Direct PCR Research Articles

A simple and cost-effective real-time PCR method using diluted and heat-treated whole blood lysate

Although DNA extraction is a crucial step before polymerase chain reaction (PCR), attempts to perform PCR without DNA isolation have been ongoing since the 1990s. While partial success has been achieved with direct conventional PCR, the DNA isolation step has remained indispensable for real-time PCR. Here, we developed a method that does not require complete DNA isolation by lysing EDTA-treated whole blood samples through the application of osmotic pressure and heat, followed by centrifugation to get a clear lysate. Blood samples were mixed with distilled water, incubated at 95 °C for 20 min before centrifugation at 14,000 rpm for 5 min. The resulting lysates were used as templates for real-time PCR. Real-time PCRs were performed under identical conditions with lysates and DNA samples using 9 different primer sets. Target genes were successfully amplified using 1:10 and 1:5 diluted blood lysates both at 60 °C and 61 °C. The PCR efficiency for ACTB and PIK3CA differed by 20% and 14%, respectively, between the DNA samples and blood lysates. We successfully amplified all selected genes using “GG-RT PCR”, greater temperature, greater speed method, without the need for additional enzymes or buffers. GG-RT PCR presents a cost effective option for applications such as SNP analysis and deletion detection if appropriate primer designs are made.

First Report of Fusarium proliferatum Causing Pinellia ternata Corm Rot in Henan, China.

Pinellia ternata (Thunb.) Makino is a perennial herb of the genus Pinellia in the Araceae family, which is a kind of precious Chinese herbal medicine (Bai et al. 2022). In May 2023, rotting was observed on P. ternata corm in the Tanghe planting base (3.33 ha, 32º46´6˝N, 113º 8´47˝E, 138 m), with an incidence of approximately 10%. To identify the causal agent, five rotten corms were randomly sampled from the Tanghe planting base. Symptomatic corm tissues were cut into small pieces (3 mm3) with asymptomatic tissue margins, disinfected in 75% ethanol for 30s and in 2% sodium hypochlorite for 3 min, rinsed three times with sterile distilled water, and then placed on synthetic nutrient-poor agar (SNA) plates incubated at 20℃ with a 12 h light/dark photoperiod. Hyphal-tips from the growing edge of colonies were transferred to fresh SNA to obtain pure cultures. In total, five pure isolates were obtained with similar colony morphology and white (top view) or grayish white (back view) coloration, with villous surfaces and abundant aerial hyphae. On SNA, the isolates mainly produced microconidia. Microconidia were ellipsoidal to cylindrical, thin-walled, smooth, hyaline and measured 5.40 to 11.98 (av. 7.14) µm long and 1.60 to 5.38 (av. 2.54) µm thick (n=50). Based on these morphological characteristics of colony and conidia, the causal agent was preliminarily identified as Fusarium sp. (Crous et al. 2021). To confirm pathogen identity, the total genomic DNA of 2 isolates (BX-2 and BX-3) was extracted using the 2×T5 Direct PCR kit (Beijing Tsingke Biotech Co., Ltd, Beijing, China). The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) gene were PCR-amplified using the universal primers ITS1/ITS4, 5f2/7cr, EF-1/EF-2 and BT2F/BT4R (Crous et al. 2021), respectively. Sequences have been deposited in GenBank (PP125560-PP125561 for ITS, PP782474-PP782475 for rpb2, PP782476-PP782477 for tef1, PP171436-PP171437 for tub2). BLAST search showed that all sequences were 99% to 100% homology with the corresponding sequences of Fusarium proliferatum (CBS 182.32). A phylogenetic tree was constructed based on the Maximum-Likelihood method in MEGA-X (Kumar et al. 2018) and revealed that the two isolates were closest to F. proliferatum. The pathogenicity tests were conducted with the two isolates (BX-2 and BX-3) as described by Liu et al. (2022) with slight modifications. For each treatment, five healthy P. ternata corms were inoculated with 1ml of conidial suspension (1×107 conidia/ml). Control plants (n=5) were inoculated with sterilized water. All samples were covered with plastic bags and maintained at a relative humidity greater than 90% in a greenhouse at 20℃ with a 12 h light/dark photoperiod. After 15 days, inoculated corms showed similar symptoms to those on the original diseased plants, while control plants remained symptomless. F. proliferatum was successfully reisolated from the inoculated plants and identified based on its morphological characteristics. Thus, Koch's postulates were fulfilled. The experiment was repeated thrice with similar results. Morphological characteristics, sequence alignment and Koch's postulates test confirmed that F. proliferatum was the cause of P. ternata corm rot disease. To our knowledge, this is the first report of F. proliferatum causing P. ternata corm rot in Henan province, China. Our findings are important for informed surveillance of the disease.

FASTER AND COST-EFFECTIVE EXTRACTION FREE METHOD FOR SARS-COV2 DETECTION BY RT-PCR

Background: The World Health Organization (WHO) declared an outbreak of febrile respiratory illness caused by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) in Wuhan City, China in December 2019. Within the immediate outbreak of three months, the global community was challenged with a devastating pandemic that caused heavy morbidities and mortalities around the globe. The expeditious spread of the virus, challenged the diagnostic laboratories to rapidly develop the accurate molecular diagnostic methods. As SARS CoV-2 assays became available for testing on existing molecular platforms, laboratories devoted unprecedented energy and resources to evaluating the analytical performance of the new tests and in some cases developed their diagnostic assays under FDA-EUA guidance. Aim: This study aims to compare the diagnostic efficacy of the extraction-free method of COVID-19 PCR and the conventional RT-PCR with extraction. Method: To get an accurate view of how an omission of RNA extraction step would perform in a real-world setting, a comparative study was performed using TATA MD CHECK RT-PCR DIRECT (Direct PCR) and LabGun Genomics for Conventional RT-PCR method. Result: From this study, direct RT-PCR correctly identified 92.3% of samples (n = 50) identified positive for SARS-CoV-2 RNA by conventional RT-PCR featuring an RNA extraction. Conclusion: Direct methods may represent a reasonable alternative to meet higher testing demands with low turnaround time as reverse transcription PCR includes traditionally time-consuming RNA extraction and purification procedures.

Development and optimization of immunomagnetic bead separation-convective PCR (IMBS-CPCR) for on-site detection of four common Vibrio species in aquaculture

Foodborne infectious diseases, particularly those caused by pathogenic Vibrio species in aquaculture, are a significant concern in the field of seafood safety. They not only hinder the economic development of the industry but also pose a major threat to public health. Traditional detection methods are often too slow, insensitive, or impractical for portable use, highlighting the urgent need for a sensitive, on-site detection method for Vibrio that prioritizes convenience and speed. This study developed a visual IMBS-CPCR method for the detection of four common pathogenic Vibrio species: Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, and Vibrio harveyi. The method achieved high capture rates for target bacteria (82.6%–83.3%) and low capture rates for non-target bacteria (below 11%), demonstrating good sensitivity by detecting bacteria at concentrations as low as 10 CFU/mL. Optimal amplification conditions were determined for each species, with specificity tests confirming that only target bacteria produced amplification bands. Sensitivity tests revealed detection limits of 101 CFU/mL for V. alginolyticus, V. anguillarum, and V. fluvialis, and 102 CFU/mL for V. harveyi. The combined techniques improved detection sensitivity in simulated fish samples by 10–100 fold compared to direct CPCR or PCR methods.

Comparison of qPCR and chromogenic culture methods for rapid detection of group B streptococcus colonization in Vietnamese pregnant women

IntroductionNeonatal infections can rapidly become severe, with delays in treatment often proving fatal. Streptococcus agalactiae (Group B Streptococcus, GBS) is a common cause, typically transmitted from colonized pregnant women to neonates during childbirth. In Vietnam, routine prenatal care lacks standardized GBS screening protocols. This study aims to compare enhanced qPCR methods with the culture method, evaluate the diagnostic accuracy of these qPCR procedures, and assess the frequency of GBS infection in pregnant Vietnamese women during their final trimester. Materials and methodsPregnant women aged 35 weeks gestation or more were recruited. Rectovaginal swabs were collected and analyzed for GBS using chromogenic culture, a commercial real-time PCR kit, and in-house real-time PCR assays targeting the cfb and sip genes. Clinical diagnostic values were calculated, and GBS prevalence was determined. ResultsThe study included 259 pregnant women with a mean age of 30.2 ± 5.0 years. Of these, 96.6 % had gestational ages of 37 weeks or more at delivery. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the cfb-based and sip-based qPCR assays were 94.1/92.7, 99.0/99.5, 97.1/98.5, 97.8/97.3, and 97.6 %, respectively. The Kappa values were excellent (0.940 and 0.939), with results available in under 2 h. GBS prevalence was 24.7 % and 25.5 % by cfb-based and sip-based qPCR assays, aligning with the culture method (25.5 %). ConclusionsBoth direct real-time PCR assays demonstrated high accuracy and were comparable to chromogenic culture in diagnosing GBS. A significant prevalence of GBS colonization was found among Vietnamese pregnant women in their final trimester.

Infectious Agents Associated with Abortion Outbreaks in Italian Pig Farms from 2011 to 2021

The present study retrospectively analyzed the infectious agents associated with 829 abortion outbreaks occurring from 2011 to 2021 in northern Italy. Foetuses were subjected to necropsies, and organ samples were analyzed by direct PCR to screen for six swine pathogens. In 42.0% of the examined outbreaks, at least one infectious agent was found. Porcine reproductive and respiratory syndrome virus (PRRSV) (24.9%) and porcine circovirus-2 (PCV2) (11.5%) were the most frequently detected among the known abortion-inducing pathogens. Chlamydia spp. (5.6%), porcine parvovirus (PPV) (4.0%), and Leptospira spp. (2.6%) were less common. Although its role in swine reproductive disorders is still unclear, PCV3 was detected in 19.6% of the cases. Coinfections were detected in 25.0% of positive outbreaks, and the most frequent coinfection was represented by PRRSV and PCV2 (32.2%), followed by PRRSV and PCV3 (23%). PCV2 prevalence showed a slight but consistent reduction during the study period, while PCV3 increased in frequency. Our data suggest an overall reduction in abortion outbreaks during the study period. PRRSV was confirmed as the main abortion agent detected in the examined area, while PCV2 prevalence showed a decline. Conversely, PCV3 detection has been increasing, supporting its potential role as an abortion agent. Our results highlight the importance of implementing a consistent and standardized sampling procedure, as well as a thorough diagnostic protocol, to reduce the incidence of inconclusive diagnoses.

CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia

BackgroundThe severity of infectious disease outcomes is dependent on the virulence factors of the pathogen and the host immune response. CARD8 is a major regulator of the innate immune proinflammatory response and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the CARD8 gene was investigated in relation to bacterial meningitis.MethodsA total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients.ResultsOf the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified 7 patients with bacterial meningitis caused by Neisseria meningitidis (n = 4) and Streptococcus pneumoniae (n = 3). The RT-PCR revealed 39 positive samples for N. meningitidis (n = 10) and S. pneumoniae (n = 29). A total of 332 whole-blood samples were genotyped with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild genotypes. The polymorphic gene carriers among laboratory confirmed, clinically diagnosed meningitis and healthy controls were 23(46%), 246(40%), and 1526(39%), respectively with OR = 1.27 (0.7–2.3) and OR = 1.34 (0.76–2.4). The presence of the C10X polymorphism in the CARD8 gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers.ConclusionsA greater proportion of C10X in the CARD8 gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than heterozygote gene carriers and healthy controls.

Occurrence of chlorine-resistant Pseudomonas aeruginosa in hospital water systems: threat of waterborne infections for patients

BackgroundSeveral healthcare-associated infection outbreaks have been caused by waterborne Pseudomonas aeruginosa exhibiting its ability to colonize water systems and resist conventional chlorine treatment. This study aims to investigate the occurrence of Pseudomonas aeruginosa in hospital drinking water systems and the antimicrobial resistance profiles (antibiotic and chlorine resistance) of isolated strains.MethodsWe investigated the presence of Pseudomonas aeruginosa in water and biofilms developed in nine hospital water systems (n = 192) using culture-based and molecular methods. We further assessed the survival of isolated strains after exposure to 0.5 and 1.5 ppm concentrations of chlorine. The profile of antibiotic resistance and presence of antibiotic resistance genes in isolated strains were also investigated.ResultsUsing direct PCR method, Pseudomonas aeruginosa was detected in 22% (21/96) of water and 28% (27/96) of biofilm samples. However, culturable Pseudomonas aeruginosa was isolated from 14 samples. Most of P. aeruginosa isolates (86%) were resistant to at least one antibiotic (mainly β-lactams), with 50% demonstrating multidrug resistance. Moreover, three isolates harbored intI1 gene and two isolates contained blaOXA−24,blaOXA−48, and blaOXA−58‌ genes. Experiments with chlorine disinfection revealed that all tested Pseudomonas aeruginosa strains were resistant to a 0.5 ppm concentration. However, when exposed to a 1.5 ppm concentration of chlorine for 30 min, 60% of the strains were eliminated. Interestingly, all chlorine-resistant bacteria that survived at 30-minute exposure to 1.5 ppm chlorine were found to harbor the intI1 gene.ConclusionsThe detection of antimicrobial resistant Pseudomonas aeruginosa in hospital water systems raises concerns about the potential for infections among hospitalized patients. The implementation of advanced mitigation measures and targeted disinfection methods should be considered to tackle the evolving challenges within hospital water systems.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam

ObjectiveThis study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation.ResultsThe rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Detection of Phytoplasma belonging to the 16SrXXI group in Scott pine and mountain pine

One of the reasons for the deterioration of coniferous stands condition in a number of European countries is associated with their infection with phytoplasmas (obligate intracellular pathogens) — bacteria lacking a cell wall. The aim of the work is to identify the presence of phytoplasma infection in samples of Scots pine (Pinus sylvestris L.) and mountain pine (Pinus mugo Turra) collected in the Moscow and Samara regions, which had characteristic symptoms of the disease, and to determine the taxonomic affiliation of the phytoplasm. Phytoplasma was detected using direct and nested PCR with primer pairs P1/16S-Sr and R16F2n/R16R2 respectively. Phytoplasma DNA was found in six of the seven pine specimens, including an asymptomatic one. Analysis of restriction fragment length’s polymorphism after digestion of DNA amplicons with restriction endonucleases AluI, MseI, HhaI, HpaII, HaeIII, RsaI, and TaqI indicated the similarity of Russian strains of Scotch pine and mountain pine phytoplasmas to Lithuanian strains of PineLRN and PineBLD of mountain pine phytoplasma (GenBank Accession Number MK089821 and MK089819, respectively) identified as ‘Candidatus Phytoplasma pini’ (subgroup 16SrXXI-A). A phytoplasma related to this species has been registered on the territory of the Russian Federation for the first time.

The discovery of novel variants reveals the genetic diversity and potential origin of Seoul orthohantavirus.

Seoul orthohantavirus (SEOV) has been identified as one of the main causative agents of hemorrhagic fever with renal syndrome (HFRS) in China. The virus was found circulating in rodent populations in almost all provinces of the country, reflecting the wide distribution of HFRS. Here, using the direct immunofluorescence assay (DFA) and real-time quantitative reverse transcription PCR (qRT-PCR) approach, we performed screening in 1784 small mammals belonging to 14 species of three orders captured in the main areas of HFRS endemicity in Yunnan province (southwestern China) and identified 37 SEOV-positive rats (36 Rattus norvegicus and 1 Rattus tanezumi). A 3-year surveillance of HFRS epidemics and dynamics of rodent reservoir density and virus prevalence implied a potential correlation between them. The subsequent meta-transcriptomic sequencing and phylogenetic analyses revealed three SEOV variants, among which two are completely novel. The ancestral character state reconstruction (ACSR) analysis based on both novel variants and documented strains from 5 continents demonstrated that SEOV appeared to originate near the southwestern area (Yunnan-Kweichow Plateau) of China, then could spread to other regions and countries by their rodent carriers, resulting in a global distribution today. In summary, these data furthered the understanding regards genetic diversity and the potential origin for SEOV. However, the expanding endemic foci in the province suggest that the virus is spreading over a wider region and is much more diverse than previous depicted, which means that increased sampling is necessary.

Detection of HBV DNA by direct PCR protocol and Nested PCR protocol and comparing them in chronic patients and healthy carriers of Hepatitis B virus.

Hepatitis B virus is a major public health problem, and the outcome of infection depends on the effectiveness of the virus's interaction with the host, and in particular on the strength of the natural and conditioned response of humoral and cellular immunity. This study was conducted to assess the clinical status, genetic immunity and viral (viral) traits of Iraqi patients with chronic hepatitis B virus and healthy carriers of hepatitis B surface antigen, as well as to show the relationship between the immune status and the chronic condition of the disease. The study included (60) patients with chronic hepatitis B virus type (B) and (60) healthy hepatitis B surface antigen carriers who attended the Teaching Hospital for Gastrointestinal and Liver Diseases in Nasiriya City Al-Hussein Teaching Hospital, Thalassemia Center and the Central Blood Bank in Nasiriya Citysouthern of Iraq. for the period from the first period. From February 2022 until the end of February 2022, the average age of patients infected with chronic hepatitis B virus was 64.5 years, and the average age of healthy carriers of hepatitis B surface antigen was 35. 1 year, and the ratio of males to females was (12.2:) for chronic patients and (13.6:) for carriers of the disease. As for the deoxyribonucleic acid, it was (100%) in chronic patients and healthy carriers using the Nested type polymerase chain reaction, and at a rate of (30.0%) and (46.7%) in chronic patients and healthy carriers, respectively using the polymerase chain reaction technique. direct with a significant difference between the two interactions. The current results confirm that a nested PCR protocol to detect HBV DNA is a more sensitive way of detecting HBV in Iraq CHB patients and carriers. Recent advanced techniques such as nucleic acid assays, quantitative assays for viral load and RT-PCR for Iraqi viral hepatitis laboratories are recommended.

Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study.

To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance. This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)]. A total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity. This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).

Rapid Detection of Severe Fever with Thrombocytopenia Syndrome Virus in the Acute Phase of Infection by Direct Real-Time Reverse Transcription without RNA Extraction.

No specific treatment has been developed for severe fever with thrombocytopenia syndrome (SFTS). However, the prognosis can improve with early plasma exchange. Therefore, rapid and accurate detection of SFTS virus is important for diagnosis and prognosis. Direct real-time reverse transcription polymerase chain reaction (RT-PCR) testing is easier and more time-efficient than conventional real-time RT-PCR. Our study compared direct real-time RT-PCR efficiency without the RNA extraction and purification of conventional real-time RT-PCR. Samples were collected from 18 patients with SFTS and five without SFTS. A strong correlation (r = 0.774, 95% CI: 0.652-0.857, P <0.01) was found between conventional and direct real-time RT-PCR assays. Direct real-time RT-PCR showed 84.4% sensitivity and 92.0% specificity for viral detection. Direct real-time RT-PCR is an effective diagnostic tool for patients with acute phase SFTS, but further optimization is required for viral detection.

Long-range and real-time PCR identification of a large SERPINC1 deletion in a patient with antithrombin deficiency.

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods.

A Rare Combination of Compound Heterozygous Mutations in the PAH Gene in Three Unrelated Consanguineous Iranian Families with Classical Phenylketonuria.

The PAH gene mutations have been linked to the development of phenylketonuria (PKU), which is recognized as the most common inborn metabolic disorder, and is caused by a deficiency in the phenylalanine hydroxylase (PAH) enzyme. The Iranian population, known for its diversity and high consanguinity, offers a valuable sample for studying autosomal recessive disorders. Our study investigated three unrelated families with PKU from Iran, utilizing clinical, laboratory, and computational approaches. We performed direct PCR sequencing for 13 exons of the PAH gene on three Iranian patients who were diagnosed with PKU. Then, Sanger sequencing confirmed the segregation of the mutations from parents to probands. Pathogenicity predictor tools, including ACMG, CADD, SIFT, Polyphen-2, and Mutation Taster, were utilized to analyze the identified genetic variants. The three-dimensional structure of the mutant forms of the protein was predicted. We also analyzed the protein-protein interactions of PAH using the STRING database. All three patients exhibited rare compound heterozygosity rearrangements in the PAH gene (NM_000277.3). These included three missense variants: [c.533A>G/c.1222C>T], [c.526C>T/c.1222C>T], and [c.533A> / c.526C>T]. This study adds to the body of evidence establishing the association between PAH mutations and the development of PKU. We speculated that the conjunction of a high consanguinity rate in populations such as Iran, coupled with the founder effect, can give rise to atypical genetic profiles, as observed in the rearrangement of compound heterozygosity in this study Moreover, our research underscores the significance of genetic testing in the precise diagnosis of individuals affected by inborn errors of metabolism.

Outcomes of kidney transplant recipients exposed to Chagas disease under Benznidazole prophylaxis. A single center 10-year experience.

Chagas disease (ChD) is endemic in many parts of the world and can be transmitted through organ transplantation or reactivated by immunosuppression. Organs from infected donors are occasionally used for transplantation, and the best way of managing the recipients remains a subject of debate. We present a single-center cohort study describing a 10-year experience of kidney transplantation in patients at risk of donor-derived ChD and or reactivation. Patients received prophylactic treatment with Benznidazole and were monitored for transmission or reactivation. Monitoring included assessing direct parasitemia, serology, and polymerase chain reaction (PCR). Fifty-seven kidney transplant recipients (KTRs) were enrolled in the study. Forty-four patients (77.2%) were at risk of primary ChD infection, nine patients (15.8%) were at risk of disease reactivation, and four patients (7.0%) were at risk of both. All patients received Benznidazole prophylaxis, starting on the first day after transplantation. Parasitemia was assessed in 51 patients (89.5%), serology also in 51 patients (89.5%), and PCR in 40 patients (70.2%). None of the patients exhibited clinically or laboratory-detectable signs of disease. A single patient experienced a significant side effect, a cutaneous rash with intense pruritus. At 1-year post-transplantation, the patient and graft survival rates were 96.5% and 93%, respectively. In this study, no donor-derived or reactivation of Trypanosoma cruzi infection occurred in KTRs receiving Benznidazole prophylaxis.

Sample-to-answer direct real-time PCR detection of Anaplasma phagocytophilum, Ehrlichia spp., and Babesia spp. infections in whole-blood specimens.

This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance.

Comparison of methods for sampling and detection of carbapenemase-producing organisms in clinical handwash basin drains in health care

There are currently no standardised methods for the sampling and testing of clinical hand wash basin (HWB) samples for the detection of carbapenemase-producing organisms (CPO). Methods of sampling (drain aspirate versus swab from top of drain) and detection of CPO in clinical HWB drains in two different healthcare settings, one which was dealing with a hospital wide outbreak of CPO (hospital A) and another with no reported outbreaks (hospital B) were compared. Drain aspirates and swabs from HWB drains were tested using multiplex PCR together with culture-based methods. No significant difference in detection of CPO was found between drain aspirate or swab methods of sampling. Direct PCR on samples detected significantly more carbapenemase genes than culture on CARBA agar (p < 0.0001 and 0.0045 respectively). A higher percentage of HWB drains were positive in hospital A both by culture and direct PCR, and a significantly higher number of carbapenemase genes were detected in hospital A HWB drain aspirate both by PCR and culture (p = 0.014 and 0.0071 respectively). There was a high correlation between drain swab positivity by PCR and culture in hospital A (91%) compared to hospital B where it was only 33%. No difference could be found in drain contamination rates when HWB with rear drain was compared against those with drain directly below the tap. Colonisation of HWB at the top of the drain may be related to risk of cross transmission of CPO from healthcare environment to patients.

Circulation patterns and molecular characteristics of respiratory syncytial virus among hospitalized children in Tianjin, China, before and during the COVID-19 pandemic (2017–2022)

Respiratory syncytial virus (RSV) is the main pathogen that causes hospitalization for acute lower respiratory tract infections (ALRIs) in children. With the reopening of communities and schools, the resurgence of RSV in the COVID-19 post-pandemic era has become a major concern. To understand the circulation patterns and genotype variability of RSV in Tianjin before and during the COVID-19 pandemic, a total of 19,531 nasopharyngeal aspirate samples from hospitalized children in Tianjin from July 2017 to June 2022 were evaluated. Direct immunofluorescence and polymerase chain reaction (PCR) were used for screening RSV-positive samples and subtyping, respectively. Further analysis of mutations in the second hypervariable region (HVR2) of the G gene was performed through Sanger sequencing. Our results showed that 16.46% (3215/19,531) samples were RSV positive and a delayed increase in the RSV infection rates occurred in the winter season from December 2020 to February 2021, with the average RSV-positive rate of 35.77% (519/1451). The ON1, with H258Q and H266L substitutions, and the BA9, with T290I and T312I substitutions, are dominant strains that alternately circulate every 1–2 years in Tianjin, China, from July 2017 to June 2022. In addition, novel substitutions, such as N296Y, K221T, N230K, V251A in the BA9 genotype, and L226I in the ON1 genotype, emerged during the COVID-19 pandemic. Analysis of clinical characteristics indicated no significant differences between RSV-A and RSV-B groups. This study provides a theoretical basis for clinical prevention and treatment. However, further studies are needed to explore the regulatory mechanism of host immune responses to different lineages of ON1 and BA9 in the future.