LC–HRMS determination of piperine on rat dried blood spots: A pharmacokinetic study

A liquid chromatography-high resolution mass spectrometry （LC-HRMS） method was developed and validated for the determination of piperine （PPR） on dried blood spots （DBS）. DBS samples were prepared by spiking the whole blood with analyte to produce 30 μL of blood spots on specimen collection cards. Chromatographic separation was achieved on an Atlantis dC18 column using acetonitrile and water （0.1% formic acid） （85：15, v/v） as mobile phase in an isocratic mode of elution at a flow rate of 0.75 mL/min. MS detection was carried out in electrospray positive ion mode for the target ions and monitored at m/z 286.1465 for PPR and 272.1303 for the internal standard （IS）. The developed method exhibited a linear dynamic range over 0.01-2000 ng/mL for PPR on DBS. The overall extraction recovery of PPR from DBS was 92.5%. Influence of hematocrit and spot volume on DBS was also evaluated and found to be well within the acceptable limits. The method was successfully applied to pharmacokinetic studies of PPR in rats.

Dried blood spots,valid screening for viral hepatitis and human immunodeficiency virus in real-life

AIM To detect chronic hepatitis B(CHB),chronic hepatitis C(CHC) and human immunodeficiency virus(HIV) infections in dried blood spot(DBS) and compare these samples to venous blood sampling in real-life.METHODS We included prospective patients with known viral infections from drug treatment centers,a prison and outpatient clinics and included blood donors as negative controls. Five drops of finger capillary blood were spotted on filter paper,and a venous blood sample was obtained. The samples were analyzed for HBs Ag,antiHBc,anti-HBs,anti-HCV,and anti-HIV levels as well as subjected to a combined nucleic acid test(NAT) for HBV DNA,HCV RNA and HIV RNA.RESULTS Samples from 404 subjects were screened(85 CHB,116 CHC,114 HIV and 99 blood donors). DBS had a sensitivity of > 96% and a specificity of > 98% for the detection of all three infections. NAT testing did not improve sensitivity,but correctly classified 95% of the anti-HCV-positive patients with chronic and past infections. Anti-HBc and anti-HBS showed low sensitivity in DBS(68% and 42%).CONCLUSION DBS sampling,combined with an automated analysis system,is a feasible screening method to diagnose chronic viral hepatitis and HIV infections outside of the health care system.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV,HCV,and Syphilis from a Single Dried Blood Spot

Objective The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA,HIV-1 DNA,and HCV RNA using one dried blood spot(DBS)as an alternative sample to plasma.Method A total of 571 paired DBS/plasma samples were collected from men who have sex with men(MSM)and injection drug users(IDUs),and serological and molecular assays were performed.Using plasma results as the reference standard,the performance of DBS tests for HIV-1 RNA,HIV-1 DNA,and HCV RNA was evaluated.Pearson’s correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.Results Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA,five samples(5/32)were not detectable in DBS,while measurable HIV-1 RNA levels were present in plasma(1.44 to3.99 log10 copies/m L).There were two samples(2/94)with undetectable HCV RNA in DBS,while measurable HCV RNA levels were present in plasma(-5 to 5.99 log10 copies/m L).The correlation between HIV-1 RNA light chain variable region(VL)values obtained from plasma and DBS showed that r=0.683(P<0.01),n=27 and r=0.612(P<0.01),n=89 in HCV RNA.Bland-Altman analysis revealed that in HIV-1 RNA,the mean(±SD)difference between HIV-1 RNA in plasma and DBS was 1.00±1.01 log10 copies/m L,and all samples were within±1.96 SD(-0.97 to 2.97 log10 copies/m L)for DBS.The mean difference(±SD)in HCV RNA was 0.15±1.08 log10 copies/m L,and 94.38%(84/89)were within±1.96 SD(-1.96 to 2.67 log10 copies/m L).Overall,HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma.HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma.HIV-1 DNA RT-PCR using a DBS showed acceptable performance.Conclusion The performance of the simultaneous detection of HIV-1 RNA,HIV-1 DNA,and HCV RNA using one DBS was acceptable.DBS,as an alternative sample to plasma,may be a viable option for the simultaneous detection of HIV-1 RNA,HIV-1 DNA,and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Dried blood spot sampling as an alternative for the improvement of hepatitis B and C diagnosis in key populations

BACKGROUND To achieve the elimination of hepatitis B and C,there is an urgent need to develop alternative strategies to increase the access of diagnosis,particularly among key populations such as people living with human immunodeficiency virus(HIV),individuals with coagulopathies and chronic kidney disease(CKD)patients.AIM To evaluate the use of dried blood spot(DBS)in the detection of hepatitis B virus(HBV)and hepatitis C virus(HCV)markers.METHODS A total of 430 individuals comprised of people living with HIV,coagulopathies and CKD provided paired serum and DBS samples.HBsAg,anti-HBc and anti-HCV were tested in those samples using a commercial electrochemiluminescence.Demographic and selected behavioral variables were evaluated to assess possible association with HBV and HCV positivity.RESULTS Using DBS,HBsAg prevalence varied from 3.9%to 22.1%,anti-HBc rates varied from 25.5%to 45.6%and anti-HCV positivity ranged from 15.9%to 41.2%in key populations.Specificities of HBV and HCV tests using DBS varied from 88.9%to 100%.The HBsAg assay demonstrated the best performance in CKD and coagulopathy individuals and the anti-HCV test had a sensitivity and specificity of 100%in people living with HIV.Accuracy of HBV and HCV detection in DBS varied from 90.2%to 100%.In the CKD group,HBsAg positivity was associated with infrequent use of condoms,and anti-HBc positivity was associated with sharing nail cutters/razors/toothbrushes.Anti-HCV reactivity was positively associated with a history of transplantation and length of time using hemodialysis in both specimens.In people living with HIV,only the male gender was associated with anti-HBc positivity in serum and DBS.CONCLUSION DBS with electrochemiluminescence are useful tools for the diagnosis and prevalence studies of hepatitis B and C among key populations and may increase the opportunity to foster prevention and treatment.

Screening of amino acids in dried blood spots by stable isotope derivatization-liquid chromatography-electrospray ionization mass spectrometry

Direct infusion mass spectrometry(DIMS) is a powerful technique in clinical diagnosis for screening neonatal amino acid metabolic disorders from dried blood spots(DBS).However,DIMS sometimes generated false-positive results for analysis of amino acids.In this work,we utilized a stable isotope derivatization method,combining with liquid chromatography tandem mass spectrometry(SID-LC-MS),to improve the specificity for screening amino acids in DBS specimens.A pair of isotope reagents,p-(dimethylamino)phenyl isothiocyanate(DMAP-NCS) and 4-isothiocyanato-N,N-bis(methyl-[2H2])aniline([2H4]DMAP-NCS),was synthesized and used to label amino acids in DBS specimens.The [2H4]DMAP-NCS labelled amino acid standards were used as internal standards to compensate the matrix effect.This method was validated by measuring linearity,recovery and accuracy.The results showed that the developed SID-LC-MS method can be used for sensitive and selective determination of 12 diagnostically important amino acids in DBS specimens.

Detection of Schistosoma mansoni DNA using polymerase chain reaction from serum and dried blood spot card samples of an adult population in North-western Tanzania

Background:Real-time polymerase chain reaction(PCR)is a sensitive and specific method for diagnosing schistosomiasis.However,this method should be performed in a laboratory,usually located distant from the sample collection site.Therefore,it is important to have fast sampling preservation methods,which allow simple transport prior to DNA extraction and amplification.The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR.Methods:A cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria,fanzania.Serum samples and ethylenediaminetetraacetic acid(EDTA)-anticoagulated whole blood for preparation of dried blood spots(DBS)were collected to test for Schistosoma mansoni infection by real-time PCR.A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz(KK)method was used for analysis.Sensitivity and negative predictive value(NPV)were calculated.The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold(Ct)values from serum and DBS.Results:According to the reference,92.5%S.mansoni positive samples were determined.The serum-based real-time PCR performed excellently with 95.4%sensitivity,whereas the DBS-based real-time PCR showed a low sensitivity(45.4%).The Ct-values were significantly higher in DBS(median:37.3)than in serum samples(median:27.5,P<0.001),reflecting a lower parasite-specific DNA load on the filter cards.With increasing egg counts,an increase in sensitivity was observed for all methods.The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100%for medium and severe infections.The DBS real-time PCR showed a sensitivity of only 85.7%even for severe infections.Conclusions:DBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage,storage duration,use of different filter papers and extraction methods before it is used in future studies.In contrast,our results showed that the POC-CCA test is a sensitive and precise test for detecting S.mansoni infections.

Comparative study on anti-HCV testing using plasma, dried plasma spots (DPS), and dried blood spots (DBS)

The aim of this study was to evaluate the performance of an assay using dried plasma spot(DPS)and dried blood spot(DBS)samples for the serological detection of anti-hepatitis C virus(HCV)antibodies.Between January and July 2019,plasma,DPS and DBS specimens were collected from individuals at high-risk for HCV infection.Samples were tested for anti-HCV by ELISA,and the performance of DPS and DBS specimens was examined using results from the plasma testing,as the standard.Blood samples were collected from 329 persons,including 129 men who have sex with men and 200 intravenous drug users.Results from the plasma testing indicated that 118 samples(59.0%)were HCV positive.Data from the DPS sample testing showed sensitivity as 99.2%(95%confidence interval[CI]:0.95-1.00)and specificity as 100%(95%CI:0.98-1.00)for HCV detection,with Kappa of 99.3%(95%CI:0.98-1.00)while in DBS sample testing the sensitivity as 98.3%(95%CI:0.93-1.00)and specificity as 100%(95%CI:0.98-1.00),with Kappa of 98.7%(95%CI:0.97-1.00),respectively.Spearman’s correlation coefficients for the comparisons between plasma and DPS specimen,plasma and DBS specimens,DPS and DBS specimens were 0.857,0.750,and 0.739,respectively.Compared with the results in plasma,1 sample was not detected using the DPS specimens,and 2 samples were failed for the positive detection,using the DBS specimens.Both DPS and DBS samples were promising alternatives to plasma,for the detection of anti-HCV antibodies.

Performance of clinical signs and symptoms,rapid and reference laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea:a prospective diagnostic accuracy study

Background Passive diagnosis of human African trypanosomiasis(HAT)at the health facility level is a major component of HAT control in Guinea.We examined which clinical signs and symptoms are associated with HAT,and assessed the performance of selected clinical presentations,of rapid diagnostic tests(RDT),and of reference laboratory tests on dried blood spots(DBS)for diagnosing HAT in Guinea.Method The study took place in 14 health facilities in Guinea,where 2345 clinical suspects were tested with RDTs(HAT Sero-K-Set,rHAT Sero-Strip,and SD Bioline HAT).Seropositives underwent parasitological examination(reference test)to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay(ELISA)/Trypanosoma brucei gambiense,trypanolysis,Loopamp Trypanosoma brucei Detection kit(LAMP)and m18S quantitative PCR(qPCR).Multivariable regression analysis assessed association of clinical presentation with HAT.Sensitivity,specificity,positive and negative predictive values of key clinical presentations,of the RDTs and of the DBS tests for HAT diagnosis were determined.Results The HAT prevalence,as confirmed parasitologically,was 2.0%(48/2345,95%CI:1.5–2.7%).Odds ratios(OR)for HAT were increased for participants with swollen lymph nodes(OR=96.7,95%CI:20.7–452.0),important weight loss(OR=20.4,95%CI:7.05–58.9),severe itching(OR=45.9,95%CI:7.3–288.7)or motor disorders(OR=4.5,95%CI:0.89–22.5).Presence of at least one of these clinical presentations was 75.6%(95%CI:73.8–77.4%)specific and 97.9%(95%CI:88.9–99.9%)sensitive for HAT.HAT Sero-K-Set,rHAT Sero-Strip,and SD Bioline HAT were respectively 97.5%(95%CI:96.8–98.1%),99.4%(95%CI:99.0–99.7%)and 97.9%(95%CI:97.2–98.4%)specific,and 100%(95%CI:92.5–100.0%),59.6%(95%CI:44.3–73.3%)and 93.8%(95%CI:82.8–98.7%)sensitive for HAT.The RDT’s positive and negative predictive values ranged from 45.2–66.7%and 99.2–100%respectively.All DBS tests had specificities≥92.9%.While LAMP and m18S qPCR sensitivities were below 50%,trypanolysis and ELISA/T.b.gambiense had sensitivities of 85.3%(95%CI:68.9–95.0%)and 67.6%(95%CI:49.5–82.6%).Conclusions Presence of swollen lymph nodes,important weight loss,severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in HAT endemic areas in Guinea.Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy.Trypanolysis on DBS may discriminate HAT patients from false RDT positives.

Performance of the procedure for ultra-rapid extraction and loop-mediated isothermal amplifcation (PURE-LAMP) method to detect malaria in Haiti

Background Malaria continues to cause burden in various parts of the world.Haiti,a Caribbean country,is among those aiming to eliminate malaria within a few years.Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction-loop-mediated isothermal amplifcation(PURE-LAMP)method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission.Methods Febrile and afebrile people were recruited from three administrative divisions within Haiti:Nippes,Sud and Grand’Anse,during the summers of 2017(early August to early September)and 2018(late July to late August).Their blood samples were tested by microscopy,rapid diagnostic tests(RDT),PURE-LAMP and nested PCR to detect Plasmodium infection.Sensitivity,specifcity,positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard.Results Among 1074 samples analyzed,a positive rate of 8.3%was calculated based on the nested PCR results.Among febrile participants,the rates in 2017 and 2018 were 14.6%and 1.4%,respectively.Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR,and all three were from the same locality.There was no afebrile participants recruited in 2017.The PURE-LAMP,RDT and microscopy had respective sensitivities of 100%,85.4%and 49.4%.All of the testing methods had specifcities over 99%.Conclusions This study confrmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.

Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions

This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using direct polymerase chain reaction(PCR)amplification workflows.The commercially available PowerPlex21(PP21)System(Promega,Wisconsin,USA),which follows similar direct workflows,was used as a reference.Anticoagulate blood applied to chemically impregnated FTATM Micro Cards(GE Healthcare UK Limited,Amersham Place,Little Chalfont,Buckinghamshire,HP79NA,UK)was used to represent a complex biological sample.Allelic concordance,first‑pass success rate,average peak heights,heterozygous peak height ratios(HPHRs),and intracolor and intercolor peak height balance were determined.In reduced volume PCR reactions,the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System.The level of performance was maintained at PCR reaction volumes,which are 40%of that recommended.The EX22 and PP21 System kits possess comparable overlapping genome coverage.This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTATM Micro Cards.Allelic concordance,first‑pass success rate,average peak heights,HPHRs,and intracolor and intercolor peak height balance were determined.A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full,half,and reduced PCR reaction volumes.The PP21 System(Promega)was used as a reference kit.Where appropriate,the distributions of data were assessed using the Shapiro‑Wilk test.For normally‑distributed data,statistics were calculated using analysis of variance(ANOVA)and for nonparametric data the Wilcoxon/Kruskal‑Wallis test was used.Statistical significance was set at P<0.05.Confidence intervals for mean values were set at 95%.On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards,both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases.The performance of both EX16 and EX22 was comparable to that of the PP21 System.This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.

HIV screening and retention in care in people who use drugs in Madrid, Spain: a prospective study

Background::The burden of human immunodeficiency virus(HIV)infection in people who use drugs(PWUD)is significant.We aimed to screen HIV infection among PWUD and describe their retention in HIV care.Besides,we also screen for hepatitis C virus(HCV)infection among HIV-seropositive PWUD and describe their linkage to care.Methods::We conducted a prospective study in 529 PWUD who visited the"Ca?ada Real Galiana"(Madrid,Spain).The study period was from June 1,2017,to May 31,2018.HIV diagnosis was performed with a rapid antibody screening test at the point-of-care(POC)and HCV diagnosis with immunoassay and PCR tests on dried blood spot(DBS)in a central laboratory.Positive PWUD were referred to the hospital.We used the Chi-square or Fisher’s exact tests,as appropriate,to compare rates between groups.Results::Thirty-five(6.6%)participants were positive HIV antibodies,but 34 reported previous HIV diagnoses,and 27(76%)had prior antiretroviral therapy.Among patients with a positive HIV antibody test,we also found a higher prevalence of homeless(P<0.001)and injection drug use(PWID)(P<0.001),and more decades of drug use(P=0.002).All participants received HIV test results at the POC.Of the 35 HIV positives,28(80%)were retained in HIV medical care at the end of the HIV screening study(2018),and only 22(62.9%)at the end of 2020.Moreover,12/35(34.3%)were positive for the HCV RNA test.Of the latter,10/12(83.3%)were contacted to deliver the HCV results test(delivery time of 19 days),5/12(41.7%)had an appointment and were attended at the hospital and started HCV therapy,and only 4/12(33.3%)cleared HCV.Conclusions::We found almost no new HIV-infected PWUD,but their cascade of HIV care was low and remains a challenge in this population at risk.The high frequency of active hepatitis C in HIV-infected PWUD reflects the need for HCV screening and reinforcing the link to care.