Molecular Xenomonitoring Using Mosquitoes to Map Lymphatic Filariasis After Mass Drug Administration in American Samoa

Background: Mass drug administration (MDA) programs have dramatically reduced lymphatic filariasis (LF) incidence in many areas around the globe, including American Samoa. As infection rates decline and MDA programs end, efficient and sensitive methods for detecting infections are needed to monitor for recrudescence. Molecular methods, collectively termed ‘molecular xenomonitoring,’ can identify parasite DNA or RNA in human blood-feeding mosquitoes. We tested mosquitoes trapped throughout the inhabited islands of American Samoa to identify areas of possible continuing LF transmission after completion of MDA. Methodology/Principle Findings: Mosquitoes were collected using BG Sentinel traps from most of the villages on American Samoa’s largest island, Tutuila, and all major villages on the smaller islands of Aunu’u, Ofu, Olosega, and Ta’u. Real-time PCR was used to detect Wuchereria bancrofti DNA in pools of #20 mosquitoes, and PoolScreen software was used to infer territory-wide prevalences of W. bancrofti DNA in the mosquitoes. Wuchereria bancrofti DNA was found in mosquitoes from 16 out of the 27 village areas sampled on Tutuila and Aunu’u islands but none of the five villages on the Manu’a islands of Ofu, Olosega, and Ta’u. The overall 95% confidence interval estimate for W. bancrofti DNA prevalence in the LF vector Ae. polynesiensis was 0.20–0.39%, and parasite DNA was also detected in pools of Culex quinquefasciatus, Aedes aegypti, andAedes (Finlaya) spp. Conclusions/Significance: Our results suggest low but widespread prevalence of LF on Tutuila and Aunu’u where 98% of the population resides, but not Ofu, Olosega, and Ta’u islands. Molecular xenomonitoring can help identify areas of possible LF transmission, but its use in the LF elimination program in American Samoa is limited by the need for more efficient mosquito collection methods and a better understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes and infection and transmission rates in humans

Molecular Xenomonitoring Using Mosquitoes to Map Lymphatic Filariasis after Mass Drug Administration in American Samoa

BACKGROUND: Mass drug administration (MDA) programs have dramatically reduced lymphatic filariasis (LF) incidence in many areas around the globe, including American Samoa. As infection rates decline and MDA programs end, efficient and sensitive methods for detecting infections are needed to monitor for recrudescence. Molecular methods, collectively termed \u27molecular xenomonitoring,\u27 can identify parasite DNA or RNA in human blood-feeding mosquitoes. We tested mosquitoes trapped throughout the inhabited islands of American Samoa to identify areas of possible continuing LF transmission after completion of MDA. METHODOLOGY/PRINCIPLE FINDINGS: Mosquitoes were collected using BG Sentinel traps from most of the villages on American Samoa\u27s largest island, Tutuila, and all major villages on the smaller islands of Aunu\u27u, Ofu, Olosega, and Ta\u27u. Real-time PCR was used to detect Wuchereria bancrofti DNA in pools of ≤ 20 mosquitoes, and PoolScreen software was used to infer territory-wide prevalences of W. bancrofti DNA in the mosquitoes. Wuchereria bancrofti DNA was found in mosquitoes from 16 out of the 27 village areas sampled on Tutuila and Aunu\u27u islands but none of the five villages on the Manu\u27a islands of Ofu, Olosega, and Ta\u27u. The overall 95% confidence interval estimate for W. bancrofti DNA prevalence in the LF vector Ae. polynesiensis was 0.20-0.39%, and parasite DNA was also detected in pools of Culex quinquefasciatus, Aedes aegypti, and Aedes (Finlaya) spp. CONCLUSIONS/SIGNIFICANCE: Our results suggest low but widespread prevalence of LF on Tutuila and Aunu\u27u where 98% of the population resides, but not Ofu, Olosega, and Ta\u27u islands. Molecular xenomonitoring can help identify areas of possible LF transmission, but its use in the LF elimination program in American Samoa is limited by the need for more efficient mosquito collection methods and a better understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes and infection and transmission rates in humans

Evaluating Molecular Xenomonitoring as a Tool for Lymphatic Filariasis Surveillance in Samoa, 2018–2019

Molecular xenomonitoring (MX), the detection of filarial DNA in mosquitoes using molecular methods (PCR), is a potentially useful surveillance strategy for lymphatic filariasis (LF) elimination programs. Delay in filarial antigen (Ag) clearance post-treatment is a limitation of using human surveys to provide an early indicator of the impact of mass drug administration (MDA), and MX may be more useful in this setting. We compared prevalence of infected mosquitoes pre- and post-MDA (2018 and 2019) in 35 primary sampling units (PSUs) in Samoa, and investigated associations between the presence of PCR-positive mosquitoes and Ag-positive humans. We observed a statistically significant decline in estimated mosquito infection prevalence post-MDA at the national level (from 0.9% to 0.3%, OR 0.4) but no change in human Ag prevalence during this time. Ag prevalence in 2019 was higher in randomly selected PSUs where PCR-positive pools were detected (1.4% in ages 5–9; 4.8% in ages ≥10), compared to those where PCR-positive pools were not detected (0.2% in ages 5–9; 3.2% in ages ≥10). Our study provides promising evidence for MX as a complement to human surveys in post-MDA surveillance

Pooling as a Strategy for the Timely Diagnosis of Soil-Transmitted Helminths in Stool: Value and Reproducibility

Background: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a \u27pooling approach\u27 can yield a low frequency of \u27missed\u27 infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in \u27pools-of-five\u27. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method

What Does Soil-Transmitted Helminth Elimination Look Like? Results From a Targeted Molecular Detection Survey in Japan

Background: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods: Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions: To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.[Figure not available: see fulltext.

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1 in juvenile diabetes

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.The authors wish to thank patients and subjects who participated in this study, as well as nurses and staff of the Pediatric Clinic of S. Maria della Misericordia Hospital (Perugia), Juvenile Diabetes Center-Anna Meyer Children's Hospital (Florence), Unit of Endocrinology and Diabetes-'Bambino Gesu' Children's Hospital (Rome), Hopital Necker-Enfants Malades (Paris), and Diabetes and Metabolism Service-University Hospital Centre of Coimbra (Coimbra). The authors wish also to thank Roberto Gerli for the gift of TCZ, Giovanni Ricci for histologies, and Francisco Carrilho and Eduarda Coutinho for providing and processing, respectively, DNA samples from the Portuguese cohorts. This work was supported by the European Research Council (338954-DIDO to UG) and, in part, by Associazione per l'Aiuto ai Giovani con Diabete Italia e dell'Umbria (to UG) and the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013 to AC) and the Fundacao para a Ciencia e Tecnologia (contracts IF/00735/2014 to AC, and SFRH/BPD/96176/2013 to CC).info:eu-repo/semantics/publishedVersio

Widespread Regulation of miRNA Biogenesis at the Dicer Step by the Cold-Inducible RNA-Binding Protein, RBM3

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

Background: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods: Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions: To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.[Figure not available: see fulltext.

Loss of p53 in quaking viable mice leads to Purkinje cell defects and reduced survival

The qkv mutation is a one megabase deletion resulting in abnormal expression of the qkI gene. qkv mice exhibit hypomyelination of the central nervous system and display rapid tremors and seizures as adults. The qkI locus on 6q26-27 has also been implicated as a candidate tumor suppressor gene as the qkI locus maps to a region of genetic instability in Glioblastoma Multiforme (GBM), an aggressive brain tumor of astrocytic lineage. As GBM frequently harbors mutations affecting p53, we crossbred qkv and p53 mutant mice to examine whether qkv mice on a p53−/− background have an increased incidence of GBM. qkv/v; p53−/− mice had a reduced survival rate compared to p53−/− littermates, and the cause of death of the majority of the mice remains unknown. In addition, immunohistochemistry revealed Purkinje cell degeneration in the cerebellum. These results suggest that p53 and qkI are genetically linked for neuronal maintenance and survival

The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation

We identify new mRNA targets for the QKI-6 RNA binding proteins using an unbiased approach. We show that AIP-1 mRNA is bound by QKI-6 within its 3′-UTR. This regulation is observed in oligodendrocytes and it is essential for oligodendrocyte process outgrowth