scholarly journals Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays

2008 ◽  
Vol 57 (9) ◽  
pp. 1099-1105 ◽  
Author(s):  
N. Jothikumar ◽  
A. J. da Silva ◽  
I. Moura ◽  
Y. Qvarnstrom ◽  
V. R. Hill
Keyword(s):  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Laboratory Diagnosis ◽  
Taqman Assay ◽  
Cryptosporidium Hominis ◽  
Specific Assay ◽  
Outbreak Investigations ◽  
Stool Specimens

Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1–10 oocysts in a 300 μl stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94 %. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.

scholarly journals Circulating Gut-Homing (α4β7+) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

2016 ◽  
Vol 23 (7) ◽  
pp. 610-617 ◽  
Author(s):  
Anuradha Sinha ◽  
Ayan Dey ◽  
Giulietta Saletti ◽  
Pradip Samanta ◽  
Partha Sarathi Chakraborty ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Surface Protein ◽  
Protein Antigens ◽  
Magnetic Cell Sorting ◽  
Content Type ◽  
Recent Onset ◽  
Microbiological Methods ◽  
Stool Specimens ◽  
Antibody Secreting Cells

Developing countries are burdened withShigelladiarrhea. Understanding mucosal immune responses associated with naturalShigellainfection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens,pan-Shigellasurfaceprotein antigen 1 (PSSP1) and PSSP2, common to all virulentShigellastrains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α4β7+) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive forShigellainfection by microbiological and real-time PCR assays, respectively. The frequency of α4β7+IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of theShigellaserotype isolated from these patients. Thus, α4β7+ASC responses to Ipas may be considered an indirect marker ofShigellainfection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by naturalShigellainfection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.

scholarly journals Cryptosporidium infection in non-human hosts in Malawi

2009 ◽  
Vol 76 (4) ◽  
Author(s):  
Z. Banda ◽  
Rosely A.B. Nichols ◽  
A.M. Grimason ◽  
H.V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Rainy Season ◽  
18S Rrna ◽  
Cryptosporidium Oocyst ◽  
Dry Season ◽  
Cool Season ◽  
Cryptosporidium Hominis ◽  
Adult Cattle ◽  
Faecal Samples ◽  
Cryptosporidium Oocysts

Of 1 346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3 % were from cattle (29.8 % of these were from calves < 6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6 % of adult cattle and 11.7 % of calves were infected, compared to 28.9 % of adult cattle and 36.7 % of calves in Thyolo. Dependent on season, between 7.8 % and 37.7 % (Chikwawa) and 16.7 % and 39.3 % (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n = 225], pigs [n = 92], sheep [n = 6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6 % of goat samples contained oocysts in Chikwawa, compared to between 16.7 % and 39.3 % in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7 %), whereas in Thyolo, infections occurred in all three seasons (17.9 % in the rainy season, 25 % in the cool season and 60 % in the dry season). Often diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and / or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.

scholarly journals Evaluation of a Sample Pooling Strategy for Sars-cov-2 Using Real Time PCR

2021 ◽  
Author(s):  
Annet M Nankya ◽  
Luke Nyakarahuka ◽  
Stephen Balinandi ◽  
John Kayiwa ◽  
Julius Lutwama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Virus Disease ◽  
Laboratory Diagnosis ◽  
Turnaround Time ◽  
Limited Resources ◽  
Asymptomatic Patients ◽  
Sample Pooling ◽  
Sample Testing ◽  
Pooling Strategy

Abstract Back ground: Corona Virus Disease 2019 (COVID 19) in Uganda was first reported in a male traveler from Dubai on 21st March, 2020 shortly after WHO had announced the condition as a global pandemic. Timely laboratory diagnosis of COVID -19 for all samples from both symptomatic and asymptomatic patients was observed as key in containing the pandemic and breaking the chain of transmission. However, there was a challenge of limited resources required for testing SARS-COV-2 in low and middle income countries. To mitigate this, a study was conducted to evaluate a sample pooling strategy for COVI-19 using real time PCR. The cost implication and the turn around time of pooled sample testing versus individual sample testing were also compared.Methods: In this study, 1260 randomly selected samples submitted to Uganda Virus Research Institute for analysis were batched in pools of 5, 10, and 15. The pools were then extracted using a Qiagen kit. Both individual and pooled RNA were screened for the SARS-COV-2 E gene using a Berlin kit. Results: Out of 1260 samples tested, 21 pools were positive in pools of 5 samples, 16 were positive in pools of 10 and 14 were positive in pools of 15 samples. The study also revealed that the pooling strategy helps to save a lot on resources, time and expands diagnostic capabilities without affecting the sensitivity of the test in areas with low SARS-COV-2 prevalence.Conclusion: This study demonstrated that the pooling strategy for COVID-19 reduced on the turnaround time and there was a substantial increase in the overall testing capacity with limited resources as compared to individual testing.

scholarly journals Sequential Quadriplex Real-Time PCR for Identifying 20 Common emm Types of Group A Streptococcus

2020 ◽  
Vol 59 (1) ◽  
pp. e01764-20
Author(s):  
Srinivasan Velusamy ◽  
Katherine Jordak ◽  
Madeline Kupor ◽  
Sopio Chochua ◽  
Lesley McGee ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Group A Streptococcus ◽  
High Specificity ◽  
The United States ◽  
Rapid Identification ◽  
Invasive Group ◽  
Outbreak Investigations ◽  
Group A ◽  
Culture Negative

ABSTRACTWe developed a sequential quadriplex real-time PCR-based method for rapid identification of 20 emm types commonly found in invasive group A Streptococcus (iGAS) strains recovered through the Centers for Disease Control and Prevention’s Active Bacterial Core surveillance. Each emm real-time PCR assay showed high specificity and accurately identified the respective target emm type, including emm subtypes in the United States. Furthermore, this method is useful for rapid typing of GAS isolates and culture-negative specimens during outbreak investigations.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

scholarly journals Validation of Real-Time PCR for Laboratory Diagnosis of Acanthamoeba Keratitis

2008 ◽  
Vol 46 (10) ◽  
pp. 3232-3236 ◽  
Author(s):  
P. P. Thompson ◽  
R. P. Kowalski ◽  
R. M. Q. Shanks ◽  
Y. J. Gordon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Laboratory Diagnosis ◽  
Acanthamoeba Keratitis
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