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Increased level of suPAR in acute Epstein-Barr virus infection
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
Abstract No: 1684 Presentation at ESCV 2015: Poster 2 Improvement of the diagnosis of infectious mononucleosis with a modification of the threshold of the Vidas® EBV IgM M. Baccard 1,∗ , A. Foussadier 2 , P. Gradon 2 , P. Morand 3 1 Virology Laboratory, Universitary Hospital Grenoble, France 2 bioMérieux, Marcy l’Etoile, France 3 Virology Laboratory, Universitary hospital Grenoble, University Joseph Fourier Grenoble, France
Background: The diagnosis of Infectious Mononucleosis (IM) linked to an Epstein Barr virus (EBV)-primary infection relies on a specific serological profile showing the presence of IgM and IgG against VCA antigens together with the lack of IgG against EBNA antigens. Sensitivity and specificity of VCA IgM are of special importance to accurately classified EBV infections as primary (PrI); past (PaI); or absence (AI) of EBV infections. The aim of the study was to determine the performance of the commercially available automated VIDAS® EBV IgM reagents with modification of its threshold and grey zone in order to improve the sensitivity of the assay during IM. Methods: 589 samples were classified as PrI (189), PaI (244) or AI (166) on the basis of EBV serology routinely performed in our lab and used as “reference assay” (Enzygnost® for IgG and IgM detection and BMD® for EBNA IgG detection). All sera were tested with VIDAS VCA EBV IgM, EBV VCA/EA IgG and EBNA IgG tests. The Vidas EBV IgM sensitivity and specificity were evaluated versus EBV IgM enzygnost either with the previous recommended threshold and gray zone (i.e. negative: <0.12; equivocal: 0.12–10.18, positive >0.18) or with a new lower threshold (i.e. negative: <0.10; equivocal: 0.10–10.18, positive: >0.18). The agreement between the EBV profiles obtained with 3 Vidas reagents and the reference assays was evaluated. Results: With the new Vidas EBV IgM threshold, the sensitivity was increased from 90.7% [84.9–94.8] to 93.4% [88.2–96.8] without modification of the specificity: 98% [94.2–99.6] for AI and 95.8% [91.2–98.5] for PaI. With the new threshold, the agreements between the Vidas assay and reference assays were 90.0% [84.8–93.8] for PrI, 94.3% [90.6–96.8] for PaI and 95.2% [90.7–97.9] for AI. Conclusions: The new threshold and grey zone of the Vidas VCA IgM is helpful to improve the sensitivity of this marker during EBVprimary infections without any impact on specificity. http://dx.doi.org/10.1016/j.jcv.2015.07.261 Abstract No: 1685 Presentation at ESCV 2015: Poster 2 Development of useful generic tools for the development of real time PCR LDT M. Vignoles ∗ , E.A. Delariviere, P. Marechal, M. Dube, C. Barranger, M. Joannes bioMérieux, Parc Technologique Delta Sud, 09340 VERNIOLLE, France Background: The development of real time PCR Laboratory Developed Test (LDT) implies the combination of a core kit, an Internal control kit and primers and probes specific of the targeted pathogen. bioMérieux developed two generic kits combining internal control and core kits to be used in combination with
S113
either proprietary or commercial primers and probes: DNA Internal Control r-gene® and RNA internal control r-gene® (bioMerieux) associated with specific primers and probes constitute ready to use premixes for the detection of targeted pathogens. We present here two applications: BK primers and probe (LDT for internal purpose) combined with DNA internal control and MERS-HCoV primers and probe (available soon) combined with RNA internal control. Methods: For n tubes of Ready to use premix to prepare, n X 10 L of the initial premix, n X 2.5 L of primers and probe respectively, and reverse transcriptase for RNA parameter were added. These reconstituted premix may be dispastched in plate with easySTREAMTM (bioMérieux). The internal control, added before the extraction step, allows to check both extraction efficiency and presence of inhibitors. MERS-HCoV and BK virus sets were evaluated in combination with a ready-to-use premix for RNA internal control and DNA internal control respectively to constitute a duplex RT PCR assay for MERS-HCoV or BK virus detection. Results: The capability to efficiently detect the inhibition was evaluated for each target (BK, IC2, MERS/HCoV, IC1) with an inhibitor of Real Time PCR. Between 7% and 8% EtOH, MERS/HCoV is not detected at 530 nm whereas a positive result for IC1 is observed at 560 nm which deviates over 3 cycles. At 7% EtOH, the IC2 is not detected whereas a signal at 530 nm is detected. However, delta CT of BK virus compared to the reference is around 9.2 cycles, showing a significant inhibition of the sample. The analytical sensitivity was determined by Probit analysis (MiniTab). The LoD (95% Hit rate detection) was 2.89 log Copies/mL of Invitro transcript for MERSHCoV parameter. The LoD (95% Hit rate detection) was 2.144 log Copies/mL of reconstituted sample for BK virus parameter. Conclusion: The high quality in analytical sensitivity was demonstrated. The Generic Internal Control (DNA and RNA) readyto-use premix will allow to quickly provides a real time detection tool as the simple design of primers and probes is now sufficient to achieve this development. This new tool clearly represents a useful tool in case of outbreak situations or for LDTs application. http://dx.doi.org/10.1016/j.jcv.2015.07.262 Abstract No: 1686 Presentation at ESCV 2015: Poster 2 Increased level of suPAR in acute Epstein-Barr virus infection C.B. Christiansen ∗ , A.K. Ishøy Department of Clinical Microbiology, Rigshospitalet, Denmark Background: When the immune system is activated, cells like monocytes, activated T-cells and macrophages a protein called suPAR is excreted. Increased level of suPAR is found in acute Epstein-Barr virus, EBV, infection.EBV herpes virus found in 90–95% of the Danish population. Method: The level of suPAR was detected in 97 plasma samples from The Department of Clinical Microbiology, Rigshospitalets Biobank, and meassured using an ELISA assay, suPARnostic® . The results was used to calculate a median and compared to biochemical data such as CRP, leukocyte and lymphocyte count, liver enzymes. These data was measured by Department of Clinical Biochemistry, Rigshospitalet. Results: An increased level of suPAR was detected in acute EBV infection, and a median at 5.4 ng/ml was calculated (0.9–26.9). The median for suPAR in healthy persons is <3.0 ng/ml. There was not found any correlations between the biochemical parameters ALAT, ASAT, CPR or lymphocyte count and suPAR. We saw however, a correlation between suPAR and LDH, a correlation between suPAR
S114
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
and EBV DNA level (copies/mL), a correlation between suPAR and the clinical score and a correlation between the clinical score and leukocyte count/lymphocyte count. Conclusion: Measurement of suPAR in plasma can be used as a supplemental marker when evaluating the severity of illness in patients with acute EBV. http://dx.doi.org/10.1016/j.jcv.2015.07.263 Abstract No: 1689 Presentation at ESCV 2015: Poster 2 Interlaboratory comparison of BK virus DNA load assays M. Solis 1,2,∗ , M. Meddeb 1 , C. Sueur 1,2 , P. Domingo-Calap 2 , E. Soulier 2 , A. Chabaud 1 , P. Perrin 3 , B. Moulin 4 , S. Bahram 4 , S. Caillard 3 , F. Stoll-Keller 1 , S. Fafi-Kremer 1 1 Laboratoire de Virologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 2 Inserm UMR S1109, LabEx Transplantex, Fédération de Médecine Translationnelle de Strasbourg (FMTS), France 3 Département de Néphrologie – Transplantation, Hôpitaux Universitaires de Strasbourg, France 4 LabEx Transplantex, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, France
Background: International guidelines recommend screening of kidney transplant recipients for BK virus (BKV) replication and define BKV viremia ≥4 log10 copies/ml as presumptive BKV-associated nephropathy (BKVN) and a cutoff for therapeutic intervention. Hence, BKV DNA load (BKVL) assays need to be comparable to ensure appropriate patient care. Methods: To assess interlaboratory variability in BKV viruria and viremia testing, 27 laboratories were sent 15 (5 urine, 5 whole blood and 5 plasma) and 8 (4 urine, 2 whole blood and 2 plasma) clinical specimens in 2013 and 2014, respectively. Results: Nine BKVL assays were used, including 5 commercial kits and 4 in-house methods. The majority (71%) of laboratories targeted the StAg while the LTAg was targeted by 17% of the laboratories and 3 other techniques used a different target gene (VP1, VP2-VP3 or VP1 + StAg). Assuming that ± 0.5 log10 variation relative to the expected result is acceptable, 68% of the reported results fell within this range. Laboratories using commercial assays reported significantly more results within the acceptable range (76%) than laboratories using in-house assays (39%) (p < 0.0001). High interlaboratory variability was observed, with a variation ranging from 1.73 to 4.65 log10 copies/ml (mean = 2.55 log10 copies/ml). The number of mutations and the distance from the 3 end of the primers largely contributed to this variability, and most mutations (88.2%) were genotype-specific. Genotype II and IV samples displayed higher variability due to polymorphism on the target gene and were incorrectly quantified by all in-house assays. The calibration material also contributed to interlaboratory variability. Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. Furthermore, the extraction apparatus had only a limited impact on interlaboratory variability. Conclusion: Polymorphism of the amplification target gene and the use of different calibration material contribute largely to interlaboratory variability. This variability may significantly impact patient care and calls for presumptive BKVN cutoff reevaluation. The development of an International Standard for BKVL assay calibration and increased awareness of BKV polymorphism would
reduce interlaboratory variability and allow adequate BKV infection monitoring. http://dx.doi.org/10.1016/j.jcv.2015.07.264 Abstract No: 1695 Presentation at ESCV 2015: Poster 2 Emergence of E138 mutations among treatment naïve HIV-infected patients in Istanbul Kenan Midilli 1,∗ , Mert Ahmet Kuskucu 1 , Bilgul Mete 2 , Ozlem Altuntas Aydin 3 , Nergis 1 , Fehmi Tabak 2 ´ Ymamova 1 I.U. Cerrahpasa School of Medicine, Department of Medical Microbiology, Turkey 2 I.U. Cerrahpasa School of Medicine, Department of Infectious Diseases, Turkey 3 Haseki Research and Teaching Hospital Infectious Diseases and Clinical Microbiology Clinic, Turkey
The second generation NNRTIs rilpivirine and etravirine is recently introduced to Turkey and their use is still limited. Although mutations at the position E138 related with resistance against rilpivirine and etravirine result also with decreased susceptibility to other NRTIs which are widely included first line ART, they are not included by the list of WHO 2009 list of mutations for surveillance. We evaluated changes at this position in treatment naïve patients tested between 2009 and early 2015. At our center no changes were detected at E138 until 2009. Beginning from this time-point the incidence of E138 changes tend to increase and make the half of the NNRTI resistance mutations. The ARV resistance testing results of 590 naïve patients were screened for E138 changes retrospectively. E138 changes were limited to genotypes B/F, CRF02AG and B. The distribution of E138 mutations according the years was 1/42 in 2009, 3/68 in 2010, 3/43 2011 + 2012, 3/62 in 2013, 15/175 in 2014 and, 11/141 early 2015. Out of 36 changes at E138, 30 were A, 5 K, and 1G. Five of these 36 changes were accompanied by the changes of K101E (n = 2), V179E (n = 2), and G190E (n = 1). Despite to limited use of rilpivirine and etravirine, the incidence of changes at position E138 increased during the last two years. The increasing trend and restricted genotypic distribution of the E138 changes may be related with the transmission dynamics of HIV-1 in our region suggesting the establishment of transmission networks in certain risk groups. http://dx.doi.org/10.1016/j.jcv.2015.07.265 Abstract No: 1697 Presentation at ESCV 2015: Poster 2 Influence of HCV quantification methods for the detection of low residual viremia in the new treatment era A. Chelly 1,∗ , S. Laperche 2 , S. Akhavan 1 , E.C. Ayme 1 , J.-C. Piot 1 , V. Thibault 1 1
Virology, AP-HP GH PITIE SALPETRIERE, Paris, France 2 Virology, Institut National Transfusion Sanguine, Paris, France Background: New therapeutic combinations based on direct antivirals have revolutionized chronic hepatitis C (CHC) treatment. The high potency of such therapeutics induces a sharp drop of HCV viral load (HCV-VL) leading very often to the detection of little residual viral loads below quantification level and reported as “detected”
Abstract No: 1684 Presentation at ESCV 2015: Poster 2 Improvement of the diagnosis of infectious mononucleosis with a modification of the threshold of the Vidas® EBV IgM M. Baccard 1,∗ , A. Foussadier 2 , P. Gradon 2 , P. Morand 3 1 Virology Laboratory, Universitary Hospital Grenoble, France 2 bioMérieux, Marcy l’Etoile, France 3 Virology Laboratory, Universitary hospital Grenoble, University Joseph Fourier Grenoble, France
Background: The diagnosis of Infectious Mononucleosis (IM) linked to an Epstein Barr virus (EBV)-primary infection relies on a specific serological profile showing the presence of IgM and IgG against VCA antigens together with the lack of IgG against EBNA antigens. Sensitivity and specificity of VCA IgM are of special importance to accurately classified EBV infections as primary (PrI); past (PaI); or absence (AI) of EBV infections. The aim of the study was to determine the performance of the commercially available automated VIDAS® EBV IgM reagents with modification of its threshold and grey zone in order to improve the sensitivity of the assay during IM. Methods: 589 samples were classified as PrI (189), PaI (244) or AI (166) on the basis of EBV serology routinely performed in our lab and used as “reference assay” (Enzygnost® for IgG and IgM detection and BMD® for EBNA IgG detection). All sera were tested with VIDAS VCA EBV IgM, EBV VCA/EA IgG and EBNA IgG tests. The Vidas EBV IgM sensitivity and specificity were evaluated versus EBV IgM enzygnost either with the previous recommended threshold and gray zone (i.e. negative: <0.12; equivocal: 0.12–10.18, positive >0.18) or with a new lower threshold (i.e. negative: <0.10; equivocal: 0.10–10.18, positive: >0.18). The agreement between the EBV profiles obtained with 3 Vidas reagents and the reference assays was evaluated. Results: With the new Vidas EBV IgM threshold, the sensitivity was increased from 90.7% [84.9–94.8] to 93.4% [88.2–96.8] without modification of the specificity: 98% [94.2–99.6] for AI and 95.8% [91.2–98.5] for PaI. With the new threshold, the agreements between the Vidas assay and reference assays were 90.0% [84.8–93.8] for PrI, 94.3% [90.6–96.8] for PaI and 95.2% [90.7–97.9] for AI. Conclusions: The new threshold and grey zone of the Vidas VCA IgM is helpful to improve the sensitivity of this marker during EBVprimary infections without any impact on specificity. http://dx.doi.org/10.1016/j.jcv.2015.07.261 Abstract No: 1685 Presentation at ESCV 2015: Poster 2 Development of useful generic tools for the development of real time PCR LDT M. Vignoles ∗ , E.A. Delariviere, P. Marechal, M. Dube, C. Barranger, M. Joannes bioMérieux, Parc Technologique Delta Sud, 09340 VERNIOLLE, France Background: The development of real time PCR Laboratory Developed Test (LDT) implies the combination of a core kit, an Internal control kit and primers and probes specific of the targeted pathogen. bioMérieux developed two generic kits combining internal control and core kits to be used in combination with
S113
either proprietary or commercial primers and probes: DNA Internal Control r-gene® and RNA internal control r-gene® (bioMerieux) associated with specific primers and probes constitute ready to use premixes for the detection of targeted pathogens. We present here two applications: BK primers and probe (LDT for internal purpose) combined with DNA internal control and MERS-HCoV primers and probe (available soon) combined with RNA internal control. Methods: For n tubes of Ready to use premix to prepare, n X 10 L of the initial premix, n X 2.5 L of primers and probe respectively, and reverse transcriptase for RNA parameter were added. These reconstituted premix may be dispastched in plate with easySTREAMTM (bioMérieux). The internal control, added before the extraction step, allows to check both extraction efficiency and presence of inhibitors. MERS-HCoV and BK virus sets were evaluated in combination with a ready-to-use premix for RNA internal control and DNA internal control respectively to constitute a duplex RT PCR assay for MERS-HCoV or BK virus detection. Results: The capability to efficiently detect the inhibition was evaluated for each target (BK, IC2, MERS/HCoV, IC1) with an inhibitor of Real Time PCR. Between 7% and 8% EtOH, MERS/HCoV is not detected at 530 nm whereas a positive result for IC1 is observed at 560 nm which deviates over 3 cycles. At 7% EtOH, the IC2 is not detected whereas a signal at 530 nm is detected. However, delta CT of BK virus compared to the reference is around 9.2 cycles, showing a significant inhibition of the sample. The analytical sensitivity was determined by Probit analysis (MiniTab). The LoD (95% Hit rate detection) was 2.89 log Copies/mL of Invitro transcript for MERSHCoV parameter. The LoD (95% Hit rate detection) was 2.144 log Copies/mL of reconstituted sample for BK virus parameter. Conclusion: The high quality in analytical sensitivity was demonstrated. The Generic Internal Control (DNA and RNA) readyto-use premix will allow to quickly provides a real time detection tool as the simple design of primers and probes is now sufficient to achieve this development. This new tool clearly represents a useful tool in case of outbreak situations or for LDTs application. http://dx.doi.org/10.1016/j.jcv.2015.07.262 Abstract No: 1686 Presentation at ESCV 2015: Poster 2 Increased level of suPAR in acute Epstein-Barr virus infection C.B. Christiansen ∗ , A.K. Ishøy Department of Clinical Microbiology, Rigshospitalet, Denmark Background: When the immune system is activated, cells like monocytes, activated T-cells and macrophages a protein called suPAR is excreted. Increased level of suPAR is found in acute Epstein-Barr virus, EBV, infection.EBV herpes virus found in 90–95% of the Danish population. Method: The level of suPAR was detected in 97 plasma samples from The Department of Clinical Microbiology, Rigshospitalets Biobank, and meassured using an ELISA assay, suPARnostic® . The results was used to calculate a median and compared to biochemical data such as CRP, leukocyte and lymphocyte count, liver enzymes. These data was measured by Department of Clinical Biochemistry, Rigshospitalet. Results: An increased level of suPAR was detected in acute EBV infection, and a median at 5.4 ng/ml was calculated (0.9–26.9). The median for suPAR in healthy persons is <3.0 ng/ml. There was not found any correlations between the biochemical parameters ALAT, ASAT, CPR or lymphocyte count and suPAR. We saw however, a correlation between suPAR and LDH, a correlation between suPAR
S114
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
and EBV DNA level (copies/mL), a correlation between suPAR and the clinical score and a correlation between the clinical score and leukocyte count/lymphocyte count. Conclusion: Measurement of suPAR in plasma can be used as a supplemental marker when evaluating the severity of illness in patients with acute EBV. http://dx.doi.org/10.1016/j.jcv.2015.07.263 Abstract No: 1689 Presentation at ESCV 2015: Poster 2 Interlaboratory comparison of BK virus DNA load assays M. Solis 1,2,∗ , M. Meddeb 1 , C. Sueur 1,2 , P. Domingo-Calap 2 , E. Soulier 2 , A. Chabaud 1 , P. Perrin 3 , B. Moulin 4 , S. Bahram 4 , S. Caillard 3 , F. Stoll-Keller 1 , S. Fafi-Kremer 1 1 Laboratoire de Virologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 2 Inserm UMR S1109, LabEx Transplantex, Fédération de Médecine Translationnelle de Strasbourg (FMTS), France 3 Département de Néphrologie – Transplantation, Hôpitaux Universitaires de Strasbourg, France 4 LabEx Transplantex, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, France
Background: International guidelines recommend screening of kidney transplant recipients for BK virus (BKV) replication and define BKV viremia ≥4 log10 copies/ml as presumptive BKV-associated nephropathy (BKVN) and a cutoff for therapeutic intervention. Hence, BKV DNA load (BKVL) assays need to be comparable to ensure appropriate patient care. Methods: To assess interlaboratory variability in BKV viruria and viremia testing, 27 laboratories were sent 15 (5 urine, 5 whole blood and 5 plasma) and 8 (4 urine, 2 whole blood and 2 plasma) clinical specimens in 2013 and 2014, respectively. Results: Nine BKVL assays were used, including 5 commercial kits and 4 in-house methods. The majority (71%) of laboratories targeted the StAg while the LTAg was targeted by 17% of the laboratories and 3 other techniques used a different target gene (VP1, VP2-VP3 or VP1 + StAg). Assuming that ± 0.5 log10 variation relative to the expected result is acceptable, 68% of the reported results fell within this range. Laboratories using commercial assays reported significantly more results within the acceptable range (76%) than laboratories using in-house assays (39%) (p < 0.0001). High interlaboratory variability was observed, with a variation ranging from 1.73 to 4.65 log10 copies/ml (mean = 2.55 log10 copies/ml). The number of mutations and the distance from the 3 end of the primers largely contributed to this variability, and most mutations (88.2%) were genotype-specific. Genotype II and IV samples displayed higher variability due to polymorphism on the target gene and were incorrectly quantified by all in-house assays. The calibration material also contributed to interlaboratory variability. Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. Furthermore, the extraction apparatus had only a limited impact on interlaboratory variability. Conclusion: Polymorphism of the amplification target gene and the use of different calibration material contribute largely to interlaboratory variability. This variability may significantly impact patient care and calls for presumptive BKVN cutoff reevaluation. The development of an International Standard for BKVL assay calibration and increased awareness of BKV polymorphism would
reduce interlaboratory variability and allow adequate BKV infection monitoring. http://dx.doi.org/10.1016/j.jcv.2015.07.264 Abstract No: 1695 Presentation at ESCV 2015: Poster 2 Emergence of E138 mutations among treatment naïve HIV-infected patients in Istanbul Kenan Midilli 1,∗ , Mert Ahmet Kuskucu 1 , Bilgul Mete 2 , Ozlem Altuntas Aydin 3 , Nergis 1 , Fehmi Tabak 2 ´ Ymamova 1 I.U. Cerrahpasa School of Medicine, Department of Medical Microbiology, Turkey 2 I.U. Cerrahpasa School of Medicine, Department of Infectious Diseases, Turkey 3 Haseki Research and Teaching Hospital Infectious Diseases and Clinical Microbiology Clinic, Turkey
The second generation NNRTIs rilpivirine and etravirine is recently introduced to Turkey and their use is still limited. Although mutations at the position E138 related with resistance against rilpivirine and etravirine result also with decreased susceptibility to other NRTIs which are widely included first line ART, they are not included by the list of WHO 2009 list of mutations for surveillance. We evaluated changes at this position in treatment naïve patients tested between 2009 and early 2015. At our center no changes were detected at E138 until 2009. Beginning from this time-point the incidence of E138 changes tend to increase and make the half of the NNRTI resistance mutations. The ARV resistance testing results of 590 naïve patients were screened for E138 changes retrospectively. E138 changes were limited to genotypes B/F, CRF02AG and B. The distribution of E138 mutations according the years was 1/42 in 2009, 3/68 in 2010, 3/43 2011 + 2012, 3/62 in 2013, 15/175 in 2014 and, 11/141 early 2015. Out of 36 changes at E138, 30 were A, 5 K, and 1G. Five of these 36 changes were accompanied by the changes of K101E (n = 2), V179E (n = 2), and G190E (n = 1). Despite to limited use of rilpivirine and etravirine, the incidence of changes at position E138 increased during the last two years. The increasing trend and restricted genotypic distribution of the E138 changes may be related with the transmission dynamics of HIV-1 in our region suggesting the establishment of transmission networks in certain risk groups. http://dx.doi.org/10.1016/j.jcv.2015.07.265 Abstract No: 1697 Presentation at ESCV 2015: Poster 2 Influence of HCV quantification methods for the detection of low residual viremia in the new treatment era A. Chelly 1,∗ , S. Laperche 2 , S. Akhavan 1 , E.C. Ayme 1 , J.-C. Piot 1 , V. Thibault 1 1
Virology, AP-HP GH PITIE SALPETRIERE, Paris, France 2 Virology, Institut National Transfusion Sanguine, Paris, France Background: New therapeutic combinations based on direct antivirals have revolutionized chronic hepatitis C (CHC) treatment. The high potency of such therapeutics induces a sharp drop of HCV viral load (HCV-VL) leading very often to the detection of little residual viral loads below quantification level and reported as “detected”