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

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Mitsuko Hasegawa ◽  
◽  
Nils Pilotte ◽  
Mihoko Kikuchi ◽  
Arianna R. Means ◽  
...  
Keyword(s):  
Real Time ◽  
Ascaris Lumbricoides ◽  
Real Time Pcr ◽  
Population Level ◽  
Molecular Techniques ◽  
Control Measures ◽  
Detection Methods ◽  
High Standards ◽  
Epidemiological Characteristics ◽  
Transmission Status

Abstract 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.

scholarly journals Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona

2021 ◽  
Vol 15 (11) ◽  
pp. e0009884
Author(s):  
Aroa Silgado ◽  
Mayuli Armas ◽  
Adrián Sánchez-Montalvá ◽  
Lidia Goterris ◽  
Maria Ubals ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Skin Lesions ◽  
Tertiary Hospital ◽  
Molecular Techniques ◽  
Cutaneous Lesions ◽  
Imported Cases ◽  
Epidemiological Characteristics

Background Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. Methodology/Principal findings Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). Conclusions/Significance An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

scholarly journals Ability of Real-time PCR in diagnose Differentiation Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

2019 ◽  
Author(s):  
Maryam Fekri Soofi Abadi ◽  
Meisam Fekri ◽  
alireza moradabadi ◽  
Reza Vahidi ◽  
Simin Shamsi Meymandi ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Relative Number ◽  
Parasite Load ◽  
Detection Methods ◽  
Tissue Samples ◽  
Microscopic Evaluation ◽  
Histopathological Evaluation ◽  
Histopathological Studies

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

scholarly journals Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR

2015 ◽  
Vol 21 (1-2) ◽  
Author(s):  
N. Czotter ◽  
E. Manduláné Farkas ◽  
R. Lózsa ◽  
I. Ember ◽  
G. Szûcsné Varga ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Amplification ◽  
Molecular Techniques ◽  
Latent Infections ◽  
Quantitative Real Time Pcr ◽  
Detection And Identification

Several grapevine pathogens are disseminated by propagating material as systemic, but latent infections. Their detection and identification have a basic importance in the production and handling of propagating stocks. Thus several sensitive and reliable diagnostic protocols mostly based on molecular techniques have been developed. Of these methods quantitative real-time PCR (q-PCR) has recently got an emerging importance. Here we collected primer data for the detection and identification of grapevine pathogens which are important in the production of propagating stocks by q-PCR. Additional novel techniques that use DNA amplification, hybridization and  sequencing are also briefly reviewed.

scholarly journals Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

2006 ◽  
Vol 72 (3) ◽  
pp. 2031-2042 ◽  
Author(s):  
Linda N. Ward ◽  
Asim K. Bej
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Detection Methods ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Reading Frame ◽  
Initial Inoculum ◽  
Hemolysin Gene ◽  
Pure Cultures

ABSTRACT We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

scholarly journals Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Beatrice Barda ◽  
Rahel Wampfler ◽  
Somphou Sayasone ◽  
Khampheng Phongluxa ◽  
Syda Xayavong ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Content Type ◽  
Stool Samples ◽  
Dna Extraction Methods ◽  
Baermann Method

ABSTRACT Strongyloides stercoralis is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against S. stercoralis in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for S. stercoralis diagnosis.

scholarly journals Congenital Malaria in Newborns Delivered to Mothers with Malaria-Infected Placenta in Blue Nile State, Sudan

2020 ◽  
Vol 66 (4) ◽  
pp. 428-434
Author(s):  
Samia A Omer ◽  
Ishag Adam ◽  
Ali Noureldien ◽  
Hadeel Elhaj ◽  
Laura Guerrero-Latorre ◽  
...  
Keyword(s):  
Cord Blood ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Molecular Techniques ◽  
Blue Nile ◽  
Blood Smears ◽  
Polymerase Chain ◽  
Thick Smear ◽  
Congenital Malaria

Abstract Diagnosis of congenital malaria is complicated by the low density of the parasite circulating in the cord blood and/or the peripheral blood of the newborns. Molecular techniques are significantly more sensitive than blood smears in detecting low-level parasitemia. This study investigated the prevalence of congenital malaria by the use of the real-time polymerase chain reaction (real-time PCR) in 102 babies born to mothers with microscopically confirmed infected placenta from Blue Nile state, Sudan. At delivery time, placental, maternal peripheral and cord blood samples in addition to samples collected from the newborns’ peripheral blood were examined for malaria infection using Giemsa-stained thick smear and parasite DNA detection by real-time PCR. The overall prevalence of congenital malaria includes the total babies with cord blood parasitaemia and peripheral blood parasitaemia was 18.6 and 56.8% using microscopy and real-time PCR, respectively. Even though all the neonates were aparasitaemic by microscopy, 19 (18.6%) of the babies had congenital malaria detected by real-time PCR, 15 (25.9%) of the babies with congenital malaria were born to mothers with both placental and peripheral blood malaria infections detected using the two techniques. Congenital malaria was significantly associated with cord blood malaria infections, maternal age and maternal haemoglobin level (p &lt; 0.001). This first study investigating congenital malaria in Blue Nile state, Sudan shows that malaria-infected placenta resulted in infant and cord blood infections.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

2008 ◽  
Vol 56 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Jitu Patel ◽  
Arvind Bhagwat
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Molecular Beacon ◽  
False Negative ◽  
Detection Methods ◽  
Time Data ◽  
Pcr Assay ◽  
Serovar Typhimurium ◽  
Meat Samples

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

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