In-house Polymerase Chain Reaction Research Articles

Oral Chagas disease outbreak by bacaba juice ingestion: A century after Carlos Chagas' discovery, the disease is still hard to manage.

Orally transmitted acute Chagas disease (ACD) primarily affects low-visibility and low-income individuals in tropical and subtropical zones. Managing ACD remains challenging even after more than 100 years of its discovery. Its spread to non-endemic areas has made it a global health issue. The aim of this work is to demonstrate the difficulties encountered in handling a real-life situation. This report examines an outbreak of 39 cases of ACD due to oral transmission by bacaba juice ingestion that occurred in Pedro do Rosário, Maranhão, Brazil. A clinical and epidemiological investigation, including an entomological search, was conducted. Diagnosis criteria included positive peripheral blood smear (PBS), seroconversion of IgG, and a two-fold increase in IgG titer (laboratory criteria); and clinical findings, epidemiological exposure, and at least one positive IgG test (clinical-epidemiological criteria). In-house conventional polymerase chain reaction (PCR) was performed on 33 samples. All patients were treated with benznidazole. After 4.5 years, IgG levels were reassessed in 26 individuals. The mean age was 33.6 years, with no gender difference. The mean incubation period was 13.8 days, and the mean between symptom onset and treatment was 16.6 days. The most common symptoms were fever and lymphadenopathy (90%). Diagnostic success rates were 66.6% (laboratory criteria), 23% (clinical-epidemiological criteria), and 10.2% (high clinical suspicion despite negative tests). Test positivity rates were 69.7% (PBS), 91.4% (serology), and 100% (PCR). There were no deaths. Serological cure was achieved in 34.6% of cases, and IgG titers decreased in 15.3%. We encountered several barriers in managing ACD, including population vulnerability, reliance on outdated diagnostic techniques, lack of standardized molecular biology methods, and limited therapeutic options. This report underscores the importance of rapid surveillance and early treatment to prevent fatalities. We recommend the standardization of conventional PCR in diagnostic routines.

VISTA: an integrated framework for structural variant discovery

Abstract Structural variation (SV) refers to insertions, deletions, inversions, and duplications in human genomes. SVs are present in approximately 1.5% of the human genome. Still, this small subset of genetic variation has been implicated in the pathogenesis of psoriasis, Crohn’s disease and other autoimmune disorders, autism spectrum and other neurodevelopmental disorders, and schizophrenia. Since identifying structural variants is an important problem in genetics, several specialized computational techniques have been developed to detect structural variants directly from sequencing data. With advances in whole-genome sequencing (WGS) technologies, a plethora of SV detection methods have been developed. However, dissecting SVs from WGS data remains a challenge, with the majority of SV detection methods prone to a high false-positive rate, and no existing method able to precisely detect a full range of SVs present in a sample. Previous studies have shown that none of the existing SV callers can maintain high accuracy across various SV lengths and genomic coverages. Here, we report an integrated structural variant calling framework, Variant Identification and Structural Variant Analysis (VISTA), that leverages the results of individual callers using a novel and robust filtering and merging algorithm. In contrast to existing consensus-based tools which ignore the length and coverage, VISTA overcomes this limitation by executing various combinations of top-performing callers based on variant length and genomic coverage to generate SV events with high accuracy. We evaluated the performance of VISTA on comprehensive gold-standard datasets across varying organisms and coverage. We benchmarked VISTA using the Genome-in-a-Bottle gold standard SV set, haplotype-resolved de novo assemblies from the Human Pangenome Reference Consortium, along with an in-house polymerase chain reaction (PCR)-validated mouse gold standard set. VISTA maintained the highest F1 score among top consensus-based tools measured using a comprehensive gold standard across both mouse and human genomes. VISTA also has an optimized mode, where the calls can be optimized for precision or recall. VISTA-optimized can attain 100% precision and the highest sensitivity among other variant callers. In conclusion, VISTA represents a significant advancement in structural variant calling, offering a robust and accurate framework that outperforms existing consensus-based tools and sets a new standard for SV detection in genomic research.

A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

Investigation of Factors Associated with Subclinical Infections of Giardia duodenalis and Cryptosporidium canis in Kennel-Housed Dogs (Canis lupus familiaris).

Giardia duodenalis and Cryptosporidium spp. are zoonotic protozoal pathogens, spread by a fecal-oral route, which can infect a wide range of hosts including but not limited to dogs and humans. Giardia was recently estimated to be present in 37% to 50% of kennel-housed dogs. Cryptosporidium infections in kennel-housed dogs have been reported in 7% to 21% of the population. The goal of this study was to define demographic factors and fecal scores associated with positive screening test cases of Giardia and Cryptosporidium in kennel-housed laboratory dogs in the state of Texas. Fecal samples were collected from 153 clinically normal laboratory dogs at an academic research facility and a local laboratory dog supplier. We used 3 diagnostic tests evaluated in parallel to determine test positivity to each organism: a human point-of-care coproantigen test, a direct immunofluorescent assay, and an in-house polymerase chain reaction. Dogs were significantly more likely to test positive for Giardia (45%) than Cryptosporidium (7%) (P < 0.01). Dogs that were 18 mo of age or younger had 3 times the odds (P = 0.009) of subclinical Giardia infection compared with older dogs. We found no significant relationship between age and Cryptosporidium prevalence. Dogs with hard feces (fecal score 1-2) at the time of screening had 0.34 times lower odds ( P = 0.049) of testing positive for Giardia than dogs with normal feces, but no statistically significant relationship was found between fecal score and Cryptosporidium -positive test status. With these findings, we demonstrated the value of considering age and fecal score when choosing which dogs to screen for subclinical Giardia. Additional studies with larger sample sizes should be conducted to determine the relationship between age and fecal score and subclinical Cryptosporidium infection.

Treponema pallidum PCR with blood and cerebrospinal fluid of newborns exposed and not exposed to syphilis during pregnancy.

Congenital syphilis (CS) has severe adverse outcomes, including abortion and death. Diagnosis of CS in asymptomatic newborns remains difficult. This study aims to evaluate an in-house polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) and blood samples (BS) to identify T. pallidum DNA in newborns. We performed an exploratory cross-sectional study that included newborns exposed to syphilis during pregnancy (SEG) and non-exposed (SNEG) newborns, between 2019 and 2020. In-house conventional PCR for T. pallidum targeting the tpp47 gene was used to analyze CSFS and dried blood spots. BS was obtained from 54 newborns (33 SEG/21 SNEG) and CSF from 55 newborns (33 SEG/22 SNEG). Twenty-five (71.4%) SEG newborns had reactive BS rapid plasmatic reagins (RPR), and all of them had RPR titers less than or equal to the corresponding maternal titers. All RPR CSF tests were negative. PCR for T. pallidum DNA was positive in 19/33 (57.6%) BS, and in 22/33 CSF. The only SEG newborn with clinical signs of early CS had a positive CSF PCR and a negative BS PCR. Conversely, among SNEG newborns, PCR was positive in 2/21 BS and 5/22 (22.7%) CSF. T. pallidum DNA was identified using our PCR tests. The exposed group did not present abnormalities that would indicate CS. This prevented conclusions regarding sensitivity and specificity. Dried spot permitted bedside collection, easy transportation, and storage. Further research is needed to evaluate and improve the accuracy of CS low-cost PCR tests, especially for limited resource settings.

Performance evaluation of the SMG HHV-6 Q Real-Time PCR Kit for quantitative detection and differentiation of human herpesvirus 6A and 6B.

The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.

Integron distribution and relationship to antimicrobial resistance in E. coli isolated from blood culture

PurposeThe aim of this study was to evaluate the distribution of integrons in strains of E. coli isolated from blood culture and the relationship between integrons and antimicrobial resistance. MethodsThe study included 100 E. coli strains sent to the Medical Microbiology Laboratory from different clinics between September 2022 and June 2023. Antibiotic susceptibility was evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The presence of integrons was determined by the inhouse polymerase chain reaction (PCR). ResultsIntegron positivity was detected in 45 (45%) of isolates, and class 1 integrons were found in 41 (41%), class 2 integrons in 2 (2%), and both class 1 integrons and class 2 integrons in 2 (2%). Class 3 integron positivity was not detected. In total, 63 cases of community origin and 37 cases of hospital origin were identified. When antibiotic resistance was evaluated, the highest sensitivity was noted for amikacin (1%), meropenem (5%), imipenem (6%), and the highest resistant antibiotics were ampicillin (82%), cepfuroxime sodium (65%), and amoxicillin/clavulanate (62%), respectively. Of the 16 antimicrobial substances evaluated, 10 had an antibiotic resistance rate of over 45%. In class 1 integron-positive samples, ampicillin resistance and trimethoprim/sulfamethoxazole resistance were higher than in negative samples (p = 0.02, p = 0.0001, respectively). Fifty-one (51%) samples were found to have multiple drug resistance (MDR). In total, 59.5% of hospital-acquired isolates and 46% of community-acquired isolates were considered to be MDR. The class 1 integron positivity in MDR samples was high (p = 0.038). ConclusionThe high MDR rates in both hospital-acquired and community-acquired isolates are alarming. In particular, class 1 integron monitoring is very important to prevent the spread of MDR isolates.

Chronic sexually acquired reactive arthritis secondary to Chlamydiatrachomatis

Reactive arthritis is included in the spectrum of seronegative spondyloarthritides, occurring secondary to triggers of genitourinary and gastrointestinal tract infections. We describe two cases of sexually acquired reactive arthritis secondary to genital infection by Chlamydia trachomatis, diagnosed by in-house polymerase chain reaction performed on the first void urine. Both patients were managed with a combined approach of short course antibiotics, immunosuppressive agents, biologicals and surgical intervention.

Impact of In-house Candida auris Polymerase Chain Reaction Screening on Admission on the Incidence Rates of Surveillance and Blood Cultures With C. auris and Associated Cost Savings.

The impact of strategies for rapid diagnostic screening of Candida auris on hospital operations has not been previously characterized. We describe the implementation of in-house polymerase chain reaction (PCR) testing on admission for screening of colonization with C. auris, associated process improvements, and financial impact. This study was conducted across an integrated health system. Patients were tested based on risk factors for C. auris carriage. Pre-intervention, the PCR was sent out to a reference laboratory, and postintervention was performed in-house. Changes in the incidence rates (IRs) of C. auris present on admission (CA-POA) and C. auris hospital-onset fungemia (CA-HOF) were assessed using interrupted time series analysis. The economic impact on isolation and testing costs was calculated. Postintervention, the IR of CA-POA doubled (IRR, 2.57; 95% CI, 1.16-5.69; P = .02) compared with the pre-intervention period. The baseline rate of CA-HOF was increasing monthly by 14% (95% CI, 1.05-1.24; P = .002) pre-intervention, while during the postintervention period there was a change in slope with a monthly decrease in IR of 13% (95% CI, 0.80-0.99; P = .02). The median turnaround time (TAT) of the results (interquartile range) was reduced from 11 (8-14) days to 2 (1-3) days. Savings were estimated to be between $772 513.10 and $3 730 480.26. By performing in-house PCR for screening of C. auris colonization on admission, we found a doubling of CA-POA rates, a subsequent decrease in CA-HOF rates, reduced TAT for PCR results, and more efficient use of infection control measures. In-house testing was cost-effective in a setting of relatively high prevalence among individuals with known risk factors.

No Varicella Zoster Virus Infection among Mpox Cases in Antwerp, Belgium.

Several studies in tropical settings have reported that ∼20% of patients infected with Monkeypox virus (MPXV) also tested polymerase chain reaction (PCR) positive for Varicella zoster virus (VZV). Researchers have hypothesized that VZV infection predisposes to monkeypox (mpox), or vice versa, or that MPXV triggers the reactivation of VZV. We tested samples for VZV from a cohort of patients infected with clade IIb MPXV diagnosed between May 23 and October 14, 2022 in Antwerp, Belgium. Leftover DNA extracts of skin lesion samples from 108 mpox patients were tested with in-house PCR for VZV. No VZV infections were found. The absence of concurrent VZV-MPXV infections in our cohort indicates that VZV did not cocirculate in the population at risk for MPXV during the Belgian 2022 outbreak, but also that MPXV does not commonly trigger reactivation of latent VZV in adult men.

A-308 The Molecular Diagnosis of Varicella Zoster Virus (VZV): A Validation of a Real-Time PCR (qPCR) Assay for the Qualitative Detection

Abstract Background Varicella zoster virus (VZV) is the etiological agent of chicken pox and herpes zoster. After the primary infection, which usually occurs in childhood, VZV remains latent in sensory nerve roots for life. When reactivation occurs, especially in immunocompromised patients, the virus migrates to the skin and causes painful rashes that can evolute to a dermatome. Even after the end of the viral acute infection, some individuals may experience continued pain. Furthermore, VZV reactivation can lead to a wide spectrum of clinical manifestations such as self-limited radicular pain and central nervous system involvement. During acute illness, 90% of patients experience pain and 12% have symptoms similar to influenza infection (influenza-like). Immunocompromised patients may have an unfavorable course with disseminated infection, pneumonitis, hepatitis, and encephalitis. The cerebrospinal fluid polymerase chain reaction (PCR) test is indicated for suspected involvement of the central nervous system and complications of ocular or periocular lesions. Compared to other diagnostic methods, real-time PCR has great advantages as its high throughput, efficiency, and sensitivity. Therefore, the detection of VZV by real-time PCR presents great importance for the diagnosis and monitoring of the disease due to its precision and agility. Thus, we intended to validate a qualitative method for VZV detection by real-time PCR. Methods We evaluated the performance of the inventoried TaqMan assay (Vi06439647_s1) (Thermo Fisher Scientific). For this validation, we used 64 samples - whole blood (n = 31), cerebrospinal fluid (n = 32), vaginal lesion scraping (n = 1) - from the Genetics sector of the Pardini Group, previously tested by in-house PCR for the qualitative detection of VZV. Samples were extracted using the MagNA Pure 24 System platform (Roche Diagnostics) with the MagNA Pure 24 Total NA Isolation Kit, according to the manufacturer's instructions. All assays were performed using Taqman Universal Master Mix (Thermo Fisher Scientific). The inventoried B-actin Assay (assay ID: Hs01060665_g1) (Thermo Fisher Scientific) was used as endogenous control. Assay verification tests, evaluation of reaction efficiency, and determination of the detection limit (LOD) were performed using a synthetic control of known concentrations and a pool of negative samples (healthy volunteers). At the end of the tests, were made a comparison of the previous results with the results obtained in the ThermoFisher Real-Time PCR Assay. Results The Inventoriad Taqman Assay (VZV) had a limit of detection of 10 copies/µL and the Cq &amp;gt; 35 was defined as a cut-off to consider a sample as positive. Whole blood samples had higher Cts than cerebrospinal fluid samples, indicating lower viremia. Whitin all evaluated samples, 11 samples had positive results detected by in-house PCR (9 from whole blood and 2 from cerebrospinal fluid). Of these, 10 had a result detected in Real-time PCR and the agreement with the positive results between compared methodologies was 91.0%. The divergent sample was whole blood and can be related to DNA degradation. The 47 negative samples evaluated were concordant in both methods. Conclusion The inventoried TaqMan Assay with Taqman Universal Master Mix protocol evaluated was sensitive, fast, and applicable as a tool for the qualitative detection of VZV.

A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype.

The extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein. Four unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation. Anti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3'-end of intron 6, c.946 -2a>g in all samples. RHD/RHCE showed no alterations. A novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U- but also have qualitative changes in their s antigen expression.

Zoophilic dermatophytes during coronavirus pandemic in Germany

During the coronavirus pandemic, significantly more pets were probably bought and kept. This study focuses on whether more zoophilic dermatophytes have subsequently been isolated and which species predominate. In the 1‑year period from March 2020 through February 2021, all zoophilic dermatophytes from all submissions to the Mölbis laboratory were recorded. Both the cultural and the molecular evidence of fungal detection from skin scrapings, hair roots, and, in single cases, from nails, were considered. For dermatophyte DNA(Deoxyribonucleic acid) detection, an in-house polymerase chain reaction (PCR)- enzyme-linked immunosorbent assay (ELISA) was used. In distinct cases, identification of dermatophytes was confirmed by sequencing of the internal transcribed spacer (ITS) region of the rDNA, and of the gene of the translation elongation factor (TEF)-1α. In 579 (2.56%) of 22,575 samples studied in the year 2020/2021, zoophilic dermatophytes were detectable with PCR-ELISA and/or by cultivation. In comparison, the proportion of zoophilic dermatophytes was 2.03% in the 1‑year period 2014/2015, and only 1.6% in 2018/2019. The 579 zoophilic dermatophytes were identified as follows: Trichophyton(T.) benhamiae 186 (32.1%), T.mentagrophytes 173 (29.9%), T.quinckeanum 110 (19.0%), Microsporum(M.) canis 78(13.5%), T.verrucosum 22(3.8%), Nannizzia(N.) persicolor 8(1.4%), T.erinacei 1(0.2%), and T.equinum 1(0.2%). T.benhamiae had the highest prevalence from June to September 2020, then again in December. T.quinckeanum is associated with asharp increase in the mice population in Germany in 2020; asignificant increase was found in the months September 2020 to January 2021. T.mentagrophytes had aconspicuous peak in September. Compered with that M. canis in November. Up to 50% of the dermatophytoses caused by T.mentagrophytes, T.quinckeanum, and M.canis affected children and adolescents, while in the case of T.benhamiae it was as much as two thirds. Tinea corporis was the most common, followed by tinea faciei and tinea capitis. M.canis infections affected the capillitium more frequently than the face. Zoophilic dermatophytes were increasingly isolated during the coronavirus pandemic in Germany when compared to previous year periods. In first place, the dermatophyte T.benhamiae from guinea pigs was found in children and adolescents. Asignificant proportion of dermatophytoses concerned adults. T.quinckeanum is an emerging pathogen in Germany with unprecedented high infection rates in 2020.

Detection of human papillomavirus in fresh and dried urine through an automated system for cervical cancer screening in low- and middle-income countries.

The majority of cervical cancer cases and associated deaths occur in low- and middle-income countries (LMICs), where sociocultural barriers, poor access to prevention and care, and technical and practical difficulties hinder screening coverage improvement. Using urine specimens for human papillomaviruses (HPV) molecular screening through automated testing platforms can help to overcome these problems. We evaluated the high-risk (HR) HPV detection performance of the Xpert® HPV test on GeneXpert® System (Cepheid), on fresh and dried urine (Dried Urine Spot [DUS]) samples as compared to an in-house polymerase chain reaction (PCR) genotyping assay. Forty-five concentrated urine samples collected from women with known cytological and HPV infection status, determined through in-house PCR and genotyping assays, were tested "as is" and as DUS with the Xpert® HPV test. This system detected HR-HPV in 86.4% of fresh and in 77.3% of dried urine samples collected from HPV+ women, correctly identifying HR-HPV infection in 100% of women with low- and high-grade lesions. High concordance (91.4%, k = 0.82) was found between PCR test and Xpert® HPV Test from urine. Urine-based Xpert® HPV test seems to be a suitable screening test for detection of HR-HPV infections associated with low- and high-grade lesions requiring follow-up monitoring or treatment. This methodology, relying on noninvasively collected samples and on available rapid testing platforms, could facilitate large, at-scale screening programs, particularly in LMICs and rural areas, thus reducing adverse outcomes of HPV infection and facilitating achievement of the WHO cervical cancer elimination goal.

BK Polyomavirus Diversity After Hematopoietic Stem Cell Transplantation.

BK polyomavirus (BKPyV) infection is common after hematopoietic stem cell transplantation (HSCT) and is associated with the development of hemorrhagic cystitis (HC). The role that BKPyV plays in the pathogenesis of HC is not well characterized. We investigated the impact of BKPyV diversity on the development of HC using a previously established cohort of pediatric HSCT patients. There were 147 urine samples with quantifiable BKPyV at month 1 after HSCT; 137 (93.2%) were amplified using our in-house polymerase chain reaction approach and sent for next-generation sequencing. Subtype Ia was most frequent (61.3%), followed by subtype Ib1 (31.4%). The median viral load of subtype Ia samples was higher than for subtype Ib1 at month 1. Across the protein coding regions, APOBEC-induced mutations and signature patterns associated with HC were identified. This is the largest sequencing study of a single cohort of HSCT patients, providing a vast resource of sequence data for future analyses.

First case of mpox diagnosed in Queensland, Australia: clinical and molecular aspects.

First case of mpox diagnosed in Queensland, Australia: clinical and molecular aspects.

364. Diagnosis of Amebic Liver Abscess by a Commercial Multiplex Stool Amplification Assay. A Potentially Useful Alternative in Limited-Resources Settings.

Abstract Background Amoebic liver abscess (ALA) is caused by the protozoan parasite Entamoeba histolytica. Clinical and radiological features of ALA often mimic those of pyogenic liver abscess (PLA). Currently, differential diagnosis between ALA and PLA relys on microscopic examination, anti-amoebic IgG serology, and culture of aspirate for pyogenic organisms. Therefore, there is a need for new, rapid and more accurate microbiological methods for diagnosing ALA. Unfortunately, with few exceptions, available reports involved the use of inhouse or laboratory-tailored polymerase chain reaction (PCR) assays. We herein report our recent experience with the successful use of a commercial multiplex, nested PCR assay designed for stool, in the diagnosis of two cases of ALA. Methods Two severely ill adult Latin American migrants presented with fever, malaise, non-bloody diarrhea, abdominal pain and leukocytosis. CT scans revealed single large hypodense, inflammatory, liver cystic lesions. Results Necrotic material obtained by needle aspiration under guidance and stool samples were processed by FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City), designed to detect 13 bacterial, 5 viral and 4 parasitic pathogens, both amplified only for E. hystolitica DNA. ELISA serology for amoeba was also positive. Bacterial cultures were sterile. Treatment with Metronidazole and paromomycin resulted in complete recovery. Conclusion Analysis of liver abscess content using a commercially available, multiplex, nested PCR assay designed for stool, may represent a convenient alternative to currently available diagnostic methods, providing rapid results and allowing for immediate choosing of specific therapy targeting single pathogens on more rational basis. Since there are several commercially available multiplex PCR panels containing primers for E. histolytica, their use for evaluation of liver abscess samples has some theoretical advantages for the diagnosis of ALA, such as simplicity, ample availability, rapid processing time and capacity to exclude simultaneously several potential pathogens. Nevertheless, further testing is necessary to validate their use in liver abscess diagnosis and define their false positivity and negativity rates. Disclosures All Authors: No reported disclosures.

High Occurrence of Emerged Lophomonas Infection among Patients Suspected of Having Pulmonary Tuberculosis: In-House PCR-Based Evidence.

Lophomonas infection is a respiratory disease in humans that is associated with symptoms of cough, sputum, dyspnea, and sometimes hemoptysis, which shows the importance of differentiating this disease from tuberculosis and asthma. This study was performed on 216 participants suspected of having tuberculosis who had symptoms of fever, chronic cough, or sputum and were referred to tuberculosis laboratories in three cities in Golestan Province, northeastern Iran, during 2019-2020. A sputum sample was taken from the suspected patients. DNA was extracted from the frozen samples, and an in-house polymerase chain reaction was performed to detect the Lophomonas DNA. Out of 216 subjects, 47 (21.75%) were infected with Lophomonas spp. Moreover, 9 patients (4.2%) were infected with tuberculosis. Also, 2 patients had a comorbidity of tuberculosis and Lophomonas infection (P=0.63). There was no significant difference in the comparison of symptoms and the rate of Lophomonas infection (P=0.84), but in the comparison of the set of symptoms of cough, sputum, and fever with those of cough and sputum, cough with fever, sputum with fever, and the rate of Lophomonas infection, there was a significant difference (P=0.012). Lophomonas infection was relatively high in patients suspected of having tuberculosis and due to the similar clinical symptoms of Lophomonas infection and tuberculosis; it is recommended that the sputum samples of subjects suspected of having tuberculosis be examined for this parasite in order to make a correct diagnosis and the patients receive timely treatment and the appropriate medication.

Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population.

Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent agreement was 99%. GPP, a multi-analyte platform can easily be implemented into a routine diagnostic laboratory for detecting enteric pathogens including C. difficile.

Rapid identification of Mycobacterium tuberculosis in cultures by molecular and immunological methods

Objective : The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods : A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR- CTAB as a reference method. Results :The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions : Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosis.