Multi-locus sequence typing (MLST) is widely used to investigate genetic relationships among eukaryotic taxa, including parasitic pathogens. MLST analysis workflows typically involve construction of alignment-based phylogenetic trees - i.e., where tree structures are computed from nucleotide differences observed in a multiple sequence alignment (MSA). Notably, alignment-based phylogenetic methods require that all isolates/taxa are represented by a single sequence. When multiple loci are sequenced these sequences may be concatenated to produce one tree that includes information from all loci. Alignment-based phylogenetic techniques are robust and widely used yet possess some shortcomings, including how heterozygous sites are handled, intolerance for missing data (i.e., partial genotypes), and differences in the way insertions-deletions (indels) are scored/treated during tree construction. In certain contexts, 'haplotype-based' methods may represent a viable alternative to alignment-based techniques, as they do not possess the aforementioned limitations. This is namely because haplotype-based methods assess genetic similarity based on numbers of shared (i.e., intersecting) haplotypes as opposed to similarities in nucleotide composition observed in an MSA. For haplotype-based comparisons, choosing an appropriate distance statistic is fundamental, and several statistics are available to choose from. However, a comprehensive assessment of various available statistics for their ability to produce a robust haplotype-based phylogenetic reconstruction has not yet been performed. We evaluated seven distance statistics by applying them to extant MLST datasets from the gastrointestinal parasite Cyclospora cayetanensis and two species of pathogenic nematode of the genus Strongyloides. We compare the genetic relationships identified using each statistic to epidemiologic, geographic, and host metadata. We show that Barratt's heuristic definition of genetic distance was the most robust among the statistics evaluated. Consequently, it is proposed that Barratt's heuristic represents a useful approach for use in the context of challenging MLST datasets possessing features (i.e., high heterozygosity, partial genotypes, and indel or repeat-based polymorphisms) that confound or preclude the use of alignment-based methods.

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photo-induced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/μL (95% confidence interval (CI): 3-793p/μL) for whole blood (WB) samples and 385p/μL (95% CI: 31-2133 p/μL) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities.

The Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens.

Malaria in the first trimester of pregnancy is associated with adverse pregnancy outcomes. Artemisinin-based combination therapies (ACTs) are a highly effective, first-line treatment for uncomplicated Plasmodium falciparum malaria, except in the first trimester of pregnancy, when quinine with clindamycin is recommended due to concerns about the potential embryotoxicity of artemisinins. We compared adverse pregnancy outcomes after artemisinin-based treatment (ABT) versus non-ABTs in the first trimester of pregnancy.