Semi-nested PCR Research Articles

Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran.

Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mechanism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and preserved in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infantum using a semi-nested polymerase chain reaction (PCR) assay. The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India.

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4×106/μg of sample) and least in the brain (9×103/μg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.

Molecular evidence of Borrelia spp. in bats from Córdoba Department, northwest Colombia

BackgroundThe genus Borrelia is composed of two well-defined monophyletic groups, the Borrelia burgdorferi sensu lato complex (Bb) and the relapsing fever (RF) group borreliae. Recently, a third group, associated with reptiles and echidnas, has been described. In general, RF group borreliae use rodents as reservoir hosts; although neotropical bats may also be involved as important hosts, with scarce knowledge regarding this association. The objective of this study was to detect the presence of Borrelia spp. DNA in bats from the department of Córdoba in northwest Colombia.MethodsDuring September 2020 and June 2021, 205 bats were captured in six municipalities of Córdoba department, Colombia. Specimens were identified using taxonomic keys and DNA was extracted from spleen samples. A Borrelia-specific real-time PCR was performed for the 16S rRNA gene. Fragments of the 16S rRNA and flaB genes were amplified in the positive samples by conventional PCR. The detected amplicons were sequenced by the Sanger method. Phylogenetic reconstruction was performed in IQ-TREE with maximum likelihood based on the substitution model TPM3+F+I+G4 with bootstrap values deduced from 1000 replicates.ResultsOverall, 10.2% (21/205) of the samples were found positive by qPCR; of these, 81% (17/21) and 66.6% (14/21) amplified 16S rRNA and flaB genes, respectively. qPCR-positive samples were then subjected to conventional nested and semi-nested PCR to amplify 16S rRNA and flaB gene fragments. Nine positive samples for both genes were sequenced, and seven and six sequences were of good quality for the 16S rRNA and flaB genes, respectively. The DNA of Borrelia spp. was detected in the insectivorous and fruit bats Artibeus lituratus, Carollia perspicillata, Glossophaga soricina, Phyllostomus discolor, and Uroderma sp. The 16S rRNA gene sequences showed 97.66–98.47% identity with “Borrelia sp. clone Omi3,” “Borrelia sp. RT1S,” and Borrelia sp. 2374; the closest identities for the flaB gene were 94.02–98.04% with “Borrelia sp. Macaregua.” For the 16S rRNA gene, the phylogenetic analysis showed a grouping with “Candidatus Borrelia ivorensis” and “Ca. Borrelia africana,” and for the flaB gene showed a grouping with Borrelia sp. Macaregua and Borrelia sp. Potiretama. The pathogenic role of the Borrelia detected in this study is unknown.ConclusionsWe describe the first molecular evidence of Borrelia spp. in the department of Córdoba, Colombia, highlighting that several bat species harbor Borrelia spirochetes.Graphical

Avian gastric yeast (macrorhabdosis) in cockatiel, budgerigar and grey parrot: a focus on the clinical signs, molecular detection and phylogenetic evaluation.

Macrorhabdus ornithogaster is a microorganism that causes nonspecific and general clinical symptoms and to this day, diagnosis and also treatment have been yet hard. The present study was conducted to survey the prevalence of macrorhabdosis and to characterize M. ornithogaster phylogenetically in Psittaciformes suspected of macrorhabdosis from January 2018 to May 2019 in Ahvaz, Iran. For this purpose, fecal samples were collected from Psittaciformes with signs of the disease. Wet mounts were prepared from fecal samples and examined carefully using a light microscope. Samples from parrots with gastrointestinal symptoms of the disease were chosen for molecular diagnosis of the organism and DNA was extracted from these samples. For detection of M. ornithogaster, primer sets (BIG1, Sm4) and (AGY1, Sm4) which target the 18S rDNA gene were selected and Semi-nested polymerase chain reaction (Semi-nested PCR) was performed. The PCR method confirmed the presence of M. ornithogaster in 14.00% of the samples. Purified PCR products were sequenced for more accurate confirmation and according to the gene sequence all sequences were owned by M. ornithogaster. The results disclosed a 96.03 - 100% identity when compared to other sequences of M. ornithogaster which had previously been deposited in the GenBank® from Germany and the USA. The results of this study proved the circulation of M. ornithogaster between cockatiel, budgerigar and grey parrot. The prevalence of macrorhabdosis was higher in cockatiel compared to budgerigar and grey parrot. As far as the authors know, this was the first record of macrorhabdosis in African grey parrots.

First Report of Powdery Mildew Caused by Podosphaera fuliginea on Veronica spicata in China.

Veronica spicata L. (syn. Pseudolysimachion spicatum Opiz), Plantaginaceae, is a perennial herb and frequently cultivated in gardens as an ornamental plant in China. In June 2017, powdery mildew infections were observed on V. spicata in Jingyuetan National Forest Park (43.80°N, 125.46°E), Changchun, China. A voucher specimen was deposited in the Herbarium of Mycology of Jilin Agricultural University under the accession no. HMJAU-PM91763. The incidence of the disease on leaves and stems was about 30 to 50%. The disease initially appeared as thin white colonies, which subsequently developed into dense effuse white colonies on the plant. Hyphae were flexuous to straight, septate, 3.0 to 8.0 μm wide. Hyphal appressoria were indistinct or nipple-shaped, solitary. Conidiophores (n=30) arise from the upper surface of hyphal mother cells, erect to straight, 133.4 to 176.2 × 7.8 to 10.7 μm. Foot-cells (n=40) were cylindrical, straight or slightly flexuous, 39.1 to 78.5 × 7.4 to 9.7 μm, and followed by 1 to 3 short cells. Conidia (n=45) were catenescent, ellipsoid, oval, or doliiform, with fibrosin bodies, 17.8 to 27.8 × 12.2 to 17.4 μm, length/width ratio 1.3 to 2.1. Germ tubes were produced at the subterminal to lateral part of conidia, straight or sinuous, without a distinct terminal appressorium. The sexual morph was not observed in the collected samples. The morphological characteristics of the asexual morph were consistent with Podosphaera fuliginea (Schltdl.) U. Braun & S. Takam. (Braun and Cook 2012). To confirm the identification, the complete internal transcribed spacer (ITS) region and partial 28S rRNA gene sequences of the pathogen were amplified by semi-nested PCR with the primers ITS5/P3 followed by ITS5/ITS4, and LSU1/TW14 followed by LSU1/LSU2, respectively. The sequences of 566 bp ITS (MF543026) and 609 bp 28S rDNA (MF543027) were obtained and showed 100% identity with P. fuliginea (AB046986, ON073893) on V. spicata from USA (Hirata et al. 2000). Based on the morphological and molecular characteristics, the fungus was identified as P. fuliginea. To perform pathogenicity assays, three healthy annual plants of P. spicatum were inoculated by gently pressing a diseased leaf onto the leaves, with three non-inoculated plants as controls. All plants were placed in a greenhouse at 21 to 29 °C, 60% relative humidity, with 16 h/8 h light/dark cycle. Nine days after inoculation, typical powdery mildew colonies started to appear on the inoculated plants, while the control plants remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Powdery mildews on V. spicata (P. spicatum) were previously referred to as Erysiphe (Golovinomyces) orontii in Italy (Garibaldi et al. 2006) and Sphaerotheca (Podosphaera) fuliginea in many countries, such as Poland, Russia, Switzerland, Wisconsin, Ukraine, etc. (Amano 1986; Braun and Cook 2012; Farr and Rossman 2022; Heluta et al. 2011). To our knowledge, this is the first record of P. fuliginea on V. spicata from China and the first report of this species at all. The results of this study provide important information for horticultural management and plant protection in China. Acknowledgements This work was supported by the National Natural Science Foundation of China (31970019, 31670022).

ESTABLISHMENT OF MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF SALMONELLA SPP., SHIGELLA SPP. AND ENTAMOEBA HISTOLYTICA

Objective: To establish a novel multiplex PCR assay for simultaneous detectionof foodborne pathogens, including Salmonella spp., Shigella spp., and Entamoebahistolytica, with high sensitivity, specificity, cost-effectiveness and time-saving.Method: Multiplex PCR, Semi-Nested PCR and melting analysis for specificallyamplifying and distinguishing three highly conserved target gene regions (invA,ipaH, 18S rRNA), in order to simultaneously detect Salmonella spp., Shigella spp.,Entamoeba histolytica in a single reaction tube. Specific pathogens are characterizedbased on the melting profiles of the PCR products collected on a realtime PCRinstrument following the second round of amplification.Results: The established multiplex PCR assay is capable of simultaneously detectingSalmonella spp., Shigella spp., and Entamoeba histolytica at a limit of detection of5.86 copies/µl, 24.4 copies/µl và 53.6 copies/µl, respectively (confidence interval of95%), the specificity reached 100% when evaluated with multiple microbial speciesprovided by the Department of Microbiology, 103 Military Hospital.Conclusion: The novel multiplex PCR assay for simultaneous detection of Salmonellaspp., Shigella spp., Entamoeba histolytica has been successfully established.

Genetic diversity of Bartonella infection in residential and field rodents in Hebei, China.

Rodents are the primary natural reservoirs of Bartonella spp., and some of which are zoonotic causative agents. Hence, surveillance of Bartonella sp. infection in rodents is very important for the prevention of human bartonellosis caused by them. In this study, rodents were captured, and their spleen samples were collected for Bartonella sp. DNA detection and identification by amplifying the 16S rRNA, gltA, and ftsz genes using semi-nested polymerase chain reaction (PCR). The results indicated that Bartonella sp. DNA was detected in seven Rattus norvegicus individuals with a detection rate of 6.7% in Chengde City and bacterial DNA in 31 Apodemus agrarius individuals with a detection rate of 28.4% in Handan City. The DNA detection rate across the genders and ages of rodents was not found to be statistically significant. Furthermore, sequence analysis of the above-mentioned three genes demonstrated that at least eight Bartonella species were circulating in Hebei Province, of which three, including Bartonella rattimassiliensis, Bartonella grahamii, and Bartonella tribocorum, are human pathogens, thus suggesting the existence of a major public health risk. Overall, these results revealed the detection rate and genetic diversity of Bartonella species infection in rodents in Hebei Province, which could be potentially helpful for the prevention of bartonellosis caused by rodent-associated Bartonella species. This study highlights the urgent need for the surveillance of Bartonella infections in rodents and ectoparasites that affect both rodents and humans and can cause fever of unknown origin or endocarditis.

Accompanying a semi-nested PCR assay to support histopathology findings of fungal keratitis in formalin-fixed paraffin-embedded corneal samples.

Fungal species are responsible for 40%-50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin-fixed Paraffin-embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field. Sixty-six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi-nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing. Fungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal-positive cases, 39 were PCR-positive (95%). Moreover, of 44 PCR-positive samples, 39 (88.6%) were histopathology-positive, and 5 (11.3%) were histopathology-negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively. As we reached the acceptable Kappa agreement rate, we concluded that applying the semi-nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates.

Tomato and Jute Mallow are Two New Hosts of Papaya Bunchy Top Phytoplasma, a 16SrXII-O Subgroup Strain in Nigeria.

Nigerian papaya bunchy top (NGPBT) phytoplasma was first identified in diseased papaya plants growing in Ibadan, Oyo State, Nigeria (Kazeem et al. 2021). The NGPBT phytoplasma is a 'Candidatus Phytoplasma convolvuli'-related strain and represents a subgroup lineage, 16SrXII-O (the accession number of the reference strain is MW530522, Kazeem et al. 2021). The present communication reports that NGPBT phytoplasma can also infect tomato (Solanum lycopersicum) and jute mallow (Corchorus olitorius). Since May 2020, tomato and jute mallow grwn in Ibadan have been observed to develop yellowing, little leaf, and stunting symptoms (Fig. 1). Because the symptomatic plants occurred in the region approximately 1 km adjacent to where the NGPBT disease was reported, and the symptoms of infected plants resembled those of phytoplasma infection, molecular diagnostic assays for phytoplasma detection were deployed. Total DNAs were extracted from symptomatic plants, including four tomato plants and three jute mallows, as well as from asymptomatic two tomato and two jute mallow plants. The DNA samples were subjected to semi-nested PCR using phytoplasma 16S rRNA gene-specific primers P1A and P7A, followed by P1A and 16S-SR (Lee et al. 2004). An amplicon of 1.5 kb was obtained from each of the symptomatic plants, while no amplicon resulted from DNA samples of asymptomatic plants or negative controls without DNA templates (water and PCR reagents only). PCR products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), and three clones were chosen for each sample for Sanger sequencing (Psomagen Inc., Rockville, MD, USA). The nearly full-length 16S rRNA gene sequences (1.53kb) derived from tomato (OP123558) and jute mallow (OP123559) samples were identical. Based on the iPhyClassifier phytoplasma classification web tool (Zhao et al. 2009) and the BLAST search against the NCBI nucleotide database, these phytoplasma strains showed 100% sequence identity in 16S rRNA gene with the NGPBT phytoplasma (16SrXII-O, MW530522). Moreover, two additional genetic loci including ribosomal protein genes rplV-rpsC, and rplO-secY-adk were also amplified by nested PCR or semi-nested PCR with specific primers rpStolF/rpStolR followed by rpStolF2/rpStolR (Martini et al. 2007), and SecYF1a (Xll)/MapR-703-a, followed by SecYF2a (Xll)/MapR-703-a (Lee et al. 2010). Gene fragments of rplV-rpsC (1238bp) and rplO-secY-adk (2064bp) were amplified from DNAs of diseased papaya, tomato, and jute mallow plants. The obtained sequences were deposited into GenBank, respectively: rplV-rpsC (OP123560, OP123562, and OP123563) and rplO-secY-adk (OP123565, OP123567, and OP123568). Multilocus sequence analysis (MLSA) indicated that the sequences of phytoplasmas amplified from three different plant hosts were also identical in rp, secY, and adk genes. The MLSA results demonstrate that tomato and jute mallow are two new hosts of NGPBT phytoplasmas. This also marks the first time that phytoplasma diseases are associated with tomato and jute mallow in Nigeria, as prior to this study, phytoplasma diseases were only reported in coconut palm and papaya in the country (Osagie et al. 2016; Kazeem et al. 2021). Results from the present study suggest that insect vector(s) for the transmission of the NGPBT phytoplasma are present in the region. Since both tomato and jute mallow are important vegetable crops in Nigeria, timely dissemination of emerging disease information is needed to alert growers and extension personnel in the region. In addition, ongoing incidence, and prevalence surveys of NGPBT disease indicate that more infected papaya and tomato plants have been observed in the region than in previous years. A better understanding of the NGPBT phytoplasma disease epidemiology will help devise strategies to control the diseases associated with the NGPBT phytoplasma.

Epidemiological Study of the Occurrence of Typhus Group Rickettsia Natural Infection in Domiciliated Dogs from a Rural Community in South-Eastern Mexico.

Simple SummaryRickettsioses are relevant emergent and reemergent zoonoses in the Americas, including Mexico. Murine typhus caused by typhus group (TG) Rickettsia is prevalent in humans and their companion animals, such as dogs. This study found that 23.9% of the dogs (34/142) were infected by TG Rickettsia in Maxcanú, Yucatan (southeastern Mexico). Statistical analyses showed that reduced outdoor activities, age, sex and previous antiparasitic treatment are associated factors with less risk of TG Rickettsia infection in the sampled dogs. Monitoring and controlling these factors could help to restrict the enzootic transmission risk and prevent the potential zoonotic transmission.The aim is to describe the Typhus group (TG) Rickettsia infection in dogs and to identify factors associated with this infection. We collected blood samples and gathered exposure and clinical data of 142 dogs from a rural community of Yucatan. The Rickettsia group was determined by semi-nested PCR. Generalized linear models with binomial error distribution were used to model the associated factors from the dog sample for risk ratio (RR) estimation. Thirty-four dogs (23.9%) showed molecular evidence of TG Rickettsia DNA. The multivariate model showed that mixed-breed dogs (RR = 0.06) and dogs that had received antiparasitic treatment (RR = 0.049) had a lower risk of getting infected, taking as reference the purebred group and the non-treated dogs, respectively. Looking at variable interactions, adult dogs without outdoor activities had a lower infection risk than puppies (RR = 0.26). Among dogs with antiparasitic treatment, females had a higher infection risk than male dogs (RR = 26.2). The results showed enzootic TG Rickettsia circulation in dogs of a rural community. The factors outdoor activities, age and previous antiparasitic treatment, as well as the clinical variables signs of hemorrhages and epistaxis, were associated with a less chance of natural infection in the studied dogs. Prevention and control of the enzootic transmission risk of TG Rickettsia should help to reduce the potential zoonotic transmission of this pathogen.

The Value of GeneXpert MTB/RIF for Detection in Tuberculosis: A Bibliometrics-Based Analysis and Review.

With the continuous development of medical science and technology, especially with the advent of the era of precision diagnosis and treatment, molecular biology detection technology is widely valued and applied as an aid to early diagnosis of tuberculosis. The GeneXpert Mycobacterium tuberculosis Branching (MTB) technology is a suite of semi-nested real-time fluorescent quantitative PCR in vitro diagnostic technologies developed by Cepheid Inc. It targets the rifampicin resistance gene, rpoB, and can detect both MTB and resistance to rifampicin within 2 h. This review analyzed the papers related to GeneXpert using bibliometric software CiteSpace and Bibliometrix. A total of 151 articles were analyzed, spanning from 2011 to 2021. This bibliometrics-based review summarizes the history of the development of GeneXpert in tuberculosis diagnosis and its current status. Contributions of different countries to the topic, journal analysis, key paper analysis, and clustering of keywords were used to analyze this topic.

Isolation, Propagation and Genotyping of Human Rotaviruses Circulating among Children with Gastroenteritis in Two Egyptian University Hospitals.

Simple SummaryGroup A rotaviruses are the most common cause of acute gastroenteritis affecting Egyptian children under the age of five, with symptoms ranging from asymptomatic infection to severe dehydration or death. In the present work, diarrheal samples from Egyptian children admitted to gastrointestinal pediatric wards of two main governmental hospitals were collected and molecularly analyzed for Group A rotavirus. Our findings revealed that rotaviruses accounted for more than one-sixth of all cases under study, peaking in the winter. G1P[8] was the most prevalent rotavirus genotype in this study. The two cell lines used in our work coherently isolated and propagated rotavirus strains. Continuous rotavirus detection and genome sequencing of the successfully isolated strains will be recommended in the future in order to support the control of such viruses, and tackle the problem in Egypt.The most prevalent cause of infectious neonatal diarrhea is Group A rotavirus (RVA). Unfortunately, there is a dearth of data on the incidence of rotavirus-associated infections among Egyptian children. The present study aimed to isolate, propagate, and genotype human rotaviruses circulating among Egyptian children with acute gastroenteritis admitted to two main university pediatric hospitals, Abo El-Reesh and El-Demerdash, over two consecutive winters, 2018–2020. Diarrheal samples (n = 230) were screened for Group A rotavirus RNA using RT-PCR assay. In positive samples (n = 34), multiplex semi-nested PCR was utilized to determine G and P genotypes. Thirty-four (14.8%) of the collected samples tested positive. The genotype distribution revealed that G1P[8] was the predominant rotavirus genotype throughout the current study. All rotavirus-positive fecal samples were passaged twice on human colorectal adenocarcinoma cell line (Caco-2) and rhesus monkey kidney epithelial cell line (MA104). Both cell lines could successfully isolate 14.7% (n = 5 out of 34) of the identified strains; however, Caco-2 cell line was shown to be more efficient than MA104 in promoting the propagation of human rotaviruses identified in Egyptian children’s feces.

P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesWe aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.MethodsThis prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).ResultsAmong 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.ConclusionsThe molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).

P411 Genus specific real-time PCR-a promising technique for rapid diagnosis of fungal keratitis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectiveComparison of different existing molecular methods for diagnosis of fungal keratitis (FK) and to develop and validate genus-specific PCR for identification of most predominant FK causative agents.MethodA prospective multicentric study was performed between November 2019 to August 2021 from four centers across India. Corneal tissue/scraping samples were collected from patients with suspected keratitis for preliminary microbiological workup at respective centers and molecular diagnosis at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. A total of 87 corneal button samples were used for standardization. All samples were subjected to DNA extraction followed by molecular diagnosis using pan-fungal primers by conventional PCR, semi-nested PCR, and real-time PCR targeting the internal transcribed spacer (ITS) region of rDNA. The genus-specific primers for the most common causative agents of FK (Aspergillus sp., Fusarium sp., Alternaria sp., and Curvularia sp.) were designed in ITS2 region and standardized for real-time PCR. The best performing protocol was validated in 145 corneal samples.ResultA total of 68 patients out of 87 were diagnosed with FK of which 91.17% (n = 62/68) were microscopy positive and 82.3% (n = 56/68) were culture positive. Among the culture positive, the most common isolate was Aspergillus sp. (26, 46.42%) followed by Fusarium sp. (21, 37.5%) while the remaining samples grew dematiaceous fungi. Real-time PCR targeting ITS2 region proved to be most sensitive (52.94%) and specific (84.21%) compared with conventional PCR and semi-nested PCR. Genus-specific real-time PCR for Aspergillus sp. and Fusarium sp. showed improved sensitivity and specificity of 82.76%, 87.18%, and 90.91%, 93.48% respectively compared with all other diagnostic methods used in the study. The positive (PPV) and negative predictive value (NPV) for Aspergillus sp. specific PCR were 82.76% and 87.18% while Fusarium sp. specific PCR showed PPV of 86.96% and NPV of 95.56%. Genus-specific real-time PCRs did not show any amplification of 19 FK negative samples while faint amplification was observed in conventional PCR which on sequencing proved to be non-specific. No cross-reactivity was observed in clinical sample standardization. Due to the lack of Alternaria sp. and Curvularia sp. positive clinical samples, both PCRs were standardized using respective culture DNA which showed a positive result. Aspergillus sp. and Fusarium sp. genus-specific PCRs were further validated in 145 corneal samples, of which 91 were FK positive and showed similar results as that of standardization data. Genus-specific PCRs also reduced the turnaround time (˂24 h) by decreasing the need for the identification of causative agents.ConclusionReal-time PCR targeting ITS 2-region, particularly the genus-specific PCRs proved to be the most efficient for molecular diagnosis of FK. The genus-specific PCRs reduce the turnaround time by avoiding the need for sequencing and thus facilitating in rapid diagnosis of FK.

Hemoplasmas and ticks in cattle from Somalia

Hemotropic mycoplasmas (hemoplasmas) are small Gram-negative bacteria that parasitize red blood cells and can cause mild to severe anemia in a wide range of vertebrates, including ruminants. Cattle population in Somalia is around 3.9 million heads, with animals more concentrated around the river areas, mainly in the Juba River and Shabelle River Valleys. Information on hemoplasmas in Sub-Saharan Africa are scarce, mainly in Somalia, where no studies have been performed to date. Accordingly, this study aimed to assess the molecular occurrence of hemoplasmas in 131 cattle blood samples from Somalia. Thirty out of 131 (22.90%; 95% CI: 16.54-30.81%) cattle were infested by ticks: Rhipicephalus pulchellus (68.18%), Amblyomma gemma (18.18%), Amblyomma lepidum (9.09%), Hyalomma marginatum (1.51%), Hyalmomma rufipes (1.51%), and Rhipicephalus pravus (1.51%). A total of 74/131 (56.48%; 95% CI: 47.93-64.67%) cattle were positive for hemotropic Mycoplasma spp. by real-time PCR (qPCR) based on the 16S rRNA gene. Hemoplasma-positive samples were later subjected to species-specific PCR assays for Mycoplasma wenyonii and ‘Candidatus Mycoplasma haematobovis’ based on the 16S rRNA gene. A total of 34/74 (45.94%; 95% CI: 35.07-57.22%) animals were coinfected by both species; 31/74 (41.89%; 95% CI: 31.32-53.26%) and 3/74 (4.05%; 95% CI: 01.39-11.25%) cattle were solely positive to M. wenyonii and ‘Ca. M. haematobovis’, respectively. Six out of 74 (8.1%; 95% CI: 03.77-16.58%) cattle were negative on species-specific conventional PCR assays but tested positive by a semi-nested PCR assay based on the 16S rRNA gene of hemoplasmas. Sequencing of the detected hemotropic Mycoplasma sp. 16S rRNA gene confirmed that animals were infected by M. wenyonii and ‘Ca. M. haematobovis’. To the best of our knowledge, this is the first study on the detection of hemoplasmas in cattle from Somalia.

BK and JC polyomaviruses and risk of urothelial bladder carcinoma: a preliminary study in the northern shores of Persian Gulf, Iran

BackgroundBladder cancer is a challenging public health concern in South of Iran because of its high prevalence and the related medical expenses. Although the exact etiology of bladder cancer remains unknown, given the cell transforming ability and oncogenic potential of the members of Polyomaviridae families, this study was conducted to evaluate the magnitude of BK polyomavirus (BKPyV) and John Cunningham polyomavirus (JCPyV) among patients with bladder cancer residents in the northern shores of the Persian Gulf, South of Iran.MethodsTotally 211 patients with bladder cancer were enrolled in this study. Bladder biopsy samples of these patients and patients with interstitial cystitis as well as autoptic samples of healthy bladder were tested for detection of BKPyV and JCPyV by semi-nested PCR–RFLP followed by sequencing.ResultsBKPyV and JCPyV were detected in 1.7% and 6.1% of bladder cancer samples, respectively. These samples were infected with JCPyV genotypes 2, 3 and 6 and BKPyV genotypes I and IV. BKPyV and JCPyV coinfection was detected in 2 samples. Moreover, one of the healthy bladder samples was positive for BKPyV, and one of the interstitial cystitis samples was positive for JCPyV. Although the majority of infected patients were in the age group 70–79 years, male, residents in Tangestan, stage Ta–T1, and low-grade and high-grade papillary urothelial carcinoma, the prevalence of BKPyV and JCPyV among patients with bladder cancer was not statistically associated with age, gender, place of residency, and stage and grade of the tumor.ConclusionDespite identifying BKPyV and JCPyV in a number of bladder cancer biopsy specimens and the high prevalence of bladder cancer among people resident in South of Iran, it is suggested that these viruses are unlikely to be effective causative factors in bladder carcinogenesis in this region. Therefore, environmental risk factors and genetic backgrounds may have a more prominent role than human polyomaviruses in the development of bladder cancer in South of Iran.

Occurrence of Equine Foamy Virus Infection in Horses from Poland

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

Human rotavirus VP4 and VP7 genetic diversity and detection of GII norovirus in Ibadan as Nigeria introduces rotavirus vaccine.

ObjectiveThis cross-sectional study investigated the circulating strains of rotavirus and screened for noravirus in Ibadan, Nigeria as the country introduces the rotavirus vaccine into its national immunization program.MethodsSixty-five stool samples were collected from children younger than 5 years with clinically diagnosed diarrhea and screened for the presence of rotavirus and norovirus using RT-PCR. Rotavirus-positive samples were further analyzed to determine the G and P genotypes using semi-nested multiplex PCR.ResultsThe rates of rotavirus and norovirus positivity were 30.8% and 10.8%, respectively, whereas the rate of rotavirus and norovirus mixed infection was 4.6%. G1 was the predominant VP7 genotype, followed by G2, G9, and G1G2G9, whereas the predominant VP4 genotype was P[4], followed by P[6], P[8], and P[9]. The mixed P types P[4]P[8] and P[4]P[6] were also detected. G1P[4] was the most common VP4 and VP7 combination, followed by G2P[4], G1[P6], G1P[8], G2P[6], G2P[9], G9P[6], G2G9P[4], G2P[4]P[6], G1P[4]P[8], G2G9P[8], G1G2G9P[8], and G1[non-typable] P[non-typable], which were detected in at least 5% of the samples. Four samples had a combination of non-typable G and P types.ConclusionsIt is essential to monitor the circulation of virus strains prior to and during the implementation of the immunization program.

Prevalence, diversity and public health implications of Helicobacter species in pet and stray dogs

BackgroundIt is probable that humans can acquire H. pylori and nonH. pylori Helicobacter infections via domestic animals. The prevalence and risk factors of infections of Helicobacter species in canines of Taipei city were therefore analysed in this study. Materials and methodsA total of 95 canine faecal samples were collected from different animal shelters and hospitals in Taipei city. Total DNA was extracted for semi-nested PCR detection of Helicobacter species. The PCR products were sequenced for further comparative database and phylogenetic analyses. ResultsThe overall prevalence of Helicobacter species in canines of Taipei city was 75.79% (72/95). Two gastric, seven enterohepatic and two unclassified Helicobacter species were identified, all of which have been implicated in the aetiology of human diseases. The predominant species detected included H. canis (27.78%), H. pylori (26.39%), H. canicola (18.06%), and H. bilis (13.89%) in decreasing order, while H. canadensis and H. typhlonius were identified for the first time in canines. The genotypes in H. pylori and H. canicola clusters grouped together, with their respective reference strains, showed a close evolutionary distance in the phylogenetic tree, indicating a common ancestry may have existed in these clusters respectively. The residential region of canines, dog living status (pet or stray) and breed (purebred or mixed-breed) are the risk factors associated with Helicobacter infections in the canines examined. ConclusionThe high prevalence of Helicobacter infections in canines highlights a potential public health risk of zoonotic transmission among dogs, humans and other animals, and therefore, the need for proper methods in controlling the transmission routes. In addition, the 16S rRNA gene amplification method was found to be useful for bacterial identification and phylogenetic analysis.

Leptospirosis among Dengue-Negative Febrile Patients in Selangor, Malaysia.

In recent years, the number of leptospirosis cases, including the number of deaths, has exponentially increased in Malaysia. From June 2016 to February 2018, blood samples of 321 febrile patients with the presumptive diagnosis of dengue-like illness were examined for possible exposure to Leptospira. Two hundred fifty-five blood samples were tested as negative for dengue. Seminested polymerase chain reaction (PCR) and IgM ELISA for leptospirosis were performed. From the samples, an overall prevalence for leptospirosis based on PCR of 4.7% (12/255) was obtained. Eighteen percent (46/255) were positive for anti-Leptospira IgM antibodies. The genome sequences of six of 12 Leptospira PCR-positive samples showed > 97.0% similarity to Leptospira interrogans. One patient's sample consisted of Leptospira and chikungunya virus, suggesting a coinfection. Findings from the study suggest that leptospirosis is prevalent among dengue-negative febrile patients in Malaysia.