Comparative Study of Eleven Mechanical Pretreatment Protocols for Cryptosporidium parvum DNA Extraction from Stool Samples

Nowadays, many commercial kits allow the polymerase chain reaction (PCR) detection of Cryptosporidium deoxyribonucleic acid (DNA) in stool samples, the efficiency of which relies on the extraction method used. Mechanical pretreatment of the stools using grinding beads has been reported to greatly improve this extraction step. However, optimization of this key step remains to be carried out. Indeed, many parameters could influence the pretreatment performances, among which the modulation of the speed and duration of the grinding step, in addition to the physicochemical features of the grinding beads, have never been evaluated to date. In this study, eleven commercial mechanical pretreatment matrixes (Lysis matrix tubes®, MP Biomedical, Irvine, CA, USA) composed of beads with different sizes, shapes, and molecular compositions, were evaluated for their performances in improving Cryptosporidium parvum oocyst DNA extraction before amplification by using our routinely used real-time PCR method. As expected, the eleven commercial mechanical pretreatment matrixes showed varying performances depending on the composition, size, and shape. All in all, the best performances were obtained when using the Lysing matrix, including ceramic beads with a median size (diameter of 1.4 mm).

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Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

Microorganisms ◽

10.3390/microorganisms8091450 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1450

Author(s):

Nicolas Valeix ◽

Damien Costa ◽

Louise Basmaciyan ◽

Stéphane Valot ◽

Anne Vincent ◽

...

Keyword(s):

Comparative Study ◽

Real Time ◽

Dna Extraction ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Sample Pretreatment ◽

Dna Amplification ◽

Extraction Methods ◽

Mechanical Pretreatment ◽

Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

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Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

BMC Microbiology ◽

10.1186/s12866-020-01963-9 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Beatrice Barda ◽

Christian Schindler ◽

Rahel Wampfler ◽

Shaali Ame ◽

Said M. Ali ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Controlled Trial ◽

Bead Beating ◽

Novel Treatments ◽

Two Samples ◽

Stool Samples ◽

Pcr Method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

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Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP and human RP genes

10.21203/rs.3.rs-308136/v1 ◽

2021 ◽

Author(s):

Huseyin Tombuloglu ◽

Hussein Sabit ◽

Ebtesam Al-Suhaimi ◽

Hamoud Al-Khallaf ◽

Juma Kabanja ◽

...

Keyword(s):

Detection Method ◽

Limit Of Detection ◽

Virus Disease ◽

False Negative ◽

Rt Pcr ◽

Medical Centers ◽

Efficient Control ◽

Pcr Method ◽

Polymerase Chain ◽

Commercial Kits

Abstract Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative accuracy, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, some of these assays have the disadvantages of taking time to show the result and might produce false-negative and false-positive ones. Therefore, designing rapid and accurate PCR-based testing assay is of paramount importance for early detection of this virus and for more efficient control of the spread of this disease. We, here, describe a fast, reliable, easy-to- use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N2 and RdRP) and a human gene (RP) simultaneously. The performance and the accuracy of the assay was tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.25 copies/µL or 5 copies/reaction. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

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BACTERIAL DNA EXTRACTION METHOD FOR THE DETECTION OF HELICOBACTER PYLORI FROM HUMAN FECAL SAMPLES

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2020.v13i6.37344 ◽

2020 ◽

pp. 38-40

Author(s):

JHOAN GUAMÁN ◽

FAVIAN BAYAS-MOREJÓN ◽

ELENA BRITO ◽

NORMA PAREDES

Keyword(s):

Helicobacter Pylori ◽

Dna Extraction ◽

Conventional Polymerase Chain Reaction ◽

Chain Reaction ◽

Bacterial Dna ◽

Dna Extraction Method ◽

Stool Samples ◽

Chelex Resin ◽

Polymerase Chain ◽

Bacterial Dna Extraction

Objective: The work proposed the implementation of a method of DNA extraction for the detection of the pathogen from 50 stool samples. Methods: A method of DNA extraction with Chelex resin was applied and then detected by conventional polymerase chain reaction (PCR). Results: By PCR, 11 samples were positive for Helicobacter pylori. Conclusions: The Chelex extraction methodology allows obtaining DNA with quality necessary to be detected by PCR, making it a fast methodology for its diagnostic application.

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Comparison of Molecular and Parasitological Methods for Diagnosis of Human Trichostrongylosis

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.759396 ◽

2021 ◽

Vol 11 ◽

Author(s):

Mehdi Pandi ◽

Meysam Sharifdini ◽

Keyhan Ashrafi ◽

Zahra Atrkar Roushan ◽

Behnaz Rahmati ◽

...

Keyword(s):

Agar Plate ◽

Rural Populations ◽

Diagnostic Specificity ◽

Pcr Assay ◽

Northern Iran ◽

Wet Mount ◽

Stool Samples ◽

Pcr Method ◽

Polymerase Chain ◽

Fresh Stool

Human trichostrongyliasis is a zoonotic disease that is prevalent among rural populations in some countries. This study was performed to evaluate various parasitological methods and polymerase chain reaction (PCR) for the diagnosis of human trichostrongyliasis. A total of 206 fresh stool samples were collected from residents of endemic villages of Northern Iran. All samples were examined using conventional parasitological methods, including wet mount, formalin ethyl acetate concentration (FEAC), agar plate culture (APC), Harada–Mori culture (HMC), and Willis, along with the PCR technique. Among the total of 206 individuals examined, 72 people (35%) were found infected with Trichostrongylus species using combined parasitological methods. By considering the combined results of parasitological methods as the diagnostic gold standard, the Willis technique had a sensitivity of 91.7% compared with 52.8% for the APC, 40.3% for the HMC, 37.5% for FEAC, and 5.6% for the wet mount technique. The diagnostic specificity of all the parasitological methods was 100%. Furthermore, the PCR method detected Trichostrongylus spp. DNA in 79 fecal samples (38.3%) with a sensitivity of 97.2% and a specificity of 93.3%. According to the current findings, the Willis method was more sensitive than are the other parasitological methods in the diagnosis of human trichostrongyliasis. However, the PCR assay was more sensitive and more reliable in the detection of human trichostrongyliasis in comparison with the parasitological methods.

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A COMPARISON OF RAPID DIAGNOSTIC TEST AND MODIFIED ZIEHL-NEELSEN METHOD IN THE DIAGNOSIS OF CRYPTOSPORIDIOSIS.

Caliphate Medical Journal ◽

10.47837/cmj.202192.2 ◽

2021 ◽

Vol 9 (2) ◽

pp. 539-542

Author(s):

Dahal AS ◽

Daha AS ◽

Okolo OM ◽

Onyedibe IK ◽

Cosmas TN ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Diagnostic Test ◽

Rapid Diagnostic Test ◽

Cryptosporidium Parvum ◽

Severe Illness ◽

Chain Reaction ◽

Predictive Values ◽

Stool Samples ◽

Polymerase Chain ◽

Strip Test

Background: Cryptosporidium parvum as a leading cause of persistent diarrhoea in developing countries causes a more severe illness in patients infected with human immunodeficiency virus (HIV). The intermittent shedding of Cryptosporidium parvum oocysts in stool, even in patients with massive diarrhoea, makes diagnosis difficult. This study evaluated the validity of Crypto-Strip test in comparison to the modified Ziehl-Neelsen for the diagnosis of Cryptosporidium infection using polymerase chain reaction (PCR) as gold standard. Methods: This was a hospital based cross-sectional study of 100 HIV positive patients with diarrhoea at a tertiary health facility in Jos, Nigeria. We collected 15mls of stool sample and relevant information from patients who gave informed consent. The oocytes of Cryptosporidium parvum were identified in the stool samples using modified Ziehl-Neelsen stain and Rapid diagnostic test (Crypto-Strip). We also ran all samples using polymerase chain reaction (PCR). Statistical analysis was with statistical package for Social Sciences (SPSS) version 21. Result: Out of the 100 stool samples analysed, 13 (13%) were positive for cryptosporidiosis by PCR. Crypto- Strips recorded 12 (12%) positive with one (1) being false negative as compared to PCR. Conversely, eight (8%) of the 100 samples collected were positive for Cryptosporidium oocysts using modified Ziehl Neelsen (mZN) stain. Out of the eight positives by mZN stain, two were false positive as both tested negative by the PCR. The Crypto-Strip test kit had a sensitivity of 92.3% and a specificity of 100.0% and also positive and negative predictive values of 100.0% and 98.9%, respectively. Whereas, the modified Ziehl Neelsen stain had a sensitivity of 46.2% and a specificity of 97.7% with a positive and negative predictive value of 75.0% and 92.4%, respectively. Conclusion: In this study, we have shown that Crypto-Strip test was a better diagnostic method for the diagnosis of crypto

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Plasmid-mediated Quinolone Resistance Genes in Salmonella typhi from Patients Attending Selected General Hospitals in Abuja Municipal, Nigeria

Asian Journal of Biochemistry, Genetics and Molecular Biology ◽

10.9734/ajbgmb/2020/v4i230103 ◽

2020 ◽

pp. 45-59

Author(s):

R. Fasema ◽

B. E. Bassey ◽

Y. B. Ngwai ◽

I. H. Nkene ◽

R. H. Abimiku ◽

...

Keyword(s):

General Hospital ◽

Salmonella Typhi ◽

Quinolone Resistance ◽

General Hospitals ◽

Microbiological Methods ◽

Antimicrobial Resistance Profile ◽

Mar Index ◽

Stool Samples ◽

Pcr Method ◽

Polymerase Chain

This study investigated the antimicrobial resistance profile and presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella typhi from patients attending selected general hospitals in Abuja municipal, Nigeria. Four hundred stool samples from patients with suspected typhoid fever were collected from Asokoro General Hospital Abuja (AGH), Garki Hospital Abuja (GHA), Maitama General Hospital Abuja (MGHA) and Wuse General Hospital Abuja (WGHA) and S. typhi was isolated and identified using standard microbiological methods. Antimicrobial susceptibility testing of the isolates was carried out using Clinical and Laboratory Standards Institute (CLSI) method. Molecular detection of PMQR genes in the ciprofloxacin-resistant isolates was carried out using Polymerase Chain Reaction (PCR) method. The overall occurrence of the isolates was 13.3% (53/400), with the highest hospital-related occurrence at WGHA (18.0%). The occurrence was highest at age 21-30yrs in AGHA (20.0%), GHA (33.3%) and WGHA (45.0%). The occurrence was higher in females at AGHA (12.7%) and GHA (16.0%); but higher in males at MGHA (11.4%) and WGHA (18.2%). Resistance to ciprofloxacin was the least at 30.2%, distributed as follows: AGHA (20.0%), GHA (35.7%), MGHA (36.4%) and WGHA (27.8%). The most common resistance phenotype was: NA-S-XT-AMC-TE-CRO-C-CN with overall occurrence of 9.4% (5/53) observed in AGH (10.0%), GHA (16.7%) and MGHA (18.2%) but not in WGHA. All (100%) isolates were multiple antibiotic resistance (MAR) isolates, with MAR indices above 0.2; and the commonest MAR index of 0.6 (30.0%) in AGHA, 0.8 (35.7%) in GHA; 0.8 (45.6%) in MGHA, and was 0.7 (38.9%) in WGHA. Multidrug resistance (MDR) was the commonest at 96.2% (51/53), with occurrences in the selected hospitals as follows: AGHA (90.0%), GHA (100.0%) and MGHA (100.0%) and WGHA (94.4%).The PMQR genes detected had overall frequency in the order: aac(6′)-Ib-cr (50.0%) >qnrB (37.5%) >qnrS (18.8%); qnrS was absent in AGHA and WGHA. The genes co-existed with one another with the qnrB+ aac(6′)-Ib-cr combination, present in isolates from all the hospitals, being the most common at (31.3%). Ciprofloxacin was the most effective antibiotic against the isolates; most isolates were MAR with prior exposure to antibiotics; most isolates were MDR and the ciprofloxacin-resistant isolatesharbored qnrS, qnrB and aac(6′)-Ib-cr PMQR genes, with aac(6′)-Ib-cr being the most prevalent.

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Investigation of duck plague virus in hoar areas of Bangladesh

Bangladesh Journal of Livestock Research ◽

10.3329/bjlr.v26i1-2.49939 ◽

2020 ◽

Vol 26 (1-2) ◽

pp. 73-78

Author(s):

A Hossen ◽

MH Rahman ◽

MZ Ali ◽

MA Yousuf ◽

MZ Hassan ◽

...

Keyword(s):

Cytopathic Effect ◽

Clinical Sample ◽

Chorioallantoic Membrane ◽

Clinical Samples ◽

Isolation And Identification ◽

Duck Plague Virus ◽

Pcr Method ◽

Polymerase Chain ◽

The Individual ◽

Free Ranging

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE). Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

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Prevalence of SHV-extended spectrum β-lactamase producing carbapenem –resistantKlebsiellapneumoniae among patients with lower respiratory tractinfections in Babylon Province-Iraq.

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v9ispl1.1299 ◽

2018 ◽

Vol 9 (SPL1) ◽

Author(s):

Fatima Moeen Abbas

Keyword(s):

Resistance Rate ◽

Combination Method ◽

Diffusion Method ◽

Disk Diffusion Method ◽

Carbapenem Resistant ◽

Extended Spectrum ◽

Pcr Method ◽

Polymerase Chain ◽

Tract Infections ◽

Positive Results

This study was carried out to screen the prevalence of Klebsiella pneumoniae isolated from patients with lower respiratory tract infections in Babylon province.From December,2015 to the end of March,2016,a total of 100 sputum samples were collected from patients visited or hospitalized Merjan Teaching Hospital and Al- Hashimya General Hospital. Fifteenth (65%) isolates were identified as Klebsiellapneumoniae. All bacterial isolates were evaluated for extended spectrum β-lactamase (ESBL) production phenotypically using disk combination method. Eleven (73.3%) isolates were detected as ESBL-producers. Kirby-Bauer disk diffusion method was employed to determine resistance profile of ESBLs-positive isolates. Higher rates of resistance were observed for ampicillin and piperacillin antibiotics with (81.8%) and (72.7%) resistance rate, respectively, while the lowest rate was noticed for imipenem antibiotic (14.28%). Carbapenem-resistant isolates were investigated for blaSHV gene by Polymerase Chain Reaction (PCR) method, 2 (100%) isolates gave positive results.

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Seasonal Differences in Cyclospora cayetanensis Prevalence in Colombian Indigenous People

Microorganisms ◽

10.3390/microorganisms9030627 ◽

2021 ◽

Vol 9 (3) ◽

pp. 627

Author(s):

Hagen Frickmann ◽

Juliane Alker ◽

Jessica Hansen ◽

Juan Carlos Dib ◽

Andrés Aristizabal ◽

...

Keyword(s):

Indigenous People ◽

Rainy Season ◽

Gastrointestinal Symptoms ◽

Dry Season ◽

Seasonal Effects ◽

Chain Reaction ◽

Resource Limited Settings ◽

Resource Limited ◽

Stool Samples ◽

Polymerase Chain

Fecal-orally transmitted cyclosporiasis is frequent in remote resource-limited settings in Central and South America with poor hygiene conditions. In this study, we aimed at assessing seasonal effects on the epidemiology of colonization or infection with C. cayetanensis in Colombian indigenous people living under very restricted conditions. In the rainy season between July and November and in the dry season between January and April, stool samples from indigenous people with and without gastrointestinal symptoms were collected and screened for C. cayetanensis applying in-house real-time polymerase chain reaction (PCR). In the rainy season and in the dry season, positive PCR results were observed for 11.8% (16/136) and 5.1% (15/292), respectively, with cycle threshold (Ct) values of 30.6 (±3.4) and 34.4 (±1.6), respectively. Despite higher parasite loads in the rainy season, fewer individuals (2/16, 12.5%) reported gastrointestinal symptoms compared to the dry season (6/15, 40%). In conclusion, considerable prevalence of C. cayetanensis in Colombian indigenous people persists in the dry season. Low proportions of gastrointestinal symptoms along with higher parasite loads make colonization likely rather than infection.

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