Polymerase Chain Reaction Targeting Research Articles

B-276 Preservation of Proteomic Inflammatory Biomarkers signal in a Saliva Multiomic Buffer at Room Temperature

Abstract Background Saliva represents an attractive biospecimen as it is pain-free, simple for at home collections, and patients can provide multiple samples over time to provide time resolved data. However, few diagnostic tests have been developed using saliva due to poor stability and comparatively low concentration of analytes. Stabilization of biomarkers at ambient temperature in saliva would enable labs to take advantage of the benefits of saliva sample collections, while minimizing the risks posed by the degradation of already scarce analytes. Methods Matched samples of neat serum, saliva, and saliva collected with Saliva Multi-omic Device (SMD) preservative were collected from 30 volunteers (15 male & 15 female) ages 18–64 with no known morbidities. The Olink Inflammation panel was used to examine 92 inflammation-related proteins. The method utilized proximity extension assay technology, wherein antibody probe pairs reacted with their respective target proteins to generate a polymerase chain reaction target sequence through proximity-dependent DNA polymerization. This sequence was measured using real-time polymerase chain reaction and normalized in two steps, producing a semi-quantitative Normalized Protein eXpression (NPX) unit. To evaluate analyte stability specimens were measured initially and then again after 24 h at room temperature (23 ± 2°C). Results Protein expression (NPX) units for 36 of the 92 inflammatory markers had higher concentrations in saliva compared to serum. Serum (78/92) and saliva 78% (72/92) showed similar coverage for the number of markers in the panel where at least 75% of subjects had values exceeding the limit of detection (LoD). SMD preservation buffer improved the average stability of inflammatory protein biomarkers by 19.6% compared to neat saliva and 10.2% compared to serum over 24-hours at room temperature. However, the relative stability of individual between protein markers [DV1] [HM2] varied. For proteins in SMD buffer, initial concentrations of analytes were highly predictive [HM3] of values 24 h later, having Pearson correlation coefficients > 0.7, for 23 of the analytes and greater than 0.5 for 47. The change in protein marker in serum over time was only moderately predictive of the protein’s stability in saliva with or without preservative, having correlations of 0.33 ± 0.20 and 0.47 ± 0.19 (mean ± standard deviation), respectively. Conclusion This study demonstrates potential utility of saliva in proteomic applications utilizing the Olink platform. The SMD preservation buffer stabilized inflammatory biomarkers in saliva samples likely through protease inhibition and antimicrobial action. Preservation of self-collection salivary samples by subjects without cold storage enables noninvasive sampling and reliable measurements even when samples are collected by users at remote locations or under challenging conditions.

Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

BackgroundThe demonstration that the recently discovered Anopheles symbiont Microsporidia MB blocks malaria transmission in Anophelesarabiensis and undergoes vertical and horizontal transmission suggests that it is a promising candidate for the development of a symbiont-based malaria transmission-blocking strategy. The infection prevalence and characteristics of Microsporidia MB in Anopheles gambiae sensu stricto (s.s.), another primary vector species of malaria in Kenya, were investigated.MethodsField-collected females were confirmed to be Microsporidia MB-positive after oviposition. Egg counts of Microsporidia MB-infected and non-infected individuals were used to infer the effects of Microsporidia MB on fecundity. The time to pupation, adult sex ratio and survival were used to determine if Microsporidia MB infection has similar characteristics in the host mosquitoes An. gambiae and An. arabiensis. The intensity of Microsporidia MB infection in tissues of the midgut and gonads, and in carcasses, was determined by quantitative polymerase chain reaction. To investigate horizontal transmission, virgin males and females that were either Microsporidia MB-infected or non-infected were placed in standard cages for 48 h and allowed to mate; transmission was confirmed by quantitative polymerase chain reaction targeting Microsporidia MB genes.ResultsMicrosporidia MB was found to naturally occur at a low prevalence in An. gambiae s.s. collected in western Kenya. Microsporidia MB shortened the development time from larva to pupa, but other fitness parameters such as fecundity, sex ratio, and adult survival did not differ between Microsporidia MB-infected and non-infected hosts. Microsporidia MB intensities were high in the male gonadal tissues. Transmission experiments indicated that Microsporidia MB undergoes both maternal and horizontal transmission in An. gambiae s.s.ConclusionsThe findings that Microsporidia MB naturally infects, undergoes maternal and horizontal transmission, and is avirulent in An. gambiae s.s. indicate that many of the characteristics of its infection in An. arabiensis hold true for the former. The results of the present study indicate that Microsporidia MB could be developed as a tool for the transmission-blocking of malaria across different Anopheles species.Graphical

Molecular Surveillance of Yersinia pestis from Stray Dogs and Cats and their Fleas in Algiers.

In recent years, plague has re-emerged in several countries around the world and remains endemic in some regions. In a natural environment and in contact with rodents and their fleas, stray carnivores are most at risk of catching the disease and maintaining the spread of the bacillus. The present study aimed to demonstrate the presence or absence of Yersinia pestis in stray dogs and cats in the Algiers region by molecular methods and thus determine their role in epidemiology of this disease. Molecular research of Yersinia pestis has also been conducted on fleas from these carnivores. Preliminary identification of ectoparasites to genus and species level was performed. Real-time polymerase chain reaction targeting Yersinia pestis pla gene was used to survey the plague agent in fleas and carnivores captured as stray animals in Algiers (Algeria). Positive qPCR results were tested by PCR sequencing using glpD gene. Among 327 fleas captured from 107 dogs and 365 fleas from 140 cats, prevalence of Ctenocephalides felis was higher in cats (86,96%), whereas that of Ctenocephalides canis and Xenopsylla cheopis were higher in dogs (90,57% and 92,63%, respectively). While internal and external PCR positive controls were positive, none of the 107 dogs spleens and 140 cats spleens and none of the 256 analyzed fleas were positive for Y. pestis. These results suggested that stray cats and dogs are unlikely sources of plague in Algeria, contrary to what has been reported in other plague-endemic countries. This observation illustrates that the plague epidemiological chain varies from one region to another.

Designing one-step reverse transcriptase loop-mediated isothermal amplification for serotype O foot-and-mouth disease virus detection during the 2022 outbreak in East Java, Indonesia.

Various methods can detect foot-and-mouth disease (FMD) in cows, but they necessitate resources, time, costs, laboratory facilities, and specific clinical specimen submission, often leading to FMD virus (FMDV) diagnosis delays. The 2022 FMD outbreak in East Java, Indonesia, highlighted the need for an easy, inexpensive, rapid, and accurate detection approach. This study aims to devise a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique and phylogenetic analysis to detect the serotype O FMDV outbreak in East Java. Swab samples were collected from the foot vesicles, nasal secretions, and saliva of five suspected FMDV-infected cows in East Java between June and July 2022. The RT-LAMP design used hydroxy naphthol blue dye or SYBR Green I dye, with confirmatory analysis through reverse transcriptase polymerase chain reaction (RT-PCR) targeting 249 base pairs. PCR products underwent purification, sequencing, and nucleotide alignment, followed by phylogenetic analysis. The RT-LAMP method using hydroxy naphthol blue dye displayed a positive reaction through a color shift from purple to blue in the tube. Naked-eye observation in standard light or ultraviolet (UV) light at 365 nm, with SYBR Green I stain, also revealed color change. Specifically, using SYBR Green I dye, UV light at 365 nm revealed a color shift from yellow to green, signifying a positive reaction. Nucleotide alignment revealed mutations and deletion at the 15th sequence in the JT-INDO-K3 isolate from the East Java FMDV outbreak. Despite differing branches, the phylogenetic tree placed it in the same cluster as serotype O FMDV from Malaysia and Mongolia. JT-INDO-K3 exhibited distinctions from Indonesian serotype O FMDV isolates and those documented in GenBank. Then, the RT-LAMP method used in this study has a detection limit 10 times higher latter than the conventional RT-PCR limit, without any cross-reactivity among strains.

Molecular prevalence of Anaplasma spp. in cattle and assessment of associated risk factors in Northeast Thailand.

Anaplasma spp. are common rickettsia species described in ruminant hosts, including cattle. The clinical signs of anaplasmosis range from asymptomatic to mortality. However, there are insufficient studies on epidemiology surveys of this blood pathogen. This study aimed to estimate the prevalence and risk factors of anaplasmosis in beef and dairy cattle in Northeast, Thailand. A total of 187 blood samples of beef and dairy cattle were collected from five provinces in Northeast Thailand. Anaplasma spp. infections were screened by microscopic examination and polymerase chain reaction targeting specific genes (msp4 gene for Anaplasma marginale and 16S rRNA gene for Anaplasma platys and Anaplasma bovis). Moreover, the associated risk factors for the infections were evaluated. Overall, blood samples from cattle revealed that 17.6% (33/187) were positive for Anaplasma spp. by microscopic examination and 20.8% (39/187) were positive by DNA amplification. Of these 20.8%, 17.6% were A. marginale and 3.2% were A. platys. However, A. bovis infection was not detected. Infection with Anaplasma spp. and A. marginale showed a significant association with breed and gender (p < 0.05) while age and packed cell volume levels showed no significant statistical relationship between Anaplasma spp. infected and uninfected animals. This study indicated that anaplasmosis is distributed in beef and dairy cattle in Thailand; therefore, prevention and control strategies for these pathogens should be improved. This information will benefit veterinarians and cowherds by avoiding vector exposure and eliminating tick breeding sites.

Comparison of Cefotaxime-Resistant Escherichia coli and sul1 and intI1 by qPCR for Monitoring of Antibiotic Resistance of Wastewater, Surface Water, and Recycled Water.

Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal-Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk.

Identification of Candida auris by PCR and assessment of biofilm formation by crystal violet assay

IntroductionCandida auris is a notorious pathogen capable of forming biofilms on devices as well as host tissues, often culminating in infections. We evaluated characteristics of infections and the methods to diagnose C. auris over a period of three years in a tertiary care hospital. MethodsPatients admitted between 2018 and 2020, who had candidemia due to C. auris were included in the study. Identification was performed using HiCrome™ Candida Differential Agar, Vitek 2 (BioMérieux, Inc., Marcy-l’Etoile, France) and MALDI-TOF, Vitek-MS. Identification was confirmed by detection of rDNA region covering part of 5.8S, entire of ITS2, and part of 28S by polymerase chain reaction (PCR). Biofilm formation was assessed by crystal violet staining. ResultsPresence of central line and broad spectrum antimicrobials were noted in all patients whereas total parenteral nutrition was given in 82.1% of these patients. Identification by Vitek2 v8.1 correlated with MALDI-TOF MS. PCR products of length 163 ​bp were obtained in all isolates as visualized by agarose gel electrophoresis. The biofilm quantity measured as A560 of the twenty-eight C. auris isolates ranged from 0.16 to 0.80 compared to C. albicans. ConclusionsC. auris can be identified by PCR targeting specific rDNA region. Biofilm formation and quantification can be achieved by growing C. auris isolates in Mueller-Hinton broth over a duration of 48 ​h.

Molecular investigation of bovine tropical theileriosis outbreak in an organized dairy cattle farm in Madhya Pradesh, India.

The present study aimed to investigate an outbreak of Theileria annulata (T. annulata) infection in an organized dairy cattle farm in Madhya Pradesh, India, using clinical and molecular techniques. Following the deaths of two crossbred cattle in March 2021, 43 blood samples were collected from infected and apparently healthy animals and examined by blood smear and polymerase chain reaction (PCR) techniques. The blood smear examination showed that 23.25% of samples were positive for Theileria organisms, while conventional PCR targeting the 18S ribosomal RNA (18S rRNA) and T. annulata merozoite surface antigen-1 (TAMS-1) genes revealed that 32.55% of samples were positive for T. annulata. PCR targeting cytochrome b (Cytb) gene showed 46.51% of samples were positive for T. annulata. Haematological analysis confirmed clinical signs of infection in affected animals, which were treated with buparvaquone @ 2.5mg/kg body weight intramuscularly along with supportive medicine. Two 18S rRNA gene amplicons were sequenced and analysed in a phylogenetic tree and haplotype network with 54 Indian and 38 foreign sequences. The phylogenetic tree revealed two groups with a high posterior probability and bootstrap value, while the haplotype network revealed 35 haplotypes, with haplotype 1 (H1) being the most abundant and several single haplotypes clustering around it, indicating fast and widespread expansion. Genetic diversity indices and neutrality tests confirmed that the population was expanding. These studies highlight the significance of prompt and precise diagnosis and management of T. annulata outbreaks and provide insights into its evolutionary history and population dynamics of T. annulata in India, which could aid improving disease preventive and control strategies.

Isolation and Molecular Characterization of Clostridium perfringens Toxinotypes F & G in Diarrhoeic Sheep (Ovis aries) Flocks in Southeast of Iran.

Clostridial enteric diseases, called enterotoxemia, are caused by Clostridium perfringens toxinotypes in sheep and other ruminants. This study aimed to describe the molecular characterization of C. perfringens isolates in diarrhoeic sheep (Ovis aries) flocks in the southeast of Iran. Fecal/intestinal samples were collected from diarrhoeic (n=116), dead (n= 13), and healthy (n=63) sheep over four years (2016-2020) and subjected to bacteriological and molecular examinations. The C. perfringens isolates were typed by polymerase chain reaction targeting genes, namely 16SrRNA, CPA, CPB, ETX, IAP, CPE, and NetB. The overall prevalence of C. perfringens was 28.6% among the studied sheep, and there was a significant relationship between its isolation rate and diarrhea (P<0.001). The C. perfringens isolation rate also decreased with animal age (P=0.012) and was significantly higher in late winter and spring (P=0.000). The most prevalent toxinotypes were types A (52.4%), D (22.2%), and F (18.5%), in that order. Moreover, C, G, and B types were found in 4.2%, 1.6%, and 1.1% of the isolates, respectively, and no type E was detected. The CPE gene was detected in 32.3% of all isolates, and the diarrhoeic sheep were most likely to yield CPE+ strains of C. perfringens (93.1%). These findings highlight the importance of CPE+ strains of C. perfringens in sheep enteritis and suggest that the high presence of type F needs to be considered in new clostridial vaccines containing this toxinotype. It is noteworthy that the present study reported the isolation of C. perfringens type F, type G, and the CPE+ strains of type B from diarrhoeic sheep for the first time.

Mapping human pathogens in wastewater using a metatranscriptomic approach

The monitoring of cities’ wastewaters for the detection of potentially pathogenic viruses and bacteria has been considered a priority during the COVID-19 pandemic to monitor public health in urban environments. The methodological approaches frequently used for this purpose include deoxyribonucleic acid (DNA)/Ribonucleic acid (RNA) isolation followed by quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)‒qPCR targeting pathogenic genes. More recently, the application of metatranscriptomic has opened opportunities to develop broad pathogenic monitoring workflows covering the entire pathogenic community within the sample. Nevertheless, the high amount of data generated in the process requires an appropriate analysis to detect the pathogenic community from the entire dataset. Here, an implementation of a bioinformatic workflow was developed to produce a map of the detected pathogenic bacteria and viruses in wastewater samples by analysing metatranscriptomic data. The main objectives of this work was the development of a computational methodology that can accurately detect both human pathogenic virus and bacteria in wastewater samples. This workflow can be easily reproducible with open-source software and uses efficient computational resources. The results showed that the used algorithms can predict potential human pathogens presence in the tested samples and that active forms of both bacteria and virus can be identified. By comparing the computational method implemented in this study to other state-of-the-art workflows, the implementation analysis was faster, while providing higher accuracy and sensitivity. Considering these results, the processes and methods to monitor wastewater for potential human pathogens can become faster and more accurate. The proposed workflow is available at https://github.com/waterpt/watermonitor and can be implemented in currently wastewater monitoring programs to ascertain the presence of potential human pathogenic species.

Viral etiology of lower respiratory tract infections in adults in the pre-COVID-19 pandemic era: A cross-sectional study in a single center experience from Cameroon.

Respiratory viruses are responsible for the majority of lower respiratory tract infections (LRTIs) worldwide. However, there is a gap on the epidemiology of viral LRTIs in adults in sub-Saharan African countries. In Cameroon, like in other countries, the role of viral respiratory pathogens in the etiology of LRTIs in adults is helpful for clinical management. This study aimed to determine the viral aetiologies of LRTIs among hospitalized adults in a reference center for respiratory diseases in the town of Yaounde in Cameroon and its surroundings. A cross-sectional study was conducted from January 2017 to January 2018 at Jamot Hospital in Yaounde (Cameroon). Clinical and demographic information; BAL and sputa were collected from hospitalized patients meeting LRTI case definitions. The clinical samples were investigated for respiratory pathogens with a commercial Reverse Transcriptase Real-Time Polymerase Chain Reaction (RT-PCR) targeting 21 viruses, cultures for bacterial and fungal infections. The 77 included adult patients with LRTIs had an appropriate clinical sample for microbial investigations. A viral agent was detected in 22.1% (17/77) samples. The main viruses detected included rhinovirus (10/77), coronavirus (hCoV-OC43 and hCoV-229E), and influenza A virus (3/77 each). A concomitant viral and bacterial co-infection occurred in 7.8% of patients (6/77) while viral co-infection occurred in one patient (1.3%). No Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) was detected in clinical samples. Most patients were under antimicrobials before getting diagnosed. Respiratory viruses account for 22.1% of LRTIs in hospitalized patients in this study. Despite prior antimicrobial therapy and delay, rhinovirus, coronavirus and influenza A virus were the most detected in patients in the pre-COVID-19 pandemic era in a single center experience from Cameroon.

Detection of infectious agents in equine pregnancy loss, stillbirth and neonatal death

Equine abortion, stillbirth and neonatal death cause major economic losses to the equine industry worldwide. Both non-infectious and a wide range of infectious causes have been described. However, the relative contribution of pathogens to equine abortion, stillbirth and neonatal death is poorly documented, since available studies involve only a limited number of pathogens. Therefore, the objectives of the present retrospective monitoring study were to determine the prevalence of infectious agents associated with equine abortion and perinatal mortality in Belgium, and to set up a protocol usable under field conditions using polymerase chain reaction targeting. A real-time simple polymerase chain reaction for eight different abortifacient pathogens was conducted leading to the detection of at least one infectious agent in 37% of 105 analyzed cases. In the diagnosed cases, equine herpesvirus-1 was the most detected pathogen (49%), followed by Streptococcus equi subspecies zooepidemicus (28%), Coxiella burnetii (18%), Leptospira interrogans (3%) and Neospora caninum (3%). None of the analyzed cases was positive for equine viral arteritis, equine herpesvirus-4 and Chlamydophila spp. In this study, PCR targeting is shown to have its value to detect a (co)-infectious cause in equine abortion, stillbirth and neonatal death, especially in field conditions where autolysis and contamination might preclude a full post-mortem examination protocol based on classical microbiological examination.

Syndromic Panel Testing Among Patients With Infectious Diarrhea: The Challenge of Interpreting Clostridioides difficile Positivity on a Multiplex Molecular Panel.

Including Clostridioides difficile (CD) in gastrointestinal multiplex molecular panels (GIPCR) presents a diagnostic challenge. Incidental detection by polymerase chain reaction (PCR) without consideration of pretest probability (PTP) may inadvertently delay diagnoses of other treatable causes of diarrhea and lead to prescription of unnecessary antibiotics. We conducted a retrospective study to determine the frequency at which clinicians characterize PTP and disease severity in adult patients who test positive for CD by GIPCR. We organized subjects into cohorts based on the status of their CD PCR, glutamate dehydrogenase enzyme immunoassay (GDH), and toxin A/B detection, as well as by high, moderate, or low CD PTP. We used multivariable regression models to describe predictors of toxin positivity. We identified 483 patients with positive CD PCR targets. Only 22% were positive for both GDH and CD toxin. Among patients with a low PTP for CDI, 11% demonstrated a positive CD toxin result compared to 63% of patients with a high PTP. A low clinician PTP for CD infection (CDI) correlated with a negative CD toxin result compared to cases of moderate-to-high PTP for CDI (odds ratio, 0.19 [95% confidence interval, .10-.36]). Up to 64% of patients with negative GDH and CD toxin received CD treatment. Only receipt of prior antibiotics, fever, and a moderate-to-high clinician PTP were statistically significant predictors of toxin positivity. Patients with a positive CD PCR were likely to receive treatment regardless of PTP or CD toxin results. We recommend that CD positivity on GIPCR be interpreted with caution, particularly in the setting of a low PTP.

Effective microbial molecular diagnosis of periodontitis-related pathogen Porphyromonas gingivalis from salivary samples using rgpA gene.

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.

Markers of placental function correlate with prevalence and quantity of nucleated fetal cells in maternal circulation in normotensive term pregnancies.

Transplacental fetal cell transfer results in the engraftment of fetal-origin cells in the pregnant woman's body, a phenomenon termed fetal microchimerism. Increased fetal microchimerism measured decades postpartum is implicated in maternal inflammatory disease. Understanding which factors cause increased fetal microchimerism is therefore important. During pregnancy, circulating fetal microchimerism and placental dysfunction increase with increasing gestational age, particularly towards term. Placental dysfunction is reflected by changes in circulating placenta-associated markers, specifically placental growth factor (PlGF), decreased by several 100 pg/mL, soluble fms-like tyrosine kinase-1 (sFlt-1), increased by several 1000 pg/mL, and the sFlt-1/PlGF ratio, increased by several 10 (pg/mL)/(pg/mL). We investigated whether such alterations in placenta-associated markers correlate with an increase in circulating fetal-origin cells. We included 118 normotensive, clinically uncomplicated pregnancies (gestational age 37+1 up to 42+2 weeks' gestation) pre-delivery. PlGF and sFlt-1 (pg/mL) were measured by Elecsys® Immunoassays. We extracted DNA from maternal and fetal samples and genotyped four human leukocyte antigen loci and 17 other autosomal loci. Paternally inherited, unique fetal alleles served as polymerase chain reaction (PCR) targets for detecting fetal-origin cells in maternal buffy coat. Fetal-origin cell prevalence was assessed by logistic regression, and quantity by negative binomial regression. Statistical exposures included gestational age (weeks), PlGF (100 pg/mL), sFlt-1 (1000 pg/mL) and the sFlt-1/PlGF ratio (10 (pg/mL)/(pg/mL)). Regression models were adjusted for clinical confounders and PCR-related competing exposures. Gestational age was positively correlated with fetal-origin cell quantity (DRR = 2.2, P = 0.003) and PlGF was negatively correlated with fetal-origin cell prevalence (odds ratio [OR]100 = 0.6, P = 0.003) and quantity (DRR100 = 0.7, P = 0.001). The sFlt-1 and the sFlt-1/PlGF ratios were positively correlated with fetal-origin cell prevalence (OR1000 = 1.3, P = 0.014 and OR10 = 1.2, P = 0.038, respectively), but not quantity (DRR1000 = 1.1, P = 0.600; DRR10 = 1.1, P = 0.112, respectively). Our results suggest that placental dysfunction as evidenced by placenta-associated marker changes, may increase fetal cell transfer. The magnitudes of change tested were based on ranges in PlGF, sFlt-1 and the sFlt-1/PlGF ratio previously demonstrated in pregnancies near and post-term, lending clinical significance to our findings. Our results were statistically significant after adjusting for confounders including gestational age, supporting our novel hypothesis that underlying placental dysfunction potentially is a driver of increased fetal microchimerism.

SARS-CoV-2 mutations on diagnostic gene targets in the second wave in Zimbabwe: A retrospective genomic analysis

SARS-CoV-2 continues to be a major issue in resource-limited settings, particularly owing to the limited supply of vaccinescaused by inequitable distribution. To monitor diagnostic gene targets to identify potential test failures caused by mutations, which is important for public health. Here we analysed the genome sequence of SARS-CoV-2 from the second wave in Zimbabwe. A total of 377 samples weresequenced at Quadram Institute Bioscience. After quality control, 192 sequences passed and were analysed. The Beta variant was dominant during this period, contributing 77.6% (149) of the genomes sequenced and having a total of 2994mutations in diagnostic polymerase chain reaction target genes. Many single nucleotide polymorphism mutations resulted in amino acidsubstitution that had the potential to impact viral fitness by increasing the rate of transmission or evading the immune response to previousinfection or vaccination. There were nine lineages circulating in Zimbabwe during the second wave. The B.1.351 was dominant, accounting for >75%.There were over 3 000 mutations on the diagnostic genes and lineage B.1.351, contributing almost two-thirds of the mutations. The S-genehad the most mutations and the E-gene was the least mutated.

Detection of porcine parvovirus in domestic pigs in North Kerala

During the period from 2019 to 2021, a total of 45 tissue samples were collected from pigs in Kerala and tested for the presence of porcine parvovirus (PPV) by polymerase chain reaction (PCR) targeting NS1 gene of the virus. Of the samples tested, 4 (8.89 per cent) were found to be positive which was higher than the positivity reported for the virus in Kerala in 2016. Sequence analysis of the amplicons revealed a high degree of similarity to PPV sequences previously reported from India. Biosecurity measures should be adopted to control the spread of viral diseases.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare.

Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Profiling Bartonella infection and its associated risk factors in shelter cats in Malaysia.

Poor disease management and irregular vector control could predispose sheltered animals to disease such as feline Bartonella infection, a vector-borne zoonotic disease primarily caused by Bartonella henselae. This study investigated the status of Bartonella infection in cats from eight (n = 8) shelters by molecular and serological approaches, profiling the CD4:CD8 ratio and the risk factors associated with Bartonella infection in shelter cats. Bartonella deoxyribonucleic acid (DNA) was detected through polymerase chain reaction (PCR) targeting 16S-23S rRNA internal transcribed spacer gene, followed by DNA sequencing. Bartonella IgM and IgG antibody titre, CD4 and CD8 profiles were detected using indirect immunofluorescence assay and flow cytometric analysis, respectively. B. henselae was detected through PCR and sequencing in 1.0% (1/101) oral swab and 2.0% (1/50) cat fleas, while another 3/50 cat fleas carried B. clarridgeiae. Only 18/101 cats were seronegative against B. henselae, whereas 30.7% (31/101) cats were positive for both IgM and IgG, 8% (18/101) cats had IgM, and 33.7% (34/101) cats had IgG antibody only. None of the eight shelters sampled had Bartonella antibody-free cats. Although abnormal CD4:CD8 ratio was observed in 48/83 seropositive cats, flea infestation was the only significant risk factor observed in this study. The present study provides the first comparison on the Bartonella spp. antigen, antibody status and CD4:CD8 ratio among shelter cats. The high B. henselae seropositivity among shelter cats presumably due to significant flea infestation triggers an alarm of whether the infection could go undetectable and its potential transmission to humans.

Listeria monocytogenes isolated from raw milk: phenotypic and molecularcharacterization, pathogenicity testing, and multidrug resistance profiling

Foodborne infections are a worldwide public health emergency that actually impact a variety of disorders. In the current research, a total of 300 samples comprising raw milk (100),milk products (100), and chicken (100) were screened for detection of pathogenic Listeria species by using the USDA method. On conventional biochemical characterization, three isolates were identified as Listeria monocytogenes (from raw milk samples)indicating an overall prevalence of Listeria monocytogenes to the tune of 1%. All other samples comprising milk products and chicken samples (100) showed negativity for the presence of any of the Listeria species. Further, all three isolates were subjected to polymerase chain reaction (PCR) targeting genus-specific (prsA and iap) and species-specific gene (isp) in which all three were turn out positive for both the genes, endorsing their identification as Listeria monocytogenes. All three confirmed Listeria monocytogenes isolates were phenotypically assessed for in-vitro pathogenicity tests like hemolysis on 7% sheep blood agar, CAMP test, and PI-PLC assay. The results revealed their highly pathogenic nature. Subsequently, all these isolates were also assessed for their virulent nature by PCR, targeting the array ofmarkers including virulence-associated genes viz. hlyA, actA, plcA. The results endorsed pathogenic nature of all isolates showing amplification of all targeted virulence genes. The antibiotic resistance profiling revealed occurrence of multidrug-resistant pathogenic L. monocytogenes in foods of animal origin with maximum multiple antibiotic resistance (MAR) index of 0.6and a minimum MAR index of 0.48 in MDR, which is a matter of concern from public health point of view.