Swab Samples Research Articles

Nanoplasmonic microarray–based solid-phase amplification for highly sensitive and multiplexed molecular diagnostics: application for detecting SARS-CoV-2

A novel approach is introduced using nanoplasmonic microarray–based solid-phase recombinase polymerase amplification (RPA) that offers high sensitivity and multiplexing capabilities for gene detection. Nanoplasmonic microarrays were developed through one-step immobilization of streptavidin/biotin primers and fine-tuning the amplicon size to achieve high plasmon-enhanced fluorescence (PEF) on the nanoplasmonic substrate, thereby improving sensitivity. The specificity and sensitivity of solid-phase RPA on nanoplasmonic microarrays was evaluated in detecting E, N, and RdRP genes of SARS-CoV-2. High specificity was achieved by minimizing primer-dimer formation and employing a stringent washing process and high sensitivity obtained with a limit of detection of four copies per reaction within 30 min. In clinical testing with nasopharyngeal swab samples (n = 30), the nanoplasmonic microarrays demonstrated a 100% consistency with the PCR results for detecting SARS-CoV-2, including differentiation of Omicron mutations BA.1 and BA.2. This approach overcomes the sensitivity issue of solid-phase amplification, as well as offers rapidity, high multiplexing capabilities, and simplified equipment by using isothermal reaction, making it a valuable tool for on-site molecular diagnostics.Graphical abstract

Metagenomic sequencing of CRISPRs as a new marker to aid in personal identification with low-biomass samples.

The high specificity of the human skin microbiome is expected to provide a new marker for personal identification. Metagenomic sequencing of clustered regularly interspaced short palindromic repeats (CRISPRs), which we call metaCRISPR typing, was shown to achieve personal identification accurately. However, the intra-individual variability observed in previous studies, which may be due to poor DNA yields from skin samples, has resulted in non-reproducible results. Furthermore, whether metaCRISPR typing can assist in the forensic human DNA analysis of low-biomass samples, from which the information obtained is insufficient, is unknown. In the present study, we sequenced serially diluted control streptococcal CRISPRs cloned into plasmids to determine the minimum copy number required to obtain reproducible results from metaCRISPR typing. We found that at least 102 copies of CRISPRs are necessary to obtain reproducible results. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA typing. When the DNA extracted from the skin swabs was diluted, no information was obtained from six out of eight samples by human DNA typing. On the other hand, beta diversity indices of spacer sequences compared with reference samples were below 0.8 for three out of six samples, for which no information was obtained from human DNA analysis, indicating that the spacers observed in these samples were similar to those in the references. These results indicate that metaCRISPR typing may contribute to the identification of individuals from whom the samples were obtained, even in cases where human DNA yields are insufficient to perform human DNA analysis.IMPORTANCEPrevious studies have developed new personal identification methods utilizing personal differences in the skin microbiome. However, intra-individual diversity of skin microbiome may preclude the application of microbiome-based personal identification. Moreover, no study has compared microbiome-based personal identification and practical human DNA analysis. Here, we revealed that the results of metaCRISPR typing, a previously developed microbiome-based personal identification method, are stable if the copy number of the marker gene is sufficient. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA analysis. Our results indicate that metaCRISPR typing may provide additional information for personal identification using low-biomass samples that cannot be used for conventional human DNA analysis.

Prevalence, Cross Contamination, Virulence, and Multidrug Resistance Profiles of Staphylococcus aureus Isolates from Four Middle-Scale Dairy Farms in Bareilly, Northern India.

Milk, a nutritious global important food commodity, serves as an excellent medium for microbial growth as well. The foodborne pathogen Staphylococcus aureus is a commensal member of human microflora that enters the food chain through poor hygienic practices and cross contamination and causes various clinical manifestations in humans. During this study, raw milk and swab samples (milker's hand, udder, towel, milking bucket, and farm floor) were collected from four middle-scale buffalo dairy farms. The results revealed S. aureus presence in 11.6% (n = 56/448) bucket raw milk samples and 2.6% (n = 12/448) udder raw milk samples. Contrarily, S. aureus prevalence was significantly higher in farm floors (100%, n = 84/84), towel (35.7%, n = 10/28), milking bucket (11.6%, n = 56/448), milker's hand (10.7%, n = 3/28), and udder swab samples (4.0%, n = 18/448). The chi-square test yielded p values of 0.000, 0.005, and 0.0011 for udder raw milk, udder swab, and milker's hand swab, respectively. The p values of the milking bucket (p = 0.048) and farm floors (p = 0.0183) confirmed their possible role in S. aureus cross contamination. Gene amplifications of nuclease and enterotoxin A indicate potential virulence of S. aureus isolates in collected samples. Antibiotic susceptibility testing revealed multidrug resistance in 44% (n = 239) of S. aureus isolates with the highest resistance of 61% against penicillin. Resistance against ampicillin, streptomycin, and lincomycin was observed. Fewer S. aureus isolates were resistant to kanamycin and erythromycin, whereas the lowest number of resistant isolates was observed against chloramphenicol. A high prevalence of S. aureus in the farm environment and milking equipment suggested the cross contamination of potentially enterotoxin-producing and multidrug-resistant S. aureus to raw milk. Therefore, good hygiene practices should be enforced to avoid foodborne and zoonotic infections.

Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia

Bats are known reservoirs for a wide range of pathogenic microorganisms, including viruses, bacteria, fungi, helminths, and protozoa, which can be transmitted and infect other zoonotic organisms. Various studies have utilised next-generation sequencing (NGS) to describe the pathogens associated with bats. Although most have characterised microbial communities in specific body fluids, few have analysed the composition and diversity of these microbial communities across different body fluids at the individual level. In this study, we employed two next-generation sequencing techniques: amplicon-based sequencing of the V4 hypervariable region of the 16S- and 18S-rRNA genes and viral metagenomics, to describe the prokaryotic, eukaryotic, and viral communities present in blood, faeces, and oral swab samples collected from two genera of bats (Carollia and Phyllostomus) in the department of Casanare, eastern Colombia. A total of 60 samples corresponding to the three bodily fluids were processed and analysed. The results indicated that the microbial communities across the body fluids were mainly composed of bacteria, fungi, protozoa, and various DNA and RNA viruses, showing a variability of microbial genera and species. The abundances, diversity metrics, and correlations of these microorganisms displayed patterns associated with bat genus and body fluids, suggesting that the ecological characteristics of these microbial communities may be influenced by the ecological and physiological traits of the bats. Additionally, we found similar community compositions of bacteria, some fungal genera, and viruses in the three body fluids, indicating a possible circulation of these microbes within the same bat. This could be due to microbial movement from the gut microbiota to other physiological systems or transmission via blood-feeding vectors. Furthermore, our results revealed the presence of various microbes of public health concern, including Bartonella spp., Mannheimia haemolytica, Rhodotorula spp., Piroplasmida spp., Toxoplasma gondii, Alphacoronavirus spp., and Bat circovirus. The abundance of these pathogenic microbial species across the three bodily fluids suggests potential transmission routes from bats to other organisms, which may contribute to the emergence of zoonotic disease outbreaks. These findings highlight the variability of microorganisms present within the same bat and the different pathogen-host interactions that may regulate the presence and transmission of these zoonotic microbes. Further research is required to elucidate the genomic features, ecological interactions, and biological activities of these microbial communities in bats.

Occurrence, Multidrug Resistance, and Multilocus Sequence Typing of Extended-Spectrum Beta-Lactamase/AmpC-Producing Escherichia coli from Farmed Eggs.

The present study evaluated the occurrence, antibiogram profile, and sequence types (STs) of multidrug resistant (MDR) Escherichia coli from freshly laid eggs (n = 480), feed (n = 24), water (n = 24), poultry droppings (n = 24), and hand swab samples (n = 10) collected from 24 deep litter (DL) and caged poultry layer farms (12 per category) across Punjab, India. The overall E. coli contamination rate in DL and cage farms was 32% (95% confidence intervals [CI], 26.6-37.8%) and 16.7% (95% CI, 12.6-21.6%), respectively. The logistic regression analysis revealed that the DL system had higher odds of occurrence (odds ratio [OR]) of extended-spectrum beta-lactamase (ESBL) (2.195, 95% CI, 1.065, 4.522) and ESBL/AmpC coproducers (2.69, 95% CI, 1.122, 6.45) compared to the cage system. Additionally, isolates from the DL were 4.065 (95% CI, 1.477, 11.188) times more tetracycline resistant compared to the latter; however, resistance to amoxyclavulanate (OR, 0.437; 95% CI, 0.209, 0.912), and ampicillin (OR, 0.343; 95% CI, 0.163, 0.720) was lesser in DL system. Notably, around 97.7% and 87.2% of the isolates from the DL and cage system were MDR, with the DL system having 6.439 (95% CI, 1.246, 33.283) times more chances of harboring MDR E. coli. Additionally, among the resistance genes, the DL system demonstrated significantly high presence of blaAmpC (56%), qnrA/B/S (42.3%), and tetA/B (30.6%). Furthermore, multilocus sequence typing of 11 MDR isolates (n = 5, DL, and 6, cage) revealed the presence of 10 STs, of which ST10, ST155, and ST156 were found to be of public health importance. Therefore, the present study highlights the burden of MDR, ESBL, and AmpC-producing E. coli on poultry eggs and farm environment, which could be carried over to human handlers and consumers upon direct contact during handling and processing.

Prevalence, Molecular Characterization and Antibiogram Pattern of Livestock Associated Methicillin Resistant Staphylococcus aureus in Livebird Markets Gusau, Zamfara State, Nigeria

Study’s Excerpt The prevalence and antibiotic resistance patterns of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in poultry markets within the Gusau metropolis was examined. Phenotypic and molecular (PCR) detection methods were combined to identify the LA-MRSA isolates. Multidrug resistance (MDR) phenotypes were observed in 72.2% of the isolates. Full Abstract Poultry farms have been identified as hotspots for the emergence and spread of antibiotic-resistant bacteria, especially livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA). In this study, a total of 320 swab samples were randomly collected from fecal litter, Hands of poultry handlers, Soil, Feed, and Water from five different poultry markets in the Gusau metropolis. The LA-MRSA isolates involved were detected culturally and identified phenotypically by the use of PCR assay. The pooled prevalence of the LAMRSA isolates in the samples was 22(100%). The chi-square test was used to compare the prevalence of LAMRSA among the samples collected at 95% confidence interval and 0.05 level of significance. There is no statistically significant difference in the prevalence of LAMRSA among the samples. Findings from the antibiogram study conducted revealed that all the 22(100%) LAMRSA isolates were resistant to Penicillin antibiotics, with resistance to other antibiotics following the order: Nitrofurantoin, 17(77.3%), Sulphamethoxazole, 13(59.1%), Chloramphenicol 6(27.28%), Ciprofloxacin 5(22.7%) and Erythromycin 4(18.2%). A total of 14 multidrug resistant phenotypes were identified from the 22 LAMRSA isolates with 16(72.2%) demonstrating cross resistant to ≥3 of the six different classes of antibiotics used. The MDR rate in soil samples was the highest (27.3%) (p<0.05) compared to other MDR isolates from the other sources. Of all the isolates, 22(100%) demonstrated a MAR Index ranging from 0.2-0.8. These are disturbing trends with significant risks to humans and animals. Suggesting a potential for inoculation of self and transmission to others, particularly through those who sell and make daily contact with live birds. Therefore, the government should awaken the consciousness of poultry farmers/handlers by way of sensitization on the need to improve water quality and practice routine personal and environmental hygiene as it relates to poultry management, with a focus on antibiotic stewardship within the one health framework so as to lower endemicity and equally break the chain of transmission of LA- MRSA across the farm to fork continuum.

Prevalence of Leishmania infantum in felines from Heraklion region, Crete

Greece is recognized as an endemic region for various species of Leishmania, a genus of parasitic protozoa transmitted through the bites of infected female sandflies. This indicates that the country serves as a natural reservoir for this parasite, infecting both humans and animals. This study focuses on Heraklion, Crete, one of Greece's largest cities, where there is currently no available data on the prevalence of leishmaniosis in the feline population. While cases of canine leishmaniosis have been documented in various parts of the island, there have been no reported cases of feline leishmaniosis in Heraklion. This study primarily investigates the prevalence of leishmaniosis in cats as a means of detection in this species in Heraklion. To accomplish this, between 2018 – 2023, EDTA-blood samples and conjunctival swab samples were collected from a total of 253 feral cats. The cats were divided into two groups, depending on the location they lived (i.e., areas out of the city center or close to/in the city center): the initial group consisted of 112 cats, while the latter group comprised 141 cats. Both groups encompassed cats of various ages, habitats, and both genders. The samples were stored at -18 °C and analyzed with TaqMan real-time quantitative PCR for the presence of Leishmania infantum DNA.Out of the cat population included in the study, 15 were tested positive for FeL (5.93%). Despite the lower prevalence of leishmaniosis compared to dogs, it should be taken into account, especially for differential diagnosis. In conclusion, to the best of our knowledge, this is the first study that confirms the presence of FeL in felines in Heraklion.

Increased incidence of methicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus epidermidis in the skin and nasal carriage among healthcare workers and inanimate hospital surfaces after the COVID-19 pandemic

Background and Objectives: Healthcare workers in hospitals are exposed to infectious diseases that occur in the hospital making them a source of infection for the patients. It is interfaced as cross-contamination agents for MRSA and MR-CoNS, and preventive measures need to be adapted accordingly. The study aimed to assess Methicillin-Resistant Staphylococcus (MRS) on the skin and nasal cavities of healthcare workers (HCWs) and identifying isolates to the species level. Materials and Methods: Swab samples were cultured on mannitol salt agar (MSA) to obtain MRS and determine their ability to produce coagulase. Their susceptibility to antibiotics were determined by agar screening and disk diffusion methods and further identification was done at the species level. Results: The highest percentage of methicillin resistant coagulase positive Staphylococci (MRCoPS) was reported among skins of male HCWs, (71.4%) were identified as MRSA. The highest levels of methicillin resistant coagulase negative Staph-ylococci (MRCoNS) were mainly detected in both nasal cavities, (75%) were identified as MRSE. MRSA was reported from doctors (p-value 0.033), whereas the highest incidence of MRSE was obtained from the nurses (p-value 0.048). Conclusion: This study highlighted that incidence of MRSA was mainly detected in doctors and MRCoNS in both nasal cav- ities. The highest percentage of MRCoNS was recovered from the patients’ room followed by the reception table. Moreover, vancomycin is suggested to be highly effective in managing and controlling S. aureus, MRSA- and MRSE related infections.

Single nucleotide polymorphism discrimination and genotyping based on cascade strand displacement reaction mediated label-free Cas12a system

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy.

Detection of viral infectious agents causing calf diarrhea on dairy farms in Serbia

Neonatal calf diarrhea (NCD) is a multifactorial disease commonly occurring in unweaned calves, and one of the major causes of calflosses worldwide. In this study, we investigated the role of viral agents in neonatal calf diarrhoea from dairy farms with reported outbreaks in the winter of 2021-2022.For this study, we tested rectal swab samples from 20 commercial dairy farms with 100-1000 cows with reported acute diarrhoea in neonatal calves.From each farm, five to ten calves, up to 2 months of age, in total 154, with acute diarrhea were sampled by rectal swabbing. Out of 20 tested dairy farms, at 4 farms none of the testedviruses was detected whereas at least one of the tested viruses was detected at 16 farms (80%). As the most prevalent, mono BRV infection was detected at 10 farms (62.5%), followed by a single BCoV infection at 3 farms (18.75%). Coinfection of BVDV and BRV was demonstrated at 2 farms (12.5%), and simultaneous infection with BVDV, BRV and BCoV at one farm (6.25%). G typing of BRV revealed that 60% of the BRVs were G10, 33.3% were G6, and 16.7% were G8. P typing indicated that 66.7% of the BRVs were P[11] and that 33.3% were P[5]. This study revealed viral causative agents being the most often found in diarrheic neonatal calves in Serbia, thuscontributing to the development of more effective strategies for disease prevention and control.

Who threw that stone? A study on DNA transfer

Contact or touch DNA traces from stones account for around 5 % of all crime scene-related swab samples analysed in our department. These traces are often used to identify perpetrators in cases such as burglary, when a stone is used as a tool to break a window or in cases of property damage during riots. Provided that a DNA profile can be obtained in such a case, questions may arise in court regarding the possibilities of DNA transfer onto the stone. Was the subject's DNA indeed transferred onto the stone while it was being used for the crime, or was it already present as background DNA? Alternatively, could it have been transferred by other means, such as by handing over the stone to someone else who then threw it, or by touching it during an attempt to prevent someone else from throwing it? This study focused on two scenarios: experiments involving different participants throwing various stones and a handover scenario where one person touched the stone and another person threw it. We observed that the amount of DNA transferred/detected on the stone is mainly dependent on the individual handling it rather than on the properties of the stone itself or on the order in which the stones are thrown. In the handover scenario, the person who first touched the stone was found to be the main contributor to the trace as often as the person who eventually threw the stone. Our findings therefore confirm that no conclusions can be drawn about the way of interaction with the stones based solely on the obtained DNA profiles.

Microbial Sampling and Analysis Taken from Fingerprint System Devices Located at Al-Yarmouk Teaching Hospital in Baghdad - Iraq

Biometric devices (BDs) are nowadays common in use for a variety of purposes. This system involves physical contact between the skin and surface of the device, which is likely to be contaminated by microorganisms of multiple users. However, it can be a source of multiple microbiological contaminations. This study was done at AL-Yarmouk Teaching Hospital-Baghdad- Iraq, during the period started from 15th November 2023 till 15th March 2024. It was designed to find out the most common bacteria and fungi that might contaminate the fingerprint devices and performing antimicrobial susceptibility tests. Forty-six sterile swab samples taken from Twenty-three fingerprint system devices distributed in twelve different hospital departments. From each device we took two swabs, one before sterilization other after, and tested sensitivity patterns against 16 antimicrobials. Four Gram positive bacteria scored on fingerprint devices, in Medicine, Surgery, Orthopedics and Obstetrics & Gynecology departments represented by Coagulase-negative Staphylococci (CoNS). Response to sensitivity test for antibiotics, especially Moxifloxacin, Linezolid, Teicoplanin, Vancomycin, Tigecycline and Rifampicin, 100%, as well other antibiotics but in varying degrees, and not any type of fungi were isolated. In conclusion, Gram positive bacteria population was the most common organisms, and highly response in varying degrees to the most Sixteen antibiotics used in sensitivity test, however, should proper maintenance and educational practices must be applied to minimize contamination.

Prevalence of Campylobacter and antibiotic susceptibility in chickens at slaughterhouses and retail markets in the Mekong Delta

This study aims to determine the prevalence of Campylobacter and its antibiotic susceptibility at slaughterhouses and live poultry markets in the Mekong Delta, Vietnam. A total of 177 samples, including 112 fecal samples, 28 cecal samples, and 37 rectal swab samples were collected. Thirty-nine samples tested positive for Campylobacter spp. by culture method, accounting for 22%. Isolation rates of Campylobacter in rectal swab samples were highest at 29.7% (11/37), followed by cecal samples at 25% (7/28) and fecal dropping samples at 18.8% (21/112). Species identification was performed by multiplex PCR and the result showed that the prevalence of Campylobacter coli (12.4%) was higher than Campylobacter jejuni (3.4%). Antibiotic susceptibility test showed that all isolated Campylobacter were susceptible to imipenem and meropenem (100%). Most Campylobacter were resistant to tetracycline (96.4%), ciprofloxacin (92.9%), nalidixic acid (89.3%), and ampicillin (67.9%). This study highlights the high contamination of Campylobacter and its antibiotic resistance in poultry in the Mekong Delta, Vietnam.

Comparing the frequency, antifungal susceptibility, and enzymatic profiles of the oral fungal composition in patients with and without Alzheimer's disease admitted to a neurology clinic.

Studies have shown that changes in the frequency of oral microorganisms may play a key role in the development of Alzheimer's disease (AD). However, no research has been conducted on the oral fungal composition in AD-patients. The present study aimed to investigate the changes in the frequency of oral fungal composition, the antifungal susceptibility, and the enzymatic profiles of oral fungal composition in patients suffering from AD compared to non-AD individuals. In the present analytical cross-sectional study during 12 months, 76 hospitalized patients with AD were matched with 76 individuals without AD. A sterile serum physiology-moistened cotton-tipped swab was used to sample the mouth area. All swabs were cultured on Sabouraud Chloramphenicol Agar. Fungal identified were confirmed through the PCR-sequencing techniques. Enzyme activity index (EAI) for important pathogenic factors including proteinase, esterase and hemolysin was measured using relevant protocols. The susceptibility to 8 antifungal agents (nystatin, voriconazole, itraconazole, fluconazole, posaconazole, amphotericin B, 5-fluorocytosine, and caspofungin) against fungal strains obtained from AD-patients was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, document M38-A2 for filamentous fungi, and document M27-A4 for yeasts. The results showed that compared to the non-AD individuals, the prevalence of oral fungal composition in AD group was 1.6 times higher. Candida albicans was the most common fungal species isolated from oral swab samples of AD group (n=53, 80%) and non-AD group (n=28, 40%), and the diversity of the oral fungal composition in AD-patients were lower than non-AD individuals. Among the 3 investigated virulence factors, a statistically significant difference was shown in terms of hemolysin activity level between the two studied groups (p<0.05) and the activity level of esterase and proteinase enzymes did not show a significant difference in the two studied groups (p>0.05). The results showed that almost all of the tested isolates were susceptible to nystatin, the most widely prescribed antifungal to treat superficial infections, and only 1.69 % (2/118) of the Candida isolates were resistant to this antifungal drug. Understanding the changes in the frequency of oral fungal composition the antifungal susceptibility, and the enzymatic profiles of oral fungal composition in patients suffering from AD compared to non-AD individuals makes it possible to better understand the etiology of this disease.

Pertussis Outbreak During 2023 in Gipuzkoa, North Spain.

Pertussis has re-emerged in many countries despite the wide use of vaccines for over 60 years. During 2023, we observed an increase in the incidence of pertussis in Gipuzkoa, north of Spain (with a population of 657,140 inhabitants), mainly affecting children between 11 and 15 years of age. This study included all confirmed cases diagnosed by PCR in nasopharyngeal swab samples. The genome of seven isolates collected in 2023 was sequenced. Between 2018 and 2023, 884 cases of whooping cough were diagnosed. Pertussis incidence (in cases per 100,000 inhabitants) decreased from 36.7 in 2018 to no cases in 2021, increasing again to 56.8 in 2023. In 2023, the age group of 11-15 years old had the highest incidence rate of 409.3. Only 2 of the 56 children < 6 years old required hospitalization, and there were no deaths. The seven isolates collected in 2023 showed the same BPagST-4 (ptxA1/ptxP3/prn2/fim2-1/fim3-1 allelic combination), with all of them expressing the pertactin antigen. Immunity waning after the last dose of vaccination at 6 years old, together with the lack of circulation of Bordetella pertussis during the COVID-19 pandemic, were probably the main reasons for the high increase in the incidence of pertussis in Gipuzkoa in 2023.

Empirical assessment of the enrichment-based metagenomic methods in identifying diverse respiratory pathogens.

Probe-based nucleic acid enrichment represents an effective route to enhance the detection capacity of next-generation sequencing (NGS) in a set of clinically diverse and relevant microbial species. In this study, we assessed the effect of the enrichment-based sequencing on identifying respiratory infections using tiling RNA probes targeting 76 respiratory pathogens and sequenced using both Illumina and Oxford Nanopore platforms. Forty respiratory swab samples pre-tested for a panel of respiratory pathogens by qPCR were used to benchmark the sequencing data. We observed a general improvement in sensitivity after enrichment. The overall detection rate increased from 73 to 85% after probe capture detected by Illumina. Moreover, enrichment with probe sets boosted the frequency of unique pathogen reads by 34.6 and 37.8-fold for Illumina DNA and cDNA sequencing, respectively. This also resulted in significant improvements on genome coverage especially in viruses. Despite these advantages, we found that library pooling may cause reads mis-assignment, probably due to crosstalk issues arise from post-capture PCR and from pooled sequencing, thus increasing the risk of bleed-through signal. Taken together, an overall improvement in the breadth and depth of pathogen coverage is achieved using enrichment-based sequencing method. For future applications, automated library processing and pooling-free sequencing could enhance the precision and timeliness of probe enrichment-based clinical metagenomics.

Sputum and tongue swab molecular testing for the in-home diagnosis of tuberculosis in unselected household contacts: a cost and cost-effectiveness analysis.

Delayed and missed diagnosis are a persistent barrier to tuberculosis control, partly driven by limitations associated with sputum collection and an unmet need for decentralized testing. Household contact investigation with point-of-care testing of non-invasive specimens like tongue swabs are hitherto undescribed and may be a cost-effective solution to enable community-based active case finding. In-home, molecular point-of-care testing was conducted using sputum and tongue specimens collected from all household contacts of confirmed tuberculosis cases. A health economic assessment was executed to estimate and compare the cost and cost-effectiveness of different in-home, point-of-care testing strategies. Incremental cost effectiveness ratios of strategies utilizing different combination testing algorithms using sputum and/or tongue swab specimens were compared. The total implementation cost of delivering the standard of care for a 2-year period was $84 962. Strategies integrating in-home point-of-care testing ranged between $87 844 - $93 969. The cost-per-test for in-home, POC testing of sputum was the highest at $20·08 per test. Two strategies, Point-of-Care Sputum Testing and Point-of-Care Combined Sputum and Individual Tongue Swab Testing were the most cost-effective with ICERs of $543·74 and $547·29 respectively, both below a $2,760 willingness-to-pay threshold. An in-home, point-of-care molecular testing strategy utilizing combination testing of tongue swabs and sputum specimens would incur an additional 10.6% program cost, compared to SOC, over a 2-year period. The increased sample yield from tongue swabs combined with immediate result notification following, in-home POC testing would increase the number of new TB cases detected and linked to care by more than 800%. Evidence before this study: We searched PubMed for original research published between January 1, 1950 and June 30, 2024 that evaluated the cost-effectiveness of in-home POC molecular testing, as part of HCI strategies for tuberculosis. PubMed search terms used included ["household contact investigation" OR "household contact tracing"] AND "tuberculosis" AND "cost-effectiveness". The search revealed 8 studies, of which one was removed as HCIs were leveraged for the provision of short course preventative therapy and not tuberculosis testing. None of the studies were conducted in South Africa. All seven remaining studies relied on a hub-and-spoke model of sputum collection and transportation with sputum tested at a centralized laboratory facility. Although active case finding strategies like HCIs are endorsed by the WHO to improve early case detection and treatment initiation, limited research has been done to assess its cost-effectiveness in low- and middle-income countries.Added value of this study: To our knowledge, this is the first example of in-home molecular point-of-care (POC) testing as part of HCI. The use of primary data to estimate and compare the incremental cost effectiveness of different combination, in-home testing strategies utilizing alternative sample types equips policy makers with a selection of strategy options to choose from. The tradeoff between sample types with high collection yield and those with increased accuracy becomes evident in the economic analysis, highlighting the need to consider both yield and accuracy in effective clinical decision making and use-case development. The success of in-home, POC tongue swab testing of all contacts, irrespective of symptom presentation shows great promise for universal testing programs.Implications of all available evidence: Results from our economic modeling provide evidence in support for the integration of in-home, POC tuberculosis (TB) testing during HCI. The use of less invasive tongue swab samples to increase sample yield in the absence of sputum expectoration highlights the value of combination testing strategies. Immediate result notification resulting from rapid, in-home POC testing shows great promise for increasing early case detection and improving treatment uptake. In-home, POC testing strategies, when incorporated into HCI could curb ongoing community transmission and reduce the overall burden of TB. Considerations for adopting novel POC testing strategies in future active case finding programs like HCI should strongly be considered. We evaluated the cost-effectiveness of in-home, point-of-care TB testing of household contacts. The findings indicate that combined testing strategies using tongue swab and sputum specimens could significantly increase TB case detection, with modest additional program costs.

The effectiveness of liver function tests in the prediction of mortality in patients with COVID-19

Objective: To evaluate the impact of liver function test abnormalities on mortality in critically ill coronavirus disease-2019 patients. Method: The single-centre, retrospective study was conducted at the Department of Anaesthesiology and Reanimation, Mersin City Training and Research Hospital, Turkiye, and comprised data from March 2020 to January 2022 of patients with positive real-time reverse transcription polymerase chain reaction test on nasopharyngeal swab sample for coronavirus disease-2019. Comorbidities, demographic data, admission to and discharge from the intensive care unit, Acute Physiology and Chronic Health Evaluation II scores, complete blood count on the first and second day of hospitalisation, coagulation parameters, biochemical analysis, 7-day, 28-day and total mortality of the patients in the intensive care unit were recorded. The patients were then divided into groups, with Group 1 having patients with normal liver enzymes, Group 2 having patients with abnormal liver enzymes, and Group 3 having patients with liver damage. Data was analysed using SPSS 20. Results: Of the 940 subjects, 587(62.4) were males and 353(37.6) were females. The overall mean age of the patients was 67.11±16.65 years. There were 395(42%) patients in Group 1, 534(56.8%) in Group 2 and 11(1.2%) in Group 3. Gamma-glutamyl transferase-1s, aspartate aminotransferase-2nd and albumin-2nd were significant predictors of mortality (p&lt;0.05). Conclusion: High gamma-glutamyl transferase, aspartate aminotransferase and low albumin levels in critically ill coronavirus disease-2019 patients could be considered indicators of high mortality rate. Key Words: Coronavirus, Critical illness, Liver function test, Pandemic.

From Mother to Child: Epigenetic Signatures of Hyperglycemia and Obesity during Pregnancy.

In utero exposure to maternal hyperglycemia and obesity can trigger detrimental effects in the newborn through epigenetic programming. We aimed to assess the DNA methylation levels in the promoters of MC4R and LPL genes from maternal blood, placenta, and buccal swab samples collected in children born to mothers with and without obesity and Gestational Diabetes Mellitus (GDM). A total of 101 Caucasian mother-infant pairs were included in this study. Sociodemographic characteristics, clinical parameters, physical activity, and adherence to the Mediterranean diet were evaluated in the third trimester of pregnancy. Clinical parameters of the newborns were recorded at birth. A negative relationship between MC4R DNA methylation on the fetal side of the GDM placenta and birth weight (r = -0.630, p = 0.011) of newborns was found. MC4R DNA methylation level was lower in newborns of GDM women (CpG1: 2.8% ± 3.0%, CpG2: 3.8% ± 3.3%) as compared to those of mothers without GDM (CpG1: 6.9% ± 6.2%, CpG2: 6.8% ± 5.6%; p < 0.001 and p = 0.0033, respectively), and it was negatively correlated with weight (r = -0.229; p = 0.035), head circumference (r = -0.236; p = 0.030), and length (r = -0.240; p = 0.027) at birth. LPL DNA methylation was higher on the fetal side of the placenta in obese patients as compared to normal-weight patients (66.0% ± 14.4% vs. 55.7% ± 15.2%, p = 0.037), and it was associated with maternal total cholesterol (r = 0.770, p = 0.015) and LDL-c (r = 0.783, p = 0.012). These results support the role of maternal MC4R and LPL methylation in fetal programming and in the future metabolic health of children.

Clinical Validation and Comparison of Bacterial Vaginosis Detection

Abstract Introduction/Objective Bacterial vaginosis (BV) is considered a top reason for women’s gynecological visits, causing symptoms such as vaginal discharge, foul odor, itching, and burning associated with the alteration of the normal vaginal microbiome. It is often presented as a decrease in Lactobacillus spp. and an increase in anaerobic organisms such as Gardnerella vaginalis and Atopobium vaginae. In response to the demand for more automated and less subjective testing, molecular methods have recently been developed to aid in BV testing. The objectives of this study are to validate and compare the molecular Aptima® Bacterial Vaginosis Assay by Hologic® to the established BV Screen via a Gram stained vaginal smear graded through Nugent scoring at RUSH University Medical Center in Chicago, IL and perform a cost analysis between the testing methods. Methods/Case Report Samples of known BV value were tested on the Panther® instrument to validate the Aptima® Bacterial Vaginosis Assay for accuracy and precision. Clinical samples of vaginal swabs were used to make a Gram stained BV screen for Nugent scoring, then afterwards were placed into Hologic Multitest Aptima® probes for molecular testing on the Panther® instrument. Nugent scoring was performed blind by the molecular test. Molecular results of negative, positive, and invalid were then compared to the Nugent scores: scores of 0-3 for negative/normal vaginal flora, 7-10 for positive BV, and 4-6 for intermediate/altered scores were screened for discrepancy. Results (if a Case Study enter NA) The Aptima® BV Assay tested 52 known samples with 97% accuracy and 100% precision. A total of 80 clinical samples of vaginal swabs used for Gram stained BV screens were run on the Aptima® BV Assay; 61 provided conclusive data for comparative analysis. When compared to the original Nugent scores, the positive percent agreement (PPA) was 77.0% and negative percent agreement (NPA) was 92.9%. Conclusion The Aptima® BV Assay proved to be both accurate and precise in its validation testing. Clinically, it was able to screen for BV in vaginal samples with a good percent agreement to Gram stained BV screens. Further testing of additional clinical samples are to be done to continue analysis. Although the supplies needed to perform a Gram stain cost less than molecular testing, the reimbursement payment for molecular testing is much greater and overall reduces employee time costs for performing the test, making it the more cost effective choice for the laboratory.