DNA Polymerase Chain Reaction Research Articles

Genotyping-driven diversity assessment of biocontrol potent Bacillus spp. strain collection as a potential method for the development of strain-specific biomarkers.

Bacillus species are among the most researched and frequently applied biocontrol agents. To estimate their potential as environmentally friendly microbial-based products, reliable and rapid plant colonization monitoring methods are essential. We evaluated repetitive element-based (rep) and Random Amplified Polymorphic DNA (RAPD) PCR (Polymerase Chain Reaction) genotyping in a diversity assessment of 251 strains from bulk soil, straw, and manure samples across Serbia, highlighting their discriminative force and the presence of unique bands. RAPD 272, OPG 5, and (GTG)5 primers were most potent in revealing the high diversity of a sizable environmental Bacillus spp. collection. RAPD 272 also amplified a unique band for a proven biocontrol strain, opening the possibility of Sequence Characterized Amplified Region (SCAR) marker design. That will enable colonization studies using the SCAR marker for its specific detection. This study provides a guide for primer selection for diversity and monitoring studies of environmental Bacillus spp. isolates.

Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of β-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

Is There an Association between Concurrent Epstein–Barr Virus Infection and Sudden Hearing Loss?—A Case—Control Study in an East Asian Population

Viral infection serves as the crucial etiology for the development of sudden sensorineural hearing loss (SSNHL). We aimed to investigate whether there is an association between concurrent Epstein-Barr virus (EBV) infection and SSNHL in an East Asian population. Patients who were older than 18 years of age and met the criteria of sudden hearing loss without an identifiable etiology were enrolled from July 2021 until June 2022, followed by the serological testing of IgA antibody responses against EBV-specific early antigen (EA) and viral capsid antigen (VCA) with an indirect hemagglutination assay (IHA) and real-time quantitative polymerase chain reaction (qPCR) of EBV DNA in serum before the treatment was initiated. After the treatment for SSNHL, post-treatment audiometry was performed to record the treatment response and degree of recovery. Among the 29 patients included during enrollment, 3 (10.3%) had a positive qPCR result for EBV. In addition, a trend of poor recovery of hearing thresholds was noted for those patients with a higher viral PCR titer. This is the first study to use real-time PCR to detect possible concurrent EBV infection in SSNHL. Our study demonstrated that approximately one-tenth of the enrolled SSNHL patients had evidence of concurrent EBV infection, as reflected by the positive qPCR test results, and a negative trend between hearing gain and the viral DNA PCR level was found within the affected cohort after steroid therapy. These findings indicate a possible role for EBV infection in East Asian patients with SSNHL. Further larger-scale research is needed to better understand the potential role and underlying mechanism of viral infection in the etiology of SSNHL.

A Case Report: False-Negative HIV Antibody Test in a Patient Presenting With an Unusual Case of Cytomegalovirus Oropharyngeal Ulcerations

Abstract Background Cytomegalovirus (CMV) oropharyngeal ulcerations are rare diagnoses that usually only occur in immunocompromised individuals. False-negative third-generation HIV tests in a patient with AIDS are also exceedingly rare and, when they occur, underscore the complex host and viral relationships involved in HIV disease mediation. Case Presentation We present a case of CMV oropharyngeal ulcerations in a patient diagnosed with advanced AIDS with a persistently negative HIV antibody test. Confirmative testing and diagnosis of HIV were performed with a qualitative DNA polymerase chain reaction and confirmed with a quantitative RNA polymerase chain reaction. Cytomegalovirus oropharyngeal ulcerations were managed with ganciclovir 5 mg/kg with significant improvement in oral ulcers. Conclusions We present a rare case of an HIV-seronegative patient clinically diagnosed with advanced AIDS and CMV oropharyngeal ulcerations. False-negative HIV tests can delay diagnosis and treatment; hence, clinical suspicion is needed for accurate diagnosis and early treatment.

Molecular detection of some genes virulent bacteria Proteus mirabilis isolated from kidney stones

The current study Included the collection of samples (68) grit kidneys of patients undergoing surgery for lifting gravel in hospitals in the city of Tikrit and for the period from the month of April 2012 to the month of May 2013. The results of the study that people with the proportion of males (64.71%) while she was infected with the proportion of females (35.29 %) outweigh any male female ratio (1.83:1), diagnostic results showed depending on the biochemical tests and API-20system showed that E.coli and Ps.aeruginosa were the most frequent among other bacterial species and by isolating (30.76%) and (21.22%), respectively, while the bacteria P.mirabilis has given isolate percentage (15.38%) Followed by bacteria and Kl.pneumoniae Citrobacter.freundii by insolation (9.6%) and (5.67%) respectively, while giving the rest of the bacterial species (Staph.aureus,, Acinetobacter. freundii, E.faecalis Serratia .marscenes) isolation rate equal (3.84%), while isolating the bacteria Staph.epidermidis did not exceed (1.92%).The results of the investigation of the presence of genes,( mrpA, zapA, ureC) using polymerase chain reaction DNA (PCR) , Revealed that all isolates bacteria of P.mirabilis owning genes ( mrpA, zapA, ureC) encoding virulence factors (urease, protease, the ability to produce pili (mrp).

Analysis of the reliability of rapid diagnostic tests for varicella, including breakthrough cases.

In the era of universal varicella vaccination, diagnosis of varicella is challenging, especially for breakthrough cases. We sought to clarify the reliability of direct varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) and DermaQuick® VZV using the immunochromatography technique as rapid diagnostic tests for varicella. In addition, the usefulness of saliva as a sample type for direct LAMP was investigated. Among the 46 enrolled patients with suspected VZV infection, 31 patients (67.3%) were positive for the nucleic acid test based on real-time PCR from skin swab samples. Direct LAMP of skin swabs was positive in 29 (63.0%) of 46 patients. DermaQuick® VZV was positive in 25 (54.3%) of 46 patients. VZV DNA was detected in only 48.4% of oral swabs with the direct LAMP method. With real-time polymerase chain reaction (PCR) as the standard for diagnosing varicella, the sensitivity and specificity of DermaQuick® VZV were 80.7% and 100%, respectively. The sensitivity and specificity of direct LAMP from skin swabs were 93.6% and 100%, respectively. The sensitivity and specificity of real-time PCR for DNA extracted from oral swabs were 74.2% and 93.3%, respectively. Thus, oral swab samples are not suitable for breakthrough varicella diagnosis. Although DermaQuick® VZV is considered the most convenient point-of-care test for varicella, its sensitivity and specificity were lower than those of direct VZV LAMP.

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and invitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.

Evaluation of the optimal sampling approach for HPV genotyping in circumcised heterosexual men with genital warts

BackgroundHuman papillomavirus (HPV) causes a serious socioeconomic burden globally. However, there is currently no consensus on the optimal sampling method for HPVDNA genotyping in circumcised heterosexual men. This study aimed to determine the diagnostic efficacy of 6 different anatomic sampling sites in HPV DNA polymerase chain reaction (PCR) testing of circumcised heterosexual men with genital warts. MethodsThe study included circumcised heterosexual men who presented to our clinic with complaints of genital warts. Swab samples were obtained from the penile shaft (PS), scrotum, coronal sulcus (CS), and external urethral meatus (EUM). First-void urine (FVU) and genital wart biopsy (GWB) were also tested for HPV DNA by PCR. ResultsA total of 32 patients (mean age: 36.9 ± 6.9 years) were included. None of the six samples studied was sufficient on its own to reveal all HPV types detected in a patient. When the samples were analyzed individually, GWB detected an average of 49.5% of total HPV types in a patient. This rate was 50.5% for PS, 40.4% for CS, 31.6% for scrotum, 26.3% for EUM, and 15.8% for FVU samples. The detection rate increased to 75.8% with combined testing of GWB and PS samples, 83.2% with GWB/PS/CS, 90.5% with GWB/PS/CS/scrotum, and 98.9% with GWB/PS/CS/scrotum/EUM samples. ConclusionNo single anatomic region or sample type can detect all HPV types present in circumcised heterosexual men by PCR assay. The detection rate approaches 99% when wart biopsy is combined with swab sampling of the penile shaft, coronal sulcus, scrotum, and external urethral meatus.

Detection of urogenital pathogens in sterile pyuria samples by polymerase chain reaction

BackgroundPatients with sterile pyuria may be infected with sexually transmitted diseases or have renal tuberculosis. This study investigated the possibility of detecting sexually transmitted diseases and Mycobacterialtuberculosis in sterile pyuria samples with polymerase chain reaction.MethodsForty-four day-3 negative urine culture samples were investigated for the presence of sexually transmitted diseases and Mycobacterialtuberculosis.ResultsAmong the 44 samples, 61.4% were positive by polymerase chain reaction (PCR) for bacterial DNA (either sexually transmitted diseases or Mycobacteria). Among the 27 positive samples, 37% were positive for Ureaplasmaurealyticum, 26% were positive for Chlamydiatrachomatis, 14.8% were positive for Neisseriagonorrhoeae, 11.1% were positive for Mycoplasmagenitalium, 7.4% were positive for Mycoplasmahominis, and only one sample (3.7%) was positive for Mycobacterialtuberculosis. No significant associations were found between PCR-positive urine samples and patient characteristics.ConclusionsIt was concluded that Ureaplasmaurealyticum was predominant in sterile pyuria followed by Chlamydiatrachomatis. There were no significant associations between PCR-positive samples and sex, symptomatic patients, or antibiotic use. PCR is an instant diagnostic tool for sexually transmitted diseases in sterile pyuria; hence, it is advised to be performed on negative culture samples as a routine laboratory screening test whenever possible.

РЕЗУЛЬТАТЫ СРАВНИТЕЛЬНЫХ ДИАГНОСТИЧЕСКИХ ИССЛЕДОВАНИЙ КРОВИ И МОЛОКА НА ЛЕЙКОЗ, С ИСПОЛЬЗОВАНИЕМ РИД И ПЦР

The purpose of the study was to substantiate the use of PCR for blood and milk analysis of cows in a farm with poor bovine enzootic leukemia. When comparing the effectiveness of serological and molecular genetic methods of laboratory diagnostics, it was found that polymerase chain reaction (PCR) is more sensitive to the detection of bovine leukemia virus infected by the method in comparison with the immune diffusion reaction (RID). A total of 271 blood samples and 271 milk samples from cows were examined. In the RID with blood serum samples obtained from 271 heads of cattle, positive results (the presence of antibodies against the leukemia virus) were detected in 43 samples, which also showed the presence of proviral DNA in PCR. The results of REED and PCR studies of blood serum were identical in 38.1% of cases. Parallel studies of PCR diagnostics additionally revealed 70 heads of cattle infected with the virus. As a result of the study of blood samples in PCR using the Leukosis test systems, the presence of proviral DNA of the virus was detected in 113 of the 271 samples studied. The DNA of the pathogen was found in both whole blood and serum, as well as in a blood clot resuspended with buffered saline solution and in whole milk from sick animals. For the study of milk in REED, lactoserum was previously obtained according to the original (developed by university staff) methodology. Of the 271 milk samples studied, 40 cows were found to be lactoseropositive. Studies in RID of whey confirmed the results obtained in RID with blood serum in 93% of cows. The results of REED and PCR studies of blood serum demonstrate a sensitivity of PCR significantly exceeding the sensitivity of REED, however, when analyzing the results of studies, the age and physiological parameters of the tested animals should be taken into account, since the level of antibodies and the amount of proviral DNA in their lifetime can vary significantly.

Requiring Reconsideration of Differences of Aeromonas Infections Between Extra-Intestinal and Intestinal in Hospitalized Patients.

The purpose of this study is to examine the variations between extra-intestinal and intestinal infections of Aeromonas in terms of strain types, risk factors, drug susceptibility results, and the distribution of drug resistance and virulence genes. A total of 188 Aeromonas strains were identified to the species level using housekeeping genes (rpoD, gyrB, and gyrA). The risk factors for Aeromonas extra-intestinal and intestinal infection, as well as mortality, were retrospectively examined in this study. The broth microdilution method was used to investigate the antimicrobial susceptibility profiles. Touchdown polymerase chain reaction (PCR) assays and DNA sequencing were employed to confirm virulence and the presence of drug resistance genes. The housekeeping genes identified 188 strains into 7 species. Extra-intestinal isolates generally contained A. caviae and A. hydrophila, while intestinal were A. veronii (p=0.0001). Extra-intestinal infections (158/188) were the main type and accounted for 24/27 of all fatalities. Malignant tumors, hepatobiliary diseases, anemia, and hypoproteinemia were linked to infections. Poor results were associated with septic shock. Using the broth microdilution method, over 80% isolates were susceptible to most antimicrobials, except for ceftazidime (79.8%) and ceftriaxone (69.7%). Except for imipenem, intestinal strains were more susceptible to other medications than extra-intestinal. Using touch-down polymerase chain reaction testing and DNA sequencing, 6 strains, 31 strains, and a strain only had bla TEM, bla CphA, and bla VIM, respectively. Two Aeromonas hydrophila each possessed bla CphA+ bla CTXM-M-9, and bla CphA + bla CTX-M-1 + bla CTX-M-15-like + bla TEM; two Aeromonas caviae each possessed bla NDM + bla CTX-M-1 +bla CTX-M-15-like + bla TEM, and bla NDM + bla TEM. Thirty-four of the 42 strains mentioned above were isolated from extra-intestinal. Act, aexT, and ascF-G, were in intestinal more frequently, but alt, hlyA, ela, and lip were in extra-intestinal more frequently. Aeromonas inside and outside intestinal differed in their clinical characteristics, drug susceptibility, drug resistance and virulence genes.

Simultaneous detection and evaluation of Prevotella intermedia, Prevotella nigrescens, Prevotella loescheii, and Prevotella melaninogenica in subgingival plaque samples of chronic periodontitis and healthy individuals through multiplex polymerase chain reaction.

Prevotella is a Gram-negative anaerobic bacilli. The phenotypic characteristics of the various species of Prevotella are similar, which often makes it difficult in routine differentiation and identification of all the species. The purpose of the study was to detect and compare presence of Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Prevotella loescheii in subgingival plaque samples of chronic periodontitis and healthy individuals. Two hundred and thirty-six subjects were considered consisting of chronic periodontitis (128) and healthy (108) individuals. Subgingival plaque sample was collected in reduced transport fluid and analyzed. DNA extraction and polymerase chain reaction (PCR) were performed for genus Prevotella followed by positive samples were considered for the detection of selected species through multiplex PCR using specific primers. Out of 236 samples, 94.1% were positive for genus Prevotella. Out of 222 cases P. nigrescens showed the highest number of cases positive (59.5%) followed by P. melaninogenica (57.2%), P. intermedia (55.4%), and P. loescheii (40.1%). Species were analyzed individually between chronic periodontitis and healthy, P. intermedia, P. nigrescens, and P. loescheii showed greater positivity in healthy compared to chronic periodontitis. Positivity for P. melaninogenica was high in chronic periodontitis compared to healthy. The number of positive cases for species, when correlated with clinical parameters showed an increase in mean score for all clinical parameters assessed, suggesting the presence of variation in the prevalence of Prevotella species and geographic variation do exist in oral microflora. Findings suggest that they can be normal commensals and opportunistic.

Human papilloma virus-related oral and oropharyngeal squamous cell carcinoma: An assessment of histomorphological features

ABSTRACT Background: Human papillomavirus (HPV) has emerged as an important cause of oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC). Cancers with HPV as a causative agent are seen to exhibit certain specific histomorphological features. Aim: This study aims to describe the histomorphology of oral and oropharyngeal cancers and discuss their association with HPV. Material and Methods: Hospital-based prospective study done over 31 months (December 2018–April 2021), including a total of 90 cases of OSCC and OPSCC. They were subjected to detailed histopathological evaluation, DNA polymerase chain reaction testing for testing of HPV association, followed by the analysis of data by statistical methods. Results: Out of a total of 90, 73 cases were OSCC and 17, OPSCC. A total of 15 cases were HPV positive. HPV status was found to be significantly associated with purely nonkeratinizing tumors and hybrid/mixed squamous cell carcinomas, pushing the pattern of invasion, absence of perineural invasion, presence of tumor infiltrating lymphocytes, moderate-to-dense host lymphocyte response, loose stromal response, absence of tumor budding, and high mitotic rate (&gt;20/10 hpf). Conclusion: HPV-positive OSCC and OPSCC have distinct histomorphological features and careful analysis of the same can assist in identifying these types of cancers better and thus help in prognostication and treatment.

Immunohistochemical Evaluation of p16 and p53 in Oral and Oropharyngeal Squamous Cell Carcinoma with Special Regard to Human Papillomavirus Status.

Incidence of human papillomavirus (HPV)-associated oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC) is on a rising trend globally and has specific therapeutic implications. HPV-related tumors have a distinct pathogenetic mechanism targeting p16 and p53 both. However, there are limited studies evaluating p16 and p53 expression in combination. The aim of the study is to evaluate p16 and p53 immunohistochemical expression pattern in OSCC and OPSCC, with special reference to HPV association. This was a hospital-based prospective study done over 22 months (September 2018 to June 2020), including a total of 54 cases of OSCC and OPSCC. They were subjected to clinicopathological evaluation, p16 and p53 immunohistochemistry, and DNA polymerase chain reaction testing for testing of HPV association, followed by analysis of data by statistical methods. Out of 54, 43 cases were OSCC and 11 cases were OPSCC. A total of nine cases were HPV positive. HPV association was found to be significant with tonsil as primary site, age range between 40 and 60 years, and absence of tobacco or alcohol habit. Presence of HPV infection was also significantly associated with p16 overexpression, in combination with p53 negativity. The findings indicate that p16 overexpression combined with a negative p53 expression can be used for HPV detection and the former alone may be used as diagnostic marker in OPSCC only. HPV-associated OSCC and OPSCC are a unique subset of cancers, and using combination of molecular biomarkers could help in diagnosis and prognosis.

Association of Melatonin receptor 1A (MTNR1A) gene polymorphism with seasonal suppression of fertility in buffaloes

The polymorphism in Melatonin receptor 1A (MTNR1A) gene and its association with reproductive cyclicity (cyclic, n=51; non-cyclic, n=50) during summer season and age at first calving (&lt;3.5 years, n=38; &gt;3.5 years, n=60) was investigated in buffaloes subjected to blood sampling. Polymerase chain reaction (PCR) of genomic DNA was carried out using three primers viz. 824 bp (primer pair 1), 856 bp (primer pair 2) and 268 bp (primer pair 3) corresponding to exon-II of MTNR1A gene. Restriction fragment length polymorphism was performed using PCR products of primer pair 1 and 2. The digestion of PCR product of primer pair 1 with restriction enzymes viz. MnII, RsaI or Eco3II generated total restriction sites respectively as 14, 4 and 5. Based upon differential restriction sites, six genotypes revealed in buffaloes using MnII were MM-1 to MM-6, four genotypes using RsaI were RR-1 to RR-4, and four genotypes using Eco3II were EE-1 to EE-4. The digestion of PCR product of primer pair 2 with MnII, RsaI or Eco3II generated restriction sites respectively as 13, 5 and 6. Based upon differential restriction sites, six genotypes revealed using MnII were mm-1 to mm-6, five genotypes using RsaI were rr-1to rr-5, and three genotypes using Eco3II were ee-1 to ee-3. For primer pair 3, single stranded conformational polymorphism analysis exhibited 5 genotypes viz. AA, BB, AB, CC and DD. The results revealed that the buffaloes during summer had polymorphic genotypes for MTNR1A gene as buffaloes showing reproductive cyclicity had MM-3, MM-4, RR-2, EE-3, mm-2, mm-4, rr-3, ee-3, CC and DD genotypes, whereas their non-cyclic counterparts had MM-2, MM-6, EE-4, mm-3, mm-6, AA, BB and AB genotypes. However, no such differences were recorded with respect to age at first calving. In conclusion, polymorphic genotypes of MTNR1A gene may be considered as genetic markers to identify buffaloes with better reproductive potential during summer season.

HPV 18 variants in women with cervical cancer in Northeast Brazil

BackgroundHuman Papillomavirus (HPV) is the main etiological factor for the development of cervical cancer. HPV 18 is the second most frequent type, accounting for up to 65% of all cases. HPV intratypic variation may influence the potential for progression to invasive cancer. The aim of this study was to evaluate the prevalence of human papillomavirus 18 intratypic variants in cervical cancer samples from women in the state of Maranhão, Brazil. MethodsThe study included 118 women over 18 years of age with a diagnosis of cervical cancer. Tumor fragments were collected and subjected to DNA extraction and Polymerase Chain Reaction (PCR) for HPV detection using the PGMY09/11 and GP+5/6 primers. Positive samples were submitted to automated sequencing for viral genotyping. To determine the HPV 18 lineages, positive samples were submitted to PCR, using specific primers to amplify the LCR and E6 regions of HPV 18 virus. ResultsHPV was present in 88 women (73.3%). Of those, 48 (54%) were HPV 16, the most prevalent, followed by 12 (13.6%) HPV 18. Histologically, squamous cell carcinoma was predominant (79.1%). Among the HPV 18 variants identified, 10 (80%) belonged to lineage A, and sublineages A1, A2, A3, and A4. Two (29%) HPV 18 B variant was also detected, with the sublineages B1 and B2. In this study, the C variant was not found. There was no statistically significant association between the HPV 18 lineages found and sociodemographic and lifestyle variables (p > 0.05). ConclusionsA higher frequency of HPV 16 and 18 were found in women with cervical cancer in the state of Maranhão, Brazil, with a high prevalence of the lineage A among women with HPV 18.

Clinical, histopathologic, immunohistochemical, and electron microscopic findings in cutaneous monkeypox: A multicenter retrospective case series in Spain

The worldwide outbreak of monkeypox has evidenced the usefulness of the dermatologic manifestations for its diagnosis. To describe the histopathologic and immunohistochemical findings of monkeypox cutaneous lesions. This is a retrospective histopathologic and immunohistochemical study of 20 patients with positive Monkeypox virus DNA polymerase chain reaction and immunohistochemical positivity for Vaccinia virus in cutaneous lesions. Four cases were also examined by electron microscopy. The most characteristic histopathologic findings consisted of full-thickness epidermal necrosis with hyperplasia and keratinocytic ballooning at the edges. In some cases, the outer root sheath of the hair follicle and the sebaceous gland epithelium were affected. Intraepithelial cytoplasmic inclusion bodies and scattered multinucleated keratinocytes were occasionally found. Immunohistochemically, strong positivity with anti-Vaccinia virus antibody was seen in the cytoplasm of ballooned keratinocytes. Electron microscopy study demonstrated numerous viral particles of monkeypox in affected keratinocytes. Small sample size. Electron microscopic study was only performed in 4 cases. Epidermal necrosis and keratinocytic ballooning are the most constant histopathologic findings. Immunohistochemical positivity for Vaccinia virus was mostly detected in the cytoplasm of the ballooned keratinocytes. These findings support the usefulness of histopathologic and immunohistochemical studies of cutaneous lesions for diagnosis of monkeypox.

Mutational Study in the Exon2 of Osteoprotegrin Gene in Osteoporotic Women in Chahar Mahal va Bakhtiari Province of Iran

Background: Osteoporosis is a multifactorial disease. Mutation in cystein-rich domain2 (CRD2) and cystein-rich domain3 (CRD3) of osteoprotegrin protein can prevent its interaction with receptor activator of nuclear factor kappa beta (RANKL) ligand and lead to osteoporosis. Objectives: This study aimed to investigate the possible alteration in exon2 of the osteoprotegrin gene encoding CRD2 and CRD3 domains in osteoporotic women in Chahar Mahal va Bakhtiari Province of Iran. Methods: The N-terminal region of human osteoprotegerin (OPG) protein was aligned with mice osteoprotegrin proteins using Clustal Omega multiple alignment tool. The genomic DNA of 72 osteoporotic women was extracted by commercial kit. The region of exon2 of the OPG gene encoding CRD2 and CRD3 was amplified by PCR and sequenced by DNA sequencing. Results: The result of multiple protein alignment showed dissimilarities among three species in terms of CRD2 and CRD3 domains. The results of polymerase chain reaction (PCR) amplification and DNA sequencing indicated that CRD2 and CRD3 sequence were intact in osteoporotic women. Conclusion: Due to the binding possibility of OPG protein with RANKL ligand, it was concluded that the expression of OPG gene may have been different in osteoporotic women. However, it was recommended that further studies should be conducted in order to confirm this finding.

924. Reducing Rates of Hospital Acquired Clostridium Difficile Infection by Switching to Two-Step Testing

Abstract Background Clostridium difficile infection (CDI) tests have various degrees of sensitivity, making diagnosis challenging. Polymerase chain reaction (PCR) positive tests in colonized patients can impact rates of hospital-acquired CDI (HACDI). A negative toxin A/B enzyme immunoassay (EIA) as a second step can help determine colonization but is not as sensitive. We aimed to study the impact of a two-step testing strategy on rates of HACDI and CDI treatment outcomes. Methods Two-step testing consisted of C. difficle DNA PCR, which if positive, was followed by toxin A/B EIA (Figure 1). We conducted a retrospective study in a 359-bed hospital from March 2020 to July 2021. Patients with positive PCR and negative EIA results were included and further classified based on CDI treatment status. The primary outcome was the number of HACDI cases avoided. Secondary outcomes included location at the time of testing, symptoms, leukocytosis, discharge disposition and readmission. Results Ninety-six patients were included. Sixty-five patients (68%) were treated while 31 (32%) were not. HACDI was avoided in 18 cases (18.8%). There were more HACDI avoided in the non-treated group compared to the treated group (32.3% vs 12.3%; p=0.026). Most non-treated patients were tested on medical-surgical units (77.4%), whereas 49.2 % of the treated patients were tested in more acute areas such as emergency or intensive care. Treated patients were more likely to have leukocytosis and/or fever (66.2%) compared to non-treated patients (29%; p= 0.0009), while there was no difference in abdominal pain, diarrhea, bloody stool, or constipation (Table 1). Most patients had an abdominal CT scan (70.8%). Of those that had a CT, colitis was found in 48.5%. Patients in the treated group were more likely to have had a CT completed that showed colitis vs the non-treated patients (43.1% vs 16.1%; p&amp;lt; 0.011). Finally, compared to the non-treated patients, treatment did not affect home disposition on discharge or 30-day readmission rate (80% vs 71%, P=0.436) and (25 % vs 29%, P= 0.63) respectively. Symptoms Associated with C. difficile infection. Schematic of C. difficile test interpretation. Conclusion CDI two-step testing led to decreased HACDI rates without affecting discharge disposition or readmission rates in patients with PCR positive and toxin EIA negative results regardless of treatment status. Disclosures All Authors: No reported disclosures.

Genetic Diversity of Candida spp. Isolates Colonizing Twins and Their Family Members.

A wide range of options for studying Candida species are available through genetic methods. Twins, particularly monozygotic ones and their families may be fitting subjects for studying those microorganisms. The question is: How specific can yeast flora be in an individual? The study aimed to analyze the strain relatedness among commensal yeasts isolated from various parts of the bodies of healthy people and to compare correlations between the genotypes of the isolates. Yeasts were isolated from 63 twins and their family members (n = 25) from the oral cavity, anus, interdigital space and navel. After species identification, Candida albicans (n = 139), C. parapsilosis (n = 39), C. guilliermondii (n = 25), C. dubliniensis (n = 11) and C. krusei (n = 9) isolates were analyzed using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) optimization method. The similarities between the strains were calculated based on the Dice (Sab) coefficient and are displayed graphically as dendrograms. Using cluster analysis, the following relatedness was distinguished: 13 genotypes and three unique (Un) patterns among C. albicans; 10 genotypes and four Un patterns among C. parapsilosis; three genotypes and one Un pattern among C. guilliermondii and C. dubliniensis; and three genotypes among C. krusei isolates. The presence of identical, similar or both genotypes among the strains isolated from family members shows the transmission of yeasts between ontocenoses in the same person and between individuals. The similarity between the genotypes of C. albicans, C. guilliermondii, C. dubliniensis and C. krusei was more remarkable than between the genotypes of C. parapsilosis in the strains isolated from ontocenoses of the same individual and their family members. The degrees of genetic similarity between Candida spp. strains isolated from monozygotic twins and those obtained from their relatives did not differ.