BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

BACKGROUND AND OBJECTIVE

Nontuberculous mycobacteria (NTM) lung disease appears like tuberculosis infection but is resistant to primary anti-tuberculosis drugs. Hence, patients whose sputum sample tests positive for acid-fast bacilli (AFB) and bacterial culture for several times should be assessed for colonization or infection with NTM in a damaged lung secondary to TB. In such cases, though drug-resistant TB may be adequately treated, treatment may need to be directed towards the NTM as well. In NTM therapy, the duration and choice of treatment agent is based upon the specific organism and disease extent. This study used one-step multiplex PCR (mPCR) assay for rapid differentiation of solid cultures in Ogawa medium as Mycobacterium tuberculosis (MTB) and/or NTM.

A total of 80 stocked isolates obtained from the Lung Center of the Philippines from January to December 2018 were screened for NTM in terms of growth in Ogawa medium, acid fastness, and MPT64 TB antigen test result. These were from sputum specimens of multidrug-resistant tuberculosis (MDR-TB) patients. DNA was extracted from cultures (n=55) grown in Ogawa medium and one-step mPCR was performed to identify NTM to the species level.

Out of 80 samples screened, a total of 55 isolates were identified as NTM. One-step mPCR identified 12.73% (7/55) as M. abscessus, 34.55% (19/55) as M. massiliense, 1.82% (1/55) as M. kansasii, and 50.91% (28/55) were identified only up to genus Mycobacteria spp. Neither M. avium complex nor M. intracellulare was identified among the samples tested.

One-step mPCR was able to identify isolates as MTB or NTM coinciding with the initial screening using MPT64 TB antigen test. Multiplex PCR has given a more specific identificati on to the species level. The use of mPCR in identifying MTB and clinically significant NTM’s is suitable for the adequate treatment of mycobacterial infection.

Human ; Bacteria ; Antimicrobial Stewardship ; Insemination, Artificial, Heterologous ; Multiplex Polymerase Chain Reaction ; Polymerase Chain Reaction

OBJECTIVES

To validate a method in detecting SARS-CoV-2 via RT-qPCR in pregnant and non-pregnant samples other than nasopharyngeal swabs and/or oropharyngeal swabs such as cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk.

We performed a validation experiment using MGI easy extraction kits and BGI PCR kits on non-conventional specimens, including cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk to detect and confirm the presence of SARS-CoV-2. In addition, we tested the validated method on 572 purposively sampled field-collected non-conventional specimens from a cohort of 109 unvaccinated pregnant and 47 unvaccinated non-pregnant women to assess which candidate non-conventional maternal- and fetal-associated specimens may contribute to maternal-fetal viral vertical transmission.

Positive detection of SARS-CoV-2 viral RNA in non-conventional specimens was demonstrated and verified. Of the 572 non-conventional samples tested, 1.8% (10/572) were positively validated by RT-qPCR for SARS-CoV-2 in the maternal-associated specimens particularly the rectal (5), placental (1), and cervical (4) swabs among six pregnant and four non-pregnant individuals. In contrast, no SARS-CoV-2 viral RNA was detected in fetal-associated specimens.

The results of the validation study may serve as an additional diagnostic screening layer to support maternal-child care. Furthermore, viral detection in these non-conventional maternal specimens may also be utilized to provide guidance in the clinical management of neonates, and pregnant women during delivery.

BACKGROUND

Broad  Panel Respiratory Multiplex PCR (Pneumonia Panel) tests a panel of bacteria and viruses associated with community acquired pneumonia (CAP) which help streamline antimicrobial therapy. Recently, pneumonia panel aids clinicians in early streamlining of antimicrobials as opposed to waiting for bacterial culture results [2].

To determine whether the use of pneumonia panel improves the overall survival rate, length of hospital stay, and number of antibiotic free days among hospitalized CAP patients.

In this RCT, adult patients admitted for CAP were randomized to perform pneumonia panel and sputum culture (pneumonia panel group) versus sputum culture only (control group). The results were relayed to the medical team and were incorporated into the medical records. Length of hospital stay, antibiotic free days in day 28, and mortality rates were the primary outcomes measured.

Eighty participants completed the study. There was no significant difference in the length of hospital stay (p-value 0.073, 95% C.I.), duration of antibiotic therapy (p-value 0.332, 95% C.I.), and mortality rates (p-value 0.570, 95% C.I.) between the 2 groups.

Routine use of pneumonia panel does not significantly reduce length of hospital stay, duration of antibiotic therapy, and mortality rates among admitted patients with moderate to severe CAP. The benefit of pneumonia panel was seen on early detection of drug resistant pathogen resulting in early antibiotic escalation and shorter duration of antibiotic therapy. Further studies are necessary to show its benefit in the high risk population.

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.
Ligustrum/genetics* ; Seeds ; Fruit ; Polymerase Chain Reaction ; Research Design

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.
Artemisia/genetics* ; Trichomes ; Polymerase Chain Reaction ; Nucleic Acid Amplification Techniques ; Plant Leaves/genetics*

Objective: To analyze the genetic characteristics of varicella-zoster virus (VZV) in people aged 20 years and under in Yichang City of Hubei Province from 2019 to 2020. Methods: Based on the Yichang Health Big Data Platform, we investigated cases 20 and under clinically diagnosed as herpes zoster in three hospitals from March 2019 to September 2020. Collecting vesicle fluid and throat swab samples of the cases and completing questionnaires to obtain basic information. Real-time fluorescent quantitative PCR was used for positive identification of the virus. PCR amplification of VZV's open reading frame (ORF) and sequencing of the products to determine the VZV genotype. Analyze mutations at some specific single nucleotide polymorphism (SNP) sites. Results: Among 46 cases of herpes zoster, the male to female ratio was 1.3∶1 (26∶20) and the age ranged from 7 to 20 years old. Fifteen cases had been vaccinated against varicella, including 13 and 2 cases of 1 and 2 doses, respectively. VZV strains were detected in 34 samples (73.91%), all belonging to Clade 2. Phylogenetic tree analysis of the nucleotide of ORF22 showed, compared with Clade 2 referenced strains, the sequence matching degree of nucleotide for all 34 samples was 99.0% to 100.0%. Conclusion: The main VZV strain causing herpes zoster in people aged 20 years and under in Yichang from 2019 to 2020 was Clade 2.
Humans ; Child ; Adolescent ; Young Adult ; Adult ; Herpesvirus 3, Human/genetics* ; Phylogeny ; Herpes Zoster/epidemiology* ; Polymorphism, Single Nucleotide ; Real-Time Polymerase Chain Reaction ; Nucleotides

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β₂ toxin (cpb₂) and atypical-cpb₂ (aty-cpb₂), analyze the epidemiological characteristics and genetic polymorphism of the cpb₂ of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb₂ of 188 Clostridium perfringens strains were examined by PCR; the cpb₂ sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb₂-library based on 110 strains carrying the cpb₂ were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb₂ (con-cpb₂) and aty-cpb₂. Results: The specificity of PCR assay for the cpb₂ and aty-cpb₂ was verified. The PCR results for cpb₂ amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb₂, 94 types A strains carried aty-cpb₂, 6 types A strains carried con-cpb₂, and 7 types F strains carried aty-cpb₂. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb₂ toxin was developed, and the previous PCR method for detecting aty-cpb₂ was improved. aty-cpb₂ is the primary gene encoding of β₂ toxin. There is a significant nucleotide sequence variance between the various cpb₂ genotypes.
Humans ; Clostridium perfringens/genetics* ; Clostridium Infections ; Bacterial Toxins/genetics* ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Genetic

Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×10² copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Humans ; SARS-CoV-2/genetics* ; COVID-19/diagnosis* ; Subgenomic RNA ; Real-Time Polymerase Chain Reaction/methods* ; RNA, Viral/genetics* ; Sensitivity and Specificity ; Nucleocapsid/chemistry* ; COVID-19 Testing