DNA Polymerase Chain Reaction Research Articles

Molecular Detection of Echinococcus granulosus Sensu Stricto in Environmental Dog Faecal Samples from the Magallanes Region, Patagonia, Chile

Echinococcus granulosus is endemic in the Magallanes region and other areas of Chile. After a successful control programme implemented between 1979 and 2004, dogs’ prevalence decreased from 70% to 0.5%. Since the end of the programme, no prevalence study of canine echinococcosis has been performed in this region. Dog faecal samples were collected from epidemiological units for DNA isolation and polymerase chain reaction (PCR) to detect E. granulosus. In addition, dog owners were required to answer a questionnaire for a risk factor analysis. We collected 1069 environmental dog faecal samples from 267 urban, 241 periurban, and 61 rural epidemiological units. E. granulosus was found in 11/61 (18%) and 1/241 (0.4%) epidemiological units from rural and periurban areas, respectively. The Ultima Esperanza province showed the highest prevalence with eight out of 16 units with faeces positive to E. granulosus showing a main spatial cluster of canine echinococcosis. None of the risk factors investigated showed a statistical significance with positive units. This study shows the active transmission of the parasite in the Magallanes region, especially in the Ultima Esperanza province, with a possible re-emergence of the parasite. Further studies focusing on the incidence in humans are required together with the reestablishment of the compulsory deworming of dogs.

RAPD Fingerprinting and Genetic Diversity of Salmonella Spp. Isolated from Broiler and Layer Flocks in Karbala, Iraq.

Salmonellosis in poultry is one of the most significant bacterial infections causing mortality, reduced production, and serious economic losses. This study aimed to study the molecular diversity among Salmonella isolates and investigate the epidemiological spread of these bacteria in broiler and layer chicken flocks in five different farms in Karbala, Iraq, using random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). In total, 217 cloac a swabs were collected from the farms, out of which 129 and 88 swabs were taken from broiler and layer chickens. The samples were screened by PCR for S. enterica subsp. enterica using primers specific for the invA gene. Afterward, RAPD-PCR with uniplex or multiplex octamer primers was applied to genotype the isolates. The incidence rate of Salmonella infections in broilers and layers was estimated to be 27.9% and 12.5%. The uniplex primers P2 and P3, along with the multiplex primers yielded discriminatory patterns. Moreover, the RAPD typing showed a diverse range of banding patterns of Salmonella spp. Dendrograms created through GelJ software revealed various Salmonella genotypes in broilers and layers. The RAPD-PCR could be used as an accurate and fast tool to identify genetic relatedness among Salmonella spp. The obtained results would assist researchers in epidemiological studies and controlling salmonellosis in poultry fields.

DNA extraction and pattern of crab and macrobentos from North Sumatran mangrove forest, Indonesia

Mangrove forest ecosystem is one of the most productive and unique ecosystems that serves to protect coastal areas from various disturbances, as well as provide habitat for various animal species. The large number of crab species and macrobentos in mangrove ecosystems results in frequent errors in the naming of species that have similarities in morphological features. This problem can be solved through a comprehensive approach by combining morphological analysis with genetic analysi.This study aims to report a DNA extraction and PCR amplification prior was used for the identification of crab and macrozoobentos from mangrove forest, North Sumatra. Primer 16S has a conserved area so it is appropriately used in Polymerase Chain Reaction (PCR) and sequencing analysis to determine taxonomy, phylogeny and diversity between specie. Visualization of PCR amplification results with primer16S from crab samples and macrobentos resulting a low DNA band with a size of 206 bp and a high of 678bp

Antibiotic resistance and phylogenetic comparison of human, pet animals and raw milk Staphylococcus aureus isolates

The present study was conducted to compare the S. aureus isolates from different sources in the basis of resistance phenotypic and genotypic features and phylogenetic differences. Total of 70 S. aureus isolates (including 25 human, 25 raw milk and 20 pet animal isolates) were subjected to the antimicrobial susceptibility testing, polymerase chain reaction (PCR) detection of the resistance genes and DNA fingerprinting using random amplification of polymorphic DNA–PCR (RAPD-PCR) to survey the variability of the isolates. Among 70 S. aureus, 55 (78.5%) isolates were MRSA. The isolates showed the highest antibiotic resistance to methicillin, ampicillin and penicillin (78.5%) and showed the lowest resistance to ciprofloxacin (12.8%). ErmB and tetM resistance genes were present in all isolates and the vanA gene was not detected in any of the isolates. Thirteen distinct clusters were identified in RAPD-PCR fingerprinting. Statistical analysis showed that the isolates without resistance to antibiotics were significantly in associated with raw milk origin (P < 0.05). According to the results of the study, S. aureus strains with pets and raw milk origin are significant sources of antibiotic-resistant isolates such as MRSA. They are also carriers of resistance genes that can be transmit to human isolates and cause drug resistance in human infections. Identifying the source of these infections is possible with a reliable genotyping method such as RAPD-PCR.

Clinical Behavior and Outcome of HPV-Positive Nasopharyngeal Carcinoma

Nasopharyngeal cancer (NPC) is traditionally EBV related. However, HPV-positive (HPV+) NPC seems to be increasing in the Western world recently. We describe the clinical behavior and outcomes of HPV+ NPC compared to EBV-positive (EBV+) NPC and HPV+ oropharyngeal cancer (OPC).All newly diagnosed non-metastatic viral-related NPC and OPC treated with IMRT from 2005-2020 were reviewed. Viral etiology was confirmed by p16 staining, supplemented by real-time polymerase chain reaction of DNA for high-risk HPV, and EBER in-situ hybridization for EBV. Clinical characteristics of HPV+ and EBV+ NPC were compared using the 2005-2020 NPC cohort, while locoregional control (LRC), distant control (DC) and overall survival (OS) were compared using the 2005-2018 cohort to allow sufficient follow-up among HPV+ NPC, EBV+ NPC and HPV+ OPC patients. Presenting signs/symptoms of HPV+ NPC and EBV+ NPC were compared by 1:1 matched pair analysis (matched to T category, N category, smoking, age, gender, WHO type IIA vs IIB, and diagnosed year). Multivariable analysis (MVA) evaluated the cohort effect adjusting for confounders.A total of 29 HPV+ NPC (25 Caucasians and 4 Asians), 422 EBV+ NPC, and 1310 HPV+ OPC were eligible. HPV genotype was available in 20/29 HPV+ NPC patients: 14 (70%) HPV-16; 6 (30%) non-HPV-16. Compared to EBV+ NPC overall, HPV+ NPC patients were older (median age: 58 vs 52 years, P = 0.006), were predominantly Caucasian (86% vs 15%, P < 0.001), and had higher T (T2-4: 86% vs 68%, P = 0.039) but similar N categories (N2-3: 66% vs 58%, P = 0.44). Within the matched cohort, more HPV+ NPC patients complained of local pain vs EBV+ NPC (28% vs 3%, P = 0.025) but a similar proportion had cranial neuropathy (21% vs 31%, P = 0.55). No significant difference existed in gross tumor volume or involved lymph node distribution (P > 0.05). Median follow-up for HPV+ NPC, EBV+ NPC and HPV+ OPC was 3.1, 6.0, and 5.2 years respectively. Compared to EBV+ NPC (n = 374), HPV+ NPC (n = 20) had similar 3-year LRC (95% vs 90%, P = 0.416) and OS (84% vs 89%, P = 0.137), but non-significantly lower DC (75% vs 88%, P = 0.13). Compared to HPV+ OPC (n = 1310), HPV+ NPC also had similar 3-year LRC (95% vs 94%, P = 0.74) and OS (84% vs 87%, P = 0.307), but lower DC (75% vs 90%, P = 0.036). The difference in DC became non-significant in MVA [HR 1.83 (0.73-4.58), P = 0.19] after adjusting for T category, N category, smoking pack-years, and chemotherapy.HPV+ NPC is an emerging entity, of which about one-third are caused by non-HPV-16 genotype. Compared with the more common EBV+ NPC cases, HPV+ NPC presents with more local pain and advanced T categories but has similar outcomes. HPV+ NPC also has similar outcomes to HPV+ OPC except that DC is lower in univariable analysis, possibly attributable to higher T category and non-HPV-16 genotypes. There may be a role for modification of systemic therapy in HPV+ NPC to address distant failure risk.

Prevalence and Molecular Epidemiology of Cryptosporidium Infection in Clarias gariepinus Fish in Egypt.

This study investigated the prevalence and molecular detection of Cryptosporidium spp. in catfish (Clarias gariepinus). A total of 300 Carias gariepinus fish were collected from two freshwater sources: the Nile River (180) and drainage canals (120). The stomach and intestine epithelium of each individual fish sample were screened by modified Ziehl-Neelsen (mZN) staining technique for the detection of Cryptosporidium oocysts followed by the serological survey for detection of Cryptosporidium antibodies using Enzyme-Linked Immunosorbent Assay (ELISA) and molecular characterization using complemented DNA polymerase chain reaction (cPCR). ELISA showed higher prevalence of 69.3% than that prevalence obtained by mZN, 64% for the total examined Clarias gariepinus fish. Also, higher prevalence of Cryptosporidium infection 65.5% and 75.8% obtained by ELISA than 61.1% and 68.3% by mZN, in both fish groups from Nile River and Drainage canal, respectively. PCR analysis revealed the expected positive bands at 1056bp. DNA sequencing and phylogenetic analysis proved that the positive-PCR Cryptosporidium isolate identified in the present study was Cryptosporidium molnari. Freshwater fishes (Clarias gariepinus) are subjected to a high infection rate with Cryptosporidium spp.; the drainage canals obtained fishes showed higher prevalence than that collected from Nile River which indicates an important public health problem and a potential risk of drainage canals in Egypt. ELISA showed higher prevalence of cryptosporidiosis than mZN, for the total examined Clarias gariepinus fish and phylogenetic analyses confirmed this protozoal organism to be a novel species of Cryptosporidium molnari.

Evaluating maternal characteristics and DNA polymerase chain reaction birth testing of neonates born with HIV in a KwaZulu-Natal referral hospital – missed opportunities?

Background. Congenital HIV infection has declined in South Africa with an expanded programme to decrease perinatal HIV transmission. Understanding the challenges within this programme can identify opportunities for improvements. There is an opportunity with birth HIV DNA polymerase chain reaction testing to expedite very early treatment of neonates born with HIV in referral hospitals. Objectives. This study was aimed at describing the characteristics of mothers of neonates born with HIV as well as the evaluation of the access and availability of birth HIV DNA PCR results within a referral hospital. Method. This study was a retrospective chart review of all documented neonates born with HIV delivered between 1 January 2017 and 30 June 2019 at King Edward VIII Hospital, Durban, KwaZulu-Natal. The birth HIV DNA PCR results provided through institutional programmes were analysed, and the clinical characteristics of mothers of neonates born with HIV were described. Results. Review of hospital-based records, during the 30-month study period, identified 6 902 (40.02% of 17 243 total live births) birth HIV DNA PCR tests having been done. During the sample period, documented positive, rejected or indeterminate results were analysed. These results indicated only 37 (0.54%) of known HIV-exposed neonates who were tested, to have a documented positive birth HIV DNA PCR result. Fifty-nine neonates had either indeterminate or rejected results. Negative HIV DNA PCR results could not be verified. Analysis of the mothers of neonates with documented HIV-positive results indicated high mean viral loads of these mothers (52 343 copies/mL) and most were diagnosed in the current pregnancy or at delivery. Conclusion. Review of the characteristics of mothers of neonates born with HIV within a public referral hospital reiterates that late HIV diagnosis is common in this high-risk group. Birth HIV DNA PCR results may fail to adequately identify all positive neonates timeously for very early initiation of antiretroviral treatment.

Cytomegalovirus colitis combined with Clostridium innocuum as a cause of lower GI bleeding in a patient with COVID-19

A 67-year-old woman who presented with fever received a diagnosis of COVID-19 infection and was admitted to the medicine ward. She had a history of hypertension and diabetes. On day 4 of hospitalization, owing to progressive dyspnea and oxygen desaturation, she received intubation and mechanical ventilator support. She received remdesivir, dexamethasone, empiric antibiotics, enoxaparin, and tocilizumab during her hospitalization. On day 24, the patient developed bloody stools, and her hemoglobin level decreased from 11.7 g/dL to 9.2 g/dL. Because of persistent bleeding, she underwent colonoscopy, which revealed colitis and ulcer bleeding at the rectum (A). CT showed wall thickening of the lower part of the rectum (B). A pathologic study of the biopsy specimen showed enlarged nuclei and viral inclusions in both the cytoplasm and the nuclei (C). Immunohistochemical stain was positive for cytomegalovirus (CMV) antigen (D). The result of a blood CMV DNA polymerase chain reaction was positive, and the diagnosis of CMV infection was confirmed. In addition, there was growth of Clostridium innocuum in the stool culture. Her bleeding subsided after ganciclovir and metronidazole treatment, and she was extubated smoothly on day 38. Here we illustrate a COVID-19 patient who presented with lower GI bleeding caused by CMV coinfection and growth of C innocuum simultaneously. Endoscopy plays a crucial role in the treatment of patients with GI bleeding. All authors disclosed no financial relationships. Commentary The healthier you are the healthier you stay; the sicker you are the sicker you get. This case reflects this axiom. This unfortunate patient experienced severe respiratory failure secondary to COVID-19 infection. Colonoscopy revealed a dramatic "punched out" ulcer, highly suggestive of a viral colitis, and indeed the patient was found to have a CMV infection. She was also found to harbor C innocuum, which, despite its name, should not be confused with C difficile. C innocuum is an anaerobic gram-positive rod that is considered to be a variant of normal gut flora. C innocuum usually behaves in a pathogenic manner in patients who are immunocompromised, as was clearly the case with this patient. C innocuum is often seen to cause illness in patients after organ transplantation, most commonly renal transplantation. Fortunately, this patient appears to have done well after aggressive therapy for her COVID-19 infection. Douglas G. Adler, MD, FASGE, GIE Senior Associate Editor, Co-Director, Center for Advanced Therapeutic Endoscopy (CATE), Porter Adventist Hospital, PEAK Gastroenterology, Denver, Colorado Mohamed O. Othman, MD, Associate Editor for Focal Points

Acute CoVID-19-associated bilateral neuropathy of the abducens nerve in a child

We have presented the first description of acute bilateral neuropathy of the abducens nerve associated with COVID-19 in a child.A boy aged 2 years 2 months acutely developed bilateral convergent strabismus, the next day fatigue, drowsiness, and a single vomiting not associated with food intake, were joined. A differential diagnosis was performed between brainstem encephalitis, other organic (non-inflammatory) lesions of the brain stem, and idiopathic bilateral neuropathy of the abducens nerves. According to laboratory data, mild lymphocytosis, mild eosinophilia were detected, according to polymerase chain reaction DNA of the herpes virus type 1/2, cytomegalovirus, herpes virus type 6, Epstein-Barr, tick-borne encephalitis, borrelia, anaplasma, RNA ehrlichia were not detected. IgM and IgG to tick-borne encephalitis were not detected. Neurophysiological and neuroimaging examinations did not reveal any pathology. On the 6th day of the disease, SARS-CoV-2 RNA was detected by polymerase chain reaction of the nasopharynx and oropharynx discharge. The final diagnosis was formulated. — bilateral neuropathy of the abducens nerves. Bilateral convergent paralytic strabismus. Concomitant diagnosis: new coronavirus infection, subclini-cal form (positive by polymerase chain reaction in the discharge from the throat and nose of the 2019-nCoV coronavirus (SARS-CoV-2) RNA). Decrease in the severity of strabismus was noted over time against the background of the therapy.Clinicians dealing with the COVID-19 should also pay attention to its ophthalmological manifestations and take into account that it can debut with them, as was in our case.

Investigations on Transfer of Pathogens between Foster Cows and Calves during the Suckling Period.

Simple SummaryRegarding the increasing importance of alternative calf rearing systems, such as the foster cow system, better knowledge about the transfer of pathogens via suckling calves would be very helpful. In this context, the present study was conducted on a large organic dairy farm practising calf rearing on foster cows. For microbiological examinations, quarter milk samples were collected both from foster cows and biological dams. Additionally, swabs were taken from the oral cavities of the associated foster calves. The concordance of pathogens between cows and calves was further examined by strain comparisons. The same strains of isolates were detected for Pasteurella multocida, Staphylococcus aureus, S. sciuri and Streptococcus (Sc.) suis. Based on the present results and the available literature, transmission of P. multocida and S. aureus via suckling calves is considered very likely. Transmission of pathogens from the dam to the foster cow with the suckling calf as a vector could not be detected.To date, there have been few studies on the health effects of foster cow systems, including the transmission of mastitis-associated pathogens during suckling. The present study aimed to compare the pathogens detected in the mammary glands of the foster cow with those in the oral cavities of the associated foster calves and to evaluate the resulting consequences for udder health, calf health and internal biosecurity. Quarter milk sampling of 99 foster cows from an organic dairy farm was conducted twice during the foster period. Oral cavity swabs were taken from 345 foster calves. Furthermore, quarter milk samples were collected from 124 biological dams to investigate possible transmission to the foster cows via the suckling calves. All samples were microbiologically examined and confirmed by MALDI-TOF (matrix-assisted laser desorption time-of-flight mass-spectrometry). Using RAPD-PCR (randomly amplified polymorphic DNA polymerase chain reaction), strain similarities were detected for Pasteurella multocida, Staphylococcus aureus, S. sciuri and Streptococcus (Sc.) suis. Transmission of P. multocida and S. aureus probably occurred during suckling. For S. sciuri and Sc. suis, environmental origins were assumed. Transmission from dam to foster cow with the suckling calf as vector could not be clearly demonstrated.

Detection and treatment of cerebral toxoplasmosis in an aplastic pediatric post-allogeneic hematopoietic cell transplant patient: a case report

BackgroundCerebral toxoplasmosis infection presents with non-specific neurologic symptoms in immunocompromised patients. With lack of measurable adaptive immune responses and reluctance to sample affected brain tissue, expedient diagnosis to guide directed treatment is often delayed.Case presentationWe describe the use of cerebrospinal fluid polymerase chain reaction and plasma cell-free DNA technologies to supplement neuroimaging in the diagnosis of cerebral toxoplasmosis in an immunocompromised pediatric patient following allogeneic hematopoietic cell transplantation for idiopathic severe aplastic anemia. Successful cerebral toxoplasmosis treatment included antibiotic therapy for 1 year following restoration of cellular immunity with an allogeneic stem cell boost.ConclusionsPlasma cell-free DNA technology provides a non-invasive method of rapid diagnosis, improving the likelihood of survival from often lethal opportunistic infection in a high risk, immunocompromised patient population.

Characterization of exopolysaccharide-producing lactic acid bacteria from Taiwanese ropy fermented milk and their application in low-fat fermented milk.

ObjectiveThe aim of this study was to characterize the exopolysaccharides (EPS)-producing lactic acid bacteria from Taiwanese ropy fermented milk (TRFM) for developing a clean label low-fat fermented milk.MethodsPotential isolates from TRFM were selected based on the Gram staining test and observation of turbid suspension in the culture broth. Random amplified polymorphic DNA-polymerase chain reaction, 16S rRNA gene sequencing, and API CHL 50 test were used for strain identification. After evaluation of EPS concentration, target strains were introduced to low-fat milk fermentation for 24 h. Fermentation characters were checked: pH value, acidity, viable count, syneresis, and viscosity. Sensory evaluation of fermented products was carried out by 30 volunteers, while the storage test was performed for 21 days at 4°C.ResultsTwo EPS-producing strains (APL15 and APL16) were isolated from TRFM and identified as Lactococcus (Lc.) lactis subsp. cremoris. Their EPS concentrations in glucose and lactose media were higher than other published strains of Lc. lactis subsp. cremoris. Low-fat fermented milk separately prepared with APL15 and APL16 reached pH 4.3 and acidity 0.8% with a viable count of 9 log colony-forming units/mL. The physical properties of both products were superior to the control yogurt, showing significant improvements in syneresis and viscosity (p<0.05). Our low-fat products had appropriate sensory scores in appearance and texture according to sensory evaluation. Although decreasing viable cells of strains during the 21-day storage test, low-fat fermented milk made by APL15 exhibited stable physicochemical properties, including pH value, acidity, syneresis and sufficient viable cells throughout the storage period.ConclusionThis study demonstrated that Lc. lactis subsp. cremoris APL15 isolated from TRFM had good fermentation abilities to produce low-fat fermented milk. These data indicate that EPS-producing lactic acid bacteria have great potential to act as natural food stabilizers for low-fat fermented milk.

Investigating DNA Damage Mechanism Induced by Monosodium Glutamate and Associated DNA Repair Cell Machinery: A Literature Review

Introduction: Monosodium Glutamate (MSG) is a widely used food additive to enhance flavours. Though commonly used, MSG’s accumulation in the body can induce genomic instabilities. These genome instabilities are detectable through various methods such as Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) and comet assay. Additionally, cells can employ DNA repair mechanisms to ameliorate this damage. The objective of this review paper is to investigate the role of prolonged MSG ingestion in DNA damage, potential downstream diseases, and DNA repair mechanisms that cells undertake to counteract these effects, such as nucleotide excision repair (NER) and base excision repair (BER). Compounds such as vitamin C, green tea extract, and Allium sativum have been shown to ameliorate the health hazards of MSG by inhibiting oxidative stress, reducing apoptosis, and increasing antioxidant availability. Methods: This review focuses on MSG-induced DNA damage mechanisms including gene suppression, chromosomal disruption, and carcinogenic effects. We conducted a comprehensive literature review of 28 peer-reviewed articles published since 2000-present. Results: Studies show that MSG consumption may lead to the formation of Reactive Oxygen Species (ROS) and micronuclei (MN), which are known as biomarkers of carcinogenesis. Furthermore, the genomic instabilities that lead to this effect were identified through the use of RAPD-PCR and comet assays. These instabilities are mainly dealt with by endogenous repair machineries such as NER and BER. Also, researchers have identified many substances which act as preventative measures towards the potential harmful impacts of MSG. Discussion: Diseases, such as cancer and obesity, may be linked to chronic intake of MSG. The efficacy of the mentioned DNA damage detection methods will be discussed. Furthermore, the endogenous mechanisms of NER and BER are outlined in this review. Substances such as vitamin C, green tea extract, and Allium sativum aid to prevent genotoxic effects induced by MSG. Conclusion: Through this research, we hope to bring awareness to the harmful impacts of MSG on genome stability and its role in disease development. We aim to educate the public about the prevalent usage of MSG in the food industry and to inform individuals to take precautions in their food consumption.

Population-Based HIV Impact Assessments Survey Methods, Response, and Quality in Zimbabwe, Malawi, and Zambia.

The population-based HIV impact assessment (population-based HIV impact assessments) surveys are among the first to estimate national adult HIV incidence, subnational prevalence of viral load suppression, and pediatric HIV prevalence. We summarize the survey methods implemented in Zimbabwe, Malawi, and Zambia, as well as response rates and quality metrics. Each cross-sectional, household-based survey used a 2-stage cluster design. Survey preparations included sample design, questionnaire development, tablet programming for informed consent and data collection, community mobilization, establishing a network of satellite laboratories, and fieldworker training. Interviewers collected demographic, behavioral, and clinical information using tablets. Blood was collected for home-based HIV testing and counseling (HBTC) and point-of-care CD4+ T-cell enumeration with results immediately returned. HIV-positive blood samples underwent laboratory-based confirmatory testing, HIV incidence testing, RNA polymerase chain reaction (viral load), DNA polymerase chain reaction (early infant diagnosis), and serum antiretroviral drug detection. Data were weighted for survey design, and chi square automatic interaction detection-based methods were used to adjust for nonresponse. Each survey recruited a nationally representative, household-based sample of children and adults over a 6-10-month period in 2015 and 2016. Most (84%-90%) of the 12,000-14,000 eligible households in each country participated in the survey, with 77%-81% of eligible adults completing an interview and providing blood for HIV testing. Among eligible children, 59%-73% completed HIV testing. Across the 3 surveys, 97.8% of interview data were complete and had no errors. Conducting a national population-based HIV impact assessment with immediate return of HIV and other point-of-care test results was feasible, and data quality was high.

Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study

The present study aimed to carry out DNA extraction and polymerase chain reaction (PCR) amplification to isolate the physiological and phenotypic traits of Candida albicans and Candida dubliniensis in denture wearer patients with and without denture-induced stomatitis. A total sample size of 160 participants were divided into two equal groups (80 each), patients in the study group having 40 males and 40 females with Newton type II denture stomatitis, and in the control group, healthy 40 males and 40 females those who wear complete denture were selected. All the samples were collected from the hard palate with a sterile swab and inoculated on CHROM agar plate; samples that displayed dark green colored colonies were selected for DNA extraction. DNA isolation was done on agarose gel using electrophoresis. Biorad gene identification was used. Strands depicting the presence of DNA in particular samples were identified, and further standardization of the procedure was done. PCR amplification was done using Candida species-specific primer, preset to the hyphal wall of the protein 1 gene with the CRR forward and reverse primers, under strict standard conditions with reverse transcriptase technique. Results showed that prevalence of C. albicans was more in females with denture stomatitis which was 67.50% than in males, i.e., 52.50%, and prevalence of C. dubliniensis was found in one female and in one male who were having denture stomatitis and it was not isolated from patients without denture stomatitis. Statistical analyses were performed using the Chi-square test. Denture stomatitis is the most common problem faced by long-term denture wearers, with C. albicans as one of the causative organisms. However, recent findings show an emerging pathogenic yeast species, C. dubliniensis, which was isolated from denture-induced stomatitis candidates in the present study, which is closely related to the C. albicans species. The identification of candidal strains causing denture stomatitis with DNA extraction and PCR amplification and its management by determination of its susceptibility to antifungals may improve the treatment outcome of the same. Candidiasis is the most frequently seen mucocutaneous infection of the oral cavity especially in denture wearers. It is caused mainly by the genus Candida. C. dubliniensis is phenotypically similar but genotypically different from C. albicans. This affects the treatment outcome drastically as there is enough literature suggesting resistance to the common antifungal drugs. Hence, drugs like fungus-specific calcineurin inhibitors should also be considered in resistant patients. Therefore, DNA identification of Candida genus plays a major role in deciding the treatment outcome. How to cite this article: Patil A, Boparai NK, Shankargouda SB, et al. Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study. J Contemp Dent Pract 2021;22(5):517-521.

Molecular Identification of Meloidogyne javanica occurring in Upper Egypt Based Polymerase Chain Reaction (PCR) Assays

Molecular diagnosis represents one of the important trends in the confirmation of morphological identification to the root-knot nematodes which one of the most global pathogens. In this context, current study aimed to describe Meloidogyne javanica occurring in cucumber plants in Assiut governorate (Upper Egypt) using the random amplified polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) assay. Four mitochondrial and specific-specific sequences characterized amplified regions (SCAR) primers were used. The results showed that the SCAR primers, Fjav/Rjav and Far/Rar were efficiently amplified with 670 bp and 517 bp, respectively. On the contrary, the mitochondrial primers did not show any results. In conclusion, the present study recommended the SCAR primers as effective identified primers for the species of Meloidogyne javanica.

A Study on the Prevalence and Source of Staphylococci and Methicillin-resistant Staphylococcus aureus Causing Superficial Incisional Surgical Site Infection

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are common causatives of superficial incisional surgical site infection (SSI). The source of the pathogens is still not fully diagnosed whether it is endogenous or exogenous particularly with regard to the extent of its resistance to antibiotics. Therefore, this study is designed to determine the rate of infection, the source of pathogens, and the extent of their resistance to antibiotics. For this purpose, pre-, intra- and post-operative swabs from the nasal and skin of patients undergoing surgeries and samples from the hospital environment have been collected and processed for isolation and identification of staphylococci. Bacterial analysis and antibiotic susceptibility profiles of the isolates are assessed by unweighted pair group method with arithmetic mean (UPGMA) analysis based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and disc diffusion test for antibiotics susceptibility profile. The microbiological and PCR results indicate that SSIs are found in 113/512 (22.07%), Staphylococcus spp. rated 67/512 (13.09%) of infections. Further analysis indicates that S. aureus, CoNS, and both of them were causes SSI with different rates 41/67 (61.2%), 23/67 (34.3%), and 3/67 (4.5%), respectively. Results of RAPD-PCR for 70 isolates reveal that 52/70 (74.28%) of SSIs are from endogenous source, followed by 10/70 (14.29%) and 8/70 (11.43%) from hospitals acquired and undetermined sources, respectively. Moreover, results of antibiotic susceptibility test reveal that 24/44 (54.5%) of isolates belong to methicillin-resistant Staphylococcus aureus; from both endogenous and exogenous sources with 13/24 (54.17%) and 11/24 (45.83%), respectively.

Congruent changes in microbial community dynamics and ecosystem methane fluxes following natural drought in two restored fens

Both the frequency and intensity of drought events are expected to increase, with unresolved alterations to peatland methane cycling and the involved microbial communities. While existing studies have assessed drought effects via experimental approaches under controlled conditions, to our knowledge, no studies have examined the in-situ effects of natural drought in restored temperate fens. In this study, we used quantitative polymerase chain reaction (qPCR) and high throughput 16S rRNA gene amplicon sequencing of DNA and complementary DNA (cDNA) to determine the abundances and community structure of total and putatively active microbial communities following the 2018 European summer drought. Together with geochemical and methane flux data, we compared these results to a non-drought reference dataset. During drought, water level and methane flux rates decreased to a new recent minimum in both fens. This corresponded with pronounced shifts in porewater geochemistry. Microbial community composition in the drought year differed markedly, and was characterized by a greater relative and total abundance of aerobic methanotrophs, and, in one of the two sites, by a decrease in total methanogen abundance. In contrast to the non-drought reference years, type I methanotrophs were clearly more dominant than type II methanotrophs in both fens. cDNA sequencing confirmed the activity of type I methanotrophs during drought, with Methylomonaceae having the highest average relative abundance of bacterial cDNA transcripts. We show that changes in microbial community dynamics, porewater geochemistry, and ecosystem methane fluxes can be substantial following natural drought in restored fens, and provide the first in-situ evidence from a natural drought which suggests type I methanotroph populations are more active than type II methanotrophs in response to drought effects. Type I methanotrophs may represent a key microbial control over methane emissions in restored temperate fens subject to natural drought. • Drought decreased water level and ecosystem methane fluxes substantially. • Microbial community dynamics reflected drought conditions at the sites. • Methanogen gene abundances decreased in one of the two fens. • Proportion of type II methanotrophs was low and similar to a non-drought year. • Type I methanotrophs dominated the active bacterial community profile at both sites.

Pro-Platelet Formation Is Impaired in Megakaryocytes of MYH9 -Related Disease Patients

IntroductionThe MYH9 gene encodes myosin-IIA, a nonmuscle myosin heavy chain that facilitates shape changes in diverse cell types. The mutation of MYH9 may lead to the MYH9 -related disease (MYH9 -RD), an autosomal dominant disorder with macrothrombocytopenia and leukocyte inclusion bodies at birth and the risk of developing nephropathy, deafness and cataracts during infancy or adult life. Currently, at least 44 different MYH9 mutations in all 40 exons have been reported. Mutated MYH9 protein expression is reduced in megakaryocytes and is clumped in a few aggregates together with the wild-type protein in neutrophils, where they form the inclusion bodies. In a mouse model, myosin-IIA protein inhibits pro-platelet formation and is regulated by Rho-associated kinase (ROCK). However, how the mutated myosin-IIA influences the pro-platelet formation (PPF) of megakaryocytes in patients has not been explored. The aim of this study is to determine the function and regulation of mutated myosin-IIA in MYH9 -RD patients' megakaryocytes.MethodsOne kindred with inherited macrothrombocytopenia was identified and 5 healthy donors were enrolled as controls. Peripheral blood smear review, slit lamp examination, pure-tone audiometry, renal ultrasonography and examination of blood (urea, creatinine, electrolytes) and urine were performed for each family member to diagnose and evaluate the disease. Bidirectional polymerase chain reaction (PCR) amplification and DNA sequence analysis were performed on all 40 MYH9 exons for each patient. Platelets from peripheral blood and megakaryocytes from bone marrow were fractionated for electron microscopy. Bone marrow tissues from patients were stained with fluorescein-labeled antibodies against CD41 and CD144 to observe the distribution of mature megakaryocytes in the vascular niche by confocal fluorescence microscopy. CD34+ cells were isolated from bone marrow samples by cell sorting for in vitro culture with TPO. On day 8, megakaryocytes were enriched using an albumin density gradient and seeded onto 96-well plates in serum-free medium with or without Y27632, the ROCK inhibitor. At the same time every day for 5 days, the percentage of proplatelet-displaying megakaryocytes was quantified by inverted microscopy.ResultsFive patients in the kindred were diagnosed with MYH9 -RD based upon peripheral blood smear review, one of whom had severe renal failure. None of the patients suffered from deafness or cataracts. A pathogenic heterozygous sequence variant in exon 39 (c.5521G>A, p.E1841K) was detected, which substitutes glutamate at codon 1841 to lysine. This mutation is one of the 44 reported pathogenic mutations. Electron microscopic results showed that platelets from patients had increased diameters and irregular shapes while megakaryocytes from patients had smaller diameters and a disorganized demarcation membrane system (DMS). Immunofluorescence results of bone marrow tissues showed that proplatelet-displaying mature megakaryocytes in MYH9 -RD patients located distally to the vascular niche, compared with the control group. CD34+ cells from bone marrow differentiated into megakaryocytes after in vitro culture with TPO. However, the PPF of megakaryocytes from MYH9 -RD patients appeared much earlier than that from controls. At the same time point, PPF megakaryocytes in MYH9 -RD patients were significantly increased compared to those of the control group. When cultured with Y27632, the PPF appearance time and proplatelet-displaying megakaryocyte percentage in the control group were advanced, while the MYH9 -RD group was not influenced.ConclusionThe mutation in exon 39 of the MYH9 gene may lead to morphological abnormalities of megakaryocytes and platelets. The regulation of myosin-IIA function by ROCK in patients may be decreased when the MYH9 gene is mutated. As a result, the timing of PPF of megakaryocytes with mutated myosin-IIA in the bone marrow niche of MYH9 -RD patients may be advanced, and their mis-location away from the vascular niche may lead to reduced platelet production and increased platelet destruction. DisclosuresNo relevant conflicts of interest to declare.

Use of Leflunomide for Treatment of Cytomegalovirus Infection in Recipients of Allogeneic Stem Cell Transplant

Background :Reactivation of cytomegalovirus (CMV) commonly occurs post allogeneic stem cell transplant (ASCT). The incidence is at least 40-50% in India, where most patients and donors are CMV seropositive. Ganciclovir, although the drug of choice, is expensive and associated with significant toxicities. Second line agents like foscarnet and cidofovir are not available in India leaving few therapeutic options. Leflunomide, an anti rheumatoid arthritis drug, acts by inhibiting virion assembly of CMV and has been effective in post renal transplant CMV infections. This drug is inexpensive and has a favourable toxicity profile. There have been only occasional case reports of leflunomide post ASCT. Here we report the efficacy of leflunomide in CMV reactivation in ASCT setting.Methods :All patients who underwent ASCT at our centre between April 2015 and February 2017 were included in this retrospective study. Serologic evaluation for CMV infections was done at baseline for all patients and donors. Conditioning regimens used were either myeloablative [total body irradiation (TBI)-cyclophosphamide (Cy)] or reduced intensity (fludarabine with either melphalan, or Cy or treosulfan +/- 2 gray TBI). Graft versus host disease (GVHD) prophylaxis used was cyclosporine or tacrolimus with methotrexate or mycofenolate mofetil for matched sibling donor (MSD) and matched unrelated donor (MUD) transplants. Post transplant Cy was used for haplo-identical transplants (HIT).Monitoring was done twice weekly using quantitative CMV DNA polymerase chain reaction (QPCR) on peripheral blood post transplant till day +120 or longer if patient was on steroids. Patients who were detected to have a positive QPCR greater than 500 copies/mL at 2 consecutive time points were started on pre-emptive therapy. Ganciclovir was the preferred agent for pre-emptive therapy, however in case of ongoing cytopenias, leflunomide was used. In case of clinical resistance to ganciclovir, leflunomide was added as second line. Leflunomide was started at a loading dose of 100 mg once a day for 3 days followed by daily maintenance dose of 20 mg till clearance of CMV (undetectable CMV DNA on QPCR at 2 consecutive time points) or intolerance. Failure of leflunomide was defined as > 1 log increase in CMV copy number at any time or if another anti CMV agent was added due to CMV disease or non-clearance. Invasive procedures such as broncho-alveolar lavage, colonoscopic biopsies, and liver biopsies were used to detect CMV disease, when clinically suspected.Results :Seventy patients underwent ASCT during this period. This included 47 MSD transplants, 18 HIT and 5 MUD transplants. There were 49 episodes of CMV reactivation in 43 patients (61%). The incidence was highest in HIT with 17 of 18 patients developing reactivation.Leflunomide was used in 24 (49%) of the 49 episodes of CMV reactivations. It was used as 1st line agent in 15 episodes and as 2nd line in 9. The median CMV copy number/ml was 0.82x 103 (range 0 to 13x103) on day of starting leflunomide. The median duration of use was 34 days (range 6 to 85 days). It was used pre-emptively in 20 episodes and as therapeutic (i.e. patient had end organ involvement) in 4. None of the patients had toxicity requiring stopping of the drug. Nine (38%) of the 24 CMV reactivation episodes cleared with leflunomide. When used as a 1st line agent, CMV infection cleared in 7 of the 15 episodes (46%) in comparison to ganciclovir as 1st line with which 12 (52%) of 23 episodes cleared.Seven of the 24 episodes had initial CMV burden more than 2 x 103 copies/mL. Leflunomide failed in all of these, as against clearance in 9 of the 17 episodes which had CMV less than 2 x 103 copies/mL. (p= 0.022). None of the patients who received leflunomide in therapeutic setting cleared CMV (0 of 4), compared to 9 of 20 with pre-emptive leflunomide. (p= NS).Conclusions:This is the largest study of using leflunomide for CMV reactivation in post ASCT setting to our knowledge. Leflunomide has considerable activity against CMV, however it is best used when CMV copies are less than 2000 /mL in pre-emptive setting. In therapeutic setting its role seems limited. A larger prospective study is warranted. DisclosuresNo relevant conflicts of interest to declare.