Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths.

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin-ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

The Genetic Polymorphisms of 24 Base Pair Duplication and Point G102S of Human Chitotriosidase to Bancroftian Filariasis at the Thai⁻Myanmar Border.

Lymphatic filariasis, caused by lymphatic filarial parasites, Wuchereria bancrofti, and Brugia malayi, causes significant morbidity and disability to 120 million people in the tropics and subtropics. Chitin has an important role for embryogenesis in adult worms and is a component of microfilaria sheath. Human chitotriosidase (CHIT1) is a chitin-degrading enzyme which provides a protective role against chitin-containing pathogens. Here, we determined the association of CHIT1 polymorphisms with susceptibility to bancroftian filariasis (BF) in 88 individuals at the Thai⁻Myanmar border. Two common polymorphisms of CHIT1, contributing inactive CHIT protein, including 24 base pair (24 bp) duplication in exon 10, and p. G102S in exon 4 were genotyped by allele-specific Polymerase Chain Reaction (PCR) and PCR sequencing, respectively. Unexpectedly, genotype frequencies of 24 bp duplication insertion homozygous (INS/INS) were significantly higher in endemic normal (EN) (40.0%) than BF patients (31.4%). In contrast, genotype frequencies of p. G102S homozygous (A/A) in BF patients (21.6%) was higher than in EN (19.0%) without statistical difference. Mutant allele frequencies of 24 bp duplication were 0.6125 (98/160) and p. G102S were 0.392 (69/176). Genotype and allele frequencies of CHIT1, 24 bp duplication, and p. G102S, showed no association with BF patients.

A Simple Genotyping Method for Rapid Differentiation of Blastocystis Subtypes and Subtype Distribution of Blastocystis spp. in Thailand.

Blastocystis spp. is one of the most common protozoa of humans and animals worldwide. The genetic diversity of Blastocystis spp. might be associated with a wide range of symptoms. However, the prevalence of each subtype is different in each country. Until now, there is no standard method for subtyping of Blastocystis spp. We developed a sequential restriction fragment length polymorphism (RFLP) analysis for the rapid differentiation of human Blastocystis subtypes. A large-scale study was also conducted to determine the subtype distribution of Blastocystis spp. in Thailand. Stool samples were collected from 1025 school-age students in four regions of Thailand. Blastocystis infections were identified by direct smear, formalin ethyl-acetate concentration technique (FECT), Boeck and Drbohlav's Locke-Egg-Serum (LES) medium culture, and polymerase chain reaction (PCR) of small-subunit ribosomal DNA (SSU rDNA). Subtypes of Blastocystis spp. were determined by RFLP. Phylogenetic tree of partial SSU rDNA sequences of Blastocystis spp. was constructed using the Maximum Likelihood (ML) method. Out of 1025 students, 416 (40.6%) were positive for Blastocystis spp. Using two steps of RFLP reactions, we could determine subtype one⁻three among these students. Subtype 3 was the most common subtype (58.72%) in Thai students, followed by subtype 1 (31.2%), and subtype 2 (10.1%). Blastocystis subtype 3 was the most prevalent in all regions of Thailand. The subtype distribution of Blastocystis spp. in Thailand was different from other countries.

The Experimental Infections of the Human Isolate of Strongyloides Stercoralis in a Rodent Model (The Mongolian Gerbil, Meriones Unguiculatus).

Strongyloidiasis is life-threatening disease which is mainly caused by Strongyloides stercoralis infection. Autoinfection of the parasite results in long-lasting infection and fatal conditions, hyperinfection and dissemination (primarily in immunosuppressed hosts). However, mechanisms of autoinfection and biology remain largely unknown. Rodent models including mice and rats are not susceptible to the human isolate of S. stercoralis. Variations in susceptibility of the human isolate of S. stercoralis are found in dogs. S. ratti and S. venezuelensis infections in rats are an alternative model without the ability to cause autoinfection. The absence of appropriate model for the human isolate of strongyloidiasis hampers a better understanding of human strongyloidiasis. We demonstrated the maintenance of the human isolate of the S. stercoralis life cycle in the Mongolian gerbil (Meriones unguiculatus). The human isolate of S. stercoralis caused a patent infection in immunosuppressed gerbils, more than 18 months. The mean number of recovery adult parasitic worms were 120 ± 23 (1.2% of the initial dose) and L1s were 12,500 ± 7,500 after day 28 post-inoculation (p.i.). The prepatent period was 9⁻14 days. Mild diarrhoea was found in gerbils carrying a high number of adult parasitic worms. Our findings provided a promising model for studying biology and searching new alternative drugs against the parasites. Further studies about the hyperinfection and dissemination would be performed.