Multiplex Real-time PCR Research Articles

Development of a multiplex real-time PCR for the simultaneous detection of monkeypox virus clades I, II, and goatpox virus

Development of a multiplex real-time PCR for the simultaneous detection of monkeypox virus clades I, II, and goatpox virus

Chronic Strongyloides stercoralis infection in Fijian migrants to the UK.

Introduction. Strongyloides stercoralis, the human threadworm, is a parasitic nematode with global distribution, estimated to infect over 600 million people. Chronic infection is often asymptomatic, but hyperinfection and dissemination syndromes can occur in the immunosuppressed with high case fatality rates. Whilst strongyloidiasis is endemic in Fiji, its prevalence in Fijian migrant groups in the UK is unknown.Gap Statement. No previous studies have been conducted on the prevalence of Strongyloides and other gastrointestinal parasites (GIPs) in Fijian migrants to the UK.Aim. We conducted a cross-sectional study of the prevalence of GIPs in a Fijian migrant population.Methodology. Participants completed a questionnaire on residence, travel and clinical symptoms and were asked to provide a serum sample for S. stercoralis IgG ELISA, venous blood samples for eosinophil count and a faecal sample for charcoal culture, multiplex real-time PCR (rtPCR) and microscopy after formalin-ethyl acetate concentration. Sequencing was performed on pooled Strongyloides larvae for nuclear 18S rRNA hyper-variable regions (HVRs) I and IV.Results. A total of 250 participants (94% male) with median (range) age 37 (20-51) years entered the study, 15 (1-24) years since leaving Fiji. S. stercoralis IgG ELISA was positive in 87/248 (35.1 %) and 14/74 (18.9 %) had a GIP detected in faeces. This included 7/74 (9.5 %) with Strongyloides and 5/74 (6.8 %) with hookworms. Dermatological symptoms were more common in those with Strongyloides, and eosinophilia (>0.5×109 cells per litre) was present in 55.6% of those with positive S. stercoralis IgG. rtPCR was the most sensitive faecal diagnostic test for Strongyloides and hookworms in faeces. Sequences of nuclear 18S rRNA for HVRs I and IV confirmed the presence of S. stercoralis.Conclusion. This first cross-sectional study in Fijian migrants found a high rate of chronic infection with GIPs, particularly S. stercoralis. Faecal microscopy was insensitive compared to charcoal culture, rtPCR or serology, demonstrating the importance of specialist parasitological tests when investigating people with a suspected chronic infection. Our study highlights an overlooked burden of strongyloidiasis in the UK and has implications for screening and treatment programmes in Fiji and for migrants from Fiji.

A multiplex qPCR followed by high-resolution melting analysis for the detection of blood-feeding sources in Culex sp. mosquitoes.

Culex species, such as Culex quinquefasciatus and Culex nigripalpus display a range of feeding habits and act as vectors for pathogens that can cause diseases in both humans and animals. Understanding their feeding habits is pivotal for enhancing disease prevention strategies. The present study introduces the application of two multiplex real-time PCR (qPCR) followed by high-resolution melting (HRM) as a cost-effective and time-efficient alternative. This investigation involved the development of two multiplex qPCR-HRM: assay 1 detects human, dog and chicken, while assay 2 detects cat, cattle and horse in Culex sp. engorged female mosquitoes. The qPCR-HRM reactions showed a detection limit of one copy of genomic DNA when performed as single and multiplex qPCR-HRM. The reaction efficiencies were 97.96% for human, 100.60% for dog, 99.03% for chicken, 99.92% for feline, 99.18% for cattle and 97.68% for horse. The qPCR-HRM method, employing multiplex 1 and 2, was applied to field-collected mosquitoes and demonstrated the ability to detect DNA from multiple blood sources within a single sample. By analysing both multiplexes, it was possible to identify up to five distinct blood sources in Cx. quinquefasciatus and Cx. nigripalpus, and up to two sources in Culex coronatus. Sequencing corroborated the qPCR-HRM results, confirming the presence of DNA from one to four different blood sources with 100% accuracy. The development of these molecular methods may contribute for identification of blood-feeding patterns in mosquitoes. It contributes to studies on the dissemination and transmission of pathogens among various animals and humans, thereby bridging the gap between epidemiology and vector monitoring.

Etiological characteristics and whole genome sequence analysis of Clostridium perfringens causing a food poisoning outbreak

This study investigated the etiological characteristics and whole genome sequencing of clostridium perfringens (CP) from a food poisoning outbreak. Multiplex real-time PCR was employed to screen pathogens in collected samples. Based on the preliminary screening results, the isolation, cultivation, and identification of suspected pathogenic CP bacteria were performed. The nucleic acid tests were conducted for CP-related virulence genes on CP isolates, anal swab specimens and food samples. The molecular typing of CP isolates was analyzed by using pulsed-field gel electrophoresis (PFGE). A phylogenetic tree was established to analyze nucleotide and amino acid homology after sequencing the PLC gene. The whole genome sequencing and gene annotation on the representative strain QD2022FB4-CP4 were performed to explore its drug resistance, toxin genes and biological characteristics. The analysis revealed that the food poisoning was triggered by F-type Clostridium perfringens, which infected the consumers by contaminating'roujiamo' sandwiches. The whole genome sequencing of the strain QD2022FB4-CP found that it had active metabolic processes, multiple virulence genes and multidrug resistance characteristics.

Evaluation of biofilm formation and carbapenem resistance in Klebsiella pneumoniae isolated from clinical samples at a rural hospital in western Uttar Pradesh

ABSTRACT Introduction: Klebsiella pneumoniae commonly causes healthcare-associated infections and shows multidrug resistance. K. pneumoniae can produce biofilm. Carbapenem resistance in K. pneumoniae is due to the production of carbapenemases mainly. This study was done to evaluate the formation of biofilm and carbapenemase resistance in K. pneumoniae isolates. Material and Methods: A total of 110 K. pneumoniae isolated from various clinical samples were taken, the antibiotic susceptibility test was done by the Kirby disk diffusion method, and biofilm detection was done by the tissue culture plate method. All the carbapenem-resistant isolates were confirmed by multiplex real-time PCR (mPCR). Those found positive for any of the carbapenemase genes were tested by the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and ethylenediamine tetraacetic acid (EDTA)-modified carbapenem inactivation method (eCIM). Results: Out of 110 isolates, 66% (72/110) were carbapenem-resistant (suggestive of carbapenemase producers) by Kirby-Bauer disk diffusion but 58% (42/72) of Klebsiella isolates were confirmed for carbapenemase production by mPCR. Maximum number of carbapenemase gene were New Delhi metallo-β-lactamase (NDM) 52% (N = 22), 29% (N = 12) coproducers (NDM+OXA-48), and lowest in oxacillinase (OXA-48), 19% (N = 8). The overall sensitivity of MHT and mCIM+eCIM was 62% and 93%, and specificity was 88% and 97%, respectively. Our study showed that moderate biofilm producers were 51% (N = 56) K. pneumoniae isolates, strong biofilm producers 27% (N = 30), and 22% (N = 30) were weak/non-biofilm producers. We also found the correlation between biofilm formation and carbapenem-resistant K. pneumoniae (CR-KP) genes was statistically significant with a P value of 0.01*<0.05. Conclusion: Most isolates of K. pneumoniae demonstrated a wide range of antibiotic resistance and were biofilm producers. Our results indicated that the combination of mCIM with eCIM showed high sensitivity and specificity to detect CR-KP compared with MHT.

Genital infections in high-risk human papillomavirus positive Paraguayan women aged 30–64 with and without cervical lesions

Objective To determine the prevalence of genital infections (GIs), including sexual transmitted STIs: Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, and opportunistic pathogens that generally do not cause STIs, non-classic STI: Ureaplasma urealyticum, Ureaplasma parvum and Mycoplasma hominis, in women with high-risk oncogenic human papillomavirus (hr-HPV) infection and their association with cervical lesions. Methods A cross-sectional study was carried out including 231 hr-HPV positive women. Of these, 46 has histologically confirmed cervical intraepithelial neoplasia 3 (CIN3) or more (including CIN3 and cervical cancer lesions-CIN3+). GIs were detected by multiplex real time PCR. Odds ratios (OR) were estimated to explore possible associations between GIs and the presence or absence of CIN3+ lesions. Additionally, we examined associations between sociodemographic, sexual, and clinical characteristics and the presence of GIs. Results In total, there were 174/231 cases of GIs corresponding to an overall prevalence of 75.3% (95%CI: 69.4–80.4), being non-classic STIs the most common (72.3%) compared to STIs (12.6%). The most prevalent non-classic STI and STI were U. parvum (49.8%) and C. trachomatis (7.4%), respectively. The odds of presenting GIs were 3 times higher in women under 46 years compared to older counterparts (OR: 3.32, 95%CI: 1.74–6.16), and in women with a normal Pap smear with inflammation compared to those without inflammation (OR: 3.31, 95%CI: 1.15–9.77). GIs were equally present in women with and without CIN3+ lesions. Conclusion We observed an association of GIs with inflammation in the Pap smear, but no association with CIN3+, as some of them are very common and likely part of the normal vaginal flora, suggesting that such infections do not appear to be cofactors in cervical carcinogenesis, although larger prospective studies are needed.

Systemic Acanthamoeba T17 infection in a free-ranging two-toed sloth: case report and literature review of infections by free-living amebas in mammals.

A free-ranging, adult female two-toed sloth (Choloepus hoffmanni) was brought to a wildlife rescue center in Costa Rica with ocular and auricular myiasis and numerous skin lesions. After one month of unsuccessful systemic and topical antimicrobial treatment, the patient died. A postmortem examination was performed, and tissues were examined histologically, confirming disseminated amebic infection with intralesional trophozoites and cysts in the lungs, liver, eye, heart, spleen, and stomach. Immunohistochemistry identified the ameba as Acanthamoeba sp. A multiplex real-time PCR assay, 18S ribosomal DNA PCR, and sequencing performed on formalin-fixed, paraffin-embedded lung tissue confirmed the Acanthamoeba T17 genotype. The Acanthamoeba genus is in the group of free-living amebas that cause infection in humans and animals, and it is ubiquitous in the environment. Acanthamoeba T17 has been isolated from water and soil, but to our knowledge, this genotype has not been implicated in infections of animals previously and has not been reported from Costa Rica. Systemic Acanthamoeba infection has not been described in sloths previously. We provide a comprehensive literature review describing infections by free-living amebas of the genus Acanthamoeba spp., Balamuthia spp., and Naegleria spp. in domestic, zoo, and wild mammals.

Epidemiology and genetic diversity of human respiratory syncytial virus in Belgium between 2011 and 2019

BackgroundHuman respiratory syncytial virus (HRSV) is worldwide one of the leading causes of acute respiratory tract infections in young children and the elderly population. Two distinct subtypes of HRSV (A and B) and a multitude of genotypes have been described. The laboratory of Clinical and Epidemiological Virology (KU Leuven/University Hospitals Leuven) has a long-standing history of HRSV surveillance in Belgium.MethodsIn this study, the seasonal circulation of HRSV in Belgium was monitored during 8 consecutive seasons prior to the SARS-CoV-2 pandemic (2011–2012 until 2018–2019). By use of a multiplex quantitative real time PCR panel, 27,386 respiratory samples were tested for HRSV. Further subtyping and sequencing of the HRSV positive samples was performed by PCR and Sanger sequencing. The prevalence and positivity rate were estimated in 4 distinct age groups and the circulating strains of each subtype were situated in a global context and in reference to the described genotypes in literature.ResultsHRSV circulated in Belgium in a yearly re-occurring pattern during the winter months and both HRSV subtypes co-circulated simultaneously. All HRSV-B strains contained the 60 nt duplication in the HVR2 region of the G gene. Strains of subtype HRSV-A with a 72 nt duplication in the HVR2 region were first observed during the 2011–2012 season and replaced all other circulating strains from 2014 to 2015 onwards.

Molecular survey of certain protozoan agents that cause diarrhea in children in Sudan

Introduction Diarrhea is a significant health problem in the Third World. Identification of the pathogen that causes diarrhea is vital for measures to prevent and control this disease. There are also very few reports of diarrhea in Sudan. Our study aimed to determine the Prevalence of specific protozoan pathogens (Entamoeba histolytica, Cryptosporidium parvum., and Giardia spp) in children in Khartoum, Sudan. Methods We conducted a cross-sectional survey among children under five years of age hospitalized with acute diarrhea between April and December 2014. Diarrheal stool samples were collected, and E. histolytica, C. parvum, and Giardia spp were examined using multiplex real-time PCR. Results Four hundred and thirty-seven children with acute diarrhea were included in this study; the higher prevalence of diarrhea was in the age ≤ 2 years old (403, 92.2%), >2–≤4 years (32, 7.3%), and >4–<5 years (2, 0.5%). The male-to-female ratio in this study was 1:1.7. Infection with intestinal parasite was found in 155 (35.5%) cases, and co-infection was detected in 16 (3.7%) cases. Giardia spp (18.8%) and C. parvum (15.8%) were the most frequently identified parasites, followed by E. histolytica (0.9). The parasite infection rate was highest and lowest in the under 2-year-old group 143 (35.5%) and the 2–4-year-old group 12 (37.5%). The infection rate was higher in boys 104 (37.7%) than in girls 51 (31.7%). The number of positive cases was higher in the rainy season (August to December) 143 (37.4%), corresponding with that in the dry Season (April to June) 12 (21.8%). Discussion Our present study demonstrated the high prevalence of Giardia spp and C. parvum in children with diarrhea in the Khartoum region and the usefulness of the multiplex real-time method in disclosing pathogenic protozoal agents. Our result highlighted the necessity of developing intervention measurement and control strategies to deal with childhood parasitic diarrhea in this region.

Evaluation of a multiplex real-time PCR targeting the β-tubulin gene for the detection and differentiation of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, S. schenckii and S. brasiliensis. The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

Comparison of Urine and Cervical Samples for Genotyping Via HPV DNA Test

<i>Background</i>: Human papillomavirus (HPV) is a key factor in cervical cancer development. This study aimed to compare the efficacy of urine and cervical samples for HPV genotyping, evaluating their diagnostic performance in detecting high-risk HPV genotypes. <i>Methods</i>: This comparative cross-sectional study conducted over one year at the Department of Gynaecological Oncology, BSMMU, Dhaka, the study enrolled 74 women aged 30-60 years with positive visual inspection with acetic acid (VIA) results or abnormal Pap test findings. Urine samples (20 ml) and cervical samples were collected from each participant. The samples were analyzed using multiplex real-time PCR to amplify high-risk HPV types (16, 18, and others). DNA was extracted using the Qiagen viral DNA extraction kit. Sensitivity and specificity of HPV detection in urine samples were compared to cervical sampling, the gold standard. Data were analyzed with SPSS 22.0, and agreement was assessed using the Kappa index. <i>Result</i>: Cervical samples detected HPV in 17.56% of participants, while urine samples identified HPV in 5.40%. The agreement between urine and cervical samples was moderate, with a kappa value of 0.743. Among 74 cases, 5 cases were detected as HPV 16 and HR (co-injection) in both cervical and urine sample, 2 cases as HPV 16 in both cervical and urine samples, 2 cases as only HR type in both cervical and urine sample. The kendall’s correlation of agreement was 0.361 and a significance of 0.002. <i>Conclusion</i>: Cervical samples are more reliable for HPV detection compared to urine samples, though urine testing shows high sensitivity.

Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR.

We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

A Novel Method for Rapid Screening of Salmonidae Ingredients and Accurate Detection of Atlantic Salmon (Salmo salar) Simultaneously Using Duplex Real-Time PCR Coupled with Melting Curve Analysis

The substitution of ingredients with Salmonidae, particularly Salmo salar, has led to widespread reports of financial losses and health risks globally, emphasizing the urgent need for the development of a rapid and precise method for species identification. The aim of the present study was to develop a novel method for the rapid screening of Salmonidae ingredients and the accurate detection of S. salar simultaneously using multiplex real-time PCR coupled with melting curve analysis. Specifically, primer sets specific for S. salar and Salmonidae were cross-confirmed. Moreover, the reaction system and conditions of a real-time duplex PCR were optimized, and the proposed methodology was verified, proving that the assay has good specificity and sensitivity. Clear and distinguishable melting peaks, with expected Tm values of around 80 °C (S. salar) and 84 °C (Salmonidae), were observed for twelve products, proving the presence of S. salar. However, four products were not derived from S. salar, but they could have belonged to another species within the Salmonidae family due to the presence of only one specific melting peak at a Tm value of about 84 °C. Therefore, the novel assay in the present study allows for the fast and accurate screening of Salmonidae ingredients and the detection of S. salar simultaneously.

Prevalence and Genotype Distribution of HPV in Gynecological Patients: A Cross-Sectional Study

Human papillomavirus (HPV) is a prevalent sexually transmitted virus that can lead to genital warts or cancer. Over 100 HPV strains have been discovered, some classified as high-risk and others as low-risk. This study aimed to analyze the prevalence of HPV genotypes in patients with gynecological problems and to compare the frequency of low-risk and high-risk HPV infections. A cross-sectional study was performed. Five hundred patients were recruited who attended a gynecological clinic due to various reasons such as genital warts, itching, bleeding, or even asymptomatic cases discovered during routine visits in the period extending from January 2nd, 2017 to December 31st, 2022. Genotyping was performed using the Hybrispot technique, targeting 27 different HPV genotypes. DNA flow technology, a rapid and sensitive method, was utilized for the analysis. Out of the 500 patients, 109 tested positive for HPV, indicating 21.8% incidence among those seeking gynecological care. Among the HPV-positive patients, 59% had low-risk HPV, and 41% had high-risk HPV. The most common low-risk genotypes were HPV6 and HPV11, while the most common high-risk genotypes were HPV53, HPV58, HPV18, and HPV16. Several infrequent HPV genotypes were missed by the multiplex real-time PCR method. The study revealed a higher frequency of low-risk HPV compared to high-risk HPV, with no significant difference between the two groups.

Long-term outcomes of high-risk human papillomavirus in anal infection support HPV men vaccination

Abstract Introduction/Objective The clinical laboratory in a local hospital has an important role in population surveillance. The clinical laboratory performs analyses on patients admitted to the hospital as inpatients or critical patients and it is also a reference point for people’s health check-ups and health surveillance. Infectious diseases normally have severe symptoms. To ensure benefits from therapeutic treatment, a timely diagnosis is necessary. The human papillomavirus (HPV) virus is among the most diagnosed sexually transmitted infections worldwide, and the male gender is not exempt from it. In fact, 1 out of 3 male individuals is infected with HPV and 1 out of 5 male individuals is infected with a high-risk strain (1). In Italy, vaccination is underway for women only. Vaccine prevention extended to male individuals could lead to a halving of HPV cases. Methods/Case Report From 2018 to 2022, we performed 7362 HPV tests using DNA extraction technique (QiaSimphony) and real-time Multiplex PCR (SeeGene 28 strains). Results (if a Case Study enter NA) 3762 out of 7362 HPV tests performed were positive for one (2059 patients) or more (1969 patients) HPV strains. Women, who are more aware of prevention, are more represented in our sample of subjects. The tests performed on women are 6499 and half (3331) are positive for at least one strain of HPV. Regarding men, 863 tests were performed. About half (431 patients) were positive for at least one HPV strain. Noteworthy, out of 175 anal HPV tests, 48% were positive and 1 patient had squamous cell carcinoma. Furthermore, in our male patients tested 2 - 12% had low or medium-risk injuries. In total, out of 7529 tests carried out from 2018 to 2022, over than 50% were found to be high-risk viral strains (5231). Of these, however, approximately half (2781) are now preventable with the monovalent HPV vaccine (strains covered: 6, 11, 16, 18, 31, 33, 45, 52, 58). Conclusion The Ospedale Isola Tiberina – Gemelli Isola is an Institution in the center of Rome, Italy. It is also a reference center for birthrates and women’s prevention. Therefore, our Institution is very efficient in raising awareness of the prevention of HPV-related cancer. Current data indicate that male cases are not negligible, being positive in almost 50% of anal tests, and associated with an increased risk of the related cancers. These data can have an important impact on the Italian population, both in terms of the spread of HPV and healthcare burden and in terms of high healthcare costs.

Molecular Survey of Parasitic Contamination of Frozen Berries.

Berries represent healthy dietary options and contain bioactive compounds associated with a decreased risk of diseases. Despite representing healthy food choices, these products can be contaminated by pathogenic microorganisms, including parasites. Among foodborne parasites, Giardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, Toxoplasma gondii, and Echinococcus multilocularis are of significant public health importance and have been recently detected in fresh berries in Europe, including Italy. Berries can be purchased fresh or frozen, and it is worrying that even frozen berries could represent a risk for the consumer. In fact, several parasites can resist freezing temperatures and have been responsible for outbreaks of infection. The aim of this study was to investigate the presence of G. duodenalis, C. parvum, C. cayetanensis, T. gondii, and E. multilocularis in frozen berries with simplex and multiplex real-time PCR protocols. A total of 108 packages of mixed frozen berries were bought from supermarkets located in a south-eastern region of Italy. The samples were tested using two simplex real-time PCR protocols targeting C. parvum and G. duodenalis, respectively, and a multiplex real-time PCR targeting C. cayetanensis, T. gondii, and E. multilocularis. None of the investigated parasites were detected in the frozen berry samples tested. This research topic is still unexplored and of great current interest. These results represent a first attempt to investigate parasitic contamination of frozen berries sold on the Italian market, but further large-scale surveys are required.

Prevalence of high-risk human papillomavirus genotypes and viral load correlated with squamous cell inflammation among women in Gabon

BackgroundHigh-risk genotypes of Human Papillomavirus are responsible for 90% of cases of cervical cancer worldwide. Inflammation of squamous cells is mainly linked to HPV. In Gabon, HPV is endemic and circulates among the female population. The study aimed to determine the prevalence of HR-HPV genotypes and to investigate the correlation between squamous cell inflammation and HPV viral load in infected women in Gabon.MethodsThe cross-sectional study was conducted at Libreville University Hospital Center (UHC) and National Public Health Laboratory from March to May 2024 among 399 women. Two cervical smears were taken. Genotype detection was carried out by multiplex fluorescence real-time PCR in the NPHL virology unit. Cytology was carried out in UHC’s anatomic-pathology laboratory. Data were analyzed by SPSS software. Graphs were plotted using Microsoft Excel 2016.ResultsThe prevalence of Human Papillomavirus was 26.1% (95% CI: 22-30.6). The prevalence of HR-HPV genotypes was 24.8%. The most common HR-HPV genotypes were HPV-16/52/18/35/56/58/53/68. The rate of multiple HPV infections was 29.8% and 95.2% for the HR-HPV infection rate. Viral load was significantly correlated with squamous cell inflammation (r = 0.977 and P = 0.001).ConclusionHR-HPV infection remains a concern in women, however early screening is necessary for optimal monitoring and management. HR-HPV viral load is a predictive marker of squamous cell inflammation.

Performance Evaluation of NovaplexTM Multiplex Real-Time PCR Assay for Detection of Streptococcus agalactiae Serotypes.

Streptococcus agalactiae, or group B streptococcus (GBS), is a Gram-positive pathogen with an extended track record of colonization in the gastrointestinal and genitourinary tracts. GBS can induce disease in individuals across all age demographics, yet it predominantly triggers infections in neonates and the elderly. Identification of the serotype is vital for effective management of the disease as it provides critical information for clinicians on the cause of the disease. In this study, we evaluated the rapid, simple, and easy-to-adopt multiplex real-time PCR technique, NovaplexTM (NovaPCR). A total of 131 clinical isolates of different serotypes were tested using NovaPCR. Observations revealed that 129 isolates showed the same observations as LA and conventional mPCR. NovaPCR accurately identified serotypes IV and V, which were first classified as serotype Ia in the LA test and mPCR, and the difference between the traditional (LA test and mPCR) and NovaPCR methods is only 1.52%. Accurate serotype identification is helpful for monitoring the epidemics and achieving optimal clinical outcomes, and NovaPCR showed a reliable, fast, easy-to-interpret, and cost-efficient performance in GBS serotyping.

Bovine tuberculosis in Central Ethiopian slaughterhouses and the identification of causative mycobacteria by multiplex real-time PCR

BackgroundBovine tuberculosis (bTB) is a chronic disease caused by members of the Mycobacterium tuberculosis complex (MTBC) that ultimately leads to the development of progressive granulomatous lesions. Although the disease is widespread, especially in crossbred cattle in Ethiopia, routine investigations and surveillance are lacking. Thus, the aim of this study was to determine the prevalence, associated risk factors, and species of mycobacteria causing bTB in slaughtered cattle at four slaughterhouses in Central Ethiopia.MethodsPostmortem examination of 7,640 cattle was conducted using a cross-sectional slaughterhouse survey. A total of 388 tuberculous-like lesions (TBLs) were collected from 173 animals and cultured. Six target genes were used to differentiate mycobacterial species using multiplex real-time PCR (mRT-PCR). Multivariate logistic regression analyses and related odds ratios (ORs) were used to gauge the strength of the associations between risk factors, TBL incidence and culture growth.ResultsThe prevalence of TBL was 2.3% (95% CI = 2.0-2.6). Logistic regression analysis indicated an increased risk of TBL in crossbred cattle (OR = 11.8, 95% CI: 6.4, 21.2, p < 0.001). Animals slaughtered at Adama (OR = 3.2, 95% CI: 1.2, 7.3, p = 0.009) or Burayu (OR = 5.8, 95% CI: 3.9, 8.9, p < 0.001) had a greater risk of TBL than those slaughtered at Sululta. There were significantly more TBL-positive lesions in the lungs and lymph nodes related to the lung (OR = 7.1; 95% CI: 2.7, 24.5, p < 0.001) and the head lymph node (OR = 5.6; 95% CI: 1.8, 21.7; p = 0.006) compared to gut associated lymph nodes. Among the 173 TBL-positive animals, 36% (95% CI = 28.8, 43.2), and among the 388 TBL-positive tissues, 24.2% (95% CI = 20, 29) were culture and mRT-PCR positive. All the culture-generated isolates were positive for M. bovis in mRT-PCR. Among them, two animals had mixed infections including one zebu cattle tested positive for both M. caprae and M. bovis, and a crossbred cow tested positive for both M. tuberculosis and M. bovis in mRT-PCR. This suggests persistent transmission within the cattle population, posing a substantial public health threat.ConclusionThis study revealed an eleven-fold greater risk of bTB-related lesions in crossbred cattle compared to local zebu cattle. This finding highlights the necessity for targeted interventions, continuous vigilance, and thorough carcass inspection to mitigate public health risks.

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.