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Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

Background Human T-cell lymphotropic virus (HTLV-1) is a retrovirus endemic in Latin America, Africa, and Asia. Increasing numbers are being reported in the United States. HTLV-1 causes Adult T-cell Leukemia/Lymphoma (ATL) in 3–5% of HTLV-1 carriers, usually only after a prolonged latent period. Co-infection with the nematode Strongyloides stercoralis (SS) is associated with early onset of ATL. The exact mechanism by which SS accelerates ATL development in HTLV-1 subjects is not understood. CADM1 has been recently identified as a surface marker of HTLV-1 infected cells; CD7 is a probable marker of early cell transformation. We hypothesize that previous SS infection will increase the proportion of infected T cells (CADM1+) and lead to transformation of infected cells (CADM1+CD7low) in HTLV-1 subjects. Methods In this pilot study, we tested seven subjects that were diagnosed with HTLV-1 between 2006 and 2008 and actively followed up at the HTLV-1 Cohort Clinic at the Tropical Medicine Institute in Lima, Peru. Five had previous SS infection. We performed surface flow cytometry staining of peripheral blood mononuclear cells with monoclonal antibodies against CADM1, CD7, CD4, and CD3. Current SS infection was ruled out by the Baermann technique in a stool sample. Results Average age was 53 years (range 30–79). Average time since diagnosis of HTLV-1 was 10 years (range 9–11). One 30-year-old patient with prior SS infection was diagnosed with ATL and hospitalized in the ICU at the time of follow-up and excluded from group comparison. There was a trend for higher proportions of CADM1+ T-cells in subjects with previous SS co-infection compared with those without SS (median 16.20% vs. 9.55%, P = 0.13, Mann–Whitney). The proportion of PBMCs that were CADM1+CD7− was also increased in some of those with previous SS (Figure 1). Conclusion Our pilot data suggest that SS co-infection is associated with prolonged effects on HTLV-1 infection. We noted trends towards increased numbers of HTLV-1 infected cells (CADM1+) and a predisposition towards malignant transformation (CADM1+CD7low). Further studies are needed to confirm this observation and to determine whether CADM1 and CD7 expression may be useful as a screening tool to monitor HTLV-1 subjects at high risk of developing ATL. Figure 1. Representative flow cytometry. Disclosures All authors: No reported disclosures.