Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock.

Background and Aim: Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and culling of infected animals. Moraxella spp. is common bacterial pathogens that can cause keratoconjunctivitis in livestock. Therefore, rapid and accurate diagnosis is crucial for effective treatment and disease control. This study aimed to develop a multiplex real-time polymerase chain reaction (mRT-PCR) assay for the detection and differentiation of Moraxella bovoculi, Moraxella ovis, and Moraxella bovis. Materials and Methods: Three reference strains of Moraxella as positive controls and 36 lacrimal swab samples collected from cattle were used to evaluate the developed mRT-PCR assay DNA extraction that was performed using the RIBO-sorb DNA/RNA extraction kit. Primers and probes were designed using the SpeciesPrimer pipeline. The annealing temperature, primer and probe concentrations, and sensitivity and specificity of the assay were optimized. Results: An mRT-PCR assay was developed to detect pathogens associated with IBK in cattle on the basis of optimized parameters. The specificity and sensitivity of this assay were confirmed using samples containing individual pathogens (O - M. ovis, B - M. bovis, and BO - M. bovoculi), combinations of two pathogens (O-B, B-BO, and O-BO), and when the DNA of all three pathogens was present in a single reaction (O-B-BO). The analytical sensitivity of mRT-PCR for detecting M. ovis and M. bovoculi DNA was 21 copies or 50 fg per reaction, whereas that for M. bovis was 210 copies or 500 fg per reaction. In addition, this assay has been tested on samples isolated from the affected eyes of cattle in the Akmola region of the Republic of Kazakhstan. Conclusion: For the first time in the Republic of Kazakhstan, the proposed mRT-PCR assay for the simultaneous detection of three Moraxella spp. pathogens has been developed. This assay exhibits the required specificity and high sensitivity for m RT-PCR, facilitating the timely implementation of effective measures for disease control and the prevention of economic losses. These losses are linked to a reduction in livestock breeding value, a reduction in meat and milk production, a reduction in the reproductive performance of heifers, resulting in fewer offspring, as well as costs related to the treatment of affected animals.

Epidemiology of Influenza-like Illness and Respiratory Viral Etiology in Adult Patients in Taiyuan City, Shanxi Province, China between 2018 and 2019.

To determine the epidemiological status of influenza and understand the distribution of common respiratory viruses in adult patients with influenza-like illness (ILI) cases in Taiyuan City, Shanxi Province, China, epidemiological data between 2018 and 2019 were retrieved from the China Influenza Surveillance Information System, and two sentinel ILI surveillance hospitals were selected for sample collection. All specimens were screened for influenza virus (IFV) and the other 14 common respiratory viruses using real-time polymerase chain reaction. The results of the 2-year ILI surveillance showed that 26,205 (1.37%) of the 1,907,869 outpatients and emergency patients presented with ILI, with an average annual incidence of 297.75 per 100,000 individuals, and ILI cases were predominant in children <15 years (21,348 patients, 81.47%). Of the 2713 specimens collected from adult patients with ILI, the overall detection rate of respiratory viruses was 20.13%, with IFV being the most frequently detected (11.79%) and at a relatively lower rate than other respiratory viruses. Further subtype analysis indicated an alternating or mixed prevalence of H1N1 (2009), H3N2, Victoria, and Yamagata subtypes. This study provides a baseline epidemiological characterization of ILI and highlights the need for a nationwide detection and surveillance system for multiple respiratory pathogens.

Association of different microbes and pathogenic factors in cases of infectious bovine keratoconjunctivitis in cattle from Eastern Kazakhstan.

Background and Aim: Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although Moraxella bovis is recognized as an etiologic agent of IBK, other bacterial and viral agents have been suspected to play a role in the pathogenesis of this disease. This study aimed to evaluate samples collected from the eyes of IBK-affected cattle in Eastern Kazakhstan at different stages of IBK for the presence of Mor. bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi, and Bovine Herpes Virus Type 1 (BHV-1) and to characterize Mor. bovoculi pilA gene sequence diversity from Mor. bovoculi positive samples. Materials and Methods: Individual ocular swabs (n = 168) were collected from cattle that had clinical signs of IBK during the summer of 2022 on farms in the Abay region of Kazakhstan. Eye lesion scores (1, 2, and 3) were assigned depending on the degree of ocular damage. Infectious bovine keratoconjunctivitis-associated organisms were detected using a multiplex real-time polymerase chain reaction assay. The Mor. bovoculi pilA gene was sequenced from Mor. bovoculi positive samples. Results: Mycoplasma bovis and BHV-1 were not detected in any of the collected samples. Mycoplasma bovoculi was identified in the majority of samples overall, usually in mixed infection with Moraxella spp. Moraxella bovoculi was detected in 76.2% of animals and predominated in animals with eye lesion scores 2 and 3. Mycoplasma bovoculi was detected only in association with Mor. bovis and/or Mor. bovoculi in animals with eye lesion scores 2 and 3. Moraxella bovis was found in 57.7% of animals and was always identified in association with another organism. Sequencing of the pilA gene in 96 samples from Mor. bovoculi positive samples identified five PilA groups. The majority belonged to PilA group A. However, three new PilA groups were identified and designated PilA groups N, O, and P. Conclusion: The results indicate a high prevalence of Myc. bovoculi and Mor. bovoculi in eyes of cattle with IBK on livestock farms in Eastern Kazakhstan. Additional novel Mor. bovoculi PilA groups were identified.

Detection of causative agents of bacterial pneumonia in hospitalized hajj and umrah cases by multiplex real-time polymerase chain reaction.

Background and Objectives: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is commonly detected in pneumonia patients who travel from the Middle East regions. Besides MERS-CoV, many other pathogenic agents cause pneumonia. Detection of such organisms must be done swiftly, especially in case of the negative MERS-CoV samples. The aim of this study was to identify the pathogenic agents that might account for bacterial pneumonia, from Hajj and Umrah pneumonia cases. Materials and Methods: We conducted a cross-sectional study, 38 pneumonia clinical samples from suffering of Hajj and Umrah in 2017 with negative MERS-CoV were selected. The laboratory testing was done at National Reference Laboratory in Jakarta and performed by multiplex real-time PCR using a FTD respiratory pathogens. Results: Haemophilus influenzae (26.4%) was the most frequent bacteria detected. Other causative agents of bacterial pneumonia identified were Moraxella catarrhalis (20.8%), Klebsiella pneumoniae (13.2%), Streptococcus pneumoniae (9.4%), and Staphylococcus aureus (5.7%). From 38 samples showed that 25 (65.79%) samples were positive with bacteria, including five samples with coinfection. The coinfection were combinations among S. aureus and S. pneumoniae (1/20), S. pneumoniae and K. pneumoniae (1/20), S. pneumoniae and M. catarrhalis (2/20), S. pneumoniae and H. influenzae (2/20), K. pneumoniae and H. influenzae (5/20), and M. catarrhalis and H. influenzae (5/20). Conclusion: Haemophilus influenzae is the most recurrent bacteria to be identified in samples of pneumonia of hajj and umrah cases.

Age and sex trends of Gardnerella vaginalis infection in patients with sexually transmitted infections in Korea.

BACKGROUND AND OBJECTIVES: Gardnerella vaginalis and Candida albicans are the most common causative agents of bacterial vaginosis, and infections with these pathogens lead to inflammation, endometritis, and pruritus. The aim of this retrospective study was to determine the trends of G. vaginalis infections based on real-time PCR data according to age and sex in patients with sexually transmitted diseases. MATERIALS AND METHODS: A total of 59,381 specimens isolated at a clinical laboratory from September 2018 to December 2020 were subjected to real-time PCR for the detection of G. vaginalis DNA. Sample types included catheter, pus, tissue, swab, and urine samples. RESULTS: Among 59,381 samples, 20,718 (34.8%) were positive for G. vaginalis. Of the positive samples, 13,186 (63.7%) were from male patients and 7,532 (36.3%) were from female patients. Average patient age was 39.1 years (the average age of male and female patients was 38.34 and 40.43 years, respectively). Female patients younger than 19 years exhibited the highest incidence of G. vaginalis, at 71.57%, followed by 68.46% incidence in those aged 20-29 years; the lowest incidence was in women aged 40-49 years. Further, among specimen types, the highest number of G. vaginalis-positive specimens was obtained by the swab sampling method. CONCLUSION: From 2018 to 2020 in Korea, the number of tests conducted for bacterial vaginosis has increased, while the incidence of G. vaginalis infections appears to have decreased. the finding that female adolescents have a high tendency to carry the pathogen is important. and for effective surveillance of BV, sampling by cotton swabs and detection by multiplex PCR might be a good approach.

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women.

PURPOSE: Sexually Transmitted Diseases (STDs) can cause sterility and many other problems for women planning pregnancy. Currently, almost 340 million people worldwide suffer from Sexually Transmitted Infections (STIs). This study made attempts to quickly identify STDs' most critical infectious agents using dedicated primers and probes. METHODS: The present study was done on the cervical samples of 200 infertile women. After extracting the total DNA of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium, quantitative methods were employed to determine the rate of target bacteria using multiplex real-time PCR. RESULTS: The multiplex qPCR showed the rates of 47%, 16%, 46%, and 16.5% for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women, respectively. In some patients, there were co-infections with two or three bacteria. The diagnostic approach used in our research could be employed as an alternative detection tool to identify the four most common STD-associated bacterial agents while detecting mixed infections. CONCLUSIONS: Infertile women with no biological problems could have their genital tract checked using this newly designed identification technique and get proper treatment for their infections as quickly as possible.

COVID-19: Multiorgan Dissemination of SARS-CoV-2 Is Driven by Pulmonary Factors.

Multi-organ failure is one of the common causes of fatal outcome in COVID-19 patients. However, the pathogenetic association of the SARS-CoV-2 viral load (VL) level with fatal dysfunctions of the lungs, liver, kidneys, heart, spleen and brain, as well as with the risk of death in COVID-19 patients remains poorly understood. SARS-CoV-2 VL in the lungs, heart, liver, kidneys, brain, spleen and lymph nodes have been measured by RT qPCR using the following formula: NSARS-CoV-2/NABL1 × 100. Dissemination of SARS-CoV-2 in 30.5% of cases was mono-organ, and in 63.9% of cases, it was multi-organ. The average SARS-CoV-2 VL in the exudative phase of diffuse alveolar damage (DAD) was 60 times higher than in the proliferative phase. The SARS-CoV-2 VL in the lungs ranged from 0 to 250,281 copies. The "pulmonary factors" of SARS-CoV-2 multi-organ dissemination are the high level of SARS-CoV-2 VL (≥4909) and the exudative phase of DAD. The frequency of SARS-CoV-2 dissemination to lymph nodes was 86.9%, heart-56.5%, spleen-52.2%, liver-47.8%, kidney-26%, and brain-13%. We found no link between the SARS-CoV-2 VL level in the liver, kidneys, and heart and the serum level of CPK, LDH, ALP, ALT, AST and Cr of COVID-19 patients. Isolated detection of SARS-CoV-2 RNA in the myocardium of COVID-19 patients who died from heart failure is possible. The pathogenesis of COVID-19-associated multi-organ failure requires further research in a larger cohort of patients.

Development and Application of a Multiplex Real-Time Polymerase Chain Reaction Assay for the Simultaneous Detection of Bacterial Aetiologic Agents Associated With Equine Venereal Diseases.

Venereal diseases caused by bacteria are important to the equine industry due to economic losses caused by decline of conception rate in breeding horses. Therefore, identification of infected animals as well as the implementation of appropriate managerial procedures based on accurate diagnosis is critical. In this study, two types of multiplex real-time polymerase chain reaction with high sensitivity and specificity were developed for the simultaneous detection and differentiation of five commonly associated bacterial pathogens of venereal diseases in horses, consisting of Taylorella equigenitalis, Taylorella asinigenitalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus zooepidemicus. The assay was applied to samples collected as part of the surveillance of T.equigenitalis infection in South Korea. Swab samples collected from horses in 2015 were tested. T. equigenitalis and K. pneumoniae was detected in 21 (21.0%) and two (2.0%) samples, respectively. No samples were positive for T. asinigenitalis, P. aeruginosa, and S. zooepidemicus. Application of this assay to an existing surveillance program has allowed for an enhanced surveillance for a wider range of venereal diseases of equine to be implemented in South Korea.

Multiplex Real-Time Polymerase Chain Reaction on Sputum for the Diagnosis of Pneumocystis jirovecii Pneumonia in Children: A Retrospective Study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) is a serious opportunistic infection in immunocompromised children. Real-time polymerase chain reaction (PCR) is widely used for the diagnosis of PCP due to its good accuracy. However, the diagnostic performance of multiplex real-time PCR on sputum in children with PCP has never been explored. METHODS: Medical records of 63 consecutive pediatric patients were analyzed retrospectively, including 13 cases with PCP and 50 with non-PCP pneumonia. Pneumocystis jirovecii (P. jirovecii) and other co-pathogens detected by multiplex real-time PCR in sputum samples were summarized. Using clinical composite diagnosis as the reference standard, we further compared the diagnostic performance of multiplex real-time PCR to combined serological markers (1,3)-ß-D-glucan plus lactate dehydrogenase. Additionally, modifications of antimicrobial treatment for pediatric PCP patients after the report of multiplex real-time PCR results were reviewed. RESULTS: In children with PCP, nonproductive cough and shortness of breath were more common, lymphocyte count in peripheral blood was markedly lower, and serum levels of (1,3)-ß-D-glucan and lactate dehydrogenase were much higher than non-PCP group. Multiplex real-time PCR reached a sensitivity of 100% in diagnosing PCP, which was better than serum (1,3)-ß-D-glucan plus lactate dehydrogenase (76.9%). Its specificity (98.0%) significantly surpassed serum (1,3)-ß-D-glucan plus lactate dehydrogenase (84.4%). Furthermore, multiplex real-time PCR showed a good performance in identifying co-pathogens in sputum of pediatric PCP patients. Cytomegalovirus, Epstein-Barr virus and Streptococcus pneumoniae were the most common co-pathogens in these patients. Initial antimicrobial treatment was modified in 76.9% of children with PCP after the report of PCR results. CONCLUSION: Multiplex real-time PCR on sputum is a diagnostic tool with good performance for the identification of P. jirovecii as well as co-pathogens in children with PCP. Sputum may be an alternative to bronchoalveolar lavage fluid for PCR assay in children when bronchoscopic examination is not feasible.

Viral Load and Patterns of SARS-CoV-2 Dissemination to the Lungs, Mediastinal Lymph Nodes, and Spleen of Patients with COVID-19 Associated Lymphopenia.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18-1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810-250281), lymph nodes-832 copies (range 96-11586), and spleen-71.5 copies (range 0-2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

Utility of a multiplex real-time polymerase chain reaction for combined detection and serotyping of dengue virus in paediatric patients hospitalised with severe dengue: A report from Chennai.

Objective: Molecular detection and serotyping are rapid, sensitive and accurate techniques for early diagnosis of paediatric dengue. The present study evaluates multiplex real-time polymerase chain reaction (PCR) for diagnosis of dengue virus in children hospitalised with severe dengue (SD) and attempts to establish an association of clinical severity with specific serotypes. Methods: Four hundred and eighty-five samples were received from hospitalised paediatric patients with suspected dengue from March 2019 to February 2020. Multiplex real time PCR was employed for diagnosis. An in-house real-time PCR that combined diagnosis and serotyping was established. Non-structural protein 1 (NS1) assay and real-time PCR were assessed for their accuracy in diagnosing severe paediatric dengue. Results: Three hundred and twenty-five (67%) patients were positive for dengue RNA by real-time PCR. All four serotypes were identified throughout the year; dengue serotype 2 (DEN-2) was predominant (61%) followed by DEN-3, 20%. Compared to the commonly used NS1 testing, multiplex real-time PCR showed greater sensitivity in diagnosing SD. Conclusions: Compared to NS1, multiplex real-time PCR is a rapid and accurate diagnostic test for children hospitalised with SD. DEN-2 was the predominant serotype in severe cases. Continued surveillance of serotypes should be carried out year-round in endemic areas.

Co-detection of bocavirus and bacteria in a respiratory specimen from a pregnant woman using multiplex real time PCR; a pathogenic role, or a bystander?

A pregnant woman presented by cough and dyspenia. Employing a respiratory multiplex real-time PCR, Human bocavirus (HBoV), Haemophilus influenza and Staphylococcus aureus were positive at cycle thresholds (CTs) of 21, 35 and 33.5, respectively. The patient was diagnosed for bacterial respiratory infection superimposed by bocavirus due to a relative high CT value. Patient's condition improved using bronchodilators and corticosteroid without any further antibiotic treatment. HBoV is not exclusively a bystander pathogen in some patients.

Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study.

Introduction: Various pathogens cause respiratory tract infections in children of <5 years of age causing severe morbidity and mortality. The profile of causative agents varies from place to place. Aims: The objectives of our study were to detect the profile and trends of respiratory pathogens causing acute respiratory tract infection in children using a custom multiplex real-time polymerase chain reaction (RT-PCR) and to develop a diagnostic algorithm. Materials and Methods: A total of 997 children with clinical manifestations of respiratory infections were included in the study. Their nasopharyngeal aspirate and throat swab samples were subjected to nucleic acid extraction followed by multiplex RT-PCR for eighteen viruses and six bacteria. Statistical Analysis Used: Chi-square test was employed to study the P value of different viruses and bacteria. Results: A total of 765 (76.73%) samples were found to be positive for one of the respiratory pathogens. Viruses were detected in 598 (59.98%) and bacteria in 167 (41.85%) samples, respectively. The prevalence of single and co-infections among viruses and bacteria were 77.76% and 22.24%, 81.44% and 18.56% each, respectively. Respiratory syncytial virus (RSV) A/B and Streptococcus pneumoniae were the most predominant pathogens detected in the study and were associated with lower respiratory tract infections. Conclusion: RSV and S. pneumoniae were the most common pathogens detected, higher prevalence was observed in children <1 year of age. Viruses were predominant during winter months. The study helped to prepare diagnostic algorithm which will help in reducing diagnostic costs. However, further studies are required to assess whether viruses are bystander or real pathogens and include larger panel of bacteria and viruses for diagnosis.

Evaluation of multiplex real-time PCR for identifying dermatophytes in clinical samples-A multicentre study.

The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.

Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.

Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

Development of a multiplex real-time PCR assay for the detection of Bordetella pertussis and Bordetella parapertussis in a single tube reaction.

Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at a particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2-4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagent (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect B. pertussis and B. parapertussis. The sensitivity and the specificity of the assay for B. pertussis were 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of B. pertussis and B. parapertussis.

Triplex real-time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus.

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT-PCR method was established. This triplex real-time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real-time PCR across a broad dynamic range of 10(2) -10(7) copies/assay using plasmid DNA standards. The limit of detection was 10(2) copies/assay. The quantitative value was comparable with that of monoplex real-time PCR of stool samples. Our triplex real-time PCR is useful for detection of NoV and SaV infections.

Simultaneous detection of Chlamydia spp., Coxiella burnetii, and Neospora caninum in abortion material of ruminants by multiplex real-time polymerase chain reaction.

Chlamydia spp., Coxiella burnetii, and Neospora caninum are responsible for reproductive diseases and are closely linked with high abortion rates in ruminants. Furthermore, C. burnetii and Chlamydia spp. have zoonotic potential. A real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of Chlamydia spp., C. burnetii, and N. caninum. The detection of beta-actin as internal control in the same PCR reaction provides additional information about sample quality by detecting the presence of PCR inhibitors. The multiplex real-time PCR developed in the current study shows a greater sensitivity compared to previously used single-target PCR reactions with a reproducible detection limit of 0.13 plasmid copies per PCR for each target. Additional parallel amplification of all detectable pathogens did not adversely impact sensitivity. This new multiplex PCR allows the highly sensitive, cost-effective, and rapid detection of 3 important pathogens and has the potential to be a useful time-saving tool in the routine diagnosis of abortion cases in ruminants.

Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study

Introduction: Various pathogens cause respiratory tract infections in children of <5 years of age causing severe morbidity and mortality. The profile of causative agents varies from place to place. Aims: The objectives of our study were to detect the profile and trends of respiratory pathogens causing acute respiratory tract infection in children using a custom multiplex real-time polymerase chain reaction (RT-PCR) and to develop a diagnostic algorithm. Materials and Methods: A total of 997 children with clinical manifestations of respiratory infections were included in the study. Their nasopharyngeal aspirate and throat swab samples were subjected to nucleic acid extraction followed by multiplex RT-PCR for eighteen viruses and six bacteria. Statistical Analysis Used: Chi-square test was employed to study the P value of different viruses and bacteria. Results: A total of 765 (76.73%) samples were found to be positive for one of the respiratory pathogens. Viruses were detected in 598 (59.98%) and bacteria in 167 (41.85%) samples, respectively. The prevalence of single and co-infections among viruses and bacteria were 77.76% and 22.24%, 81.44% and 18.56% each, respectively. Respiratory syncytial virus (RSV) A/B and Streptococcus pneumoniae were the most predominant pathogens detected in the study and were associated with lower respiratory tract infections. Conclusion: RSV and S. pneumoniae were the most common pathogens detected, higher prevalence was observed in children <1 year of age. Viruses were predominant during winter months. The study helped to prepare diagnostic algorithm which will help in reducing diagnostic costs. However, further studies are required to assess whether viruses are bystander or real pathogens and include larger panel of bacteria and viruses for diagnosis.