Pathogen Nucleic Acid Research Articles

Advances in visual detection of pathogen nucleic acids by CRISPR-Cas

Visual detection is a technique for evaluating the results through visual judgment without relying on complex optical detection systems. It obtains results quickly based on signals, such as visible light, changes in air pressure, and migration distance, that can be directly observed by naked eyes, being widely used in the in vitro diagnostics industry. The CRISPR-Cas system has the potential to be used in the development of point of care testing (POCT) technologies due to the advantages of mild reaction conditions, no need for thermal cycling or other control measures, and a robust signal amplification capability. In recent years, the combination of visual detection and CRISPR-Cas has significantly reduced the need for laboratory infrastructures, precision instruments, and specialized personnel for nucleic acid detection. This has promoted the development of POCT technology and methods for nucleic acids. This article summarizes the signal output modes and characteristics of the visual detection of nucleic acid by CRISPR-Cas and discusses the issues in the application. Finally, its future clinical translation is envisioned with a view to informing the development of CRISPR-Cas visualization assays.

Foodborne Pathogens Across Different Food Matrices in Sicily (Southern Italy).

Foodborne diseases result from the consumption of foods contaminated with pathogens or their toxins and represent a serious public health problem worldwide. This study aimed to assess the presence of Rotavirus (RoV), Adenovirus (AdV), Norovirus (NoV), Hepatitis A and Hepatitis E viruses (HAV and HEV, respectively), Toxoplasma gondii, Coxiella burnetii and Leptospira spp. across various food matrices in Sicily. The analysis concerned 504 samples, including mussels, farmed meat, game meat, vegetables and bulk milk. Following appropriate pre-treatment, acid nucleic extraction was carried out and amplification of pathogen nucleic acids was carried out by molecular methods. The mussels tested positive for NoVs (3/51, 5.9%) and farm meat resulted positive for T. gondii (1/34, 2.9%). The game offal samples tested positive for HEV, which was detected in 17 out of 222 samples (7.7%), and T. gondii (18/318, 5.7%) and Leptospira spp. (2/318, 0.6%). The milk samples tested positive for C. burnetii (15/85, 17.6%), T. gondii (2/85, 2.4%) and Leptospira spp. (1/85, 1.2%). This study highlights the variability in the risk of contamination of different food matrices, confirming the importance of vigilance in the consumption of potentially contaminated food products.

Plastic bronchitis associated with human bocavirus 1 infection in children.

Plastic bronchitis (PB) is a clinical-pathological syndrome characterized by the abnormal accumulation of endogenous substances in the bronchial airways, causing partial or complete obstruction and resulting in impaired lung ventilation. In this retrospective analysis, we aim to summarize the clinical manifestations, imaging characteristics, diagnostic methods, and treatment approaches to enhance clinicians' ability to detect children who are infected withhuman bocavirus 1(hBoV 1) and develop PB. In the period from January 2021 to January 2024, a total of six hBoV 1 infection children were diagnosed with PB through bronchoscopy. The onset of the condition was mainly concentrated between June and December. The detection methods used included metagenomic next-generation sequencing for pathogen identification (three cases) and respiratory pathogen nucleic acid 13-plex detection (oropharyngeal swab) (three cases), both of which confirmed the presence of hBoV 1. Out of the six children with PB, two were girls and four were boys. Their ages ranged from 10 months to 4 years old. Common symptoms reported by all patients included fever, cough,and wheezing. Chest high-resolution computed tomography scans revealed atelectasis in six cases, in addition to pneumonia. After the removal of the plastic bronchi via bronchoscopy, the airway obstruction symptoms in the children were relieved, and no recurrence was observed during the follow-up period. Pathological findings indicated cellulose exudation and inflammatory cell infiltration, consistent with nonlymphatic PB. When children infected with hBoV 1 exhibit persistent or worsening symptoms such as cough, fever, and wheezing despite treatment, clinicians should remain highly vigilant for the potential occurrence of PB. Bronchoscopy plays a crucial role not only in diagnosing the presence of a plastic bronchus but also in effectively treating PB.

Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States

We measured concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, enterovirus D68, human parainfluenza types 1, 2, 3, 4a, and 4b in aggregate, norovirus genotype II, rotavirus, Candida auris, hepatitis A virus, human adenovirus, mpox virus, H5 influenza A virus, and pepper mild mottle virus nucleic acids in wastewater solids prospectively at 191 wastewater treatment plants in 40 states across the United States plus Washington DC. Measurements were made two to seven times per week from 1 January 2022 to 30 June 2024, depending on wastewater treatment plant staff availability. Measurements were made using droplet digital (reverse-transcription–) polymerase chain reaction (ddRT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater.

B-195 Accelerating Molecular Diagnostics: Development of a novel Rapid, Integrated PCR System for Acute Respiratory Infections Detection

Abstract Background The COVID-19 pandemic and acute respiratory infections have underscored the necessity for high-throughput and timely molecular diagnostics. Existing molecular diagnostic methods are labor-intensive, a challenge particularly evident in large-scale testing. This study aims to streamline the nucleic acid testing process by integrating extraction and detection in PCR testing, thereby optimizing efficiency. Methods We developed 'EXP-160R', an integrated PCR system capable of rapid thermal cycling. This innovation employs advanced temperature control technology to markedly reduce heating, cooling, and annealing-extension times. The PCR reagent-kit (Zybio Inc.) was optimized to complement the instrument's capabilities, aiming for pathogen nucleic acid detection in acute respiratory infections within 25 minutes. The precision of in-tube temperature during rapid amplification was validated by using temperature-sensitive molecular probes in system development stage. Performance evaluation used the WHO International Standard for SARS-CoV-2 RNA to assess sensitivity and detection limits. We also analyzed 500 clinical samples from multiple Chinese hospitals, including 295 COVID-19 positives and 205 negatives, comparing the EXP-160R system's performance with an NMPA-approved PCR kit. Results The innovative system significantly improved PCR cycle times, enabling detection of 16 samples in 22 minutes and a throughput of 2400 tests per day, without compromising sensitivity or specificity. Validation confirmed a detection limit of 200 copies/mL, and the system achieved a 100% detection rate for medium and high viral load samples, with a coefficient of variation (CV) for Ct values ≤ 5%. Clinical trial comparisons with clinical standard diagnostics revealed a sensitivity of 98.05%, specificity of 100%, accuracy of 99.2%, and a Kappa coefficient of 0.984. When compared to the established PCR kit, our system showed comparable efficacy, with a sensitivity of 98.50%, specificity of 98.67%, accuracy of 98.6%, and a Kappa of 0.971. Conclusions The rapid PCR system represents a significant advancement in molecular diagnostics, offering rapid, high-throughput, and accurate detection of SARS-CoV-2. This system is particularly beneficial in urgent testing scenarios, such as airports and fever clinics. It addresses current pandemic needs and sets a new benchmark for future respiratory and infectious disease diagnostics, enhancing both efficiency and user experience.

Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges.

The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.

Bacteria and RNA virus inactivation with a high-irradiance UV-A source

Disinfection with LED lamps is a promising ecological and economical substitute for mercury lamps. However, the optimal time/dose relationship needs to be established. Pathogen inactivation by UV-A primarily relies on induced reactive oxygen species (ROS) formation and subsequent oxidative damage. While effective against bacteria and enveloped viruses, non-enveloped viruses are less sensitive. In this study, we explored the disinfection properties of 10 W UV-A LED, emitting in the 365–375 nm range. UV-A at high values of irradiance (~ 0.46 W/cm2) can potentially induce ROS formation and direct photochemical damage of the pathogen nucleic acids, thus improving the disinfection. The UV-A inactivation was evaluated for the bacterium Escherichia coli (E. coli), non-enveloped RNA bacteriophage MS2, and enveloped mammalian RNA virus—Semliki Forest virus (SFV). The 4 log10 reduction doses for E. coli and SFV were 268 and 241 J/cm2, respectively. Furthermore, in irradiated E. coli, ROS production positively correlated with the inactivation rate. In the case of MS2 bacteriophage, the 2.5 log10 inactivation was achieved by 679 J/cm2 within 30 min of irradiation. The results demonstrate significant disinfection efficiency of non-enveloped virus MS2 using high-irradiance UV-A. This suggests a potential strategy for improving the inactivation of UV-A-unsusceptible pathogens, particularly non-enveloped viruses. Additionally, the direct UV-A irradiation of self-replicating viral RNA from SFV led to a significant loss of viral gene expression in cells transfected with the irradiated RNA. Therefore, the virus inactivation mechanism of high-irradiance UV-A LED can be partially determined by the direct damage of viral RNA.Graphical abstract

Respiratory pathogen dynamics in community fever cases: Jiangsu Province, China (2023–2024)

BackgroundRespiratory infectious diseases have the highest incidence among infectious diseases worldwide. Currently, global monitoring of respiratory pathogens primarily focuses on influenza and coronaviruses. This study included influenza and other common respiratory pathogens to establish a local respiratory pathogen spectrum. We investigated and analyzed the co-infection patterns of these pathogens and explored the impact of lifting non-pharmaceutical interventions (NPIs) on the transmission of influenza and other respiratory pathogens. Additionally, we used a predictive model for infectious diseases, utilizing the commonly used An autoregressive comprehensive moving average model (ARIMA), which can effectively forecast disease incidence.MethodsFrom June 2023 to February 2024, we collected influenza-like illness (ILI) cases weekly from the community in Xuanwu District, Nanjing, and obtained 2046 samples. We established a spectrum of respiratory pathogens in Nanjing and analysed the age distribution and clinical symptom distribution of various pathogens. We compared age, gender, symptom counts, and viral loads between individuals with co-infections and those with single infections. An autoregressive comprehensive moving average model (ARIMA) was constructed to predict the incidence of respiratory infectious diseases.ResultsAmong 2046 samples, the total detection rate of respiratory pathogen nucleic acids was 53.37% (1092/2046), with influenza A virus 479 cases (23.41%), influenza B virus 224 cases (10.95%), and HCoV 95 cases (4.64%) being predominant. Some pathogens were statistically significant in age and number of symptoms. The positive rate of mixed infections was 6.11% (125/2046). There was no significant difference in age or number of symptoms between co-infection and simple infection. After multiple iterative analyses, an ARIMA model (0,1,4), (0,0,0) was established as the optimal model, with an R2 value of 0.930, indicating good predictive performance.ConclusionsThe spectrum of respiratory pathogens in Nanjing, Jiangsu Province, was complex in the past. The primary age groups of different viruses were different, causing various symptoms, and the co-infection of viruses did not correlate with the age and gender of patients. The ARIMA model estimated future incidence, which plateaued in subsequent months.

2D Janus carrier-enabled trojan horse: Gallium delivery for the sequential therapy of biofilm associated infection

Biofilm-associated infections (BAIs) continue to pose a major challenge in the medical field. Nanomedicine, in particular, promises significant advances in combating BAIs through the introduction of a variety of nanomaterials and nano-antimicrobial strategies. However, studies to date have primarily focused on the removal of the bacterial biofilm and neglect the subsequent post-biofilm therapeutic measures for BAIs, rendering pure anti-biofilm strategies insufficient for the holistic recovery of affected patients. Herein, we construct an emerging dual-functional composite nanosheet (SiHx@Ga) that responds to pHs fluctuation in the biofilm microenvironment to enable a sequential therapy of BAIs. In the acidic environment of biofilm, SiHx@Ga employs the self-sensitized photothermal Trojan horse strategy to effectively impair the reactive oxygen species (ROS) defense system while triggering oxidative stress and lipid peroxidation of bacteria, engendering potent antibacterial and anti-biofilm effects. Surprisingly, in the post-treatment phase, SiHx@Ga adsorbs free pathogenic nucleic acids released after biofilm destruction, generates hydrogen with ROS-scavenging and promotes macrophage polarization to the M2 type, effectively mitigating damaging inflammatory burst and promoting tissue healing. This well-orchestrated strategy provides a sequential therapy of BAIs by utilizing microenvironmental variations, offering a conceptual paradigm shift in the field of nanomedicine anti-infectives.

Light-Switch Electrochemiluminescence-Driven microfluidic sensor for rapid and sensitive detection of Mpox virus

Rapid, real-time, and accurate detection of pathogenic nucleic acids is crucial for the control and treatment of Mpox virus infection; however, classical detection methods are limited by time-consuming amplification steps, cumbersome equipment, and the requirement for professional operators. Herein, we developed an electrochemiluminescence (ECL)-driven microfluidic detection chip based on a light-switch molecule, which when coupled with rapid recombinase polymerase amplification (RPA) enabled the detection of Mpox within 30 min. [Ru(phen)2dppz]2+ was used as the ECL light-switch molecule owing to its significant ECL signal enhancement property when intercalated into double-stranded RPA products. The microfluidic chip included two independent liquid storage tanks preloaded with an RPA reagent mixture and [Ru(phen)2dppz]2+, which were mixed via centrifugation to produce ECL signals. The developed sensing platform effectively detected Mpox virus with high sensitivity, and the limit of detection was as low as 10 copies/µL. Moreover, it showed 100 % sensitivity and 100 % specificity when validated against the quantitative polymerase chain reaction assay using clinical samples from the pustules, throat, urine, blood, saliva, and anus of Mpox-positive patients. Combined with a hand-held detection device, this platform can facilitate the early diagnosis of Mpox cases in point-of-care testing without trained personnel and specialized equipment.

Rapid, sensitive and multiplexed detection of SARS-CoV-2 viral nucleic acids enabled by phase-based surface plasmon resonance of metallic gratings.

A rapid, sensitive and easy-to-implement approach is proposed for the detection of pathogenic nucleic acids based on phase-based plasmonic spectroscopy of metallic gratings. The plasmonic sensors were fabricated using interference lithography and functionalized with single-stranded DNA probes to specific target SARS-CoV-2. The biosensor achieved the detection of 40 fM viral nucleic acids within 5 min; furthermore, a detection capability of 1 aM (0.6 copies/µL) was acquired when combining with the recombinase polymerase amplification. Additionally, the multiplexed sensing system was demonstrated to simultaneously detect three genomic sequences on a single sensor chip, thereby enhancing diagnostic accuracy and enabling high-throughput detection.

Minute level ultra-rapid and thousand copies level high-sensitive pathogen nucleic acid identification based on contactless impedance detection microsensor

Early screening for pathogens is crucial during pandemic outbreaks. Nucleic acid testing (NAT) is a valuable method for keeping pathogens from spreading. However, the long detection time and large size of the instruments involved significantly limited the efficiency of detection. This work described an integrated NAT microsensor that facilitated rapid and extremely sensitive detection based on nucleic acid amplification (NAA) on a chip. The biochip consisted of two layers incorporating a heater, a thermometer, an interdigital electrode (IDE) and a reaction chamber. The Pt electrode based heater and thermometer were utilized to maintain a specific temperature for the sample in the chamber. The thermometer exhibited a good linear correlation with a sensitivity of 9.36 Ω/°C and the heater achieved a heating efficiency of approximately 6.5 °C/s. Multiple ions were released during NAA, resulting in a decrease in the impedance of the amplification system solution. A large signal of impedance was generated by the released ions due to its linear correlation with the logarithm of the ion concentration. With this detection principle, IDE was employed for real-time monitoring of the in-chip reaction system impedance and NAA process. Specific nucleic acids from two pathogens (SARS-CoV-2, Vibrio vulnificus) were detected with this microsensor. The samples were qualitatively analyzed on microchip within 3 min, with a limit of detection (LOD) of 103 copies/μL. The proposed sensor presented several advantages, including reduced NAT time and increased sensitivity. Consequently, it has shown significant potential in rapid and high-quality nucleic acid testing for the field of epidemic prevention.

Electrochemical sensing strategies for on‐site testing of pathogenic nucleic acids

AbstractRapid and reliable on‐site pathogen testing is crucial for diagnosing and managing human health. Nucleic acids (NAs) containing genetic information are valuable target molecules for pathogen testing, and sensitive and rapid detection of NAs using electrochemical approaches has been intensively investigated. Detection approaches for NAs are diverse and compatible with current gene amplification methods and continue to expand with the development of novel functional materials and molecules. The variety of electrochemical sensing devices also continues to expand, and more practical testing is being pursued. This review outlines the latest detection approaches and basic guidelines for NA detection. Furthermore, this review provides an overview of electrochemical sensing devices that utilize novel and unique materials and functions and comprehensively discusses their advantages.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses.

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

Microglial- neuronal crosstalk in chronic viral infection through mTOR, SPP1/OPN and inflammasome pathway signaling.

HIV-infection of microglia and macrophages (MMs) induces neuronal injury and chronic release of inflammatory stimuli through direct and indirect molecular pathways. A large percentage of people with HIV-associated neurologic and psychiatric co-morbidities have high levels of circulating inflammatory molecules. Microglia, given their susceptibility to HIV infection and long-lived nature, are reservoirs for persistent infection. MMs and neurons possess the molecular machinery to detect pathogen nucleic acids and proteins to activate innate immune signals. Full activation of inflammasome assembly and expression of IL-1β requires a priming event and a second signal. Many studies have demonstrated that HIV infection alone can activate inflammasome activity. Interestingly, secreted phosphoprotein-1 (SPP1/OPN) expression is highly upregulated in the CNS of people infected with HIV and neurologic dysfunction. Interestingly, all evidence thus far suggests a protective function of SPP1 signaling through mammalian target of rapamycin (mTORC1/2) pathway function to counter HIV-neuronal injury. Moreover, HIV-infected mice knocked down for SPP1 show by neuroimaging, increased neuroinflammation compared to controls. This suggests that SPP1 uses unique regulatory mechanisms to control the level of inflammatory signaling. In this mini review, we discuss the known and yet-to-be discovered biological links between SPP1-mediated stimulation of mTOR and inflammasome activity. Additional new mechanistic insights from studies in relevant experimental models will provide a greater understanding of crosstalk between microglia and neurons in the regulation of CNS homeostasis.

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology.

High-throughput sequencing (HTS) has revolutionised the field of pathogen genomics, enabling the direct recovery of pathogen genomes from clinical and environmental samples. However, pathogen nucleic acids are often overwhelmed by those of the host, requiring deep metagenomic sequencing to recover sufficient sequences for downstream analyses (e.g., identification and genome characterisation). To circumvent this, hybrid-capture target enrichment (HC) is able to enrich pathogen nucleic acids across multiple scales of divergences and taxa, depending on the panel used. In this review, we outline the applications of HC in human pathogens-bacteria, fungi, parasites and viruses-including identification, genomic epidemiology, antimicrobial resistance genotyping, and evolution. Importantly, we explored the applicability of HC to clinical metagenomics, which ultimately requires more work before it is a reliable and accurate tool for clinical diagnosis. Relatedly, the utility of HC was exemplified by COVID-19, which was used as a case study to illustrate the maturity of HC for recovering pathogen sequences. As we unravel the origins of COVID-19, zoonoses remain more relevant than ever. Therefore, the role of HC in biosurveillance studies is also highlighted in this review, which is critical in preparing us for the next pandemic. We also found that while HC is a popular tool to study viruses, it remains underutilised in parasites and fungi and, to a lesser extent, bacteria. Finally, weevaluated the future of HC with respect to bait design in the eukaryotic groups and the prospect of combining HC with long-read HTS.

Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection

Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod)

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. ImportancePertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Context-dependent accuracy of the cobas plasma separation card for HCV RNA viral load measurement.

Collection and preservation of plasma are challenging in remote or under-resourced settings. The cobas® Plasma Separation Card (PSC) is an alternative specimen type for blood-borne pathogen nucleic acid quantitation. We assessed PSC as a specimen type for HCV RNA quantitation in Pakistan. Plasma from venous blood and PSC from finger prick blood were prepared at two sites: Site 1 (in Lahore, n = 199) consisted of laboratory-based outpatient clinics. Specimens were prepared in the same facility and stored frozen. Site 2 was a catchment area within a resource-limited, semi-urban locality of Islamabad with limited access to healthcare services (n = 151). Community public health outreach staff collected blood and prepared the PSC in the participants' homes. Specimens were transported to the central hepatitis laboratory in Lahore to be stored frozen until tested. HCV RNA testing was performed using the cobas HCV RNA test in a central laboratory. Concordance with respect to RNA detectability was high at Site 1 (97.4%), but lower at Site 2 (82.4%). At Site 1, HCV viral load in plasma and PSC were well correlated across the linear range with a 0.21 log10 IU/mL mean bias toward higher concentrations in PSC. At Site 2, HCV viral load in plasma and PSC were poorly correlated. There was a 0.11 log10 IU/mL mean bias toward higher concentrations in PSC. PSC performance can be excellent in underserved settings where refrigerated transport of traditional specimens is difficult. In very challenging field settings, extra support must be provided to ensure correct specimen collection and handling.

Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review

Nucleic acid extraction from biological sources has evolved with time and is currently one of the most important contributors to the development of modern human molecular diagnostics. Pure and sufficient quantity of DNA and RNA from limited quantity of cells, which may be fresh or archived, holds key to successful detection of source and cause of many diseases. The challenges of cell disruption to release nucleic acids and the wide range of strategies applied to purify them by removing various enzyme inhibitors are discussed. The demographic spread of the disease of tuberculosis, hurdles faced during transportation of the clinical samples for nucleic acid extraction of Mycobacterium tuberculosis pathogen and different detection technologies supported by the extracted pathogen nucleic acid are reviewed.