PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia.

BACKGROUND: Malaria remains a main parasitic disease of humans. Although the largest number of cases is reported in the African region, there are still endemic foci in the Americas. Central America reported 36,000 malaria cases in 2020, which represents 5.5% of cases in the Americas and 0.015% of cases globally. Most malaria infections in Central America are reported in La Moskitia, shared by Honduras and Nicaragua. In the Honduran Moskitia, less than 800 cases were registered in 2020, considering it an area of low endemicity. In low endemicity settings, the number of submicroscopic and asymptomatic infections tends to increase, leaving many cases undetected and untreated. These reservoirs challenge national malaria elimination programmes. This study aimed to assess the diagnostic performance of Light Microscopy (LM), a nested PCR test and a photoinduced electron transfer polymerase chain reaction (PET-PCR) in a population of febrile patients from La Moskitia. METHODS: A total of 309 febrile participants were recruited using a passive surveillance approach at the Puerto Lempira hospital. Blood samples were analysed by LM, nested PCR, and PET-PCR. Diagnostic performance including sensitivity, specificity, negative and positive predictive values, kappa index, accuracy, and ROC analysis was evaluated. The parasitaemia of the positive samples was quantified by both LM and PET-PCR. RESULTS: The overall prevalence of malaria was 19.1% by LM, 27.8% by nPCR, and 31.1% by PET-PCR. The sensitivity of LM was 67.4% compared to nPCR, and the sensitivity of LM and nPCR was 59.6% and 80.8%, respectively, compared to PET-PCR. LM showed a kappa index of 0.67, with a moderate level of agreement. Forty positive cases by PET-PCR were not detected by LM. CONCLUSIONS: This study demonstrated that LM is unable to detect parasitaemia at low levels and that there is a high degree of submicroscopic infections in the Honduran Moskitia.

Efficacy and Safety of Albendazole and High-Dose Ivermectin Coadministration in School-Aged Children Infected With Trichuris trichiura in Honduras: A Randomized Controlled Trial.

BACKGROUND: The efficacy of currently available anthelminthics against Trichuris trichiura infections is significatively lower than for other soil-transmitted helminths. The combination of ivermectin (IVM) and albendazole (ALB) has shown significant improvements in efficacy. METHODS: Safety and efficacy randomized controlled clinical trial comparing 3 experimental regimens against ALB monotherapy for the treatment of T. trichiura infections in northern Honduras. Infected children were randomized to 4 treatment arms: arm 1, single-dose ALB (400 mg); arm 2, single-dose ALB (400 mg) plus IVM (600 µg/kg); arm 3, ALB (400 mg) for 3 consecutive days; or arm 4, ALB (400 mg) plus IVM (600 µg/kg) for 3 consecutive days. Efficacy was measured based on the egg reduction and cure rates, both assessed 14-21 days after treatment, using the Kato-Katz method. Safety was evaluated by analyzing the frequency and severity of adverse events. RESULTS: Of 176 children randomized to 1 of the 4 treatment arms, 117 completed treatment and follow-up. The egg reduction rates for arms 1, 2, 3, and 4 were 47.7%, 96.7%, 72.1%, and 100%, respectively; with P values <.001 for comparisons between IVM groups and ALB-only arms. The cure rates were 4.2%, 88.6%, 33.3%, and 100%, respectively. A total of 48 adverse events (85.4% mild) were reported in 36 children. CONCLUSIONS: The combined use of ALB and high-dose IVM is a highly effective and well tolerated treatment for the treatment of T. trichiura infections, offering significantly improved treatment for the control of this infection. CLINICAL TRIALS REGISTRATION: NCT04041453.

Colorimetric Polymerase Chain Reaction Enabled by a Fast Light-Activated Substrate Chromogenic Detection Platform.

Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.

Genetic variability of Plasmodium falciparum histidine-rich proteins 2 and 3 in Central America.

BACKGROUND: Malaria is an important disease in many tropical countries. Rapid diagnostic tests (RDTs) are valuable tools for diagnosing malaria in remote areas. The majority of RDTs used for the diagnosis of Plasmodium falciparum are based on the detection of the specific histidine-rich proteins (PfHRP2 and PfHRP3). During the last decade, the threat posed by the lack of expression of these antigens and the variability of the proteins on the diagnosis of malaria has been widely discussed. The aim of this study was to evaluate the genetic diversity of pfhrp2 and pfhrp3 of P. falciparum isolates collected in three Central American countries. METHODS: DNA samples were amplified and sequenced to assess the diversity of nucleotides and amino acids. A search for known epitopes within the amino acid sequence was carried out, and the sensitivity of the sequences was evaluated according to a predictive model. A phylogenetic analysis was carried out including homologous sequences from different regions of the world. Protein structures were predicted in silico. RESULTS: Five different patterns for PfHRP2 and one pattern for PfHRP3 were identified. Isolates from Central America show a high level of genetic diversity in pfhrp2; however, the amino acid sequences seem to contain enough motifs to be detected by the RDTs currently available. CONCLUSION: It is unlikely that the variability of the pfhrp2 and pfhrp3 genes has a significant impact on the ability of the RDTs to detect the PfHRP antigens in Central America.

Deletions of pfhrp2 and pfhrp3 genes of Plasmodium falciparum from Honduras, Guatemala and Nicaragua.

BACKGROUND: Malaria remains a public health problem in some countries of Central America. Rapid diagnostic tests (RDTs) are one of the most useful tools to assist in the diagnosis of malaria in remote areas. Since its introduction, a wide variety of RDTs have been developed for the detection of different parasite antigens. PfHRP2 is the most targeted antigen for the detection of Plasmodium falciparum infections. Genetic mutations and gene deletions are important factors influencing or affecting the performance of rapid diagnostic tests. METHODS: In order to demonstrate the presence or absence of the pfhrp2 and pfhrp3 genes and their flanking regions, a total of 128 blood samples from patients with P. falciparum infection from three Central American countries were analysed through nested or semi-nested PCR approaches. RESULTS: In total, 25.8 and 91.4% of the isolates lacked the region located between exon 1 and exon 2 of pfhrp2 and pfhrp3 genes, respectively. Parasites from the three countries showed deletions of one or both genes. The highest proportion of pfhrp2 deletions was found in Nicaragua while the isolates from Guatemala revealed the lowest number of pfhrp2 deletions. Parasites collected from Honduras showed the highest proportion of phfrp3 absence (96.2%). Twenty-one percent of isolates were double negative mutants for the exon 1-2 segment of both genes, and 6.3% of isolates lacked the full-length coding region of both genes. CONCLUSIONS: This study provides molecular evidence of the existence of P. falciparum isolates lacking the pfhrp2 and pfhrp3 genes, and their flanking regions, in Honduras, Guatemala and Nicaragua. This finding could hinder progress in the control and elimination of malaria in Central America. Continuous evaluation of RDTs and molecular surveillance would be recommended.

First Detection of Acinetobacter baumannii in Pediculus humanus capitis from Latin America.

Several studies have documented the presence of Acinetobacter baumannii, a known multi-drug-resistant pathogen, in the human head louse, Pediculus humanus capitis. Since no reports from countries in Latin America have been published, the aim of the present study was to determine whether A. baumannii was present in head lice specimens collected in this geographic region. Head lice specimens from Argentina, Colombia, and Honduras were analyzed. PCR assays were performed to confirm the specimens' species and to investigate whether the DNA of A. baumannii was present. The products of the latter were sequenced to confirm bacterial identity. Altogether, 122 pools of head lice were analyzed, of which two (1.64%) were positive for A. baumannii's DNA. The positive head lice had been collected at the poorest study site in Honduras. The remaining specimens were negative. This study is the first to report the presence of A. baumannii in human head lice from Latin America. Further investigations are required to elucidate whether these ectoparasites can serve as natural reservoirs or even effectively transmit A. baumannii to humans.

Population genomics of ancient and modern Trichuris trichiura.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum.

INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the ß-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the ß-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the ß-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

An Integrated Study of Toxocara Infection in Honduran Children: Human Seroepidemiology and Environmental Contamination in a Coastal Community.

(1) Background: Infections caused by Toxocara canis and T. cati are considered zoonoses of global importance. Reports from North and South America indicate that human infections are widespread in both continents, but epidemiological information from Central America is still lacking. (2) Methodology: In the present cross-sectional multi-year study, we aimed to undertake the first seroepidemiological and environmental study on toxocariasis in Honduras. This included the determination of seroprevalence of anti-Toxocara spp. antibodies in children using a Toxocara spp. purified excretory-secretory antigens enzyme-linked immunosorbent assay (TES-ELISA) and a confirmatory Western blot. As well, through statistical analysis including logistic regression we aimed at identifying relevant biological and epidemiological factors associated with seropositivity. The study also entailed detection of parasites' eggs in the soil samples both through Sheather's concentration method and a nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. (3) Results: The study was undertaken in a coastal community of Honduras in 2 different years, 2015 and 2017. A total of 88 healthy schoolchildren completed the study, with participation of 79% (73/92) and 65% (46/71) of the student body in 2015 and 2017, respectively. Thirty-one children participated in both years (i.e., dual participants). Through both serological tests, seropositivity was confirmed in 88.6% (78/88) of children. Due to the high number of seropositives, logistic regression analysis was not possible for most socio-economic and epidemiological variables. Eosinophilia, on the other hand, was associated with seropositivity, independently of other intestinal helminthic infections. Continued seropositivity was observed in most of the dual participants, while seroconversion was determined in 8 of these children. Microscopic examination of soil samples did not yield any positive results. Through nested PCR-RFLP, 3 of the 50 samples (6%) were positive for Toxocara spp.; two were identified as T. canis and one as T. cati. (4) Conclusions: This work documents for the first time, high levels of human exposure to Toxocara spp. in Honduras. These findings, along with the country's favorable epidemiological conditions for this zoonosis, emphasize the need for more research to determine whether this infection is underreported in the country.

Expanding detection windows for discriminating single nucleotide variants using rationally designed DNA equalizer probes.

Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.

Use of Multi-Parallel Real-Time Quantitative PCR to Determine Blastocystis Prevalence and Association with Other Gastrointestinal Parasite Infection in a Rural Honduran Location.

To determine whether the presence of Blastocystis is associated with other gastrointestinal parasite infections, stool samples from 95 Honduran rural children were analyzed using multi-parallel quantitative real-time polymerase chain reaction (PCR) and Kato-Katz. Combined results detected the following prevalence: Blastocystis, 71.6%; Trichuris trichiura, 63.2%; Giardia lamblia, 40.0%; Ascaris lumbricoides, 15.8%; and Necator americanus, 4.2%. Age was found associated with the quantity of both Blastocystis DNA (r s = 0.524, P < 0.001) and T. trichiura DNA in the stool (fg/µL) by quantitative PCR (r s = 0.272, P < 0.001). In addition, there was an association with T. trichiura and Blastocystis infection (odds ratio [OR] = 4.72; 95% CI = 1.83, 12.20; P < 0.001). These findings demonstrate a high prevalence of Blastocystis and other intestinal parasites in a rural location in Honduras.

High Endemicity of Soil-Transmitted Helminths in a Population Frequently Exposed to Albendazole but No Evidence of Antiparasitic Resistance.

INTRODUCTION: Soil-transmitted helminths (STHs) are gastrointestinal parasites widely distributed in tropical and subtropical areas. Mass drug administration (MDA) of benzimidazoles (BZ) is the most recommended for STH control. These drugs have demonstrated limited efficacy against Trichuris trichiura and the long-term use of single-dose BZ has raised concerns of the possible emergence of genetic resistance. The objective of this investigation was to determine whether genetic mutations associated with BZ resistance were present in STH species circulating in an endemic region of Honduras. METHODS: A parasitological survey was performed as part of this study, the Kato-Katz technique was used to determine STH prevalence in children of La Hicaca, Honduras. A subgroup of children received anthelminthic treatment in order to recover adult parasite specimens that were analyzed through molecular biology techniques. Genetic regions containing codons 200, 198, and 167 of the -tubulin gene of Ascaris lumbricoides and Trichuris trichiura were amplified and sequenced. RESULTS: Stool samples were collected from 106 children. The overall STH prevalence was 75.47%, whereby T. trichiura was the most prevalent helminth (56.6%), followed by A. lumbricoides (17%), and hookworms (1.9%). Eighty-five sequences were generated for adjacent regions to codons 167, 198, and 200 of the -tubulin gene of T. trichiura and A. lumbricoides specimens. The three codons of interest were found to be monomorphic in all the specimens. CONCLUSION: Although the inability to find single-nucleotide polymorphisms (SNPs) in the small sample analyzed for the present report does not exclude the possibility of their occurrence, these results suggest that, at present, Honduras's challenges in STH control may not be related to drug resistance but to environmental conditions and/or host factors permitting reinfections.

Head lice infestations in rural Honduras: the need for an integrated approach to control neglected tropical diseases.

BACKGROUND: Pediculosis capitis is a neglected tropical disease (NTD) that receives little attention in countries where it continues to be endemic. This study aimed to understand the impact of Pediculus humanus capitis infestations in the lives of Honduran children living in extreme poverty. METHODS: A qualitative study on head lice infestation was conducted in June 2016 in a rural community in Honduras. Parents were invited to bring their children for head lice inspection using a dry-combing technique with a stainless steel-toothed comb with suction power. A semistructured questionnaire was administered to participants. Questions were broadly grouped into knowledge about transmission, control practices, barriers to treatment, and the overall impact of these infestations in children's wellbeing. Responses were coded, categorized, and organized through a theme-based approach. RESULTS: In total, 52 children aged 2-14 years (42 girls) and their mothers were enrolled in the study. The overall proportion of children with an infestation was 83%. Response analysis revealed a lack of understanding regarding lice transmission and stigmatization of infested children and the widespread belief that head lice were acquired during bathing in the slow-flowing river running through the village. An agricultural plaguicide was commonly used to rid children of head lice. CONCLUSIONS: The study underscores the dire situation of the rural poor, their physical and mental health affected by pediculosis capitis as well as other NTDs. These results highlight the need to reassess approaches and action towards combating NTDS under an integrated framework.

Bionomic aspects of Lutzomyia evansi and Lutzomyia longipalpis, proven vectors of Leishmania infantum in an endemic area of non-ulcerative cutaneous leishmaniasis in Honduras.

BACKGROUND: Some Lutzomyia species are the vectors of human leishmaniasis in the Americas. Visceral and cutaneous leishmaniasis are both endemic in the Pacific region of Honduras, but the non-ulcerative form is the more frequent clinical manifestation in this region, where Lutzomyia longipalpis is the most abundant and the only incriminated vector. Taxonomic identification and distribution studies of sand flies are important to understand the epidemiology and to control these neglected tropical diseases. RESULTS: Here, we identified more than 13,000 Lutzomyia specimens captured in Isla del Tigre, Honduras, through a classical morphological approach. The two most common species were Lutzomyia evansi and Lu. longipalpis, and this is the first report of three Lutzomyia species on this island. The blood meal source was successfully identified for five sand fly species. A barcode analysis using the cox1 mitochondrial marker proved to be effective in discriminating between species and seems to be a valuable tool for future epidemiological studies including a wider geographical area. CONCLUSION: This study updates the diversity and blood meal sources of Lutzomyia species in an island endemic for non-ulcerative cutaneous leishmaniasis in the Pacific region of Honduras, and determines the effectiveness of the barcoding approach to discriminate species, as a complementary tool to classical morphology.

Paper-Based DNA Reader for Visualized Quantification of Soil-Transmitted Helminth Infections.

Soil-transmitted helminth (STH) infections are a global health issue affecting nearly one-third of the world's population. As most endemic areas of STH are impoverished countries or regions with limited healthcare resources, the accurate diagnosis of STH requires analytical tools that are not only quantitative, but also portable, inexpensive, and with no or minimal demand for external instrument. Herein, we introduce a novel paper-based diagnostic device, termed quantitative paper-based DNA reader (qPDR), capable of quantifying STH at the molecular level by measuring distance as readout, thus eliminating the need for external readers. On the basis of the unique interfacial interaction of a DNA intercalating dye, SYBR Green I, with native cellulose on a chromatographic paper, qPDR allows the distance-based quantification of minute amounts of double-stranded DNA as short as 6 min. By integrating qPDR with polymerase chain reactions that were performed using a smartphone-controlled portable thermal cycler, we were able to quantify minute amount of genetic markers from adult worms of an STH (Trichuris trichiura) that were expelled post-treatment by infected children living in the rural areas of Honduras.

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum

Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.