The ethnobotanical heritage of Lotkuh, a high-altitude tribal haven of Chitral, the Eastern Hindu Kush, Pakistan.

BACKGROUND: In northwestern Pakistan, Lotkuh is a high-altitude terrain nestled within the eastern Hindu Kush region. Enclaved by towering peaks and harboring a unique culture, the region mirrors the geographical and cultural diversity of Pakistan. In this geographically isolated region, a treasure trove of ethnobotanical knowledge unfolds through generations of interaction between the inhabitants and indigenous plants, resulting in a profound understanding of the plant uses in nutritional, medicinal, cultural, and ritual contexts. Thus, the study seeks to gather, analyze, and document the indigenous knowledge of plant utilization of the distinct tribal culture. METHODS: Through semi-structured questionnaires, inventory interviews, and participatory workshops, data were collected by engaging a cohort of 120 local respondents. The collected data were then classified into nine distinct use categories, following which quantitative indices were calculated. RESULTS: The research identified a total of 150 plant species spanning across 59 different families and categorized them into 9 distinct usage groups. Among these, Astragalus oihorensis, Astragalus owirensis, Cicer nuristanicum, Geranium parmiricum, and Rochelia chitralensis stand out as novel species with distinctive applications. Notably, medicinal use garnered 600 reports, while animal feed, veterinary applications, human consumption, and toxicity recorded 500, 450, 425, and 104 reports, respectively. Informant consensus was high ranging between 0.8 and 0.9 with most agreement on human food and animal feed category. Platanus orientalis and Juglans regia, with RFC 0.91, were the most cited. The Family Importance Value (FIV) of Juglandaceae and Platanaceae, each with an FIV of 0.91, and Capparidaceae with an FIV of 0.83 indicate the intricate role the families play. CONCLUSIONS: In this study, we explore 150 ethnobotanical species, uncovering novel entries within ethnobotanical literature. Among these, several species showcase unique uses previously undocumented in Pakistani literature. Our research sheds light on the intricate interaction between plants and the distinct cultural landscape of the Lotkuh region.

Identification of Four Novel Candidate Genes for Non-syndromic Intellectual Disability in Pakistani Families.

Intellectual disability, a genetically and clinically varied disorder and is a significant health problem, particularly in less developed countries due to larger family size and high ratio of consanguineous marriages. In the current genetic study, we investigate and find the novel disease causative factors in the four Pakistani families with severe type of non-syndromic intellectual disability. For genetic analysis whole-exome sequencing (WES) and Sanger sequencing was performed. I-TASSER and Cluspro tools were used for Protein modeling and Protein-protein docking. Sanger sequencing confirms the segregation of novel homozygous variants in all the families i.e., c.245 T > C; p.Leu82Pro in SLC50A1 gene in family 1, missense variant c.1037G > A; p.Arg346His in TARS2 gene in family 2, in family 3 and 4, nonsense mutation c.234G > A; p.Trp78Term and missense mutation c.2200G > A; p.Asp734Asn in TBC1D3 and ANAPC2 gene, respectively. In silico functional studies have found the drastic effect of these mutations on protein structure and its interaction properties. Substituted amino acids were highly conserved and present on highly conserved region throughout the species. The discovery of pathogenic variants in SLC50A1, TARS2, TBC1D1 and ANAPC2 shows that the specific pathways connected with these genes may be important in cognitive impairment. The decisive role of pathogenic variants in these genes cannot be determined with certainty due to lack of functional data. However, exome sequencing and segregation analysis of all filtered variants revealed that the currently reported variants were the only variations from the respective families that segregated with the phenotype in the family.

Structural and functional implications of SLC13A3 and SLC9A6 mutations: an in silico approach to understanding intellectual disability.

BACKGROUND: Intellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients' learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient's mental capacity, physical skills, and adaptive behavior. METHODS AND RESULTS: Two Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant (c.1478 C > T; p. Pro493Leu) in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant (c.1342G > A; p.Gly448Arg) in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations. CONCLUSION: We have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.

Optimization of different growth parameters for maximum production of bioactive crude metabolites by Aspergillus fumigatus.

Aspergillus fumigatus is a green echinulate with greenish phialides and 2.5-3 mm conidia. The diverse biological functions of A. fumigatus secondary metabolites make them interesting. The ethyl acetate extract of A. fumigatus was tested for antibacterial activity. Culture media, temperature, incubation and pH were optimized for A. fumigatus growth. Continuous 150rpm agitation incubated the fungus at 28°C for 10 days. Potato Dextrose Broth at 28°C in shaking incubator at pH 04 produced the most biomass and secondary metabolites. Metabolite antibacterial activity was tested. Salmonella flexneri had the greatest zone of inhibition at 100µl (25.66mm) while Staphylococcus aureus had the least (16.33mm). At 75µg/mL, S. flexneri showed 23.66mm activity and S. typhi 14.66mm. At 50µg/mL, S. flexneri was 21.33mm and S. typhi 12.33mmMBC was 0.01µg/µl and MIC50 varied. At 100µg/mL, the metabolites showed antifungal efficacy against Penicillium chrysogenum (26.33mm) but not A. flavus (21.33mm). A. oryzae was significantly inhibited at 75µg/mL (26.33mm) and 50µg/mL (20.33mm). 1000µl demonstrated 100% phytotoxicity, 100µl 60% and 10µl 50%. Bactrocera cucurbitae, Sitotroga cerealella and Callosobruchus maculatus were killed at 150, 100 and 75µl. Metabolites and antibiotics synergized well. Metabolites have alkanes, esters and ethers in their infrared spectra.

Synthesis, characterization and biological investigation of zinc nanoparticles using Acacia modesta Wall. leaves.

The study aimed to synthesize zinc nanoparticles (Zn-NPs) using an aqueous extract derived from Acacia modesta Wall. leaves. Several characterization techniques were employed to confirm the successful formation of zinc nanoparticles. UV-visible spectrophotometry indicated a peak at 374 nm, validating the bioreduction process. Scanning electron microscopy (SEM) was utilized to analyze the morphology, transmission electron microscopy (TEM) to determine particle size and shape, X-ray diffraction (XRD) for crystalline structure analysis, energy-dispersive X-ray spectroscopy (EDX) for elemental composition and Fourier-transform infrared spectroscopy (FTIR) to identify functional groups. The synthesized Zn-NPs demonstrated remarkable antibacterial activity against Escherichia coli (95% inhibition) and moderate antifungal activity against Candida albicans (70% inhibition). In phytotoxicity tests, the Zn-NPs exhibited a 55% reduction in the growth of Lamina minor at the highest dilution (1000 µl). Based on these findings, the study concluded that the green-synthesized Zn-NPs hold great potential as effective antibiotics against pathogenic bacteria and could be utilized in various industrial and agricultural applications.

Natural history of non-functioning pituitary microadenomas: results from the UK non-functioning pituitary adenoma consortium.

OBJECTIVE: The optimal approach to the surveillance of non-functioning pituitary microadenomas (micro-NFPAs) is not clearly established. Our aim was to generate evidence on the natural history of micro-NFPAs to support patient care. DESIGN: Multi-centre, retrospective, cohort study involving 23 endocrine departments (UK NFPA consortium). METHODS: Clinical, imaging, and hormonal data of micro-NFPA cases between January, 1, 2008 and December, 21, 2021 were analysed. RESULTS: Data for 459 patients were retrieved [median age at detection 44 years (IQR 31-57)-152 males/307 females]. Four hundred and nineteen patients had more than two magnetic resonance imagings (MRIs) [median imaging monitoring 3.5 years (IQR 1.71-6.1)]. One case developed apoplexy. Cumulative probability of micro-NFPA growth was 7.8% (95% CI, 4.9%-8.1%) and 14.5% (95% CI, 10.2%-18.8%) at 3 and 5 years, respectively, and of reduction 14.1% (95% CI, 10.4%-17.8%) and 21.3% (95% CI, 16.4%-26.2%) at 3 and 5 years, respectively. Median tumour enlargement was 2 mm (IQR 1-3) and 49% of micro-NFPAs that grew became macroadenomas (nearly all >5 mm at detection). Eight (1.9%) patients received surgery (only one had visual compromise with surgery required >3 years after micro-NFPA detection). Sex, age, and size at baseline were not predictors of enlargement/reduction. At the time of detection, 7.2%, 1.7%, and 1.5% patients had secondary hypogonadism, hypothyroidism, and hypoadrenalism, respectively. Two (0.6%) developed hypopituitarism during follow-up (after progression to macroadenoma). CONCLUSIONS: Probability of micro-NFPA growth is low, and the development of new hypopituitarism is rare. Delaying the first follow-up MRI to 3 years and avoiding hormonal re-evaluation in the absence of tumour growth or clinical manifestations is a safe approach for micro-NFPA surveillance.

Nutritional and mineral analysis of the ultimate wild food plants of Lotkuh, Chitral, the Eastern Hindukush Pakistan.

Wild food plants (WFPs) are designated as functional foods owing to their nutritional potential and as a source of bioactive compounds vital for human health. In times of geopolitical upheaval and nutritional imbalance in mountainous areas of the world, the contribution of WFPs is extraordinary. Lotkuh is a remote mountainous region in the Eastern Hindukush that supports distinctive global plant biodiversity. The documentation and nutritional analysis of the wild edible plants have not yet been subjected to scientific investigation, even though WFPs make up a significant component of the inhabitant's diet. The current study is the first scientific investigation of the nutritional profile of 16 WFPs in the Hindukush region of Pakistan. Plants were collected from different parts of the study area and were subjected to proximate analysis adhering to the standard protocols of AOAC international. Proximate analysis revealed higher moisture in Rheum webbianum (91.5 g/100 g FW) and Oxyria digyna (90.5 g/100 g FW), while Elaeagnus angustifolia had the lowest (25.4 g/100 g FW). Mentha longifolia and Pinus gerardiana had (23.2 g/100 g) and (14.0 g/100 g) protein, whereas Berberis lyceum contained (3.6 g/100 g). Pinus gerardiana had the highest lipid (56.50 g/100 g), followed by Hippophae rhamnoides (45.50 g/100 g), and Berberis lyceum (0.91 g/100 g). Crataegus songarica with high carbohydrate (87.50 g/100 g) was followed by Eremurus stenophyllus (80.83 g/100 g), whereas Berberis lyceum had the least (18.51 g/100 g). High crude fiber (19.33 g/100 g) was found in Ziziphora clinopodiodes followed by Cotoneaster nummularia with (15.50 g/100 g). Pinus gerardiana and Prunus prostrata had low fiber of 1.387 and 1.377 g/100 g. Vitamin C was high in Mentha longifolia (90.63 mg/100 g), Eremurus stenophyllus (86.96 mg/100 g), and Ziziphora clinopodiodes (90.45 mg/100 g). Ca concentration was (948.33 mg/100 g) in Oxyria digyna followed by Cotoneaster nummularia whereas the lowest Ca (20.03 mg/100 g) was recorded in Diospyros lotus. Mg was high in Oxyria digyna (994.00 mg/100 g) and lowest (10.01 mg/100 g) in Diospyros lotus. Berberis lyceum (54.30 mg/100 g), Oxyria digyna (34.33 mg/100 g), and Rheum webbianum (26.04 mg/100 g) had the maximum iron. Mn was high in Berberis lyceum (14.33 mg/100 g), Pinus gerardiana (6.33 mg/100 g), and Elaeagnus angustifolia (4.60 mg/100 g). Prunus prostrata (12.16 mg/100 g), Oxyria digyna (10.30 mg/100 g), and Pinus gerardiana (4.16 mg/100 g) were the leading in Zn concentration whereas Ziziphora clinopodiodes (0.22 mg/100 g). The current study establishes the hitherto unidentified nutritional profile of the WFPs in the area. The prospect of nutritional research on WFPs in the Eastern Hindukush is established by this study.

Effect of growth medium nitrogen and phosphorus on nutritional composition of Lemna minor (an alternative fish and poultry feed).

Duckweed (Lemna minor L.) is an aquatic macrophyte and grows profoundly on the surface of polluted water reservoirs of Pakistan. The plant can be used as a potential alternative for the fish and poultry industry to meet the promptly growing demand for feed. Our study investigates the effect of varying concentrations (ppm) of nutrients like N, P, and their combination, NP on biomass production, carbohydrate, lipid, protein, and mineral (Ca, Mg, Fe, Mn & Zn) contents of L. minor. The varying concentrations of N and P substantially affected the above-stated parameters. The highest biomass yield was recorded in the 30 ppm NP tank as 172 g/m2 day in comparison with the control tank. Higher protein, lipid, and carbohydrate contents were recorded for 30 ppm NP, 20 ppm NP, and 10 ppm NP respectively. Minerals like Ca, Mg, Fe, Mn & Zn increased in 20 ppm P and all N concentrations. The combined application of NP was more effective in boosting the protein, carbohydrate, and lipid content whereas less effective in increasing the mineral contents. A rise in the concentration of N and P showed a positive correlation with the nutritional composition of L. minor.

Novel insights into Pinus species plastids genome through phylogenetic relationships and repeat sequence analysis.

Pinus is one of the most economical and ecological important conifers, model specie for studying sequence divergence and molecular phylogeney of gymnosperms. The less availability of information for genome resources enable researchers to conduct evolutionary studies of Pinus species. To improve understanding, we firstly reported, previously released chloroplast genome of 72 Pinus species, the sequence variations, phylogenetic relationships and genome divergence among Pinus species. The results displayed 7 divergent hotspot regions (trnD-GUC, trnY-GUA, trnH-GUG, ycf1, trnL-CAA, trnK-UUU and trnV-GAC) in studied Pinus species, which holds potential to utilized as molecular genetic markers for future phylogenetic studies in Pinnus species. In addition, 3 types of repeats (tandem, palindromic and dispersed) were also studied in Pinus species under investigation. The outcome showed P. nelsonii had the highest, 76 numbers of repeats, while P. sabiniana had the lowest, 13 13 numbers of repeats. It was also observed, constructed phylogenetic tree displayed division into two significant diverged clades: single needle (soft pine) and double-needle (hard pine). Theoutcome of present investigation, based on the whole chloroplast genomes provided novel insights into the molecular based phylogeny of the genus Pinus which holds potential for its utilization in future studies focusing genetic diversity in Pinnus species.

Isolated splenic sarcoidosis: a rare cause of hypercalcaemia in a patient with type 1 diabetes.

A 30-year-old man was admitted to Intensive Therapy Unit (ITU) with status epilepticus secondary to severe hypoglycaemia on a background of type 1 diabetes. CT of the brain showed generalised cerebral oedema. He was successfully stepped down to a medical ward after 2 weeks. He was noted to have persistently high calcium, which required multiple doses of pamidronate. Parathyroid hormone level was appropriately low. CT of the thorax, abdomen and pelvis showed mild hepatosplenomegaly with small pathological lymph nodes throughout the abdomen. Bone marrow biopsy was unremarkable. Lymph node biopsy was difficult to achieve, and therefore a positron emission tomography scan was arranged. This showed an enlarged and hypermetabolic spleen. Differential diagnoses at this point included lymphoma and sarcoidosis. He underwent diagnostic splenectomy, and the diagnosis of sarcoidosis was confirmed histologically. Calcium level remained normal 17 months after splenectomy.

Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters.

Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.

Effect of salt stress on proximate composition of duckweed (Lemna minor L.).

The shortage of conventional feedstuff is one of the rising issues faced by the developing countries of the world. To bridge the gap between supply and demand of the major feedstuff it is desirable to practice the use of non-conventional feed resources. Duckweeds are the aquatic macrophytes growing in stagnant water bodies that offer a choice to be used as an alternate feed. Before the use of any alternate feed, it is vital to know the nutritional composition of the feed under diverse environmental conditions. The objective of this study was to investigate the influence of salinity, abiotic stress, on the proximate composition of duckweed (Lemna minor L.). The experiment was laid out in Completely Randomized Design (CRD) with 3 repeats. Data was collected on protein, lipid, carbohydrate, and mineral contents. In the laboratory trial plants were grown under the saline condition of different concentrations ranging from 2 g NaCl L-1 to 12 g NaCl L-1 for a growing period of 20 days. The biomasses obtained were tested for proximate composition. ANOVA of the result exhibited a significant effect of salinity on the proximate composition of the plant. Protein residues of the plant started declining above the concentration of 4 g NaCl L-1 until the lowest value was obtained at 12 g NaCl L-1. Lipid composition showed more sensitivity to the stress with a sharp decline above 2 g NaCl L-1 having a minimum value at 12 g NaCl L-1. Carbohydrate contents increased with increasing salinity up to 6 g NaCl L-1 above which a decrease was observed. The highest accumulation of the macronutrients i.e., Ca, Mg, took place in the lower range of concentration of the salt. The percentage compositions of micronutrients such as Fe, Mn, and Zn percentage were reduced at a higher range of salinity while the optimum level was recorded in plants treated with 2 g NaCl L-1, followed by control. The total accumulation of both macro and micronutrients was higher in the plant material treated with a lower level of salt concentration, concluding a significant effect of salinity on proximate composition. As for the Indus water salinity level, the plant has the capacity of tolerance and can be grown without affecting its proximate composition.

Antiviral Activity of Interferon Alpha-Inducible Protein 27 Against Hepatitis B Virus Gene Expression and Replication.

Despite the availability of effective vaccines, hepatitis B virus (HBV) is still a major health issue, and approximately 350 million people have been chronically infected with HBV throughout the world. Interferons (IFNs) are the key molecules in the innate immune response that restrict several kinds of viral infections via the induction of hundreds of IFN-stimulated genes (ISGs). The objective of this study was to confirm if interferon alpha-inducible protein 27 (IFI27) as an ISG could inhibit HBV gene expression and DNA replication both in cell culture and in a mouse model. In human hepatoma cells, IFI27 was highly induced by the stimulation of IFN-alpha (IFN-α), and it potentiated the anti-HBV activity. The overexpression of IFI27 inhibited, while its silencing enhanced the HBV replication in HepG2 cell. However, the knocking out of IFI27 in HepG2 cells robustly increases the formation of viral DNA, RNA, and proteins. Detailed mechanistic analysis of the HBV genome showed that a sequence [nucleotide (nt) 1715-1815] of the EnhII/Cp promoter was solely responsible for viral inhibition. Similarly, the hydrodynamic injection of IFI27 expression constructs along with the HBV genome into mice resulted in a significant reduction in viral gene expression and DNA replication. In summary, our studies suggested that IFI27 contributed a vital role in HBV gene expression and replication and IFI27 may be a potential antiviral agent for the treatment of HBV.

The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression.

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens.

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.