Overview

Reference diagnostics at the SNCR for HIV and other retroviruses
Retroviral diagnostics deals with the following three basic questions:
  • Is an individual infected with a retrovirus?
  • If yes, how active is the infection i.e. how high is the viral load?
  • What are the genetic and biological characteristics of the virus?
The first two questions can be answered by means of either direct or indirect test methods. Direct tests detect specific viral components e.g. viral proteins, DNA or RNA. In addition to answering diagnostic questions, these tests can also provide information on viral replicative activity in a confirmed infection. Viral components can also be detected following in vitro virus cultivation, a procedure which simultaneously determines viral replicative capacity. In contrast, indirect tests examine the immune response of an infected person, particularly the appearance of specific antibodies.
Virus characterization consists of HIV-subtype determination and analysis of antiretroviral drug resistance. The following summarizes methodologies currently implemented at the SNCR.
 
Note: The SNCR does NOT undertake HIV-screening (with the exception of paediatric HIV-infection).
 
Review articles published by the SNCR regarding retroviral diagnostics:
  1. Schüpbach J. Biologie und Diagnostik der HIV-Infektion. Labor und Medizin 1998; 25:204-209.
  2. Schüpbach J. Human Immunodeficiency Viruses. In: Manual of Clinical Microbiology, P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken eds., ASM PRESS, 7th edition, 1999, pp. 847-870.
  3. Schüpbach J. and Gallo R.C. Human Retroviruses. In: Clinical Virology Manual, Specter S., Hodinka R.L .and Young S.A. eds., ASM PRESS, 3rd edition, 2000, pp. 513-560
  4. Schüpbach J. Human Immunodeficiency Viruses. In: Manual of Clinical Microbiology, P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken eds., ASM PRESS, 8th edition, 2003, pp. 1253-1281.
  5. Schüpbach J. SHCS and the laboratory diagnosis of HIV infection - from the development of the HIV Western blot to virus quantification and clinically relevant individual virus characterization. Ther. Umsch. 61: 603-607, 2004.




Indirect (confirmation) tests for detecting retroviral infection
Western Blot (for HIV-1, HIV-2, HTLV-2, HTLV-2).
 
This method allows sensitive detection of antibodies against a range of viral proteins and is used worldwide as a test to confirm retroviral infection. For the evaluation of results, four different organizations in the United States have established recommendations: the CDC (Centers for Disease Control), the CRSS (Consortium for Retrovirus Serology Standardization), the ARC (American Red Cross) and the FDA (Federal Drug Administration). The SNCR automatically tests HIV-1 Western Blots based on all criteria suggested by these organizations. The SNCR considers a result positive when at least three of four recommendations score a specimen positive.
 
Western Blots are suitable for confirmation of infection with HIV-1, HIV-2, HTLV-1, and HTLV-2 in adults. In contrast, applicability to paediatric infection is limited by the fact that maternal IgG is passed into the child's blood through active transplacental transport i.e. IgG Western Blots during the first year will be heavily influenced by the presence of maternal antibodies. Therefore, the diagnosis of HIV in children from HIV-positive mothers is almost exclusively based on direct virus testing.
 
Line Immunoassay InnoLia HIV-I/II Score.

Use of recombinant HIV proteins as test antigens has led to marked improvement in the sensitivity and specificity of ELISA-based screening tests (so-called "2nd generation tests"). The use of defined concentrations of recombinant proteins or synthetic peptides should conceivably also lead to equivalent improvement with Western Blots, although the diagnostic industry has yet to fully embrace this possibility. An exception is the InnoLia HIV-I/II Score Test, from the company Innogenetics, which we have applied increasingly since 2004 to both confirm and differentiate HIV-1 and HIV-2 infections.
Original work published by SNCR staff regarding development and application of Western Blots for retroviral diagnostics:
  1. Schüpbach, J., Popovic, M., Gilden, R.V., Gonda, M.A., Sarngadharan, M.G., & Gallo, R.C. Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS. Science 224: 503-505, 1984.
  2. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpbach, J., & Gallo, R.C. Antibodies reactive with human T-Lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224: 506-508, 1984.
  3. Safai, B., Sarngadharan, M.G., Groopman, J.E., Arnett, K., Popovic, M., Slisky, A., Schüpbach, J., & Gallo, R.C. Seroepidemiological studies of HTLV-III in AIDS. Lancet June 30 1(8392): 1438-1440, 1984.
  4. Schüpbach, J., Haller, O., Vogt, M., Lüthy, R., Oelz, O., Joller, H., Sarngadharan, M.G., & Gallo, R.C. Antibodies to HTLV-III in Swiss patients with AIDS, pre-AIDS, and in groups at risks for AIDS. N. Engl. J. Med. 312: 265-270, 1985.
  5. Schüpbach, J., Vogt, M., Bhushan, R., Lüthy, R., Haller, O., Joller, H., Ferber, T., Büchner, S., Schuppli, R., Schädelin, J., Hirschel, B., Cruchaud, A., Stroun, J., Frei, P., & Glauser, M.P. Prävalenz von Antikörpern gegen HTLV-III in verschiedenen Regionen der Schweiz. Schweiz. med. Wschr. 115: 1048-1054, 1985.
  6. Resnick, L., di Marzo-Veronese, F., Schüpbach, J., Tourtellotte, W.W., Ho, D.D., Müller, F., Shapshak, P., Vogt, M., Groopman, J.E., Markham, P.D., & Gallo, R.C. Intra-blood-brain-barrier synthesis of HTLV-III specific IgG in patients with AIDS or AIDS-related complex. N. Engl. J. Med. 313: 1498-1504, 1985.
  7. Schüpbach, J., and Tanner, M. Specificity of human immunodeficiency virus (LAV/HTLV-III)-reactive antibodies in African sera from southeastern Tanzania. Acta Tropica 43: 195-206, 1986.
  8. Schüpbach, J., Baumgartner, A., & Tomasik, Z. HTLV-1 in Switzerland: Low prevalence of specific antibodies in HIV risk groups, high prevalence of cross-reactive antibodies in normal blood donors. Int. J. Cancer 42: 857-862, 1988.
  9. Schüpbach, J., Wunderli, W., Kind, C., Kernen, R., Baumgartner, A., & Tomasik, Z. Frequent detection of HIV- and IgG-specific IgM and IgA antibodies in HIV-positive cord blood sera: fine analysis by Western blot. AIDS 3: 583-589, 1989.
  10. Schüpbach J, Tomasik Z, Jendis J, Böni J, Seger R, Kind C. IgG, IgM and IgA seroconversion to HIV in infants born to HIV-1 infected mothers. J. AIDS 1994; 7:421-427.
  11. Böni J, Bisset LR, Burckhardt JJ, Joller-Jemelka HI, Bürgisser P, Perrin L, Gorgievski M, Erb P, Fierz W, Piffaretti J-C, Schüpbach J. Prevalence of human T-cell leukemia virus (HTLV) types I and II in Switzerland. J. Med. Virol. 2004 ; 72:328-37.




Direct tests for detecting retroviral infection
A. Ultrasensitive antigen test for HIV-1 p24.
 
In the presence of HIV-specific antibodies, the formation of immune complexes (p24/anti-p24) makes testing difficult (unduly low concentrations or underdetection). Immunoglobulin-specific antibodies, with similarity to rheumatoid factor, can also cause problems as they result in overly high concentrations or overdetection. Using a simple but highly efficient method developed at the SNCR (boiling diluted plasma for five minutes) all interfering antibodies are eliminated, releasing p24 so that it can be detected by ELISA at its true concentration.To measure p24, the SNCR employs a highly sensitive method incorporating signal amplification. In the presence of 50 µL of plasma, the detection limit of this approach is approximately 0.5 pg/mL.
 
Positive results from antigen tests used for diagnostic purposes are confirmed by means of a neutralization test (>50% neutralization = positive), a procedure which allows achievement of practically 100% specificity. In a retrospective study undertaken at the SNCR with HIV-1 infected adults at every stage of disease progression, test sensitivity using 50µL of plasma was 97.8% and was equivalent to a RNA-PCR with a detection limit of 400 copies/mL. Similarly with pediatric HIV-infection, the diagnostic sensitivity of this approach is equivalent to RNA-PCR (100% diagnostic sensitivity at >10 days; 50% diagnostic sensitivity at <10 days). By means of systematic application of neutralization testing, a virtual 100% specificity is reached. However, with newborns a few weeks old, an isolated positive result with the antigen test (in conjunction with negative DNA-PCR and RNA-PCR results) should be interpreted with caution; p24, in the form of immune complexes with IgG antibodies originating from transfer via the placenta or through microtransfusion from the mother, may be present in the newborn's circulation without conferring an infection.
 
Work published by the SNCR regarding the development and application of HIV-1 p24 antigen tests:
  1. Schüpbach J and Böni J. Quantitative and sensitive detection of immune-complexed and free HIV antigen after boiling of serum. J. Virol. Methods 1993; 43:247-256.
  2. Schüpbach J, Böni J, Tomasik Z, Jendis J, Seger R, Kind C, and the Swiss Neonatal HIV Study Group. Sensitive detection and early prognostic significance of antigen p24 in heat-denatured plasma of human immunodeficiency virus type 1-infected infants. J. Infect. Dis. 1994; 170:318-324.
  3. Schüpbach J, Flepp M, Pontelli D, Tomasik Z, Lüthy R, Böni J. Heat-mediated immune complex dissociation and ELISA signal amplification render antigen p24 detection in plasma as sensitive as HIV-1 RNA detection by polymerase chain reaction. AIDS 1996; 10:1085-1090.
  4. Böni J, Opravil M, Tomasik Z, Rothen M, Bisset L, Grob PJ, Lüthy R, Schüpbach J. Simple monitoring of antiretroviral therapy with a signal-amplification–boosted human immunodeficiency virus type 1 p24 antigen assay with heat-denatured plasma. AIDS 1997; 11:F47-F52.
  5. Schüpbach J, Böni J. Sensitivity of heat-denatured p24 antigen in the diagnosis of pediatric HIV infection. J. AIDS Hum. Retrovirol. 1998;18:399-400.
  6. Nadal D, Böni J, Kind C, Varnier OE, Steiner F, Tomasik Z, Schüpbach J. Prospective evaluation of amplification–boosted ELISA for heat-denatured p24 antigen for diagnosis and monitoring of paediatric HIV-1 infection. J. Infect. Dis. 1999;180:1089-1095.
  7. Schüpbach J and Varnier OE. HIV-1 p24 antigen - a sensitive and precise, yet inexpensive alternative to PCR for viral DNA or RNA. International AIDS Society Newsletter Nr. 15, April 2000, 9-10.
  8. Schüpbach J, Tomasik Z, Nadal D, Flepp M, Ledergerber B, Opravil M, Böni J. Use of HIV-1 p24 as a sensitive, precise and inexpensive marker for infection, disease progression and treatment failure. Int. J. Antimicrob. Agents 2000; 16: 441-445.
  9. Ledergerber B, Flepp M, Böni J, Tomasik Z, Lüthy R and Schüpbach J. HIV-1 p24 concentration measured by boosted ELISA of heat-denatured plasma correlates with CD4+ decline, progression to AIDS and survival: Comparison with viral RNA. J. Infect. Dis. 2000; 181:1280-1288.
  10. Schüpbach J, Böni J, Flepp M, Tomasik Z, Joller H, Opravil M. Antiretroviral treatment monitoring with an improved HIV-1 p24 antigen test: an inexpensive alternative to tests for viral RNA. J. Med. Virol. 2001; 65:225-232.
  11. Schüpbach J. Measurement of HIV-1 p24 antigen by signal-amplification-boosted ELISA of heat-denatured plasma is a simple and inexpensive alternative to tests for viral RNA. AIDS Rev. 2002;4(2):83-92.
  12. Sterling TR, Hoover DR, Astemborski J, Vlahov D, Bartlett JG, Schüpbach J. Prognostic value of heat-denatured HIV-1 p24 antigen and correlation with plasma HIV-1 viral load and CD4+ T-lymphocyte level in adults. J. Infect. Dis. 2002; 186:1181-1185.
  13. Schüpbach J, Böni J, Bisset LR, Tomasik Z, Fischer M, Günthard H, Ledergerber B, Opravil M for the Swiss HIV Cohort Study. HIV-1 p24 antigen is an independent correlate of CD4 T-cell change in patients on successful long-term antiretroviral therapy. J. AIDS 2003; 33:292-299.
  14. Ribas SG, Ondoa P, Schüpbach J, van der Groen G, Fransen K. Performance of a quantitative human immunodeficiency virus type 1 p24 antigen assay on various HIV-1 subtypes for the follow-up of human immunodeficiency type 1 seropositive individuals. J. Virol. Methods 2003; 113:29-34.
  15. Schüpbach J. Viral RNA and p24 antigen as markers of HIV disease and antiretroviral treatment success. Int. Arch. Allergy Immunol. 2003; 132:196-209.
  16. Schüpbach J, Günthard H, Joos B, Fischer M, Böni J, Tomasik Z, Yerly S., Perrin L, Battegay M, Furrer H, Vernazza P, Bernasconi E, Hirschel B. HIV-1 p24 may persist during long-term highly active antiretroviral therapy, increases little during short treatment breaks, and its rebound after treatment stop correlates with CD4+ T cell loss. J. Acquir. Immune. Defic. Syndr. 2005; 40:250–256.
  17. Jennings C, Fiscus SA, Crowe SM, Danilovic AD, Morack RJ, Scianna S, Cachafeiro A, Brambilla DJ, Schüpbach J, Stevens W, Respess R, Varnier OE, Corrigan GE, Gronowitz JS, Ussery MA, Bremer JW. Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay. J. Clin. Microbiol. 2005; 43:5950-5956.
  18. Knuchel MC, Tomasik Z, Speck RF, Luthy R, Schüpbach J. Ultrasensitive quantitative HIV-1 p24 antigen assay adapted to dried plasma spots to improve treatment monitoring in low-resource settings. J. Clin. Virol. 2006; 36:64-67.
  19. Brinkhof MWG, Böni J, Steiner F, Tomasik Z, Nadal D, Schüpbach J. Evaluation of p24-based antiretroviral treatment monitoring in pediatric HIV-1 infection: prediction of the CD4+ T cell changes between consecutive visits. J. Acquir. Imm. Defic. Syndr. 2006; 41:557-562.
  20. Schüpbach J, Tomasik Z, Knuchel M, Opravil M, Gunthard HF, Nadal D, Böni J. Optimized virus disruption improves detection of HIV-1 p24 in particles and uncovers a p24 reactivity in patients with undetectable HIV-1 RNA under long-term HAART. J. Med. Virol. 2006;78:1003-1010.
  21. Tehe A, Maurice C, Hanson DL, Borget MY, Abiola N, Maran M, Yavo D, Tomasik Z, Böni J, Schüpbach J, Nkengasong JN. Quantification of HIV-1 p24 by a highly improved ELISA: an alternative to HIV-1 RNA based treatment monitoring in patients from Abidjan, Cote d'Ivoire. J. Clin. Virol. 2006;37:199-205.
  22. Knuchel MC, Jullu B, Shah C, Tomasik Z, Stoeckle MP, Speck RF, Nadal D, Mshinda H, Böni J, Tanner M, Schüpbach J. Adaptation of the ultrasensitive HIV-1 p24 antigen assay to dried blood spot testing. J. Acquir. Immune Defic. Syndr. 2007;44:247-253.

B. DNA-PCR (for HIV-1, HIV-2, HTLV-1, HTLV-2).
 
Verification of viral DNA (e.g. cell-associated virus) using the Polymerase Chain Reaction. Using this approach, virus-specific DNA is selectively amplified and the product analyzed using techniques based on hybridization with specific probes (ELISA or real time detection technologies). In order to optimize the sensitivity and specificity of this approach, it is possible to analyse several regions within a viral genome.
 
The detection limit of DNA-PCR carried out at the SNCR for all human retroviruses is in the vicinity of 1 copy/reaction. For HIV-1, the specificity of a positive result is 100% and the diagnostic sensitivity among HIV-positive adults is 96 - 98%. With pediatric HIV-infection (newborns/children >10 days old), the diagnostic sensitivity, demonstrated by the SNCR in a prospective study, was 100%. In view of a low prevalance in Switzerland, no definitive statistical analysis has been undertaken for DNA-PCR specific for other retroviruses.
 
For the verification of HIV-2, HTLV-1 and HTLV-2 infections, DNA-PCR is the method of choice.
 
 
C. MEGA PCR for HIV-1 DNA.
 
Simple calculation shows that, even at the highest achievable analytical PCR sensitivity of a single copy, millions of HIV-infected cells could conceivably still be present in the immune system despite a negative test result. With development at the SNCR of the so-called MEGA-PCR, an additional significant increase in DNA-PCR sensitivity has been achieved. The analytic detection limit of this method lies at 5 copies of HIV-1 DNA in a sample consisting of up to 500 ug DNA. When compared to standard DNA-PCR approaches capable of detecting 1 copy of HIV-1 DNA in 1 µg DNA, this results in a 125 - 250-fold increase in analytic sensitivity. The MEGA-PCR is thus suitable for confirmation of HIV-infection in adults even in the presence of a low viral load. The MEGA-PCR is also suitable for analyzing tissue. However, in children this method is of limited practical value in view of the difficulty of obtaining large blood samples.

Furthermore, the SNCR has developed a MEGA-PCR for animal retroviruses. Of particular importance with regard to xenotransplantation, in which animal tissue/organs (particularly from pigs) are transplanted into humans, is the MEGA-PCR for PERV, the porcine endogenous retrovirus. Within the framework of future clinical studies, the SNCR has developed this test as a means to virologically control both patients undergoing porcine xenotransplantation and close contacts.

A MEGA-PCR for the detection of lentiviruses in sheep and goats (SRLV, small ruminant lentiviruses) has also been developed for veterinary medicine purposes.


D. RNA-PCR for HIV-1, HIV-2 (HTLV-1, HTLV-2).

Unlike DNA-PCR, RNA-PCR is used to detect either virus particles in various body fluids or intracellular viral RNA. The method developed at the SNCR guarantees a detection limit of 5 - 10 RNA copies. However, this approach is not suitable for diagnosing HTLV-1 and HTLV-2 as these infections are not normally associated with a viremia.

a. HIV-1 Virus Load. Quantification of HIV-RNA in plasma (viral load determination for prognosis, therapy initiation and monitoring) is currently a standard procedure employed in the care of HIV-infected patients. At the SNCR we work with a standard plasma sample of 200 µL. Generally, this volume is also available from small children, for whom larger volumes would be problematic to obtain. Incorporating this sample volume and using the Roche Amplicor HIV-1 Monitor tests (version 1.5), the guaranteed limit of of this approach is between 25 - 50 copies/mL. However, in practise, smaller virus concentrations may also be detectable. A further decrease in the detection limit can be achieved by enlarging the sample volume. The precision of this method is the order of a factor of 3 i.e. half a log10.

b. Virus Load for HIV-isolates that are not recognized by commercial tests. Despite considerable improvement in the ablitiy of newer versions of the Roche Amplicor HIV-1 Monitor tests (version 1.5) to recognizing divergent subtypes, some HIV-isolates can still not be adequately quantified. In particular, viruses of the group O, as well as HIV-2, are systematically not detected. In addition, different group M subtypes (subtypes A-K) demonstrate occasional isolates which are measured at too low a concentration. Such cases can be suspected when, despite a low HIV RNA-concentration in the plasma, a clinical progression is documented or an advanced stage of disease is present. In these cases, testing with a PERT Assay can be applied to determine the concentration of particles. A PERT Assay quantifies HIV particles based on the Reverse Transcriptase contained in them (see below).

SNCR publications regarding refinement of PCR tests:
  1. Böni J and Schüpbach J. Primer extension analysis provides a sensitive tool for the identification of PCR-amplified DNA from HIV-1. J. Virol. Methods 1993; 42:309-322.
  2. Böni J, Leib SL, Emmerich B, Wiestler OD, Schüpbach J, Kleihues P. PCR identification of viral DNA in brain tissue of patients with HIV-1 encephalopathy. Neurology 1993;43:1813-1817.
  3. Böni J and Schüpbach J. Sensitive and quantitative detection of PCR-amplified HIV-1 DNA products by an enzyme linked immunoassay following solution hybridization with two differently labeled oligonucleotide probes. Mol. Cell. Probes 1993; 7:361-371.
  4. Günthard HF, Gowland P, Schüpbach J, Fung MSC, Böni J, Liou R-S, Chang NT, Grob P, Graepel P, Braun DG, Lüthy R. A phase I/IIA clinical study with a monoclonal chimeric mouse/human antibody to the V3 loop of HIV-1 gp120. J. Infect. Dis. 1994;170:1384-1393.
  5. Schüpbach J, Böni J, Tomasik Z, Jendis J, Seger R, Kind C and the Swiss Neonatal HIV Study Group. Sensitive detection and early prognostic significance of antigen p24 in heat-denatured plasma of human immunodeficiency virus type 1-infected infants. J. Infect. Dis. 1994; 170:318-324.
  6. Böni J. PCR detection of HIV. Methods in Mol. Biol. 1996;50:93-107.
  7. Nadal D, Böni J, Kind C, Varnier OE, Steiner F, Tomasik Z, Schüpbach J. Prospective evaluation of amplification–boosted ELISA for heat-denatured p24 antigen for diagnosis and monitoring of paediatric HIV-1 infection. J. Infect. Dis. 1999;180:1089-1095.
  8. Ledergerber B, Flepp M, Böni J, Tomasik Z, Lüthy R and Schüpbach J. HIV-1 p24 concentration measured by boosted ELISA of heat-denatured plasma correlates with CD4+ decline, progression to AIDS and survival: Comparison with viral RNA. J. Infect. Dis. 2000; 181:1280-1288.
  9. Bisset LR, Bosbach S, Tomasik Z, Lutz H, Schüpbach J, Böni J. Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation. J. Virol. Methods. 2001; 91:149-55.
  10. Shah CA, Böni J, Bisset LR, Seebach JD, Schüpbach J. Ultra-sensitive and specific detection of porcine endogenous retrovirus (PERV) using a sequence-capture real-time PCR approach. J. Virol. Methods 2003; 109:209-216.
  11. Böni J, Shah C, Flepp M, Luthy R, Schüpbach J. Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR). J. Med. Virol. 2004; 72:1-9.
 
E. PERT Assay (Product-Enhanced Reverse Transcriptase Assay; dectects all infectious retroviruses).
Developed at the SNCR, the PERT assay allows detection and quantification of all infectious retroviruses based on the reverse transcriptase enzyme present in virus particles. The sensitivity of the test is roughly equivalent to RNA-PCR (400 copies/mL) and is used to clarify cases of suspected HIV-infection not detectable with more traditional tests or in the case of suspected infection with an unknown retrovirus. Applicable sample materials include plasma (-70°C), cerebrospinal fluid and other body fluids. The PERT assay can be further used in the control of biomedical products (vaccines, coagulating agents, etc.) for the absence of retroviruses. It should be noted that the test can only detect retrovirus particles, not unexpressed proviruses.
 
SNCR publications regarding the PERT Assay:
  1. Pyra H, Böni J, and Schüpbach J. Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc. Natl. Acad. Sci. USA 1994; 91:1544-1548.
  2. Böni J, Pyra H, Schüpbach J. Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1 infected individuals by the product-enhanced reverse transcriptase assay. J. Med. Virol. 1996; 49:23-28.
  3. Schüpbach J, Pyra H, Jendis J., Tomasik Z, Böni J. Isolation of an in vitro transmissible agent with reverse transcriptase activity from a blood donor with a borderline-positive HIV-1 serology for more than five years. Clin. Diagn. Virol. 1996; 5: 197-203.
  4. Böni J, Stalder J, Reigel F, Schüpbach J. Detection of reverse transcriptase activity in live attenuated virus vaccines. Clin. Diagn. Virol. 1996; 5:43-53.
  5. Weissmahr RN, Schüpbach J, Böni J. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA. J. Virol. 1997; 71: 3005-3012.
  6. Conrad B, Weissmahr RN, Böni J, Arcari R, Schüpbach J, Mach B. A human endogenous retroviral superantigen as candidate autoimmune gene in type 1 diabetes. Cell 1997; 90:303-313.
  7. Böni J and Schüpbach J. Reverse transcriptase assay based on product enhancement for assessing the drug susceptibility of retroviruses. In: Methods in Molecular Medicine, vol. 24: Antiviral Methods and Protocol. D. Edited by: D. Kinchington and R.F. Schinazi. HUMANA PRESS, Totowa NJ. 1999, pp.301-312.
  8. Bürgisser P, Vernazza P, Flepp M, Böni J, Tomasik Z, Hummel U, Pantaleo G, Schüpbach J and the Swiss HIV Cohort Study. Performance of five different assays for the quantification of viral load in subjects infected with various subtypes of HIV-1. J. AIDS 2000; 23:138-144.
  9. Deichmann M, Huder JB, Kleist C, Naher H, Schüpbach J, Böni J. Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant. Arch. Dermatol. Res. 2005; 296:345-352.

F. Virus culture (for all infectious retroviruses).
 
Cells from the specimen are cultivated in vitro and the culture supernatant tested twice weekly using the PERT Assay. In the case of suspected HIV-infection, the HIV-1 p24 antigen test is carried out in addition. The overall test result is regarded as positive when at least two independently harvested supernatants demonstrate reactivity in one or both of the tests. Cultures are maintained for a maximum of four weeks. As the diagnostic sensitivity lies between 75 - 80%, use of virus culture is clearly inferior to PCR and antigen tests. Consequently, virus culture is only applied at the SNCR when looking for other retroviruses than HIV.
 
SNCR work published regarding virus culture:
  1. Jendis JB, Tomasik Z, Hunziker U, Nadal D, Seger R, Wetzel J-C, Kind C, Schüpbach J. Evaluation of diagnostic tests for HIV infection in infants born to HIV-infected mothers in Switzerland. AIDS 1988; 2:273-279.
  2. Schüpbach J, Jendis JB, Bron C, Böni J, Tomasik Z. False-positive HIV-1 virus cultures using whole blood. AIDS 1992; 6:1545-1546.
  3. Schüpbach J, Pyra H, Jendis JB, Tomasik Z, Böni J. Isolation of an in vitro transmissible agent with reverse transcriptase activity from a blood donor with a borderline-positive HIV-1 serology for more than five years. Clin. Diagn. Virol. 1996; 5: 197-203.
  4. Seemayer CA, Kolb SA, Neidhart M, Ohshima S, Gay RE, Michel BA, Gay S, Böni J, Schüpbach J. Absence of inducible retroviruses from synovial fibroblasts and synovial fluid cells of patients with rheumatoid arthritis. Arthritis Rheum. 2002; 46:2811-3.
  5. Seemayer CA, Böni J, Steiger J, Schüpbach J, Mihatsch MJ. No indication for activation of exogenous retroviruses in patients with renal allograft rejection. Clin. Nephrol. 2006; 65:324-7.




Virus characterization
A. HIV-subtype determination.
 
Mononuclear cells are isolated from EDTA-blood, DNA is extracted from the cells and an HIV-specific fragment located in the region C2-V5 of the HIV env gene is amplified using a "nested PCR". The virus subtype is determined using sequence analysis and comparison made with reference sequences belonging to a range of known subtypes (subtypes A-H, J, K of the group M, groups O and N) and circulating recombinant forms (CRF).
 
 
B. Phylogenetic analysis.

This test is primarily carried out in connection with forensic situations and deals with the question of whether or not an individual has passed HIV on to a third person. This approach requires DNA isolated from mononuclear cells of the victim, the accused, as well as a number of other DNA samples derived from donors within the geographical environs of these two individuals. As with HIV-subtype determination, a 0.7 kb-long HIV fragment from the C2-V5 region of the HIV env gene is amplified and sequenced by means of "nested PCR". Sequence similarity is determined by means of the Megaline, Clustal-W or PAUP software packages using a range of different algorithms.

For scientific studies in the field of veterinary medicine, phylogenetic analysis of retroviruses in sheep, goats, and in snakes have been undertaken.
 
 
C. Genotypic drug-resistance testing for HIV-1.
 
For this test, viral RNA is extracted from plasma. An area of the pol gene encoding both the reverse transcriptase and protease enzymes is reverse transcribed and amplified with PCR. The product is then analysed with PCR population sequencing. The established sequence is tested for mutations associated with resistance against antiretroviral medication using internet-based algorithms. The results report includes a list of the found resistance mutations and a rating of the virus with regard to its sensitivity against commonly used antiretroviral medications.
 
SNCR publications on this theme:
  1. J. Schüpbach, L. Perrin, P. Bürgisser, L. Matter, W. Fierz, P. Erb, J.C. Piffaretti, P. Grob, J. J. Burckhardt und Sektion virale Krankheiten und Sentinellasysteme des BAG. Verbreitung von HIV-Subtypen in der Schweiz 1996-1998. BAG Bulletin Nr. 5 (1. Feb. 1999): 97-101.
  2. Böni J, Pyra H, Gebhardt M, Perrin L, Bürgisser P, Matter L, Fierz W, Erb P, Piffaretti J-C, Minder E, Grob P, Burckhardt JJ, Zwahlen M and Schüpbach J. High frequency of non-B subtypes in newly diagnosed HIV-1 infections in Switzerland. J. AIDS 1999; 22:174-179.
  3. Bürgisser P, Vernazza P, Flepp M, Böni J, Tomasik Z, Hummel U, Pantaleo G, Schüpbach J and the Swiss HIV Cohort Study. Performance of five different assays for the quantification of viral load in subjects infected with various subtypes of HIV-1. J. AIDS 2000; 23:138-144.
  4. Huder JB, Böni J, Hatt J-M, Lutz H, Schüpbach J. Identification and characterization of two closely related unclassifiable endogenous retroviruses in pythons (Python molurus and P. curtus). J. Virol. 2002 76:7607-7615.
  5. Hasse B, Hummel Y, Böni J, Schüpbach J, Flepp M, Gunthard HF. 25-jähriger therapienaiver Patient mit einem dreiklassenresistenten HI-Virus. Praxis 2003; 92:601-5.
  6. Haupts S, Ledergerber B, Böni J, Schüpbach J, Kronenberg A, Opravil M, Flepp M, Speck RF, Grube C, Rentsch K, Weber R, Gunthard HF. Impact of genotypic resistance testing on selection of salvage regimen in clinical practice. Antivir. Ther. 2003 8:443-54.
  7. Shah C, Böni J, Huder JB, Vogt HR, Muhlherr J, Zanoni R, Miserez R, Lutz H, Schüpbach J. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 2004; 319:12-26.
  8. Shah C, Huder JB, Böni J, Schonmann M, Muhlherr J, Lutz H, Schüpbach J. Direct evidence for natural transmission of small-ruminant lentiviruses of subtype A4 from goats to sheep and vice versa. J. Virol. 78:7518-22.
  9. Joos B, Trkola A, Fischer M, Kuster H, Rusert P, Leemann C, Böni J, Oxenius A, Price DA, Phillips RE, Wong JK, Hirschel B, Weber R, Gunthard HF. Low human immunodeficiency virus envelope diversity correlates with low in vitro replication capacity and predicts spontaneous control of plasma viremia after treatment interruptions. J. Virol. 2005; 79:9026-9037.
  10. Simcock M, Sendi P, Ledergerber B, Keller T, Schüpbach J, Battegay M, Gunthard HF. A longitudinal analysis of healthcare costs after treatment optimization following genotypic antiretroviral resistance testing: does resistance testing pay off? Antivir. Ther. 2006; 11:305-14.
  11. Böni J, Schüpbach J, Rickenbach M. Rabbit retrotransposon sequences undetectable in blood donors and lymphoma patients of the Swiss HIV Cohort Study. AIDS Res. Hum. Retrovir. 2006; 22:499-500.
  12. Sendi P, Günthard HF, Simcock M, Ledergerber B, Schüpbach J, Battegay M. Cost-effectiveness of genotypic antiretroviral resistance testing in HIV-infected patients with treatment failure. PLoS One 2007; 2:e173.
  13. Joos B, Fischer M, Schweizer A, Kuster H, Boni J, Wong JK, Weber R, Trkola A, Gunthard HF. Positive in vivo selection of the HIV-1 envelope protein gp120 occurs at surface-exposed regions. J. Infect. Dis. 2007; 96:313-20.
  14. Yerly S, von Wyl V, Ledergerber B, Böni J, Schüpbach J, Bürgisser P, Klimkait T, Rickenbach M, Kaiser L, Günthard HF, Perrin L. Transmission of HIV-1 drug resistance in Switzerland: a 10-year molecular epidemiology survey. AIDS 2007; 21:2223-9.




Sample collection, shipment and storage
A. Sample collection and shipment
  • It is possible to use EDTA-blood for all blood examinations undertaken at the SNCR. Every sample must be labeled with the patient's identity, sample type (e.g. EDTA-blood, plasma, serum, liquor, etc.), as well as the sampling date.
  • With regard to teenagers and adults, a 10 mL sample of EDTA-blood is sufficient for all tests currently offerred. For small children, a sample of 2 - 5 mL of EDTA-blood is appropriate. In the case of an expected low virus load, larger samples (e.g. 2 x 10 mL) should be sent in as a rule. For the MEGA-PCR, 4 x 10 mL of sample are required.
  • Blood should be taken into EDTA-containing tubes or the CPT-Vacutainer (purple-top) contained in our dispatch-packet. The stopper must be perforated with a needle and should under no circumstances be physically removed (sterility!). Dispatch-packets with Vacutainers can be ordered free of charge (Tel. 044-634-2632).
  • For viral load measurements, in addition to EDTA-containing Vacutainers, use of CPT-Vacutainers is possible. However, the collected sample must be centrifuged using a swing-out rotor directly after venipuncture and BEFORE dispatching (as outlined in the enclosed instructions). If you do not have access to such a centrifuge, please use ordinary EDTA-containing Vacutainers.
  • Drug utensils (syringes, needles) intended for HIV testing using Western Blot must be sent in a closed solid plastic tube, with the needle pointing down. 

  • HIV-1 antigen test and virus load (RNA), as well as PERT Assay, can also be undertaken using cerebrospinal fluid. This is sent in cerebrospinal fluid-specific tubes i.e. an anticoagulant-free Vacutainer (red top). Heparin-containing Vacutainers must not be used.
  • ALWAYS SEND USING EXPRESS MAIL - NO SAMPLING AND SHIPMENT ON FRIDAYS AND BEFORE HOLIDAYS!

B. Sample storage

All unused material from an examination is stored for a minimum of 12 months. If EDTA-blood has been sent, the plasma and cell pellets are stored at -70°C. If only plasma or serum has been sent, excess is stored at -20°C. After 12 months, all stored material is released for internal SNCR experimental use or is destroyed.

Samples which have been sent in connection with the MoCHiv-Study are stored permanently for use in scientific projects connected with the study (Coordination Center):

Prof. Dr. med. Christoph Rudin
Universitäts-Kinderspital beider Basel
Römergasse 8
CH-4005 Basel

Tel. 061/685-6565
Fax 061/685-6566



Reports and consultation
A. Final report.
 
In the final report, individual results of every test are given, as is a conclusive overall interpretation. The final report includes information on every examination carried out on the sample. If the identity of the patient is known, the overall interpretation can also take into consideration aspects of earlier examinations.
 
B. Intermediate report.
 
Should individual tests take a longer period of time (e.g. virus cultures), an intermediate report consisting of already completed results (e.g. serology, PCR) is sent out. The intermediate report does not include an overall interpretation.
 
C. Results.
 
The results of individual tests can be either POSITIVE (POS), NEGATIVE (NEG), INDETERMINATE (IND) or, in the case of a test failure, NUL (0). With quantitative tests (virus load), a quantitative value is given at the same time. Test inaccuracy with quantitative PCR can be up to a factor of 3 i.e. half a log10.
 
D: Overall interpretation.
 
The overall interpretation gives the following possible answers to a diagnostic inquiry:
  • INFECTION CLEARLY VERIFIED
  • NO INDICATION OF INFECTION
  • INFECTION POSSIBLE - must be further clarified with tests on a new sample
  • INDETERMINATE RESULTS WHICH DO NOT SPEAK FOR AN INFECTION
  • SPECIAL REMARKS
With a resistance analysis, a detailed report on the found mutations and the expected effectiveness of the individual medications is given.
 
E. Adult patients.
 
In the case of a positive Western Blot result according to SNCR criteria, a confirmed positive antigen test or a positive PCR result, infection with a specific virus in adults can be concluded, provided that the result has not been established based on an artifact (sample mixup or contamination). Because of this latter possibility, even an "INFECTION CLEARLY VERIFIED" interpretation must be confirmed by a second sample obtained freshly and sent directly to us.

A positive result only in the PERT Assay is compatible with infection by an unknown retrovirus, but also with a number of nonspecific causes. In the later cases, larger amounts of material for confirmation and further examination are necessary.
 
F. Pediatric patients.
 
To answer the diagnostic question "is a child HIV-infected?", the different methodologic variants for direct virus detection play an important role. In the majority of cases, antigen test, RNA-PCR and, alternatively, qualitative DNA-PCR results are in agreement with each other. In the case of a positive result with at least two of these three tests, the presence of HIV-1 can be regarded as confirmed. However, in order to exclude the possibility of sample mix-up, confirmation of a positive result by means of a second freshly obtained sample is indispensable. The combination of a confirmed positive antigen test and a positive DNA or RNA-PCR currently ensures the safe detection of pediatric HIV-infection within the first weeks of life. In this manner it is possible to initiate early antiretroviral therapy with infected children.




Updated: 02/2008
© 2009 - Swiss National Center for Retroviruses