Polymerase Chain Reaction Targeting Research Articles

Lack of Targetable FGFR2 Fusions in Endemic Fluke-Associated Cholangiocarcinoma.

PURPOSECholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries.MATERIALS AND METHODSIn this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non–Ov-associated CCA tumors.RESULTSCompared with non–fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers.CONCLUSIONFGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

Co-prevalence of human Papillomaviruses (HPV) and Epstein\u2013Barr virus (EBV) in healthy blood donors from diverse nationalities in Qatar

BackgroundInfections by both human oncoviruses, human Papillomaviruses (HPV) and Epstein–Barr virus (EBV) are very common in the adult human population and are associated with various malignancies. While HPV is generally transmitted sexually or via skin-to-skin contact, EBV is frequently transmitted by oral secretions, blood transfusions and organ transplants. This study aims to determine the prevalence and circulating genotypes of HPV and EBV in healthy blood donors in Qatar.MethodsWe explored the co-prevalence of high-risk HPVs and EBV in 378 males and only 7 females blood donors of different nationalities (mainly from Qatar, Egypt, Syria, Jordan, Pakistan, and India) residing in Qatar, using polymerase chain reaction (PCR). DNA was extracted from the buffy coat and genotyping was performed using PCR and nested-PCR targeting E6 and E7 as well as LMP-1 of HPV and EBV, respectively.ResultsWe found that from the total number of 385 cases of healthy blood donors studied, 54.8% and 61% of the samples are HPVs and EBV positive, respectively. Additionally, our data revealed that the co-presence of both high-risk HPVs and EBV is 40.4% of the total samples. More significantly, this study pointed out for the first time that the most frequent high-risk HPV types in Qatar are 59 (54.8%), 31 (53.7%), 52 (49.1%), 51 (48.6%), 58 (47%) and 35 (45.5%), while the most commonly expressed low-risk HPV types are 53 (50.6%), 11 (45.5), 73 (41.7%) and 6 (41.3%), with all the cases showing multiple HPVs infection.ConclusionIn this study, we demonstrated for the first time that HPV and EBV are commonly co-present in healthy blood donors in Qatar. On the other hand, it is important to highlight that these oncoviruses can also be co-present in several types of human cancers where they can cooperate in the initiation and/or progression of these cancers. Therefore, more studies regarding the co-presence of these oncoviruses and their interaction are necessary to understand their cooperative role in human diseases.

Diagnostic value of polymerase chain reaction targeting insertion sequence IS1081 for the diagnosis of pediatric tuberculosis

Background: Aim of this study was to evaluate the efficacy of PCR targeting IS1081in diagnosis of pediatric tuberculosis and compare the results with MGIT culture.Methods: This prospective study was conducted in the department of pediatrics, S.N. medical college, Agra. 100 subjects (28 pulmonary 72 extra pulmonary) were registered in study. The specimens obtained from these cases were subjected to Ziehl–Neelsen staining (ZN), MGIT 960 TB culture and PCR targeting insertion sequence IS1081. Sensitivity, specificity, PPV and NPV of PCR were calculated in pulmonary and extra pulmonary specimens. The results of PCR IS1081 were compared to MGIT culture.Results: Microscopy with ZN staining was positive in 12 (12%) samples. MGIT culture was positive in 44% samples with maximum positivity in sputum (70%). PCR IS1081 has shown 93.3% sensitivity in pulmonary tuberculosis, while PCR IS1081 has shown 93.1% sensitivity in extra pulmonary tuberculosis. In diagnosis of childhood tuberculosis PCR IS1081 was found to be statistically significant (p value <0.05) as compared with MGIT culture. Result was statistically significant (p value <0.05) in CSF samples only.Conclusions: The study concluded that the PCR targeting sequence IS1081 technique is the most sensitive technique for a quick identification of MTB in pulmonary and extra pulmonary tuberculosis.

High-throughput sequencing for confirmation of suspected 2019-nCoV infection identified by fluorescence quantitative polymerase chain reaction.

To the Editor: Identifying the pathogen from clinical samples is crucial for the diagnosis of a newly emergent infectious disease, such as 2019 novel coronavirus (2019-nCoV), which has posed great threats to global public health.[1] In some cases, despite the positive epidemiological, clinical, and radiographic evidence, coronavirus disease 2019 (COVID-19) diagnosis can still be restricted by inconclusive polymerase chain reaction (PCR) results.[2] For samples of suspicious patients, where the fluorescence quantitative PCR (FQ-PCR) results in a few respiratory tract specimens were inconclusive, high-throughput sequencing (HTS) can be an effective confirmation method. Here, we report our experience of applying HTS to confirm a suspected 2019-nCoV infection. Three upper respiratory tract specimens including throat or nasal swabs were collected from a 65-year-old pneumonia patient with suspected history of contact with COVID-19 patients in Guangzhou on January 22 and 23, 2019. Patient consent was not applicable for this study as the samples were obtained as part of a routine diagnostic procedure. All the clinical specimens were handled at the State Key Laboratory of Respiratory Disease, Guangzhou, China. The initial 2019-nCoV screening results of the sample was negative by FQ-PCR with a commercial FQ-PCR kit (cycle threshold [CT] values of open reading frame 1ab [ORF1ab] and nucleoprotein [N] genes were >40 [#1 throat swab sample], where CT > 40 is considered negative). In view of the epidemiological link and clinical symptoms of the patient, a second screening using another FQ-PCR kit was performed and the range of CT value was weakly positive for the 2019-nCoV viral RNA (CT values of ORF1ab/ envelope protein [E]/N genes of three samples were 34.44/31.58/35.50 [#1 throat swab sample], >40/36.37/38.45 [#2 nasal swab sample], and 33.40/34.36/33.10 [#3 throat swab sample]). The test results of the two kits were inconsistent; therefore, to make a definite diagnosis, we performed meta-transcriptomic sequencing using the same throat swab sample (#1 throat swab sample) from the patient. A meta-transcriptomic library was constructed for single-end (75 base pairs) sequencing on the Illumina NextSeq 550Dx (Illumina, San Diego, CA, USA). The sequencing data were analyzed with the rapid pathogen detection (RPD-seq) system of the Sagene Company in Guangzhou, China, as previously reported.[3] In total, we obtained ten unique sequence reads that completely mapped onto the 2019-nCoV genome (GenBank accession No. MN908947.3). We considered this sample to be weakly positive rather than contaminated, based on the results that the ten reads are evenly distributed throughout the entire gene sequence of the virus. It is worth noting that these regions of ORF1ab, E and N genes were used as the standard PCR targets [Figure 1].Figure 1: High-throughput sequencing for 2019 novel coronavirus (2019-nCoV) of the throat swab sample (#1) from the patient. Ten unique reads were found, and eight of them on the largest ORF1ab gene, while one of them was located on the second largest spike protein gene-S and another one on the envelope protein gene-E of the 2019-nCoV genome.According to the national diagnosis and treatment plan released by the National Health Commission of the People's Republic of China, positive viral RNA FQ-PCR or virus gene sequencing results were the diagnostic criteria of COVID-19.[4] However, the different performance of FQ-PCR kits may lead to inconsistent results. Here we reported a case with inconsistent FQ-PCR results, for which the HTS was used to make a further diagnosis of the 2019-nCoV infection. Although HTS may be too costly and labor intensive for routine diagnosis, we believe that it can be used for further diagnosis of patients with COVID-19 with unclear FQ-PCR results under the condition of strict operation and quality control. Acknowledgements The authors are greatly indebted to Prof. Sooksan Wong, for her valuable suggestions on writing of the manuscript. Funding This work was supported by grants from the National Key Research and Development Program of China (Nos. 2018YFC1200100 and 2018YFC1311900), science research project of the Guangdong Province (No. 2019B030316028), and Guangzhou Medical University High-level University Clinical Research and Cultivation Program (Nos. [2017]159 and 160). Conflicts of interest None.

In-Depth Study of a Nosocomial Outbreak Caused by Extensively Drug-Resistant Pseudomonas aeruginosa Using Whole Genome Sequencing Coupled With a Polymerase Chain Reaction Targeting Strain-Specific Single Nucleotide Polymorphisms.

In 2013-2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011-2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active.

Identification of Methicillin-Resistant Staphylococcus aureus in Bulk Tank Milk

Abstract Staphylococcus aureus is the leading cause of intoxications in humans. Also, methicillin-resistant S. aureus (MRSA) is an emerging problem in food-producing animals. The presence of MRSA in milk may create a risk to public health. The aim of this study was to evaluate the presence of MRSA in bulk tank milk samples. One hundred and twenty bulk tank milk samples were analyzed using the convenient sampling method. The isolates were confirmed by real-time polymerase chain reaction (RT-PCR) targeting nuc and mecA gene in S. aureus. Antibiotic resistance profiles of the isolates were tested by disc agar diffusion method. In the current study, S. aureus was detected in 44 (36.66%) of the samples. The presence of the mecA gene was found to be positive in 40 (75.4%) of the 53 S. aureus isolates. As a result of the study, a high incidence of MRSA was detected in bulk tank milk samples. Antibiotic-resistant bacteria are at risk of being transferred to humans via milk. For safe and healthy milk consumption, uncontrolled use of antibiotics in dairy cows should be avoided.

Novel Duplex Polymerase Chain Reaction for the Rapid Detection of Pythium insidiosum Directly From Corneal Specimens of Patients With Ocular Pythiosis.

To standardize a novel duplex polymerase chain reaction (PCR) targeting 18S rRNA gene and internal transcribed spacer region for the identification of Pythium insidiosum isolates and also to detect P. insidiosum genome directly from corneal specimens of patients with suspected ocular pythiosis. A total of 42 nonsporulating molds culturally and morphologically resembling suspected unidentified fungal isolates (corneal buttons 33 and corneal scrapings 9) and 14 clinical specimens (corneal buttons 7 and corneal scrapings 7) clinically suspected to be ocular pythiosis were included in the present study. Standardization of uniplex PCRs and duplex PCRs targeting 18S rRNA gene and internal transcribed spacer region and further application of the standardized PCRs on both clinical isolates and clinical specimens suspected to have fungal keratitis. The sensitivity and specificity of the standardized duplex PCR were calculated using Medcal.net software. The standardized uniplex and duplex PCRs were found specific for the detection of only P. insidiosum DNA, and the analytical sensitivities of the primers were 1.36 Zg. Of the 14 clinical specimens analyzed, 13 were positive in both corneal specimens and their respective P. insidiosum isolates. The specificity of the novel duplex PCR was 100% when applied on corneal specimens and clinical isolates, but the sensitivity was 92.8% (13/14) and 100% (42/42), respectively, for the clinical specimens and fungal isolates from suspected ocular pythiosis patients included in the study. The novel duplex PCR developed in this study will aid in rapid identification of P. insidiosum clinical isolates and clinical specimens from suspected ocular pythiosis specimens, which in turn will help the ophthalmologists to initiate appropriate treatment.

Detection of Zika Virus Replication in Human Semen by Reverse-Transcription Polymerase Chain Reaction Targeting of Antisense Ribonucleic Acid.

Persistence of Zika virus (ZIKV) ribonucleic acid (RNA) in semen is common after infection. We designed a reverse-transcription polymerase chain reaction assay that targets antisense ZIKV RNA (asRNA) to assess ZIKV replication competence in ZIKV RNA-positive semen samples. We detected ZIKV asRNA in semen of 9 of 19 men (47.4%) diagnosed with ZIKV infection. All asRNA-positive samples had high ZIKV loads (cycle threshold values <26) and were obtained within 21 days of symptom onset. The sensitivity of the asRNA assay for detection of ZIKV replication was higher than that of conventional virus isolation methods (47.4% vs 21.1%, P = .032).

Screening of Blast Resistance Genes from South Tapanuli Rice Cultivars, North Sumatra, Indonesia

Rice blast disease caused by a phytopathogenic fungus, Magnaporthe oryzae, has been considered as a major threat to global rice productivity, including Indonesia. South Tapanuli is a regency in North Sumatra, known for its intensive and significant rice production activities. A collection of 13 rice cultivars originating from the region, is claimed for its resistances against the rice blast disease based on the practices by the local farmers. Molecular screening of blast resistance (R) genes was initiated to the 13 accessions using the Polymerase Chain Reaction (PCR) targeting five blast resistance (R) genes, Pup1, Pi-37, Pi-d2, Pi-ta2, Pib and Pi-kh. Out of the 13 accessions, the Pi-d2, Pi-ta2 and Pib were detected in all cultivars, showing 100% of R gene frequency. The second dominant, Pup1 and Pi-37 gene frequencies were 76.9 and 61.5%, respectively while the least dominant, Pi-kh gene frequency was 15.4%. The number of R genes detected among cultivars was ranged between 3 to 6 genes. The genetic associations among thirteen germplasms were determined using unweighted pair group method with arithmetic mean (UPGMA) analysis. Cluster analysis revealed that the least blast-resistant cultivars: Putri Kembar, Silatian and Siganteng, were grouped into cluster 1, showing polymorphism for Pi-d2, Pi-ta2 and Pi-b. The high-resistant cultivars, Martabe-Sicondong and Sayuti, Sitolas, Pulo Raja, Pulo Pandan, Siporang and Pulo Manggis, were grouped into cluster 2 and 3, respectively. The medium blast-resistant cultivars, IR 64 and Sitampan were grouped into cluster 4, showing polymorphism for Pup1, Pi-d2, Pi-ta2 and Pi-b. These results indicated that the utilization of blast-resistant cultivars in North Tapanuli was supported by the presence of R genes.

Rapid detection of mycobacterium tuberculosis complex by real-time polymerase chain reaction (PCR) in pulmonary and extra-pulmonary samples in Casablanca, Morocco

Introductionthe laboratory diagnosis of tuberculosis (TB) relies mainly on conventional techniques. However, it either lacks sensitivity or it is time-consuming. This study aims to evaluate the use of real-time polymerase chain reaction (PCR) targeting IS6110 for Mycobacterium tuberculosis (TB) Complex (MTBC) in the routine diagnosis of TB in our laboratory.Methodsclinical samples were collected from the laboratory of bacteriology at Ibn Rochd University Hospital in Casablanca Morocco. Real-time polymerase chain reaction (PCR) results were compared to AFB smear and culture on Löwenstein-Jensen (LJ) solid media. Sensitivity, specificity, positive and negative predictive value (PPV and NPV) with 95% confidence intervals were calculated using GraphPad Prism.Resultson 171 clinical samples, the study showed positivity of microscopy, culture and real-time PCR for M. TB complex as 19%, 31%, and 32% respectively. Sensitivity, specificity, PPV and NPV for real-time PCR in pulmonary samples were 95.2%, 95.4%, 90.91% and 97.65% respectively. For extra-pulmonary samples, they were: 72.7%, 90.32%, 72.7%, and 90.3%.Conclusionour study shows the effectiveness of using real-time PCR IS6110 in pulmonary and extra pulmonary samples. Future multicentric studies could seek to evaluate the place of this technique on routine diagnosis for better management of TB in Morocco.

Detection of scrub typhus by real-time polymerase chain reaction and immunoglobulin M ELISA among patients with acute febrile illness

Background: Scrub typhus caused by Orientia tsutsugamushi is a vector-borne zoonotic infection endemic in several parts of the globe. The infection generally presents with fever and nonspecific clinical features but may lead to severe complications with a high mortality rate if untreated. Early diagnosis and timely management are therefore important. Serological diagnosis such as Weil–Felix test, indirect immunofluorescence assay, immunoglobulin (Ig) M/IgG ELISA, and rapid antibody detection assays are either less sensitive or laborious. Molecular detection by polymerase chain reaction (PCR) targeting specific gene targets of O. tsutsugamushi is warranted. Materials and Methods: We developed a real-time PCR assay targeting 47-KDa htrA gene for the specific diagnosis of the pathogen. The assay was evaluated in a buffy coat from whole blood or serum samples collected from patients presenting with acute febrile illness. Randomly selected samples were also tested for IgM by commercial IgM ELISA assay. Results: The real-time PCR assay was able to detect <1 genome copy per the PCR input and specific to O. tsutsugamushi on heterologous pathogens testing. The samples were negative by real-time PCR and 13 samples were positive by IgM ELISA. This study found a relatively low prevalence of scrub typhus in the population. Conclusion: The assay developed in this study could be a useful diagnostic tool for the detection of O. tsutsugamushi in clinical samples. The study also indicated the need for a wide epidemiological survey that could help determine appropriate health measures including treatment and prevention.

Association of disease severity with toll-like receptor polymorphisms in multidrug-resistant tuberculosis patients.

The disease severity in pulmonary Multidrug-resistant tuberculosis (MDR-TB) varies from mild to severe, which is determined by host and pathogen virulence factors. The difference of symptoms felt by TB patients were interesting to investigate in discovering whether its the human immune response or bacteria's virulence gene that plays the role. The aim of this research was to analyze association between disease severity degree of pulmonary MDR-TB patients with Single nucleotide polymorphisms (SNPs) found in toll-like receptors (TLRs) gene. Blood samples were obtained from pulmonary MDR-TB patients in Dr. Soetomo Hospital, Surabaya, Indonesia. Polymerase chain reaction (PCR) multiplex and target SNPs were analyze using DigiTag2 assay. The variant of esxA gene was determined using PCR and sequencing. Severity degree was determined by chest X-ray, the lesions were scored according to their severity, score of =2.5 ranking as mild, 2.5-6 as moderate and =6 as severe. Association level between SNP in TLRs gene degree of pulmonary MDR-TB was analyzed using Chi-square test. Bonferroni correction for multiple comparison was used to anticipate genotyping error. A total of 22 MDR-TB patients were classified into severe degree group, while 16 patients were moderate/mild degree. SNPs in encoding gene of TLRs were mostly found in intron, specifically in TLR-1, TLR-2, and TLR-6. HWE P value in rs5743572 was 0.841; in rs3804100 was 0.0176; and in rs5743808 was 0.562. Association analysis between SNP in TLRs genes and degree of disease revealed significant association in rs5743572, SNP of TLR-1, with P < 0.05; odds ratio [OR] = 11.67 (95% confidence interval [CI]: 3.94-34.52); rs3804100, SNP of TLR-2 had P < 0.05; OR = 37.59 (95% CI: 9.30-151.88); and rs5743808, the SNP of TLR-6 had P < 0.05; OR = 31.5 (95% CI: 8.60-115.34). We concluded that SNPs in TLR-1, TLR-2, and TLR-6 of pulmonary MDR-TB patients was found to have an association with disease severity. TLRs polymorphism had significant association was present in TLR-1 rs5743572 in intron, TLR-2 rs3804100 in exon, and TLR-6 rs5743808 in exon and among MDR-TB isolates from patients with pulmonary MDR-TB of severe and moderate/mild degree.

Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases.

Background & objectives:In India, spotted fever group rickettsiae (SFGR) are an underdiagnosed cause of acute febrile illness (AFI). The non-specific Weil-Felix test is the first diagnostic modality for the diagnosis of SFGR in many laboratories due to the lack of advanced diagnostic facilities in developing countries. The aim of this study was to detect SFGR using molecular methods in the patients, presenting with AFI in a tertiary care centre in north India.Methods:Consecutive patients (>14 yr of age) with AFI were enrolled over a six month period. Standard investigations for common pathogens causing AFI in India (malaria, dengue, scrub typhus, leptospirosis and enteric fever) were carried out. In patients who were negative for all of the above investigations, blood was subjected to polymerase chain reaction (PCR) targeting outer membrane protein A (ompA) gene of Rickettsia.Results:Of the 51 patients with an undiagnosed aetiology, three were positive by ompA PCR. Two of the PCR products produced good sequences and BLAST identification confirmed them as Rickettsia conorii. The sequences of R. conorii reported from south India clustered with two previously reported novel rickettsial genotypes. The study sequences clustered in a group different from that of Rickettsia spp. of the south Indian sequences reported earlier.Interpretation & conclusions:This study showed the existence of R. conorii in north India. Testing for SFGR may be included in the diagnostic workup of AFI for better disease management.

Benefits of Polymerase Chain Reaction Combined With Culture for the Diagnosis of Bone and Joint Infections: A Prospective Test Performance Study.

BackgroundThe microbiological diagnosis of bone and joint infections (BJI) currently relies on cultures, and the relevance of molecular methods is still debated. The aim of this study was to determine whether polymerase chain reaction (PCR) could improve the etiological diagnosis of BJI.MethodsA prospective study was conducted during a 4-year period at Lariboisiere University Hospital (Paris, France), including patients with suspicion of infectious spondylodiscitis, septic arthritis, prosthetic joint infections, and respective noninfected groups. Clinical and radiological data were collected at inclusion and during follow-up. All samples were analyzed by conventional cultures and 16S ribosomal deoxyribonucleic acid (rDNA) gene (16S-PCR). Specific cultures and PCR targeting Mycobacterium tuberculosis were also performed for spondylodiscitis samples. Case records were subsequently analyzed by an independent expert committee to confirm or invalidate the suspicion of infection and definitively classify the patients in a case or control group. The sensitivity of the combination of culture and PCR was compared with culture alone.ResultsAfter expert committee analysis, 105 cases of BJI cases and 111 control patients were analyzed. The most common pathogens of BJI were staphylococci (30%), M tuberculosis (19%), and streptococci (14%). Adding PCR enhanced the sensitivity compared with culture alone (1) for the diagnosis of M tuberculosis spondylodiscitis (64.4% vs 42.2%; P < .01) and (2) for nonstaphylococci BJI (81.6% vs 71.3%; P < .01). It is interesting to note that 16S-PCR could detect BJI due to uncommon bacteria such as Mycoplasma and fastidious bacteria.ConclusionsOur study showed the benefit of 16S-PCR and PCR targeting M tuberculosis as add-on tests in cases of suspected BJI.

Preliminary Detection of Rickettsiae Using PCR Targeting OmpA Gene Among Dogs and Horses in Cairo, Egypt

Background: Rickettsioses are intracellular bacterial infectious diseases causing high morbidity and sometime high mortality with public health importance. Aim: The aim of present study was to detect rickettsiae in dogs and horses in Cairo, Egypt by using Polymerase Chain Reaction (PCR) targeting OmpA gene. Methods: A total number of 200 blood samples (100 from dogs and 100 from horses) were collected from Cairo province, Egypt. All blood samples were investigated by PCR using OmpA gene to detect the rickettsial infection. Furthermore, hematological examination for all blood samples was performed. Results: PCR detection of rickettsiae using OmpA gene amplification revealed that the infection rate was 18 % and 72 % in dogs and horses, respectively. Regarding hematological changes, hypochromic anemia, significant increase in neutrophils, lymphopenia and monocytopenia were recorded in rickettsiae positive dogs. Meanwhile, thrombocytopenia, leucopenia and monocytosis were recorded in rickettsiae positive horses. Conclusion: The detection of rickettsiae in dogs and horses indicated that dogs and horses play a critical role as sentinels in an epidemiological approach of rickettsiae in Cairo, Egypt.

Clostridium perfringens type A Causing Necrotic Enteritis Outbreaks among Chickens in Egypt

Undoubtedly, necrotic enteritis is identified as one of the most threatening diseases which face poultry industry in Egypt and need radical solution to avoid huge economic losses. The present work was designed for typing Clostridium perfringens isolated from diseased chickens suspected to have necrotic enteritis from different outbreaks in six Governorates in Egypt (Gharbia, Dakhalia, Sharkia, Ismailia, North Sinai and Kafr El Sheikh) during the period from August to December 2018. Intestine and liver samples were obtained from eighty six diseased broiler chickens representing 60 flocks. C. perfringens was isolated and toxigenicity of the recovered isolates was determined by Nagler's and dermonecrotic reactions. Furthermore, multiplex polymerase chain reaction (PCR) targeting alpha, beta, epsilon and iota toxins' genes was performed for result confirmation and typing of the toxigenic isolates. Sixty-six C.perfringens isolates (38.37%) were recovered from 172 intestine and liver samples and twenty isolates (30.3%) were toxigenic and typed as C. perfringens type A producing alpha toxin only. These findings established the fact that alpha toxin is the only main toxin of C. perfringens type A which is basically responsible for its pathogenicity and virulence. In addition, most of the positively toxigenic isolates were isolated from hepatic lesions (15 isolates) rather than intestinal lesion (5 isolates). In conclusion, alpha toxin is a major toxin for NE development in chickens. Genotyping of Clostridium perfringens by multiplex PCR is a useful adjunct to diagnosis of necrotic enteritis in chickens.

Comparison of an in house and a commercial real-time polymerase chain reaction targeting Toxoplasma gondii RE gene using various samples collected from patients in Turkey

BackgroundToxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. In this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions.MethodsA total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples.ResultsThe compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content.ConclusionsBoth the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. In house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30–45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

Recombinant adenoviral vaccine encoding the spike 1 subunit of the Middle East Respiratory Syndrome Coronavirus elicits strong humoral and cellular immune responses in mice.

Background and Aim:Middle East respiratory syndrome coronavirus (MERS-CoV) has rapidly spread throughout the Middle East since its discovery in 2012. The virus poses a significant global public health threat with potentially devastating effects. In this study, a recombinant adenoviral-based vaccine encoding the spike 1 (S1) subunit of the MERS-CoV genome was constructed, and its humoral, and cellular immune responses were evaluated in mice.Materials and Methods:Mice were immunized initially by intramuscular injection and boosted 3 weeks later by intranasal application. Expression of the S1 protein in the lungs and kidneys was detected using conventional polymerase chain reaction (PCR) and immunohistochemistry (IHC) targeting specific regions within the S1 subunit at weeks 3, 4, 5, and 6 after the first vaccination. Antigen-specific humoral and cellular immune responses were evaluated in serum and in cell culture following in vitro stimulation with a specific 9-mer epitope within the S1 protein (CYSSLILDY).Results:S1 protein expression was only detected by IHC in the kidneys of the Ad-MERS-S1 group at week 6 from first immunization, and in both lungs and kidneys of Ad-MERS-S1 group by conventional PCR at weeks 3 and 5 post-prime. The vaccine elicited a specific S1-immunoglobulin G antibody response, which was detected in the sera of the vaccinated mice at weeks 4 and 6 from the onset of the first immunization. There was a significant increase in the amount of Th1-related cytokines (interferon-γ and interleukin [IL] 12), and a significant decrease in the Th2-related cytokine IL-4 in splenocyte cell culture of the vaccinated group compared with the control groups.Conclusion:The results of this study suggest that this recombinant adenovirus vaccine encoding the S1 subunit of MERS-CoV elicits potentially protective antigen-specific humoral and cellular immune responses in mice. This study demonstrates a promising vaccine for the control and/or prevention of MERS-CoV infection in humans.

Developing molecular diagnostics for detection of red imported fire ants using two genes, Sinv11108 and Sinv11977.

Aggressive red imported fire ants (RIFAs) are expanding their habitat due to active international trade and global warming. To prevent infestation and settlement, RIFAs must be removed during the quarantine process. Because RIFAs are social insects and have different morphological characteristics depending on their castes, non-ant taxonomists have difficulty confirming RIFAs based on their morphological characteristics alone. The disadvantages of previously reported RIFA molecular diagnostics are that they require additional steps, such as restriction enzyme digestion followed by agarose gel electrophoresis separation or DNA sequence verification for polymerase chain reaction (PCR)-amplified products. To overcome these drawbacks, two RIFA-specific genes were selected and used to develop diverse PCR-based RIFA molecular diagnostic techniques. We found that RIFAs could be confirmed by conventional PCR targeting of two RIFA-specific genes followed by agarose electrophoresis separation. In addition, TaqMan probe real-time PCR methods had the advantage of confirming RIFAs immediately after the reactions were completed by observing fluorescence indexes. Finally, multiplex PCRs enhanced RIFA specificity and sensitivity. The new molecular diagnostic methods developed in this study had the advantages of reducing false positive and negative results together with high specificity and sensitivity for RIFAs.