Sensitive Polymerase Chain Reaction Research Articles

Application of Nanomaterials to Enhance Polymerase Chain Reaction.

Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an indispensable technique in molecular biology. However, it suffers from low efficiency and specificity problems, false positive results, and so on. Although many conditions can be optimized to increase PCR yield, such as the magnesium ion concentration, the DNA polymerases, the number of cycles, and so on, they are not all-purpose and the optimization can be case dependent. Nano-sized materials offer a possible solution to improve both the quality and productivity of PCR. In the last two decades, nanoparticles (NPs) have attracted significant attention and gradually penetrated the field of life sciences because of their unique chemical and physical properties, such as their large surface area and small size effect, which have greatly promoted developments in life science and technology. Additionally, PCR technology assisted by NPs (NanoPCR) such as gold NPs (Au NPs), quantum dots (QDs), and carbon nanotubes (CNTs), etc., have been developed to significantly improve the specificity, efficiency, and sensitivity of PCR and to accelerate the PCR reaction process. This review discusses the roles of different types of NPs used to enhance PCR and summarizes their possible mechanisms.

Drivers of Deep Molecular Response and Outcomes in Patients with Core Binding Factor Acute Myeloid Leukemia

Introduction: Core-binding factor acute myeloid leukemia (CBF-AML), including t (8;21) and Inv (16), constitutes 15% of all AML cases and generally carries a favorable prognosis. However, relapse rate is ≈ 30% and predictors are not well understood. Although CBF-AML responds to induction chemotherapy, post-remission treatment can be further optimized, particularly with current next generation sequencing (NGS) and measurable residual disease (MRD) detection techniques. Impact of baseline somatic mutations on outcomes remains unclear, as does role of allogeneic stem cell transplant (allo-SCT). In this study, we investigated the potential drivers of treatment response and overall outcomes in CBF-AML. Methods: In this single center retrospective study, clinical, demographic, and molecular data were collected from adult pts with CBF-AML who were treated at Moffitt Cancer Center. We included pts with MRD by polymerase chain reaction (PCR) for CBF abnormality (CBF-MRD) results available from time of induction and consolidation. All statistical analyses were performed in SPSS. Results: Our study included 68 patients (pts), including 26 (38%) t (8;21) AML. The median age at diagnosis was 48 years, and 54% pts were male. Pts with inv (16) AML had significantly more leukocytosis than t (8;21) (10x109/L vs. 23x109/L; p=0.001). Only 5% of pts (n=3) in our cohort had extramedullary disease on presentation. Common mutations identified on baseline NGS (n=60) were KIT (35%), NRAS (27%), KRAS (15%), FLT3-ITD and FLT3-TKD (12% each; 2 pts had both types). Mutation in KIT was more common in t (8;21) AML (50% vs. 25%; p=0.04), whereas inv (16) cases were enriched in NRAS mutations (36% vs. 13%; p=0.04). Overall, 80% of CBF-AML pts had at least one kinase mutation (KIT/RAS/FLT3). Majority of pts were treated with "7+3" (44%) and "7+3+ gemtuzumab ozagamicin (GO)" (40%) as induction regimens (Table 1). Daunorubicin was the predominant anthracycline, used in 80% of pts treated with "7+3" and 48% received dose of 90 mg/m2. Overall, 31 (46%) pts received GO as part of induction treatment, with equal distribution of "day 1 only" and "day 1,4,7" schedules at 3mg/m2. Median number of consolidation cycles was 3, and GO was incorporated in consolidation (commonly with high dose cytarabine) for 29 (43%) pts. After induction, 16% pts experienced CBF-MRD negativity which increased to 73% following consolidation, with no significant difference between t (8;21) and inv (16) AML. Use of GO during induction or consolidation was associated with significantly higher CBF-MRD negativity rate at completion of induction and consolidation (83% vs. 50%; p= 0.021). However, we did not find any significant correlation between groups based on presence of baseline kinase mutation/s and CBF-MRD negativity at end of induction or consolidation. In FLT3-positive cases, 100% of pts (5/5 evaluated;4 ITD and 1 TKD) achieved FLT3-MRD negativity by PCR (sensitivity of 5X10-5) following consolidation. Overall, 35% of pts (n=24) underwent allo-SCT in our cohort, including 15 in first remission (CR1). Kinase mutations were present at baseline in majority of these pts (75%; 87% in CR1 group). Notably, 9 pts (7 in CR1 group) in the allo-SCT cohort had positive CBF-MRD at the end of consolidation. Seven pre-transplant CBF-MRD positive cases turned MRD negative following allo-SCT. Relapse rate was low (n=10; 15%) in our CBF-AML pt population. Among pts with relapse, 6 were CBF-MRD positive post-induction and 3 had MRD even after consolidation. Use of GO in induction was associated with significantly lower relapse rate (0% vs. 28%; p= 0.001). Presence of kinase (KIT/RAS/FLT3) mutation at diagnosis was associated with shorter relapse free survival (RFS) in these pts (median RFS- 7.7 vs. 12.9 months (mo); p= 0.014; Figure 1). After a median follow up of 33.4 mo, median OS was 94.1 mo in our pt cohort. There was no significant OS difference between subgroups based on CBF-AML subtype, baseline mutations, or receipt of GO during treatment. Conclusions: CBF-AML continues to be a favorable risk disease, with a median OS >7 years in our study population. Post-treatment CBF-MRD negativity rate was 73% and was predicted by the incorporation of GO in induction or consolidation. Rate of relapse rate was low at 15%, and all occurred in pts who did not receive GO during induction or consolidation. Baseline kinase mutations (KIT/RAS/FLT3) on NGS was associated with a shorter RFS in our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3-4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6-100%) and 100% specificity (93.5-100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the β-lactams combined with novel β-lactamase inhibitors.

Gauging the skin resident Leishmania parasites through a loop mediated isothermal amplification (LAMP) assay in post-kala-azar dermal leishmaniasis

Despite the availability of highly sensitive polymerase chain reaction (PCR)-based methods, the dearth of remotely deployable diagnostic tools circumvents the early and accurate detection of individuals with post-kala-azar dermal leishmaniasis (PKDL). Here, we evaluate a design-locked loop-mediated isothermal amplification (LAMP) assay to diagnose PKDL. A total of 76 snip-skin samples collected from individuals with probable PKDL (clinical presentation and a positive rK39 rapid diagnostic test (RDT)) were assessed by microscopy, qPCR, and LAMP. An equal number of age and sex-matched healthy controls were included to determine the specificity of the LAMP assay. The LAMP assay with a Qiagen DNA extraction (Q-LAMP) showed a promising sensitivity of 72.37% (95% CI: 60.91–82.01%) for identifying the PKDL cases. LAMP assay sensitivity declined when the DNA was extracted using a boil-spin method. Q-qPCR showed 68.42% (56.75–78.61%) sensitivity, comparable to LAMP and with an excellent agreement, whereas the microscopy exhibited a weak sensitivity of 39.47% (28.44–51.35%). When microscopy and/or qPCR were considered the gold standard, Q-LAMP exhibited an elevated sensitivity of 89.7% (95% CI: 78.83–96.11%) for detection of PKDL cases and Bayesian latent class modeling substantiated the excellent sensitivity of the assay. All healthy controls were found to be negative. Notwithstanding the optimum efficiency of the LAMP assay towards the detection of PKDL cases, further optimization of the boil-spin method is warranted to permit remote use of the assay.

Monitoring of Oie-Listed Parasitic Pathogens in the Pacific Oyster (Crassostrea gigas) on the South Coast of Korea Using Species-Specific PCR

Outbreaks of oyster diseases caused by the World Organization for Animal Health (OIE)-listed protozoan pathogens have been a global concern for several decades. With the wide application of species-specific and sensitive polymerase chain reaction (PCR) techniques, it is now well known that even the Pacific oyster, insusceptible to Perkinsus marinus, Bonamia ostreae, Bonamia exitiosa could act as a carrier host. Furthermore, one of the transmission pathways of oyster pathogens includes anthropogenic activities, such as the discharge of ballast water or hull attachment during international maritime shipping. In this survey, OIE-listed pathogens, including B. ostreae, B. exitiosa, and Marteilia refringens were screened in the Pacific oysters growing in small bays off the south coast of Korea using PCR with OIE-recommended species- or genus-specific (Bonamia) primers. The survey sampled 1980 oysters from 35 sites in small bays in the south, where more than 95% of the national productions are yielded. The PCR screening indicated that none of the oysters screened in this study were positive for B. ostreae, B. exitiosa, P. marinus, and M. refringens. This study first investigated the OIE-listed protozoan pathogens in the Pacific oysters culturing in Korea, and the results indicated that the Pacific oysters raised on the south coast of Korea are free from those OIE-listed pathogens. For secure and sustainable oyster farming on the south coast, however, regular monitoring of such invasive oyster pathogens is recommended, as the international maritime trade activities are increasing in Korea.

Detection of porcine DNA in Korean processed foods by real-time PCR.

In this study, a commercial DNA extraction kit and a real-time Polymerase Chain Reaction (PCR) kit were applied to detect porcine DNA in Korean processed foods. The specificity, sensitivity, applicability and inter-laboratory reproducibility were evaluated for analytical method validation. We observed no false-positive or false-negative results in the assay, confirming its specificity. The sensitivity of the real-time PCR was expressed as the limit of detection (LOD), which was determined to be 0.005ng in 35 cycles. In the applicability test, PCR was performed on 12 types of food matrices, and there were no inhibitor effects. An interlaboratory comparison showed no statistically significant (p > 0.05) differences between the results from two laboratories. We analysed 131 samples to determine the presence of porcine DNA for halal certification: 129 samples were negative, and porcine DNA was detected in one sample each of instant noodles and dumpling. The real-time PCR applied in this study is a reliable analytical method to detect porcine DNA in food and can be used easily, quickly and routinely in food facilities.

Обнаружение особо опасных вирусных болезней рыб семейства лососёвых (Salmo salar) с применением полимеразной цепной реакции

Significant economic damage to aquaculture facilities and marine aquatic organisms is caused by viral diseases of fish, almost a quarter of the detected viruses cause diseases. Pathogens mainly fall during the transportation of infected fish from disadvantaged farms to "clean" aquatic farms, as well as from "escaped" fish from cages to the sea, where contamination of natural salmon and other aquatic organisms occurs. When delivering fish planting material to Russia from countries with different epizootological situations, constant monitoring and forecasting of possible risks is necessary. To prevent the spread of viral infections of fish, diagnostics and prevention occupy a leading place. Laboratory diagnostics of fish diseases of viral etiology is based on the isolation of the pathogen and its identification by serological methods that require a long time and research in specialized specialized laboratories of large research institutes. More effective and highly sensitive are molecular diagnostic methods, in particular, polymerase chain reaction (PCR) with reverse transcription to detect a number of particularly dangerous viral diseases of fish. Infectious necrosis of hematopoietic tissue, infectious pancreatic necrosis, viral hemorrhagic septicemia and infectious anemia of salmon were identified by highly specific and sensitive diagnostic PCR method. The improved technique of PCR with reverse transcription makes it possible to detect pathogens in salmon and other fish both with obvious clinical signs and with hidden virus carrier.

A diagnostic test of real-time PCR detection in the diagnosis of clinical bloodstream infection

Blood culture remains the standard for diagnosing bloodstream infections, but it is difficult to identify bacteria directly and timeliness. The real-time polymerase chain reaction (PCR) has the potential to fill this diagnostic gap. This study intends to explore the sensitivity and specificity of PCR in detecting bloodstream infection pathogens and to compare it with routine blood culture to explore its clinical application value. A total of 126 patients with bloodstream infections collected from various clinical departments of The First Hospital of Hebei Medical University. The patient's sample was divided into two parts. The one for multiplex PCR detection was performed using the Pathogeno Elite Multiplex PCR kit. Another blood culture was a fully automatic blood culture system from Autobio company. Among the 126 patients, a total of 17 pathogens were detected by PCR and blood culture both methods. PCR detected a total of 43 positive samples and 83 negative samples. Five samples were positive with blood culture, and 81 were negative. The negative predictive value of PCR was 0.98, with a sensitivity of 0.71 and a specificity of 0.68. A total of 38 specimens were positive for PCR but negative for blood culture, and 2 samples were positive for blood culture but negative for PCR. The top 5 pathogens with PCR detection were Epstein-Barr virus (27 cases), Human herpes virus 5 (9 cases), Klebsiella pneumoniae (5 cases), Staphylococcus (5 cases), and Stenotrophomonas maltophilia (4 cases). PCR detection can rapidly identify more pathogens and even multi-pathogen infections. Therefore, PCR testing may improve pathogen detection in patients with suspected bloodstream infections, enabling targeted treatment of patients.

Evaluation of Fine-needle Aspiration Cytology (FNAC) Sensitivity Compared to PCR for Diagnosing Tuberculosis Lymphadenitis

Background: Tuberculosis (TB) is a major healthcare burden in Sudan and other developing countries, it is considered the second most common cause of death from infectious diseases after those due to AIDS. In Sudan, TB lymphadenitis (TBLA) remains one of the major health problems. This descriptive cross-sectional study was conducted at the University of Medical Sciences and Technology (UMST) and Total Labcare Diagnostic Center (TDC). The study aims to compare the sensitivity of Fine Needle Aspiration Cytology (FNAC) smears with that of the Polymerase Chain Reaction (PCR) for the diagnosis of TBLA.
 Materials and Methods: Fifty-five dry smears were obtained using fine-needle aspiration (FNA) from an enlarged lymph node. PCR was applied to detect the target gene (IS6110). May-Grunwald-Giemsa (MGG) or Diff quick stains were used.
 Results: Two (4%) patients with TBLA were non-necrotic, while fifty-three of them (96%) were necrotic. Moreover, 17 (30%) fine-needle lymph node aspiration specimens were confirmed by PCR to be positive for Mycobacterium tuberculosis complex (MTB complex) while 38 (70%) of them were negative.
 Conclusion: There was no significant difference between the sensitivity of PCR and that of FNAC (P-value = 0.33).

57-Year-Old Man With Headache, Vomiting, and Gait Instability

57-Year-Old Man With Headache, Vomiting, and Gait Instability

Follow-up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients.

Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.

Value of digital PCR in the early diagnosis of sepsis: A systematic review and meta-analysis

BackgroundWe systematically assessed whether a digital polymerase chain reaction (PCR) could detect pathogenic microorganisms in patients with sepsis early and accurately. MethodsWe searched the Cochrane Library, MEDLINE, Embase, CNKI, CBM, and Wanfang Data databases for eligible studies to compare the detection of pathogenic microorganisms in blood samples by digital PCR with the gold standard. The Quality Assessment of Diagnostic Accuracy Studies 2 was used to evaluate bias risk, and a random-effects meta-analysis approach was used for sensitivity and specificity calculations. ResultsAmong the eight articles, there were eight identified studies with a total of 1278 subjects. The pooled sensitivity of digital PCR was 94% (95% confidence interval [CI], 85%–98%), the specificity was 87% (95% CI, 76%–94%), the positive likelihood ratio was 7.3 (95% CI, 3.8–14.2), the negative likelihood ratio was 0.07 (95% CI, 0.03–0.17), the positive predictive value was 84.7%, the negative predictive value was 89.2%, the diagnostic odds ratio was 105 (95% CI, 37–303), and the area under the receiver operating characteristic curve was 0.97 (95% CI, 0.95–1.00). Digital PCR can shorten the detection time of pathogenic microorganisms in patients with sepsis. ConclusionsDigital PCR can detect pathogenic microorganisms in patients with sepsis earlier than blood culture. Therefore, digital PCR can be used as a potential strategy for the detection of pathogenic microorganisms in patients with sepsis.

High mRNA Expression Levels of Heat Shock Protein Family B Member 2 (HSPB2) Are Associated with Breast Cancer Patients' Relapse and Poor Survival.

Small heat shock proteins (sHSPs) are ubiquitous ATP-independent chaperones that contribute to the maintenance of proteome integrity and functionality. Recent evidence suggests that sHSPs are ubiquitously expressed in numerous types of tumors and have been proposed to be implicated in oncogenesis and malignant progression. Heat shock protein family B member 2 (HSPB2) is a member of the sHSPs, which is found to be expressed, among others, in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway. In this study, we investigated the potential prognostic significance of HSPB2 mRNA expression levels in breast cancer, which represents the most frequent malignancy in females and one of the three most common cancer types worldwide. To this end, malignant breast tumors along with paired non-cancerous breast tissue specimens were used. HSPB2 expression levels were quantified in these two cohorts using a sensitive and accurate SYBR green-based quantitative real-time polymerase chain reaction (q-RT-PCR). Extensive biostatistical analyses were performed including Kaplan–Meier and Cox regression survival analyses for the assessment of the results. The significant downregulation of HSPB2 gene expression was revealed in breast tumors compared to their adjacent non-cancerous breast tissues. Notably, high HSPB2 mRNA expression predicts poor disease-free survival and overall survival of breast cancer patients. Multivariate Cox regression analysis revealed that HSPB2 mRNA overexpression is a significant predictor of poor prognosis in breast cancer, independent of other clinicopathological factors. In conclusion, high HSPB2 mRNA expression levels are associated with breast cancer patients’ relapse and poor survival.

Preserving plant samples from remote locations for detection of RNA and DNA viruses.

Viral diseases in plants have a significant impact on agricultural productivity. Effective detection is needed to facilitate accurate diagnosis and characterization of virus infections essential for crop protection and disease management. For sensitive polymerase chain reaction (PCR)-based methods, it is important to preserve the integrity of nucleic acids in plant tissue samples. This is especially critical when samples are collected from isolated areas, regions distant from a laboratory, or in developing countries that lack appropriate facilities or equipment for diagnostic analyses. RNAlater® provides effective, reliable sample storage by stabilizing both RNA and DNA in plant tissue samples. Our work indicated that total RNA or DNA extracted from virus-infected leaf samples preserved in RNAlater® was suitable for reverse transcription polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, high-throughput sequencing (HTS), and enzyme-linked immunosorbent assay (ELISA)-based diagnostic analyses. We demonstrated the effectiveness of this technology using leaf tissue samples from plants with virus symptoms grown in farmers’ fields in Bangladesh. The results revealed that RNAlater® technology was effective for detection and characterization of viruses from samples collected from remote areas and stored for extended periods. Adoption of this technology by developing countries with limited laboratory facilities could greatly increase their capacity to detect and diagnose viral infections in crop plants using modern analytical techniques.

Development of PCR-based markers for the identification and detection of Lophodermella needle cast pathogens on Pinus contorta var. latifolia and P. flexilis

Morphological similarities and fastidious development of increasingly emerging fungal needle pathogens impede accurate disease diagnosis and early detection. This study analyzed the specificity and sensitivity of polymerase chain reaction (PCR)-based markers developed for emerging needle cast pathogens Lophodermella concolor and L. montivaga co-occurring on Pinus contorta var. latifolia, and Bifusella linearis and L. arcuata on P. flexilis. To design primers, we utilized sequences of the internal transcribed spacer (ITS) region and single-copy gene (RH_2175) of the TCP-1/cpn60 chaperonin family searched through genomes of related species. In addition to the DNA of target and non-target fungal species that were used for primer assays, environmental samples with next generation sequencing data were used to evaluate primer sensitivity. Direct amplification using ITS primer pairs generated 248–260 bp amplicons and successfully differentiated the needle pathogens used in this study. Nested amplification of single-copy gene RH_2175 primer pairs which produced 409–527 bp amplicons detected Rhytismataceae species and discriminated both Lophodermella pathogens on P. contorta var. latifolia, respectively. While ITS-based primers had higher sensitivity than the 2175-based primers, both primer sets for L. concolor and L. montivaga detected their respective pathogens in asymptomatic and symptomatic needles. These molecular tools can help monitor and assess needle diseases for forest management and phytosanitary regimes.

Particular qualities of laboratory approaches in complex diagnosis of human brucellosis

Brucellosis is an infectious particularly dangerous zoonotic disease caused by bacteria of the genus Brucella, among which B. melitensis, B. abortus, and B. suis have pathogenic potential, causing a severe and often chronic course of the disease.
 Laboratory diagnostics is crucial for the detection of human cases, since the clinical symptoms of human brucellosis are variable and nonspecific. Laboratory diagnosis of brucellosis is based on three different approaches: direct bacteriological method, indirect method using serological and allergic tests and direct express method in different formats of molecular polymerase chain reaction.
 Despite the accumulated experience of using serological tests and the highly sensitive polymerase chain reaction method, the isolation of Brucella culture is considered the gold standard in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. The currently available automated systems of the bacteriological method have increased its sensitivity and shortened the detection time of Brucella species.
 The main limitations of serological tests are the lack of general interpretation criteria, low specificity due to cross-reactions with other bacteria and low sensitivity at an early stage of the disease. At the same time, in Russia, serological tests account for more than 99% of all laboratory tests and remain the main diagnostic tool. This is due to their inexpensive and convenient use at the place of medical care in endemic areas and high negative prognostic value.
 Polymerase chain reaction in various formats of rapid tests diagnoses the DNA of the pathogen in a few hours with high sensitivity and specificity. Nevertheless, a positive polymerase chain reaction result requires careful interpretation and does not necessarily indicate an active infection.
 For the convenience of using diagnostic approaches to brucellosis in practical medicine and determining the activity of the infectious process, it is necessary to improve diagnostics and develop express methods.
 The review shows both the most routine and modern laboratory methods currently available for laboratory diagnosis of brucellosis.

Temporal Dynamics of Subclinical Malaria in Different Transmission Zones of Myanmar.

ABSTRACT.Countries in the Greater Mekong Subregion have committed to eliminate Plasmodium falciparum malaria by 2025. Subclinical malaria infections that can be detected by highly sensitive polymerase chain reaction (PCR) testing in asymptomatic individuals represent a potential impediment to this goal, although the extent to which these low-density infections contribute to transmission is unclear. To understand the temporal dynamics of subclinical malaria in this setting, a cohort of 2,705 participants from three epidemiologically distinct regions of Myanmar was screened for subclinical P. falciparum and P. vivax infection using ultrasensitive PCR (usPCR). Standard rapid diagnostic tests (RDTs) for P. falciparum were also performed. Individuals who tested positive for malaria by usPCR were followed for up to 12 weeks. Regression analysis was performed to estimate whether the baseline prevalence of infection and the count of repeated positive tests were associated with demographic, behavioral, and clinical factors. At enrollment, the prevalence of subclinical malaria infection measured by usPCR was 7.7% (1.5% P. falciparum monoinfection, 0.3% mixed P. falciparum and P. vivax, and 6.0% P. vivax monoinfection), while P. falciparum prevalence measured by RDT was just 0.2%. Prevalence varied by geography and was higher among older people and in those with outdoor exposure and travel. No difference was observed in either the prevalence or count of subclinical infection by time of year, indicating that even in low-endemicity areas, a reservoir of subclinical infection persists year-round. If low-density infections are shown to represent a significant source of transmission, identification of high-risk groups and locations may aid elimination efforts.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Immune- and Stemness-Related Genes Revealed by Comprehensive Analysis and Validation for Cancer Immunity and Prognosis and Its Nomogram in Lung Adenocarcinoma.

ObjectiveLung adenocarcinoma (LUAD) is a familiar lung cancer with a very poor prognosis. This study investigated the immune- and stemness-related genes to develop model related with cancer immunity and prognosis in LUAD.MethodThe Cancer Genome Atlas (TCGA) was utilized for obtaining original transcriptome data and clinical information. Differential expression, prognostic value, and correlation with clinic parameter of mRNA stemness index (mRNAsi) were conducted in LUAD. Significant mRNAsi-related module and hub genes were screened using weighted gene coexpression network analysis (WGCNA). Meanwhile, immune-related differential genes (IRGs) were screened in LUAD. Stem cell index and immune-related differential genes (SC-IRGs) were screened and further developed to construct prognosis-related model and nomogram. Comprehensive analysis of hub genes and subgroups, involving enrichment in the subgroup [gene set enrichment analysis (GSEA)], gene mutation, genetic correlation, gene expression, immune, tumor mutation burden (TMB), and drug sensitivity, used bioinformatics and reverse transcription polymerase chain reaction (RT-PCR) for verification. ResultsThrough difference analysis, mRNAsi of LUAD group was markedly higher than that of normal group. Clinical parameters (age, gender, and T staging) were ascertained to be highly relevant to mRNAsi. MEturquoise and MEblue were found to be the most significant modules (including positive and negative correlations) related to mRNAsi via WGCNA. The functions and pathways of the two mRNAsi-related modules were mainly enriched in tumorigenesis, development, and metastasis. Combining stem cell index–related differential genes and immune-related differential genes, 30 prognosis-related SC-IRGs were screened via Cox regression analysis. Then, 16 prognosis-related SC-IRGs were screened to construct a LASSO regression model at last. In addition, the model was successfully validated by using TCGA-LUAD and GSE68465, whereas c-index and the calibration curves were utilized to demonstrate the clinical value of our nomogram. Following the validation of the model, GSEA, immune cell correlation, TMB, clinical relevance, etc., have found significant difference in high- and low-risk groups, and 16-gene expression of the SC-IRG model also was tested by RT-PCR. ADRB2, ANGPTL4, BDNF, CBLC, CX3CR1, and IL3RA were found markedly different expression between the tumor and normal group.ConclusionThe SC-IRG model and the prognostic nomogram could accurately predict LUAD survival. Our study used mRNAsi combined with immunity that may lay a foundation for the future research studies in LUAD.

Impact of Cell Free DNA Mold Polymerase Chain Reaction Panel on Healthcare-Associated Infection Investigation

Background Molds are ubiquitous in the environment but can cause severe opportunistic infections in immunocompromised patients. In 2021, the Hospital Laboratory introduced a highly sensitive cell free DNA mold polymerase chain reaction (cfDNA PCR) test. Infection Prevention and Control (IPC) saw an increase in positive mold results raising concern for potential hospital exposure. A modified mold healthcare-associated infection (HAI) investigation algorithm was created, integrating clinical correlation into test interpretation. In this retrospective study we assess the effect of the new cfDNA mold PCR panel on the rate of positive tests and describe the effect of the modified algorithm on HAI investigation. Methods We used the institutional IPC database to review all positive mold tests among oncology and stem cell transplant (SCT) patients between 2017–2021. We compared the monthly rate of positive tests meeting HAI definition and investigations conducted per year before and after the introduction of the cfDNA mold PCR panel. We evaluated the effect of the modified mold HAI algorithm on the number of HAI investigations. Results Between 2017–2021 there were 15 positive mold tests meeting HAI definition in the oncology and SCT units. 50% Aspergillus species, 14.3% Rhizopus species, 14.3% Fusarium species and 21.4% (all in 2021) mucor species. The average positivity rate was 0.29 between 2017–2020 compared to 0.58 per month in 2021 after the introduction of the cfDNA mold PCR panel (P < 0.05). There were 5 HAI investigations in the past 5 years of them 4 in 2021. Mold HAI investigation was done twice after implementation of the revised algorithm. Conclusions The new cfDNA mold PCR panel caused a significant increase in the rate of positive mold infections reported in the immunocompromised units. The modified mold HAI algorithm that integrated clinical correlation led to improved identification of HAI and better utilization of IPC resources. Molds are ubiquitous in the environment but can cause severe opportunistic infections in immunocompromised patients. In 2021, the Hospital Laboratory introduced a highly sensitive cell free DNA mold polymerase chain reaction (cfDNA PCR) test. Infection Prevention and Control (IPC) saw an increase in positive mold results raising concern for potential hospital exposure. A modified mold healthcare-associated infection (HAI) investigation algorithm was created, integrating clinical correlation into test interpretation. In this retrospective study we assess the effect of the new cfDNA mold PCR panel on the rate of positive tests and describe the effect of the modified algorithm on HAI investigation. We used the institutional IPC database to review all positive mold tests among oncology and stem cell transplant (SCT) patients between 2017–2021. We compared the monthly rate of positive tests meeting HAI definition and investigations conducted per year before and after the introduction of the cfDNA mold PCR panel. We evaluated the effect of the modified mold HAI algorithm on the number of HAI investigations. Between 2017–2021 there were 15 positive mold tests meeting HAI definition in the oncology and SCT units. 50% Aspergillus species, 14.3% Rhizopus species, 14.3% Fusarium species and 21.4% (all in 2021) mucor species. The average positivity rate was 0.29 between 2017–2020 compared to 0.58 per month in 2021 after the introduction of the cfDNA mold PCR panel (P < 0.05). There were 5 HAI investigations in the past 5 years of them 4 in 2021. Mold HAI investigation was done twice after implementation of the revised algorithm. The new cfDNA mold PCR panel caused a significant increase in the rate of positive mold infections reported in the immunocompromised units. The modified mold HAI algorithm that integrated clinical correlation led to improved identification of HAI and better utilization of IPC resources.