Noninvasive Specimens Research Articles

P-2146. Comparison of Diagnostic Performance between Induced Sputum or Tracheal Suction and Bronchoalveolar Lavage Fluid for Diagnosis of Pneumocystis jirovecii Pneumonia Using Quantitative PCR

Abstract Background The prevalence of Pneumocystis pneumonia (PCP), primarily affecting immunocompromised individuals, notably those without HIV infection, has been increasing. Diagnosing PCP is challenging due to the lack of a standard definition. Its diagnosis mainly relies on clinical history, chest imaging, and treatment response. Conventional diagnostic methods for PCP have shown limitations, particularly in sensitivity, leading to delayed diagnoses and higher mortality rates, especially in non-HIV patients. Molecular diagnostic techniques like Polymerase Chain Reaction (PCR) offer enhanced sensitivity, speed, and accuracy. This study aims to determine the diagnostic performance of our in-house qPCR to distinguish between PCP and non-PCP cases. Criteria used for diagnosis PCP Methods A prospective study was conducted from 2022 to 2024. Immunocompromised patients with pneumonia who underwent bronchoalveolar lavage and could provide induced sputum or tracheal suction samples were included in the study. Fungal burdens in non-invasive specimens (induced sputum and tracheal suction fluid) and in invasive bronchoalveolar lavage fluid (BALF) specimens were assessed by our in-house qPCR targeting mitochondrial 12s rRNA (mt 12s rRNA). The diagnostic performance of this method for both non-invasive and invasive specimens was measured in comparison to the diagnostic criteria set for PCP. In addition, the correlation between non-invasive and invasive specimens collected at approximately the same time was compared. Image 1: diagnostic performance of BAL qPCR for PCP (A) Scatter plot showing P. jirovecii mtLSU-rRNA DNA copies/μl among definite PCP, probable PCP and non-PCP, Dot line: cut off value of 2,613 DNA copies/µl (B) ROC curve analysis of the BALF for the diagnosis of PCP *P-value <0.05, ****P-value <0.0001 Results Of 114 enrolled patients, 23 met PCP criteria. BAL qPCR demonstrated high sensitivity (82.6%) and specificity (96.7%), while non-invasive samples showed lower sensitivity (65.2%) and good specificity (89.0%). A BAL qPCR cutoff of 2,613 DNA copies/μl achieved high diagnostic accuracy. Tracheal suction qPCR showed a stronger correlation with BAL qPCR than induced sputum. Image 2: diagnostic performance of sputum qPCR for PCP (A) Scatter plot showing P. jirovecii mtLSU-rRNA DNA copies/μl among definite PCP, probable PCP and non-PCP, Dot line: cut off value of 2,613 DNA copies/µl (B) ROC curve analysis of the non-invasive samples for PCP diagnosis., *P-value <0.05, ****P-value <0.0001 Conclusion Quantitative PCR (mtLSU-rRNA), especially from BAL fluid, enhances PCP diagnosis accuracy compared to traditional methods. Non-invasive sample qPCR remains valuable, with tracheal suction potentially preferred. These findings advance PCP diagnostic precision in immunocompromised patients, aiding prompt intervention. Image 3: Correlation between induced sputum/tracheal suction and BALF (A) Spearman's rank correlation between induced sputum and BALF (B) and tracheal suction and BALF Disclosures All Authors: No reported disclosures

Non-Invasive Malaria Detection in Sub-Saharan Africa Using a DNA-Based Sensor System.

Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences (p < 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa.

Prospective evaluation of non-invasive saliva specimens for the diagnosis of syphilis and molecular surveillance of Treponema pallidum.

The promising diagnostic performance of molecular testing for syphilis using saliva and urine samples has been reported; however, further evaluation of its possible application for diagnosis and molecular surveillance is required. In addition, the development of a rapid and easy-to-perform molecular test for syphilis is important for its use in the clinical setting. We comprehensively evaluated the diagnostic and surveillance performance of two novel loop-mediated isothermal amplification (LAMP) assays using saliva and urine samples. Saliva, urine, and whole blood were collected from patients who underwent serological testing for syphilis at outpatient clinics. Treponema pallidum DNA in specimens was detected using quantitative PCR (qPCR), nested PCR, and novel LAMP assays. T. pallidum genotyping was conducted by multi-locus sequence typing (MLST). Of the 163 patients recruited, 98 were diagnosed with syphilis (primary: n = 35; secondary: n = 40; latent: n = 23). qPCR showed the highest sensitivity among the molecular tests performed with a sensitivity of 54.1% and 30.3% for all syphilis patients using saliva and urine samples, respectively. A novel method of LAMP combined with dry reagents and crude DNA extraction (Dry-LAMP) showed a probit detection limit of 37.4 copies/reaction within 45 min. The agreement rate between Dry-LAMP and qPCR for saliva was 95.7% (κ coefficient 0.90). The T. pallidum genotype was identified in 48 patients by MLST using saliva samples. Molecular analysis of saliva could be used as a supplementary diagnostic test for syphilis and molecular surveillance of the T. pallidum genotype. Dry-LAMP is expected to be helpful in the clinical diagnosis of syphilis.

The evaluation of earwax as a noninvasive specimen to determine livestock exposure to death camas (Zigadenuspaniculatus).

Foothill death camas (Z. paniculatus) grows on the foothill ranges of western North America and is acutely toxic to livestock grazing these ranges. The toxic alkaloids in foothill death camas are zygadenine and a series of zygadenine esters, with zygacine, the 3-acetyl ester of zygadenine, being the most abundant. In this study, earwax was evaluated as a specimen to determine livestock exposure to foothill death camas. Death camas alkaloids were detected in the earwax of sheep administered oral doses of foothill death camas alkaloids. In addition, death camas alkaloids were detected in the earwax of sheep that grazed rangeland with abundant death camas. This study demonstrates the potential of earwax as a noninvasive specimen for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by death camas. The results from this study indicate that diagnosticians should analyze for zygacine and zygadenine in the earwax of livestock suspected to have been poisoned by foothill death camas.

Photonic platform coupled with machine learning algorithms to detect pyrolysis products of crack cocaine in saliva: A proof-of-concept animal study.

The non-invasive detection of crack/cocaine and other bioactive compounds from its pyrolysis in saliva can provide an alternative for drug analysis in forensic toxicology. Therefore, a highly sensitive, fast, reagent-free, and sustainable approach with a non-invasive specimen is relevant in public health. In this animal model study, we evaluated the effects of exposure to smoke crack cocaine on salivary flow, salivary gland weight, and salivary composition using Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The exposure to crack cocaine was performed in an acrylic box apparatus with a burned activation of crack/cocaine 400mg for 10min for 14 consecutive days. Crack/cocaine exposure increased the salivary secretion without changes in parotid and submandibular weights. Hierarchical Clustering Analysis (HCA) was applied to depict subgrouping patterns in infrared spectra, and Principal components analysis (PCA) explained 83.2% of the cumulative variance using 3 PCs. ATR-FTIR platforms were coupled to AdaBoost, Artificial Neural Networks, Naïve Bayes, Random Forest, and Support Vector Machine (SVM) algorithms tool to identify changes in the infrared salivary spectra of rats exposed to crack cocaine. The best classification of crack cocaine exposure using the salivary spectra was performed by Naïve Bayes, presenting a sensitivity of 100%, specificity of 80%, and accuracy of 90% between crack cocaine and control rats. The SHAP features of salivary infrared spectra mostly indicate the vibrational modes at 1331cm-1 and 2806cm-1, representing CH2 wagging commonly linked in lipids and C-H stretch often attributed to the CH2 or CH3 groups in lipid molecules, respectively, as the main responsible vibrational modes for crack cocaine exposure discrimination. In summary, the present pre-clinical findings indicate the potential of the ATR-FTIR platform coupled with machine learning to effectively detect changes in salivary infrared spectra promoted by exposure to crack cocaine.

Antibiotic Resistance and Serotypes Distribution in Streptococcus agalactiae Bulgarian Clinical Isolates During the Years of 2021-2024.

Streptococcus agalactiae (group B Streptococcus, GBS) is an important human and animal pathogen. In recent years, the number of streptococcal isolates resistant to antimicrobial agents has increased in many parts of the world. Various mechanisms of antimicrobial resistance and capsular serotypes of GBS with different geographical distributions can be found. A prospective cross-sectional study was conducted from September 2021 to May 2024. The survey included 257 GBS isolates from Bulgarian inpatients and outpatients with streptococcal infections. Antibiotic resistance genes and capsular serotypes were detected and evaluated using polymerase chain reaction (PCR). We classified GBS isolates into groups according to their source as vaginal samples (191) and extra-vaginal samples (66), subdivided as invasive (36) and non-invasive specimens (30). The most common serotypes were Ia (26.5%), III (20.2%), and V (19.8%). Antimicrobial susceptibility testing revealed that all examined isolates were susceptible to penicillin and vancomycin. Resistance to macrolides, lincosamides, and tetracyclines was observed in 60.3%, 24.9%, and 89.1% of the isolates. The distribution of phenotypes was cMLSb 47.4%, iMLSb 30.8%, M-type 21.2%, and L-type 0.6%. PCR analysis revealed nine genes associated with macrolide and lincosamide resistance: ermB (54.2%), ermA/TR (30.3%), mefA (20.7%), ermC (18.1%), msrD (14.8%), mefE (8.4%), IsaC (8.4%), InuB (7.7%), and IsaE (6.5%). Two genes linked to tetracycline resistance tetM (89.1%) and tetO (14.4%) were detected. Compared to the previous period, we observed increased antibiotic resistance. There was no statistical significance between the distribution of serotypes and antimicrobial non-susceptibility depending on the sample source.

Analysis of serotype distribution and characteristics of nonhemolytic and nonpigmented strains among group B Streptococcus isolates in a southern Taiwan local hospital.

Group B streptococci (GBS) are Gram-positive bacteria that are a leading cause of neonatal infections. Most invasive isolates are β-hemolytic, and hemolytic activity is critical for GBS virulence. Although nonhemolytic GBS strains are occasionally isolated, they are often thought to be attenuated in virulence. Recent studies have observed that many nonhemolytic and nonpigmented (NH/NP) strains originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations causing the NH/NP phenotype are predominantly localized in the cyl operon and abx1 gene. Previous studies on group B streptococci in Taiwan have focused on the serotype and genotype distribution. In this study, we investigated the serotype distribution of the NH/NP strains and detected the mutations of abx1. One hundred clinical GBS strains from non-invasive (vaginal and rectal swabs, 69) and invasive infections (blood, urine and abscess, 31), including 10 NH/NP isolates, were collected during 2019-2021 at Fooyin University Hospital. To confirm GBS isolates, we have developed a multiplex PCR method that detects GBS isolates, virulent strain ST-17 and virulent factor Srr1 simultaneously. Molecular serotyping was performed by multiplex PCR assay using serotype specific primer sets. The genomic region containing abx1 was amplified from DNA extracts by PCR and the amplicons were directly sequenced and analyzed on an ABI prism 3730 DNA analyzer. The capsular serotypes III and VI were the most abundant in both the non-invasive specimens and invasive specimens. The ST-17 isolates were more frequently associated with invasive infections (16.1%, 5/31) than non-invasive diseases or colonization (7.2%, 5/69). The association of NH/NP strains between noninvasive diseases or colonization (10.1, 7/69) and invasive infection (9.7%, 3/31) is nearly compatible. The NH/NP strains were isolated from various serotypes (Ia, III, V and VI) and five NH/NP isolates were serotype III. The virulence factor Srr1was detected in most of the NH/NP isolates (8/10) and one NH/NP isolate was ST-17. Abx1 mutations, including transitions, transversions and deletions, were observed in some NH/NP isolates, but some mutations also observed in hemolytic isolates. Five NH/NP isolates were erythromycin and clindamycin resistant. These results indicate NH/NP GBS strains may have the potential for invasive infections and may show higher tendency to get mutated.

Development of a Direct Electron Transfer-Type Enzymatic Sensor for the Spermine in Saliva, a Marker of Pancreatic Cancer

Introduction Pancreatic cancer (PC) has one of the worst prognoses among cancers because of the scarcity of early symptoms and the delay of early diagnosis. Current most available and advanced diagnosis methods of PC are based on imaging technologies such as computed tomography, positron emission tomography, magnetic resonance imaging, and endoscopic ultrasonography. However, such imaging methods cannot diagnose PC at an early stage. Patients diagnosed with PC often have metastasized, and only less than 30% of PC patients can undergo surgical resection. Therefore, there is an urgent need for more accurate, simple, and innovative methods for PC early diagnosis. Recently, salivary spermine has attracted attention as a new biomarker for PC <Asai et.al., Cancers, 2018>, and studies aimed at detecting spermine are attempted. Thus, we focused on the electrochemical measurements of spermine using enzyme.Electrochemical enzymatic biosensors are categorized into three generation principles based on the electron acceptors used for the oxidative half reaction. The 1st generation principle uses oxygen as the electron acceptor. However, the signal is affected by the oxygen concentration and a high applied potential is required to detect hydrogen peroxide. The 2nd generation principle requires the use of synthetic electron acceptors or mediators. The 3rd generation principle utilizes enzymes which are capable of direct electron transfer (DET) to the electrode, does not require any additional electron acceptors. Notably, the 3rd generation principle-based sensor can monitor target by applying relatively lower potentials, therefore will not be influenced by redox-active interferents. Considering the salivary sample contains variety of ingredient potentially interferants for enzymatic spermine monitoring, the DET-type enzymatic sensors are ideal. However, those have never been reported. In this work, we present the development of the 3rd generation principle based enzymatic sensor for spermine by employing a unique Methods SpdH was recombinantly produced using E. coli and purified by Ni2+ affinity chromatography. An electrochemical enzymatic sensor was constructed by immobilizing SpdH on gold electrodes. The DET properties of the enzyme-immobilized electrode were evaluated by cyclic voltammetry with an Ag/AgCl reference electrode and a Pt wire counter electrode, respectively. Chronoamperometry measurement was performed with SpdH immobilized gold electrodes using artificial saliva or phosphate buffer, where spermine was spiked with different concentrations. Results and Discussions For spermine measurement, we selected SpdH which was expected to be capable of DET with electrode, which was predicted from its unique protein structure. The crystal structure of SpdH shows the presence of heme b and FAD as the co-factors of the enzyme with a distinctive character. The heme b locates on the surface of the enzyme and is expected to accept electron from FAD and can transfer electrons to the electrode. The cyclic voltammetry observation showed the spermine-concentration dependent peak current increase in the absence of any additional electron acceptor, revealing that the enzyme harbors DET ability with electrode. The spermine concentration dependent currents increase was also observed by chronoamperometry measurement. The limit of detection of the enzymatic electrode was sub-micromolar level, which covers the physiologically relevant ranges of spermine. Furthermore, the electrode did not respond to electrochemically active interferants due to its characteristic of low redox potentials of heme being measurable at low potentials.This is the first report to construct a spermine sensor with a DET-type enzyme. DET-type enzymes allow simple electrode configurations operating at lower potentials. These features are less susceptible to redox-active interferents. The current achievements promise the future realization of a simple diagnosis method for PC using a non-invasive specimen, saliva. Figure 1

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%.

Molecular epidemiology of Streptococcus pneumoniae isolates causing invasive and noninvasive infection in Ethiopia

Streptococcus pneumoniae, a medically important opportunistic bacterial pathogen of the upper respiratory tract, is a major public health concern, causing a wide range of pneumococcal illnesses, both invasive and noninvasive. It is associated with significant global morbidity and mortality, including pneumonia, meningitis, sepsis, and acute otitis media. The major purpose of this study was to determine the molecular epidemiology of Streptococcus pneumoniae strains that cause invasive and noninvasive infections in Ethiopia. A prospective study was undertaken in two regional hospitals between January 2018 and December 2019. Whole-genome sequencing was used to analyze all isolates. Serotypes and multilocus sequence types (MLST) were derived from genomic data. The E-test was used for antimicrobial susceptibility testing. Patient samples obtained 54 Streptococcus pneumoniae isolates, 33 from invasive and 21 from noninvasive specimens. Our findings identified 32 serotypes expressed by 25 Global Pneumococcal Sequence Clusters (GPSCs) and 42 sequence types (STs), including 21 new STs. The most common sequence types among the invasive isolates were ST3500, ST5368, ST11162, ST15425, ST15555, ST15559, and ST15561 (2/33, 6% each). These sequence types were linked to serotypes 8, 7 C, 15B/C, 16 F, 10 A, 15B, and 6 A, respectively. Among the noninvasive isolates, only ST15432, associated with serotype 23 A, had numerous isolates (4/21, 19%). Serotype 14 was revealed as the most resistant strain to penicillin G, whereas isolates from serotypes 3, 8, 7 C, and 10 A were resistant to erythromycin. Notably, all serotype 6 A isolates were resistant to both erythromycin and penicillin G. Our findings revealed an abnormally significant number of novel STs, as well as extremely diversified serotypes and sequence types, implying that Ethiopia may serve as a breeding ground for novel STs. Recombination can produce novel STs that cause capsular switching. This has the potential to influence how immunization campaigns affect the burden of invasive pneumococcal illness. The findings highlight the importance of continuous genetic surveillance of the pneumococcal population as a vital step toward enhancing future vaccine design.

The Importance of Proteomics in Saliva and Tears as Potential Non-invasive Methods for Identifying Biomarkers in the Prognosis and Diagnosis of Multiple Sclerosis Patients

: Over the past two decades, the advancement of analytical examinations like proteomics for investigating neuronal-related biomarkers has emerged as one of the most important and effective tools for clinical evaluation and prognosis. However, there remains an unmet need in this area. With advances in quantifying methods and detection in omics research, including proteomics and metabolomics, the close association of the salivary glands and tears with the nervous system has become increasingly evident. As noninvasive specimens, saliva and tears serve as attractive substitutes for neuronal biomarkers, containing numerous proteins and metabolites that represent various ailments in neurodegenerative illnesses like multiple sclerosis (MS). These noninvasive biomarkers might potentially correlate with susceptibility, severity, and pathogenesis of neurological disorders and could be utilized in early diagnosis and prognosis. Therefore, tear and salivary proteomics present novel insights into understanding disease progression and offer personalized treatment options with greater sensitivity. This approach helps highlight the most relevant experimental outcomes related to tear and salivary biomarkers for multiple sclerosis.

Expanding Xpert MTB/RIF Ultra® and LF-LAM testing for diagnosis of tuberculosis among HIV-positive adults admitted to hospitals in Tanzania and Mozambique: a randomized controlled trial (the EXULTANT trial)

IntroductionTuberculosis (TB) is an important cause of morbidity and mortality among people living with HIV (PLHIV). Current WHO-recommended strategies for diagnosing TB among hospitalized PLHIV rely on symptom screening and disease severity to assess eligibility for urine lipoarabinomannan lateral flow (LF-LAM) and molecular testing. Despite these recommendations, autopsy studies show a large burden of undiagnosed TB among admitted PLHIV. The EXULTANT trial aims to assess the impact of an expanded screening strategy using three specimens (sputum, stool, and urine) for TB diagnosis among PLHIV admitted to hospitals in two high HIV and TB burden African countries.MethodsThis is a multicenter, pragmatic, individually randomized controlled trial conducted across eleven hospitals in Tanzania and Mozambique. Participants in the intervention arm will be tested with Xpert MTB/RIF Ultra® from expectorated sputum, stool, and urine samples, with additional urine LF-LAM testing in the first 24 h after hospital admission, irrespective of the presence of the symptoms. The control arm will implement the WHO standard of care recommendations. Hospitalized adults (≥ 18 years) with a confirmed HIV-diagnosis, irrespective of antiretroviral (ART) therapy status or presence of TB symptoms will be assessed for eligibility at admission. Patients with a pre-existing TB diagnosis, those receiving anti-tuberculosis therapy or tuberculosis preventive treatment in the 6 months prior to enrolment, and those transferred from other hospitals will not be eligible. Also, participants admitted for traumatic reasons such as acute abdomen, maternal conditions, scheduled surgery, having a positive SARS-CoV2 test will be ineligible. The primary endpoint is the proportion of participants with microbiologically confirmed TB starting treatment within 3 days of enrolment.DiscussionThe EXULTANT trial investigates rapid implementation after admission of a new diagnostic algorithm using Xpert MTB/RIF Ultra® in several non-invasive specimens, in addition to LF-LAM, in hospitalized PLHIV regardless of TB symptoms. This enhanced strategy is anticipated to detect frequently missed TB cases in this population and is being evaluated as an implementable and scalable intervention.Trial registrationTrial reference number: NCT04568967 (ClinicalTrials.gov) registered on 2020–09-29.

Urinary biomarkers analysis as a diagnostic tool for early detection of pancreatic adenocarcinoma: Molecular quantification approach

Background and aimsPancreatic ductal adenocarcinoma (PDAC) is infrequent. Currently, non-invasive biomarkers for early detection of PDAC are not accessible. Here, we intended to identify a set of urine markers able to discriminate patients with early-stage PDAC from healthy individuals. Patients and methodsSeventy-five urine samples from PDAC patients and 50 healthy controls were assayed using quantitative real-time PCR (qPCR). The chosen biomarkers were lymphatic vessel endothelial HA receptor (LYVE-1), regenerating islet-derived 1 alpha (REG1A), and trefoil factor family (TFF1). ResultsLYVE-1, REG1A, and TFF1 expression in PDAC proved to be significantly elevated compared to healthy individuals (p < 0.05). Determination of these markers' expression might be useful for early tumor diagnosis with a sensitivity of 96 %, 100 %, and 73.33 % respectively, and a specificity of 100 %, 82 %, and 100 % respectively. ConclusionWe have recognized three diagnostic biomarkers REG1A, TFF1, and LYVE1 that can detect patients with early-stage pancreatic cancer in non-invasive urine specimens with improved sensitivity and specificity. To the best of our knowledge, there have been no prior investigations examining the mRNA expression levels of them in urine within the Egyptian population.

Potential for and challenges of menstrual blood as a non-invasive diagnostic specimen: current status and future directions.

Menstrual blood, which is often discarded as a waste product, has emerged as a valuable source of health information. The components of menstrual blood, such as endometrial cells, immune cells, proteins, and microbial signatures, provide insights into health. Studies have shown encouraging results for using menstrual blood to diagnose a variety of conditions, including hormonal imbalances, cervical cancer, endometriosis, chlamydia, diabetes, and other endocrine disorders. This review examines the potential of menstrual blood as a non-invasive diagnostic specimen, exploring its composition, promising applications, and recent advances. This review also discusses challenges to utilizing menstrual blood testing, including ethical considerations, the lack of standardized collection protocols, extensive validation studies, and the societal stigma around menstruation. Overcoming these challenges will open new avenues for personalized medicine and revolutionize healthcare for individuals who menstruate.

Monkeypox: A Viral Zoonotic Disease of Rising Global Concern

AbstractMonkeypox (mpox) is a rare viral zoonotic disease, endemic to Central and West Africa, caused by the monkeypox virus, an orthopoxvirus similar to the variola virus (smallpox). Although sporadic travel-associated cases have historically occurred outside Africa, in May 2022, mpox began spreading globally in multiple nonendemic countries across several continents. In 2024, there has been an increase in globally reported confirmed cases of mpox and deaths from mpox, making it a public health emergency of international concern. The reasons for the unusual global spread are under investigation but likely relate to increased travel and waning population immunity to orthopoxviruses. Transmission now appears to be mainly through close, intimate contact, especially among men who have sex with men. Mpox is usually a self-limited disease. Although limited approved antiviral treatments are available, such as tecovirimat, which the European Medicines Agency approved in January 2022 for the treatment of mpox, their widespread availability and effectiveness in the current outbreak remain to be investigated. Public health control measures include surveillance, case identification/isolation, contact tracing, and targeted vaccination of contacts at high risk of exposure. However, challenges remain in curtailing the current unprecedented outbreak. Critical knowledge gaps include animal reservoir(s) responsible for initial spillover events, viral mutations that may enhance transmissibility, optimal diagnostics for noninvasive specimens, effective antiviral therapies, next-generation vaccines providing longer-term immunity, and building global capacity for outbreak response. This review summarizes the current literature on mpox virology, epidemiology, pathogenesis, clinical manifestations, diagnostics, treatment, prevention, and public health control measures. Ongoing investigation and research are needed to better understand mpox’s evolving epidemiology, pathogenicity, transmissibility, and ecology to guide strategies for containing the outbreak and preventing future global emergence.

Non-Invasive Specimen Collection in COVID-19 RT-PCR Testing: An Observational Study at an Indonesian Tertiary Hospital

Background: COVID-19 is a disease of SARS-CoV2 beta coronavirus infection. One way to control the progression of COVID-19 symptoms is a rapid and accurate diagnosis. The current gold standard of COVID-19 diagnostic method uses RT-PCR from nasopharyngeal (NP) swabs and sputum specimens. Objective: To assess the efficacy of saliva and rectal swabs as non-invasive alternatives to the conventional nasopharyngeal swab for RTPCR testing in COVID-19 diagnosis, with a focus on the potential benefits for healthcare providers and patient comfort. Methods: This observational study was conducted with 80 confirmed cases at a tertiary hospital in Indonesia, the study compared RTPCR positivity rates among different collection methods. Results: A high positivity rate of 92% for nasopharyngeal swabs, with saliva and rectal swabs yielding 36% and 34%, respectively. Notably, the positivity rates for saliva and rectal samples increased to 60% and 50% when testing occurred between five to seven days post-symptom onset. Crucially, in older patients, both saliva and rectal swabs demonstrated higher positivity rates than in younger patients within the initial four days of symptom onset, with rates of 43% and 17%, respectively. Furthermore, a significant correlation was found between rectal swab positivity on day one and mortality in the older cohort, where lower Ct values on day seven were significantly associated with the deceased group. Conclusion: These findings support the potential of saliva and rectal swabs in predicting disease severity and patient outcomes, suggesting a safer and more convenient testing strategy for COVID- 19.

Abstract 2423: Linking liquids: cfDNA in urine and plasma as informative allies in metastasized prostate cancer

Abstract Background: Liquid biopsy has become a cornerstone of non-invasive cancer diagnostics. While the major analyte for detecting and monitoring tumors has been blood, examining other body fluids, such as urine or saliva, is increasingly shifting into focus. Urine is a particularly valuable specimen due to its easy accessibility. Particularly in urologic cancers, the sensitivity of liquid biopsy could be improved by proximal sampling. While there is already evidence that cell-free DNA from urine (ucfDNA) can add to the diagnostic sensitivity in renal and bladder cancer, less is known about ucfDNA in prostate cancer patients. In this study, we aimed to determine the presence, levels, and potential clinical applications of ucfDNA in patients with metastatic prostate cancer. Moreover, we compared the findings with information obtained from plasma cell-free DNA (cfDNA). Methods: Urine was collected and stabilized using the PAXgene Urine Liquid Biopsy Set or STRECK as a preservative. The collection and stabilization using PAXgene Urine Liquid Biopsy Set allows a standardized collection and stabilization of urine with fixed volumes of urine and preservatives. Blood was collected in PAXgene Blood ccfDNA Tubes (PreAnalytiX). Matched blood and urine cfDNA samples from 122 metastatic prostate cancer patients were isolated using the QIAsymphony platform. Tumor fraction and presence of somatic copy number alterations (SCNA) were assessed from shallow whole genome sequencing data using the ichorCNA algorithm. Results: In 22/122 (18%) of patients SCNAs could be detected in both, urine and blood. Although the copy number profile was highly concordant in those patients, tumor fractions varied between plasma and urines on an individual patient level. In addition, in 52/122 (43%) and 48/122 (39%) patients, tumor-derived DNA was detected only in plasma or urine. Considering the entire cohort, neither the absolute cfDNA concentration nor tumor fraction did significantly differ between urine and plasma samples. Conclusion: Our data demonstrate that ucfDNA can provide complementary information to blood about prostate tumors, which would be missed by the sole analysis of plasma cfDNA. Taking this into account and adding that urine represents a non-invasive specimen type that can be collected at great ease, ucfDNA holds promise in the clinical management of prostate cancer. Citation Format: Tina Moser, Anna Eberhard, Matthias J. Moser, Lisa Glawitsch, Sabrina Hammer, Georgios Vlachos, Isaac Lazzeri, Leandra Ziegler, Emil Bauernhofer, Nina Monsberger, Jochen B. Geigl, Thomas Bauernhofer, Ellen Heitzer. Linking liquids: cfDNA in urine and plasma as informative allies in metastasized prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2423.

Efficacy of SARS-CoV-2 detection from used surgical masks compared with standard detection method

The real-time reverse transcription-polymerase chain reaction (RT-PCR) test is the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. Proper specimen collection and obtaining a sufficient specimen are the most essential steps for laboratory diagnosis. The nasopharyngeal (NP) swab is recommended as the reference collection method. However, NP swab collection is invasive and uncomfortable for patients and poses some risk to healthcare workers. This study aimed to compare the efficacy of SARS-CoV-2 RNA detection from surgical masks with the NP swab method using RT-PCR testing. Of 269 patients, RT-PCR RNA from NP swabs was detected among 82 patients (30.5%) and was undetected among 187 patients (69.5%). All patients were tested for SARS-CoV-2 RNA from surgical masks. SARS-CoV-2 RNA was detected in 25/82 (30.5%) surgical mask filters, while undetected among 57 (69.5%). For the surgical mask with an average use time of 7.05 h, the sensitivity was 30.5%, the specificity was 100.0%, with positive predictive value of 100.0% and negative predictive value of 76.2%. Therefore, surgical masks could be an alternative non-invasive specimen source for SARS-CoV-2 RT-PCR testing. The results of our study suggest that the test could be employed after wearing surgical masks for at least 8-12 h, with increased sensitivity when used for more than 12 h.

Non-invasive specimen collections for Mycobacterium tuberculosis detection in free-ranging long-tailed macaques (Macaca fascicularis).

Surveillance of infectious diseases in free-ranging or wild animals has been widely conducted in many habitat-range countries after the COVID-19 episode. Thailand is located in the center of the distribution range of long-tailed macaques (Macaca fascicularis; Mf) where the animals have both frequent human contact and a high prevalence of human tuberculosis. For the large-scale detection of Mycobacterium tuberculosis complex (MTBC) using IS6110-nested PCR in free-ranging Mf, non-invasive sampling was developed using oral (via rope bait) and fecal (direct swabs of fresh feces) specimen collection. Firstly, the MTBC-IS6110-nested PCR was validated in non-invasively collected specimens, in terms of its specificity and sensitivity, and then compared with those of the invasively collected oral and rectal swabs in 24 captive MTBC-suspected Mf. After validation, these methods were applied to survey for the prevalence of shed MTBC (MTBCS) in four previously reported MTBC-infected populations. A total of 173 baited rope specimens and 204 freshly defecated excretions were collected. The limit of detection of the IS6110-nested PCR technique was 10 fg/μL and the 181-bp PCR amplicon showed 100% sequence similarity with the MTB H37Rv genome sequence. Comparing the MTBCS detection between the invasive and non-invasive collected specimens in captive suspected Mf revealed a significant correlation between the two types of oral specimens (oral swabs and baited ropes; n = 24, r2 = 1, p-value < 0.001), but fresh fecal swabs showed higher MTBCS frequencies than the rectal swabs. Moreover, the proportion of MTBCS-positive free-ranging Mf were significantly higher in the fresh fecal swabs (8.82%; 95% CI; 4.9-12.7%) than in the baited ropes (5.20%; 95% CI; 1.9-8.5%). This result indicates that oral sampling via baited ropes and fecal sampling via defecated excretion swabs can serve as ancillary specimens for MTBCS detection in free-ranging non-human primates.

TP53 mutation prevalence in normal airway epithelium as a biomarker for lung cancer risk

BackgroundThere is a need for biomarkers that improve accuracy compared with current demographic risk indices to detect individuals at the highest lung cancer risk. Improved risk determination will enable more effective lung cancer screening and better stratification of lung nodules into high or low-risk category. We previously reported discovery of a biomarker for lung cancer risk characterized by increased prevalence of TP53 somatic mutations in airway epithelial cells (AEC). Here we present results from a validation study in an independent retrospective case–control cohort.MethodsTargeted next generation sequencing was used to identify mutations within three TP53 exons spanning 193 base pairs in AEC genomic DNA.ResultsTP53 mutation prevalence was associated with cancer status (P < 0.001). The lung cancer detection receiver operator characteristic (ROC) area under the curve (AUC) for the TP53 biomarker was 0.845 (95% confidence limits 0.749–0.942). In contrast, TP53 mutation prevalence was not significantly associated with age or smoking pack-years. The combination of TP53 mutation prevalence with PLCOM2012 risk score had an ROC AUC of 0.916 (0.846–0.986) and this was significantly higher than that for either factor alone (P < 0.03).ConclusionsThese results support the validity of the TP53 mutation prevalence biomarker and justify taking additional steps to assess this biomarker in AEC specimens from a prospective cohort and in matched nasal brushing specimens as a potential non-invasive surrogate specimen.