate reactions were carried out in a final volume of 25 µL containing 12.5 µL of 2X Taq polymerase master mix, 1 µL of each primer (10 µM) (Table 1), 9.5 µL of nuclease-free water, and 1 µL of the product of the first PCR.
Parasite detection by nPCR was blinded to the result obtained by LM, and once the first PCR result was obtained, it was compared with that of the LM to decide whether to repeat the amplification or not.
All reactions (for genus and species) were carried out by an initial denaturation at 94 ºC for 4 min, 35 cycles of 94 ºC for 30 s, annealing temperature for 60 s (Table 1), and 72 ºC for 60 s, with a final extension at 72 ºC for 4 min. Products were visualized by 2% agarose gel electrophoresis with ethidium bromide. Positive and negative controls were included in each set of reactions.
PET-PCR
The samples were tested in duplicate using a photo-induced electron transfer PCR (PET-PCR) in order to detect Plasmodium infections and quantify the number of parasites per µL of blood [21–24]. The parasite genome was detected by amplifying a conserved segment of the 18S ribosomal gene in the four Plasmodium species. The reaction was carried out in a volume of 20 µL containing 10 µL of Go Taq® Probe qPCR Master Mix (Promega Corp. Madison, WI, USA), 0.5 µL of each primer (10 µM) (Table 1), 4 µL of nuclease-free water, and 5 µL of DNA. Reactions were run on a Mic qPCR Cycler (Bio Molecular Systems, Brisbane, Australia) and the results were visualized in the Mic qPCR Cycler Software. The amplification conditions for both genus and species detection were 95 ºC for 15 min, 45 cycles at 95 ºC for 20 s, 63 ºC for 40 s, and 72 ºC for 30 s. The correct fluorescence channel was selected for the labeled primer (6FAM). A cycle threshold (Ct) of 40 or below was used to consider samples as positive. Samples with a Ct equal to or less than 40 and a replica with a Ct greater than 40, but less than 2-digit deviation from the positive result were considered positive. The parasite species were not assessed by PET-PCR in this study.
To quantify the parasitaemia, two reference standard curves were included. The first reference curve was prepared with a serial dilution of genomic DNA from P. falciparum strain 3D7 containing 100,000 parasites per µL. The second reference curve included serial dilutions of the plasmid pMG-Amp in which a partial sequence of 200 nucleotides of the Plasmodium 18S ribosomal gene was cloned. Both standard curves established the technique's detection limit and determined the samples' parasitaemia.
Statistical Analyses
Sensitivity, specificity, positive (PPV), and negative (NPV) predictive values were calculated for LM and nPCR using the PET-PCR as the reference assay. Furthermore, these values were also calculated for LM relative to nPCR.
Sensitivity was calculated as the ratio of true positives to total positives multiplied by 100. Specificity was calculated as the ratio of true negatives to total negatives multiplied by 100. 95% confidence intervals were calculated for sensitivity and specificity. PPV was calculated as follows: true positives / (true positives + false positives) * 100; and NPV as: true negatives / (true negatives + false negatives) * 100. Diagnostic accuracy was calculated as: (true positives + true negatives) / (true positives + true negatives + false positives + false negatives) *100 [25]. The total number of samples was those on which all three assays were successfully performed. McNemar's test was calculated between LM and nPCR results.
Receiver operating curve (ROC) analysis and areas under the curve (AUC) were carried out to assess diagnostic accuracy and to compare the diagnostic performance of LM, nPCR, and PET-PCR. AUC was interpreted as follows: 0.9-1.0, excellent; 0.8–0.9, very good; 0.7–0.8, good; 0.6–0.7, sufficient; 0.5–0.6, bad; <0.5, test not useful [26].
The Cohen´s kappa coefficient of agreement of LM and nPCR using the PET-PCR as reference was also calculated as k = po – pe / 1 – pe, where po was the relative observed agreement among assays, and pe was the hypothetical probability of chance agreement. In addition, the kappa index between LM and nested PCR was calculated using nPCR as a reference. McHugh's table was used to interpret kappa values [27].