“Criteria for the interpretation of HIV Western blot may differ: the Centers for Disease Control consider a positive WB if at least two of p24, gp41, and gp120/160 proteins are present, whereas according to WHO recommendations a WB may be judged positive if only two Env bands are found. A more restrictive recommendation is the one from the American Red Cross, which demands at least three bands, one from each group (i.e. one protein from Gag, one from Pol and one from Env). Finally, the Consortium for Retrovirus Serology Standardization recommends the presence of at least one of gp120 or gp160 proteins and one of p24 or p32 proteins [40] for a positive WB.”
Buttò S et al. Laboratory diagnostics for HIV infection. Ann Ist Super Sanita. 2010;46(1):24-33.
“Although a Positive result may [!] indicate infection with the HIV-1 virus, a diagnosis of Acquired Immunodeficiency Syndrome (AIDS) can be made only if an individual meets the case definition of AIDS established by the Centers for Disease Control. A repeat test on an independent sample should be considered to control for sample mix-up or operator error, and to verify a positive test result. Individuals may present incomplete banding patterns…A person who has antibodies to HIV-1 is presumed [!] to be infected with the virus…Do not use this kit as the sole basis of diagnosis of HIV-1 infection…[When tested on ‘Low Risk’ subjects 100 out of 495 people with a negative ELISA were indeterminate on the western blot and 0 out of 14 who had a positive ELISA were positive on the Western Blot (3 were indeterminate). One person with AIDS with a negative ELISA was indeterminate on the Western Blot and 5 out of 241 who had AIDS and were ELISA positive were indeterminate on the western blot. Of 1007 people who were HIV-negative by ELISA 207 were indeterminate on this test, of which 111 were negative by another test. Of the remaining 800 with negative ELISA and this Western Blot, 295 were indeterminate on another Western Blot]
Human immunodeficiency virus type 1 (HIV-1) Western Blot kit. OraSure. 2009 Sep
[from the 1998 test manual] Persons demonstrating antibodies to HIV-1 should be referred for medical evaluation, which may include testing by other techniques…Accurate diagnosis of HIV-1 infection is important in determining an individual’s risk for developing AIDS. Accuracy is complicated by false-positive and false-negative (EIA) results…Slight ambiguities exist in the designation of the molecular weights of the HIV-1 antigens [used in this test]…Although a [Western] blot POSITIVE for antibodies to HIV-1 indicates infection with the virus, a diagnosis of Acquired Immundeficiency Syndrome or AIDS can only be made clinically if a person meets the case definition of AIDS established by the CDC. POSITIVE blot results using any specimen type (serum, plasma, or urine) should be followed with additional testing [even though the Western Blot test is usually claimed to be a 'confirmatory' test]…The clinical implications of antibodies to HIV-1 in an asymptomatic person are not known. However, a larger proportion of such persons have virus detectable in their peripheral blood and some will develop immunodeficiency. INDETERMINATE blots should not be used as the basis for diagnosis of HIV-1 infection…A negative blot does not exclude the possibility of infection with HIV-1.”
Human Immundeficiency Virus type 1 (HIV-1) Western Blot kit. Maxim Biomedical. 2009 Jun 1
Human Immundeficiency Virus type 1 (HIV-1) Western Blot kit. Maxim Biomedical. 2009 Jun 1
“The clinical and public health implication of HIV Western blot (WB) indeterminate results is yet to be appraised in sub-Saharan Africa, including Nigeria. Using HIV Tri Line Test enzyme-linked immunosorbent assay (ELISA), 1286 patients (600 males and 686 females; age range, 5-60 years) with symptoms suggestive of HIV infection were screened. A total of 1020 (79.3%) of the patients…were HIV seropositive…Western blot analysis of sera from the 1020 HIV-seropositive individuals using the BIO-RAD NEW LAV-BLOT I specifying World Health Organization (WHO) interpretive criteria, confirmed the HIV serostatus of 815 (79.9%) of them with 205 (20.1%) individuals having indeterminate results…Patients aged 11-20 years old recorded the highest percentage of indeterminate results (31.7%) while those aged 21-30 years recorded the least (14.2%)…Result confirmed the limitation of Western blot assays in HIV confirmatory serodiagnosis. After obtaining HIV indeterminate Western blot result, clinicians should consider the total profile for the patient, reassess risk factors for HIV infection, perform a HIV retesting at 3-month intervals for 6 months or use an alternate HIV antibody confirmatory assay and running antibody tests for other human retroviruses.”
Uneke CJ et al. Western blot-indeterminate results in Nigerian patients HIV serodiagnosis: the clinical and public health implication. AIDS Patient Care STDS. 2007 Mar;21(3):169-76.
“In LAVBLOT I [Western Blot] kit there are lots of differences in results when read with different interpretation criteria. To interpret the results WHO criterion was assigned by the manufacturer. There were 158 (64.7) specimens found to be positive out of 244 used in the kit. But on using the various other criteria for interpretation the number of positive results varied from 157 (64.3) to 176 (72.1), while the number of indeterminate varied between 44 (18) and 63 (25.8). No such variation was present among the negative, which implies that some of the indeterminate specimens would be considered positive if different centers used different criteria to interpret the results [but indeterminate western blots are usually treated as negative]. [The] CDC criterion was found to be very liberal to confirm positive results…Of the 23 specimens that were run on both the LAV BLOT I and the genetic systems kit, we found a lot of discrepancies in the results. Based on the criteria suggested by the manufacturer the LAV BLOT I kit identified 3 specimens as positive, 4 specimens as negative and 16 specimens as indeterminate. However, the genetic systems identified only 2 specimens as positive, 7 specimens as indeterminate but 14 of these 23 specimens as negative. This implies that the LAV BLOT I could be wrongly reporting 10 negative specimens as indeterminate. On further analysis 1 out of this 10 specimens could be read positive if CDC & CRSS criteria were used. This again proves the variations of results with respect to different criteria. Also 1 specimen indeterminate in genetic systems kit was found to be positive in LAV BLOT I kit. We also noticed that the most common band associated with an indeterminate report in the LAV BLOT I kit was the p25 band. Four of the five specimens on which the PCR was performed were reported as negative. The genetic systems kit reported three of these four as negative and one as indeterminate. The other specimen that was reported as positive by the PCR was reported as indeterminate by the genetic systems kit. The LAV BLOT I kit on the other hand reported all the five specimens as indeterminate.”
Syed IH et al. HIV-1 western blot assay: What determines an indeterminate status?. Indian J Med Sci. 2005 Oct;59(10):443-50.
“Specimens that have bands present [on Western Blot] but do not fulfill the criteria for positivity are called Western blot indeterminate, and a follow-up specimen should be requested, usually collected three to four weeks after the initial specimen. In follow-up, patients will either show a definitive pattern indicating that they have seroconverted or will demonstrate the same banding pattern as previously observed. In the latter circumstance, the vast majority of these patients are HIV-negative and have nonspecific antibody. In these cases, if the patient is considered to be at risk or is particularly anxious, a qualitative PCR may be recommended to confirm that the patient is truly HIV-negative. Because these indeterminate banding patterns may be seen in patients who are not infected, the Western blot does not make a good screening test for HIV.”
Fearon M. The laboratory diagnosis of HIV infections. Can J Infect Dis Med Microbiol. 2005 Jan;16(1):26-30.
[According to Australian guidelines] WB [Western Blot] indeterminate group 1 [occurs with] Reactivity to viral proteins, but not to p18, p24 or any envelope glycoproteins. WB indeterminate group 2 [occurs with] Reactivity to viral proteins including p18, but not to p24 or any envelope glycoproteins. WB indeterminate group 3: Reactivity to viral proteins including p24 but not to any envelope glycoproteins. WB indeterminate group 4: Reactivity to envelope glycoproteins but to less than three other viral proteins…all WB indeterminate reactivity should be followed up for 12 weeks…”
Guidelines for interpreting HIV testing results. Australian National Reference Laboratory. 2004
“In many countries laboratories employ a two-test algorithm that examines repeatedly EIA screen reactive specimens by Western blot, but in England and Wales the prevailing approach has been, and remains, to employ at least two different tests following the initial reactive screening test, as recommended by the World Health Organisation3, or an additional screening test with a line immunoassay (LIA). This approach has been called the ‘alternative confirmatory strategy’”
Parry JV et al. Towards error-free HIV diagnosis: guidelines on laboratory practice. Commun Dis Public Health. 2003 Dec;6(4):334-50.
“Between 1996 and 2000, a total of 12,124 samples were tested for HIV-1 antibodies. A total of 1,437 plasma specimens (11.9%) were HIV-1 antibody positive. Of these, 91 ( 0.8%) gave equivocal results, with discordant serological data and indeterminate WB profiles. The WB reactivities measured on 91 indeterminate test results are summarized in Table 1. Most of the indeterminate WB results were due to antibodies against the HIV-1 core antigen p24 (30.4%). In addition, reactivities to pol p51 (13.9%), pol p66 (14.6%), and env gp41 (15.2%) antigens were frequent. Isolated p17 reactivity was observed rarely (only 2.3% of the samples). A total of 12 samples (13.3%) displayed reactivity to two of the three proteins p24, gp41, and gp120/160 and thus can be considered positive by CDC criteria…Among 1,475 HIV-negative cohort participants, 31 (2.1%) had at least one indeterminate WB test result during follow-up, of which 19 (61%) were related to false-positive ELISA results only, 11 (36%) were related to false-positive HIVSPOT test results only, and 1 (3%) was related to both (Table 1). Most of the initially indeterminate WB assays (30 of 31 [97%]), including samples considered positive by the CDC criteria (6 of 6), were negative when retested. One subject with initial indeterminate WB profile was negative throughout 30 months of follow- up, at which time he seroconverted. Only two samples remained persistently indeterminate (as long as 18 and 48 months, respectively) without developing any WB reactivity that indicated seroprogression. In addition, 17 indeterminate WB assays, including the two samples that persistently gave indeterminate WB profiles, were assessed by NASBA for HIV-1 viremia. Plasma HIV-1 viremia was not detected in any of the above specimens.”
Meles H et al. Indeterminate human immunodeficiency virus Western blot profiles in ethiopians with discordant screening-assay results. Clin Diagn Lab Immunol. 2002 Jan;9(1):160-3.
“C135 is the longest infected recipient [via blood transfusion] (over 20 years)…[and] has maintained an indeterminate HIV-1 Western blot since infection and triple-nested PCR was required to detect HIV-1 DNA”
Birch MR et al. An examination of signs of disease progression in survivors of the Sydney Blood Bank Cohort (SBBC). J Clin Virol. 2001 Oct;22(3):263-70.
“From July 1, 1999, to November 30, 1999, 2,518 HIV Western blots were performed. Of these, 47 (1.9%) showed NSS [non-specific staining]…Of the 47 samples, 11 (23%) had a positive HIV-1 ELISA result, and results for 36 (77%) were negative. Presumably all of these patients had a positive HIV ELISA result in another laboratory, since samples were sent to our laboratory for confirmatory Western blot testing only. Each of the 11 samples with a positive HIV-1 ELISA result in our laboratory was only slightly above the positive cutoff for the assay. Most true-positive samples have signal/cutoff ratios greater than 5.0, while the ELISA-positive samples in our study had signal/cutoff ratios ranging from 1.02 to 2.98.”
Willman JH et al. Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results. Am J Clin Pathol. 2001 May;115(5):764-9.
Turner V. Criteria defining a positive HIV western blot. Unpublished. 2000
“Confirmatory western blot analyses were performed in urine samples of all the ESNs [exposed to HIV, but sero-negative], their HIV-seroopositive sexual partners, and low risk controls…a gp41 monoband and other HIV bands (p68, p55, p52, p34, p25, p18), but not gp120 and/or gp160, were detected in urine samples from most of the ESNs [how could uninfected people have antibodies to HIV proteins?]
Mazzoli S et al. HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals. Nat Med. 1997 Nov;3(11):1250-7.
“All blood collections from January 1994 to March 1996 were screened for anti-HIV by HIV-1/2 enzyme immunoassay (EM). Repeatably reactive results were confirmed by HIV- 1 WB. AIl WB-positive donors were interviewed regarding risk behavior and offered repeat HIV testing. HIV polymerase chain reaction (PCR) was added to repeat HIV testing for more recent donors. Results were considered false-positive when the donor denied risk factors and 1) repeat samples tested EM-negative or 2) repeat samples tested PCR-negative and either WB-negative or WB-positive only by the new criteria (formerly, WB-indeterminate). Four donors (2 men and 2 women, aged 27-47 years) were identified as having had a false-positive HIV WB interpretation for their index donations. Two donors had p24 antigen and env WB bands and two donors had env bands only. Two donors tested negative by EM and PCR, 13 and 19 months after donation. One donor was persistently WB positive (new criteria only), but was PCR negative 5 weeks after donation. Tho samples taken from one donor 1 month after donation were negative in EM. These donors represent 11 percent of all WB-positive donors during the study period. This experience is consistent with a recent estimate, based on a 4-year database from 5 blood centers in the United States, that 4.5 percent of WB results in blood donors are false positive [and Western Blot is the ‘confirmatory’ test that is rarely questioned]
Cable RG et al. Interpretation of false-positive human immunodeficiency virus type 1 western blot assays in blood donors. Transfusion. 1997 Jun;37(6):670.
“TABLE 3. Conditions associated with WBi [Western Blot Indeterminate] result: Incomplete generation or loss of antibodies; Early seroconversion (33, 73, 106); Late-stage disease (8); Massive proteinuria (85); Passive transfer of antibodies from: Infected mothers to noninfected children (18); Unscreened immunoglobulin preparations (48, 68, 109); Cross-reactivity with: Normal cellular constituents: nucleoproteins (99); and HLA (35); Other retroviruses: human T-cell leukemia virus type 1 and HIV-2 (33); Bacteria (Mycobacterium leprae [61]); Interfering factors or sample preparation In vitro hemolysis, elevated bilirubin, rheumatoid factor (17); Heat inactivation (24, 47); Antibodies generated by influenza virus vaccine (75); African sera (34a); Disease states Polyclonal gammopathies (17); Systemic lupus erythematosus (4)”
Nuwayhid NF. Laboratory tests for detection of human immunodeficiency virus type 1 infection. Clin Diagn Lab Immunol. 1995 Nov;2(6):637-45.
“HIV serology was determined using enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. Subjects were categorized as positive in accordance with current [CDC] criteria [for Western Blot] if positive for p24 or p31 plus gp41 or gp120/160 until 1 November 1989, or if positive for two or more of p24, p41 and gp120/160 after 1 November 1989.”
Moss AR et al. HIV seroconversion in intravenous drug users in San Francisco, 1985-1990. AIDS. 1994 Feb;8(2):223-31.
“The Western blot (WB) is the most commonly used test to confirm the presence of antibodies against the human immunodeficiency virus type 1 (HIV-1). Different criteria of interpretation of the band profile have been proposed with there being no unanimity as to its reliability…The presence of antibodies against HIV-1 was prospectively studied in 8,073 samples of subjects with risk of infection. A total of 1,993 (25%) were reactive by ELISA and 1,261 were analyzed by WB, with a semiquantitative reading of the bands with a point scale from 0 to 2 being performed. The final interpretation of the WB (negative, doubtful, or positive) was carried out following 5 recommendations of usage…In order of frequency, the most frequent bands in HIV-1 + individuals were gp160 (99%), gp120, p24, p31, p55, p68, gp41, and p17 (68%). In non infected individuals, the recognized bands were, in decreasing order, p24, p17, p55, p68, p31, and glucoproteins. Different criteria of interpretation of the Western blot provide different degrees of sensitivity and specificity. The Western blot is a non standardized, expensive, laborious technique of subjective interpretation which provides an appreciable number of undetermined results.”
Soriano V et al. [Evaluation of various criteria for the interpretation of western blot for the diagnosis of human immunodeficiency virus infection. Spanish Group for the Study of HIV-2]. Med Clin (Barc). 1993 Apr 17;100(15):561-6.
“The specificity of immunoassays for detecting HIV antibodies has major shortcomings. Almost all reactions, especially in low risk populations, represent false-positive results. This has been observed with Western blot (WB), which is widely used as a confirmatory test…A serum panel of 21 blood-bank donors and two serum samples from patients with primary Sjögrem's syndrome were analysed…All sera were positive on two commercial WB assays, as well as on home-made blots using recombinant p24 proteins. All sera appeared to be HIV-negative on a third-generation ELISA. Specimens of these sera were also HIV-1-antigen negative and [by] PCR. Infection could not be detected after cocultivation of donor-derived lymphocytes with permissive cells. Furthermore, in a retrospective study of blood donations, seroconversion was not observed in any of the recipients of this blood. We performed a radioimmunoprecipitation assay (RIPA) using recombinant p24 proteins…No reaction was observed…herpes simplex virus type 1 (HSV-1), strain 17, may be the agent that caused the cross-reactivity in…two cases.”
Langedijk JP et al. Identification of cross-reactive epitopes recognized by HIV-1 false-positive sera. AIDS. 1992 Dec;6(12):1547-8.
“HIV-1 antibodies were detected in blood and semen of all 28 sample pairs. Blood and semen samples of three known seronegative controls were consistently negative…Western blot analysis of 13 paired samples showed different blood and semen IgG antibody patterns…Antibody reactivity against the gp160 band was almost as intense in seminal plasma fractions as in blood plasma, whereas antibody reactivity against p55, p24, and p17 bands was undetectable when seminal plasma was run at dilutions comparable to those of blood plasma.”
Wolff H et al. A comparison of HIV-1 antibody classes, titers, and specificities in paired semen and blood samples from HIV-1 seropositive men. J Acquir Immune Defic Syndr. 1992;5(1):65-9.
“Several different ways of interpreting a Western blot test for degrees of positivity have been established by government agencies and by consortia and are variously in use”
Swets JA. The science of choosing the right decision threshold in high-stakes diagnostics. Am Psychol. 1992 Apr;47(4):522-32.
“The initial study group consisted of 1,129 addicts (949 men and 180 women) consecutively admitted to the National Institute of Mental Health’s former Clinical Research Center at Lexington, KY, between May 15, 1971, and May 14, 1972…The WB results from the earlier study, which had employed a technique enhanced by the use of an avidin-biotin system, were reanalyzed. The Centers for Disease Control issued diagnostic criteria in 1985 recommending that a WB be considered positive if either band p24 or band gp4l was present alone or in combination with other bands. On rereading, blots with isolated p24 bands were considered to be indeterminate. One 1985 WB, with bands at both the 24 and 55 kilodalton regions, was included among the positives, since the interpretation of this pattern had previously been unclear. The 1971-72 serum specimens were not available for retesting… The two former patients whose 1971-72 WB results were most strongly reactive had current ELISA and WB assays that were negative. Their immune function parameters were inconsistent with immune suppression… The results of the ELISA and WB assays performed on the 1971-72 specimens remain an enigma. Some were interpretable as positive, although only weakly reactive. One explanation is that the results observed in 1985 were true positives, that PDAs in the early 1970s had antibodies to an HIV-like agent that was nonpathogenic, and that the WBs subsequently converted from positive to negative. Loss of HIV antibodies in asymptomatic homosexual men has been reported. It is possible that antibodies to a nonpathogenic virus would have disappeared during the 17 to 18 years between admission to Lexington and the 1989 followup. Although this potential cannot be ruled out, it is more likely that the earlier results were false positives. The reasons for false positivity are unclear, but cross reactivity with related retroviruses may be one possibility. The HIV Western blot assay may cross-react with antibodies to HTLV-I in the p24 and p55 antigen regions, but not the gp4l region. These serum specimens were tested for the presence of HTLV-I/HTLV-II antibodies by other investigators, and a 6.3 percent seropositivity rate for the entire cohort was observed. It is not known if these 10 persons were seropositive for this related retrovirus; however, it is unlikely that this type of cross reactivity accounted for the previous results, given the distribution of the bands and the fact that reactivity was not detected during selected 1989 followup WB screening. The earlier false positivity could be the consequence of either the state of the serum specimens or the test kit or assay employed. It has been suggested that artifactual findings may occur as a consequence of frequent thawing and refreezing of serum aliquots, and that frequent refreezing might affect the physical properties and serologic characteristics of the serum protein moieties. The available evidence would suggest that long-term storage and repeated thawing and refreezing does not affect subsequent testing for serum constituents [the possibility that the 1971-72 Western Blot results were valid and were due to exposure to IV drugs, was not considered, nor was the possible explantion for later survival and negative HIV tests being removal of exposure to IV drugs]
Lange WR et al. Followup study of possible HIV seropositivity among abusers of parenteral drugs in 1971-72. Public Health Rep. 1991 Jul-Aug;106(4):451-5.
“The DNA equivalent of 200 microliters of blood was assayed using the pol, gag, and nef primer pairs in a single PCR, and by performing a nested gag PCR…[on] 11 Western blot anti-p24(gag)-indeterminate blood donors. No positive signal was found for any of these donors.”
Bruisten SM et al. Enhanced detection of HIV-1 sequences using polymerase chain reaction and a liquid hybridization technique. Application for individuals with questionable HIV-1 infection. Vox Sang. 1991;61:24-9.
“Indeterminate patterns on Western blot are common in non-HIV_infected people…Western blot testing should not be done routinely as a screening test for HIV-1 infection because…it would result in a large increase in the number of indeterminate specimens, nearly all of which would not be from persons with HIV-1 infection…Possible explanations for the indeterminate reactions include exposure to an unidentified immunogen [immune system stimulator], such as another retrovirus, the presence of nonspecific comigrating antigens in the viral lysate used to prepare the Western blot strips and the presence of epitopes, posttranscriptionally modified antigens, or new antigenic sites in the viral antigen that are not present on the recombinant p24 antigen.”
Povolotsky J et al. Differences in human immunodeficiency virus type 1 (HIV-1) anti-p24 reactivities in serum of HIV-1-infected and uninfected subjects: analysis of indeterminate western blot reactions. J Infect Dis. 1991 Feb;163(2):247-51.
“As part of a phase 1 trial of a candidate AIDS vaccine, blood specimens were collected from 168 healthy adult volunteers at minimal or no risk for becoming infected with human immunodeficiency virus type 1 (HIV-1). These specimens were screened for evidence of HIV-1 infection by enzyme immunoassay (EIA) and the Biotech/Du Pont Western blot (168), culture (122), and polymerase chain reaction assay (20). None of the subjects had a positive test result by any of these assays, but 32% had indeterminate Western blot tests, most of which demonstrated a single band of low intensity. The most common bands were p24 (47%), p55 (34%), and p66 (36%); envelope bands were unusual (gp41, 2%; gp120, 2%) [meaning that none of these bands are unique to HIV, yet multiple bands would be interpreted]. No serum specimen collected after 2-11 months from individuals with indeterminate Western blot results was positive by EIA or Western blot. There was 91% agreement in the test results of the first and second serum samples when the same lot of Western blot kit was used but only 36% agreement when different lots were used.”
Midthum K et al. Frequency of indeterminate Western Blot tests in healthy adults at low risk for HIV infection. J Infect Dis. 1990 Dec;162(6):1379-82.
“We produced three murine monoclonal antibodies (mAbs) against the HIV-1 proteins. These three mAbs, namely CA-1, CA-2, CA-4, were IgG1 and all reacted with p24 on the HIV-1 Western blot. One of the mAbs, CA-4, also recognized p13, p21, p28, p29, p32, p39, p47, p55 on the Biotech/Du Pont HIV-1 Western blot strips and p21, p24, p28, p29, p39, p47, p55, p68, p80, p96; p110 on the Bio-Rad strips. CA-4 did not react with H-9 cell lysate nor with other retroviral antigens such as HTLV-1 or HIV-2 proteins. The binding of CA-4 to HIV-1 proteins was not blocked by deglycosylation. All three mAbs reacted with recombinant DNA derived capsid protein (p24) of HIV-1. These results suggest that many proteins in the HIV-1 Western blot contain antigenic epitope(s) similar to that of p24. [This shows that a single protein can cause multiple reactions on a Western Blot so that it cannot be assumed that multiple reactions are more likely to reflect infection than single reactions]
Liang CM et al. An anti-p24 monoclonal antibody shows cross-reactivity with multiple HIV-1 proteins. J Immunol Methods. 1990 Aug 28;132(1):57-62.
[In a study of healthy persons with repeatedly positive ELISA antibody tests but 'indeterminate' Western Blot 'confirmatory' tests]…After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66%) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92%) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2…we were unable to discern an association between any measure studied and indeterminate status with respect to Western blot results or a specific band.”
Jackson JB et al. Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV-1. N Engl J Med. 1990 Jan 25;322(4):217-22.
“The criterion for a Western blot assay with positive results was a speciment that exhibited at least two of three bands at p24, gp41 and gp120/160.”
Garland FC et al. Incidence of human immunodeficiency virus seroconversion in US Navy and Marine Corps personnel, 1986 through 1988. JAMA. 1989 Dec 8;262(22):3161-5.
“The currently licensed Du Pont Western blot test specifies that the test result should be interpreted as positive only when the detected bands include p24 and p31, and gp41 or gp120/160. Conversely, a negative Du Pont Western blot test result requires the absence of any and all bands–not just viral-bands. All other patterns are regarded as indeterminate…Alternative criteria have been proposed by various groups. ASTPHLD has proposed that a positive test result be defined by the presence of any two of the following bands: p24, gp41, and gp120/160 (13). The Consortium for Retrovirus Serology Standardization (CRSS) has defined a positive test result as the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160) (14). The American Red Cross has defined a positive test result as greater than or equal to 1 band from each of the GAG, POL, and ENV gene-product groups (15). These three groups and DuPont all agree that an indeterminate result is the presence of any other band or bands that fail to meet the positive criteria, and that a negative result is the absence of all bands…For all three categories with repeatedly reactive EIA test results, the Western blot results demonstrate that the ASTPHLD definition gives the highest percentage of positive and the lowest percentage of indeterminate results [note that this has nothing to do with the accuracy of the test but…] On the basis of the results described above, CDC concurred with the ASTPHLD criteria and recommends their use in public health and clinical practice…A person whose Western blot test results continue to be consistently indeterminate for at least 6 months–in the absence of any known risk factors, clinical symptoms, or other findings–may be considered to be negative for antibodies to HIV-1. Such persons should be reassured that they are almost certainly not infected with HIV-1. However, no large-scale studies have been done to provide virologic data to confirm independently the serologic findings from the studies of clients whose Western blot test results are consistently indeterminate. In contrast, an asymptomatic person who has an indeterminate Western blot test result and a history of possible exposure to or symptoms compatible with HIV infection requires additional diagnostic follow-up. This should include conducting serial Western blot testing, assessing the function of the individual’s immune system, and eliciting the cooperation of the person’s sexual and needle-sharing partners to determine whether they are infected.”
Interpretation and use of the western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. MMWR Morb Mortal Wkly Rep. 1989 Jul 21;38(Suppl 7):1-7.
“The currently licensed Du Pont Western blot test specifies that the test result should be interpreted as positive only when the detected bands include p24 and p31, and gp41 or gp120/160. Conversely, a negative Du Pont Western blot test result requires the absence of any and all bands–not just viral-bands…[Three] Alternative criteria have been proposed by various groups. ASTPHLD has proposed that a positive test result be defined by the presence of any two of the following bands: p24, gp41, and gp120/160 (13). The Consortium for Retrovirus Serology Standardization (CRSS) has defined a positive test result as the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160) (14). The American Red Cross has defined a positive test result as greater than or equal to 1 band from each of the GAG, POL, and ENV gene-product groups (15). These three groups and DuPont all agree that an indeterminate result is the presence of any other band or bands that fail to meet the positive criteria, and that a negative result is the absence of all bands. The criteria for a negative Western blot interpretation specify “no bands.” This interpretation is essential because some observed bands may reflect the presence of antibodies to HIV regulatory proteins or may indicate partially processed or degraded viral structural proteins. Furthermore, different Western blots (commercial, as well as “in-house” preparations) and different virus-antigen preparations used to prepare Western blots may contain different numbers and concentrations of both viral-specific and contaminating cellular proteins that may have unpredictable molecular weights…the ASTPHLD definition gives the highest percentage of positive and the lowest percentage of indeterminate results…On the basis of the results described above, CDC concurred with the ASTPHLD criteria and recommends their use in public health and clinical practice.”
CDC. Interpretation and Use Of Western Blot Assay for Serodiagnosis of Human Immunodeficiency Virus Type 1 Infections. MMWR. 1989;38(S-7):1-7.
“To interpret human immunodeficiency virus (HIV) Western blots, the specific nature of each band must be understood. Bands near the top of each strip, for example, were thought to reflect the reactivity of antibodies with HIV gp160 envelope precursor and the gp120 envelope glycoprotein. However, we have unexpectedly found that viral antigens, migrating with mobilities of 160 and 120 kd [molecular weight] on commercial Western blot strips, are primarily multimers of the HIV transmembrane glycoprotein gp41 that react with antibodies to gp41. Confusion over the identification of these bands has resulted in incorrect conclusions in experimental studies. Similarly, some clinical specimens may have been identified erroneously as seropositive, on the assumption that these bands reflected specific reactivity against two distinct viral components and fulfilled a criterion for true or probable positivity.”
Zolla-Pazner S et al. Reinterpretation of human immunodeficiency virus western blot patterns. N Engl J Med. 1989 May 11;320(19):1280-1.
“The pattern of bands on protein or Western immunoblot (IB) for antibodies to antigens of human immunodeficiency virus (HIV) is not always reproducible…positivity may be simulated by the reactivity of serum with nonviral proteins of molecular sizes similar to viral components. Finally, technical vagaries may result in apparent rather than real loss of antibody to HIV capsid antigen (p24)…Routine Quality Control with Two Positive QC specimens…Only 101 (56%) of the 179 assays showed the presence of p24, p32, and gp41 and/or gp120, a pattern that would be reported as positive by all sets of criteria in use at reference laboratories…Routine Quality Control with Two Negative QC Specimens…An absence of all bands…was reported for only 67 assays (67%) [i.e. 33% of tests on negative western blot controls showed one or two positive bands]…Physicians should remember that any given IB result can be erroneous and seek further confirmation by a later sample if applicability of the result to the patient seems questionable. The criteria for positivity when only some bands are seen continues to be a problem. Reactivity at either p24 or gp41, without p32 or gp120 has sometimes been considered adequate for confirmation of anti-HIV positivity. This pattern was seen 10 times with one of the positive samples inn two laboratories but a combined total of 19 times in three laboratories with the two negative samples. It is obvious that p24 alone or gp41 alone, without p32 or gp120, must be reported as indeterminate and the IB repeated on the same or a subsequent specimen. On the other hand, requiring three bands among p24, p32, and gp41 or gp120 would have permitted a positive interpretation of only 101 or 179 assays (56%) of QC(+) [positive quality control] specimens. Requiring two of the three would result in a correct interpretation for 166 of 179 (93%) assays of QC(+) samples and 99 of 101 (98%) assays of QC(-) specimens. The criteria for positivity and negativity for any given IB assay, however, must minimize false positivity and false negativity, even if reassay of specimens is necessary for a moderate number of sera. An indeterminate pattern (i.e.,, not interpretable as necessarily positive or negative) tended to occur in clusters on a given day of testing or on successive dates, particularly for the negative QC samples. This seems attributable to a systematic technical problem at that particular time. Whenever we observed this occurrence, we resubmitted to the same facility after discussion of the problem or checked reproducibility in another laboratory.”
Edwards VM, Mosley JW. Reproducibility in quality control of protein (Western) immunoblot assay for antibodies to human immunodeficiency virus. Am J Clin Pathol. 1989 Jan;91(1):75-8.
“Although the Western blot is commonly used as a confirmatory test or as a reference standard against which other tests are evaluated, criteria for what constitutes a positive result have changed over time and still remain unsettled. Major bands of diagnostic significance are p24/25 (a core protein); gp41, gp110/120, and gpl60 (envelope proteins); and p31/32 (a regulatory protein). The Association of State and Territorial Health Officers defines a positive test result as resolution of any two of p24/25, gp41, and either gp110/120 or gpl60. The American Red Cross defines a positive test result as resolution of at least one of each type (core, envelope, and regulatory) of protein. Du Pont Pharmaceuticals, Inc, producer of the only commercially available Western blot assay, requires detection of antibodies to p24/25, either gp41 or gpl20, and p31/32 for a positive result. Incomplete band patterns are considered equivocal. Because it lacks a standardized protocol, the Western blot is particularly subject to intra-observer and inter-observer variability in its performance. The recently licensed Western blot kit may improve the procedure by standardizing the test substrate and criteria, but it will not eliminate the problems associated with the test’s interpretation…these reference standards might more appropriately be considered “supporting” rather than “confirmatory.” When evaluating a diagnostic test, correction must be made for the imperfect sensitivity and specificity of the reference standards; it is best to use at least two different supporting tests.”
Schwartz JS, Dans PE, Kinosian BP. Human immunodeficiency virus test evaluation, performance, and use. Proposals to make good tests better. JAMA. 1988 May 6;259(17):2574-9.
“Applicants for United States military service, October 1985 to March 1986…were screened with a commercial enzyme-linked immunosorbent assay (ELISA), and positive samples were immediately retested in duplicate with the ELISA. All samples positive on repeat ELISA were tested with the Western blot method for reactivity with HIV proteins. Specimens were classified as positive if antibodies to HIV encoded protein gp41 or proteins p24 and p55 were detected.”
Burke DS et al. Human immunodeficiency virus infections among civilian applicants for United States military service, October 1985 to March 1986. Demographic factors associated with seropositivity. N Engl J Med. 1987 Jul 16;317(3):131-6.
“A 55-year-old asymptomatic woman was advised by the Montreal Red Cross that she was seropositive for HIV, as determined by an enzyme-linked immunosorbent assay (ELISA) and confirmed by a Western blot test. The most probable source of contamination was her husband, who had received 3 units of blood while undergoing surgery in our hospital two years previously. The husband’s serum was sent to our laboratory, but no antibodies against HIV were detected by either ELISA or indirect immunofluorescence. At the same time, we confirmed the apparent positivity of the patient’s serum both by ELISA and by an indirect immune-fluorescence test, in which H9 cells, infected by the human T-cell lymphotropic virus type IIIB strain of HIV, are used as targets. Reactivity on the latter test partially disappeared after pre-absorption of the patient’s serum with non-infected H9 cells. We pursued the search for HIV-specific antibodies in the husband’s serum by the Western blot test, using both non-infected and infected H9 cells. We screened the patient’s serum in the same way. The Western blot test failed to detect antibodies against viral proteins in the husband’s serum. These results, supported by previous reports of false seropositivity in asymptomatic blood donors, emphasize the need to be certain of viral antigen specificity when screening for HIV antibodies. We suggest that blood banks use both HIV-infected and non-infected cell lines when confirming seropositivity by the Western Blot test and that the presence of bands on such tests not be automatically considered to indicate positive status”
Roy S et al. Need for caution in interpretation of Western blot tests for HIV. JAMA. 1987 Feb 27;257:1047.
“In the absence of a 41-kd band, a blot must show both a 24-kd and a 55 kd band to be deemed positive…A second sample must be drawn and confirmed to be blot positive before a diagnosis is considered established.”
Burke DS, Redfield RR. False-positive western blot tests for antibodies to HTLV-III. JAMA. 1986 Jul 18;256(3):347.
“Of 1,027,786 units of donated blood tested, 10,385 (1.0%) were reactive on the initial EIA test for HTLV-III [HIV] antibody. Of these, 1723 (0.17%) were repeatably reactive, designated 'EIA-positive' (EIA+), and not released for transfusion. Of 1455 EIA-positive blood units, representing approximately 868,000 donors, that were tested by Western blot analysis, 333 were positive (WB+) [i.e. 83% of initial positive EIA's were false positive based on a second EIA and only 3.8% of initial EIAs were confirmed by both a second EIA and Western Blot, the other 96% were false positives]
Schorr JB et al. Prevalence of HTLV-III antibody in American blood donors. N Engl J Med. 1985 Aug 8;313(6):384-5.
“Reactions in the HTLV-III [HIV] antibody test were positive in 21% of the 692 subjects. Another 24% had borderline reactions…Analysis of the sera by Western blots revealed that 27 (66%) of 41 sera from the seropositive subjects reacted with viral protein bands from disrupted, purified HTLV-111, compared to 4 (24%) of 17 sera from seronegative subjects.”
Biggar RJ et al. Regional variation in prevalence of antibody against human T-lymphotropic virus types I and III in Kenya, East Africa. Int J Cancer. 1985 Jun 15;35(6):763-7.
“Based on available data, the Western blot should be considered positive for antibody to HTLV-III if band p24 or gp41 is present (alone or in combination with other bands).”
Provisional Public Health Service Inter-Agency Recommendations for Screening Donated Blood and Plasma for Antibody to the Virus Causing Acquired Immunodeficiency Syndrome. MMWR. 1985 Jan 11;34(1):1-5.
“After blood grouping and cross-match, the HIV negative blood from donor to recipient mice showed bands corresponding to gp120 and gp160 were observed in nine mice. Two mice had both gp120 and gp160 bands and one unidentified peak, two showed only one band gp160 and five had weak gp160 bands the so called indeterminate bands for positive but not complete absence of bands…One would think that if there really were HIV proteins, and that the HIV antibodies were truly specific, then just having one band light up would be proof that HIV is present. But according to experts, that is not the case, you need more than one3. The intriguing thing is, even if one or two bands are not sufficient to diagnose HIV infection, there must still be a reason why they are there. In fact cross-reaction is the explanation given by all HIV experts for “noninfected” Western Blots3. But if one or two bands in the WB can be caused by non-HIV, cross reacting antibodies why cant three or four or five or all of the ten bands be caused by cross reacting non-HIV antibodies. Around the world different combinations of two or three or four bands of the possible ten bands are deemed proof of infection. In Africa you need only two, envelop proteins without gag or pol to prove HIV infection, (WHO Criteria). FDA and Red Cross rules you need three bands. We conclude therefore that the HIV antibody proteins in the Western Blot antibody test are not specific.”
Kabati CIA et al. Testing for HIV Specific Proteins in Otherwise Western Blot Negative Theiller

Albino Mice. TaJONAS.

“Specific guidelines for interpretation may differ depending on the local policies. MPD recommends following the accepted policy to be in accordance with local regulations. Listed below are some of the criteria guidelines recommended by different international regulatory bodies:

Centers for Disease Control (CDC) and ASTPHLD – At least one ENV (gp41 and gp120/160) and p24;

American Food and Drug Administration (FDA) – p24 and p31 and gp41 or gp120/gp160;

Center Nationale Transfusion Sanguine – Two ENV bands with GAG or POL;

World Health Organization (WHO) – Two ENV bands with or without GAG or POL;

Consortium for Retrovirus Serology Standardization – One band of p24 or p31 and ENV band;

American Red Cross (ARC) – One band each of GAG, POL and ENV;

German Association for Control of Viral Diseases (DVV) – One ENV and at least one GAG or POL band, see also DIN 58 969, part 41.”

HIV blot 2.2: Western blot assay. MP Diagnostics.



There you have it. No “handful of wild-eyed conspiracy theorists.” No “right-wing racists,” as the Aids industry’s spinmeisters would have you believe. Just 19 very serious, concerned, highly educated people from every corner of the globe who sense that an enormous tragedy is unfolding due to the medical establishment’s unwillingness to face the evidence that the Hiv-Aids theory is a mistake.

The people on this page were intellectually curious enough to have sought out and studied the arguments that discredit the Hiv-Aids theory. Since the mass media and professional journals censor these arguments, the vast majority of doctors and scientists, although decent people who want to do the right thing, have never been exposed to them, and so accept the biased conclusions of politicized bureaucracies like the CDC and WHO, whose coziness with the drug industry is legendary and whose recommendations always seems to dovetail perfectly with drug industry marketing plans.

Were it not for the massive media blackout of information that contradicts the Hiv theory, many more people would be asking tough questions.

The next time you hear the media say, “only a handful of scientists doubt Hiv’s role in Aids,” refer them to this page. Explain to them that it is wrong to misrepresent the fact that there is enormous dissent to the Hiv-Aids paradigm.

The next time you hear the media drone, “Hiv, the virus that causes Aids,” remind them that journalists are supposed to distinguish between what is a theory and what is a fact. That Hiv-Aids is only a theory and has never been proven, is admitted by top scientists even in the Aids establishment.

The next time the media announce that tens of millions of people are dying from Hiv in Africa, ask them how they know that. Remind them that journalists are supposed to question dubious assertions from powerful, drug-industry funded agencies like the WHO, not parrot them as if they were indisputable. Ask them why they report these numbers as if they were actual Aids cases, when in fact they are projections made by WHO’s computer programs, based on very questionable statistical methodologies and contradicted by many facts including the continual large population increases experienced in the countries supposedly worst affected.

Request that the media stop twisting the truth in support of a politicized, entrenched Aids establishment that profits financially by terrorizing people, pokes its nose shamelessly into people’s private sex lives, compels people to submit to inaccurate tests and literally forces mothers and babies to swallow toxic, unproven chemotherapy drugs with horrific, often-fatal side effects.

Explain to them that this is irresponsible, and that such actions cause needless anxiety, shatter people’s lives, tear families apart, destroy hope and trigger countless suicides. And that while we realize that sensational headlines about “killer viruses” sell newspapers, the social cost of these profits is unacceptable.

Make the media understand that keeping people in the dark about the large number of credentialed dissenters to the Hiv-Aids dogmas, and the financial conflicts of interest that are rampant among Hiv-Aids scientists and NGOs, is a violation of everyone’s human right to informed consent and freedom of information.



Note: Affiliation with an organization does not imply that the organization supports the individual HIV/AIDS skeptic’s position.


Does Hiv cause Aids? Lots of scientists say ‘no.’ Read more.

Rethinking AIDS

Alive And Well

HEAL Toronto

Dr. Peter Duesberg

The Perth Group

Treatment Information Group

Immunity Resource Foundation

Alberta Reappraising Aids Society



Last updated January 30, 2013.