Patient 98PTHEM104, a 21-year-old woman, was an intravenous drug user and sex worker. She presented at the hospital on May 1996 with extrapulmonary tuberculosis and oropharingeal candidiasis. She tested positive for HIV (Fig. 1). Her CD4 cell count was 40 cells/μl. In November 1996, she had an HIV-1 viral load measurement of 61 978 copies/ml and started antiretroviral therapy with indinavir, didanosine and zidovudine, which resulted in clinical and virological improvement. In September 1997, she attended the hospital with diarrhea, fever, oral candidiasis and peripheral neuropathy. She died of AIDS on February 1998.
Serological tests may yield false negative results in HIV-1 group N infections, or infection by a new and highly divergent HIV-1 variant . There is also the possibility of infection by a particularly aggressive viral strain that may not permit the development of a host immune response. It is therefore important to analyze the genotype of the viruses from all seronegative HIV-1 infections. A nested PCR was performed to obtain a 409 bp fragment from the C2-V3 env region and a 582 bp fragment from the p17 gag region of viral isolates from both patients. Thermal cycling conditions for PCR and primers have been described previously . For patient 98PTHEM103, PCR was done on chromosomal DNA extracted from sections of different postmortem tissues using a universal extraction method previously described . For patient 98PTHEM104, serum collected in 1996 was the only available biologic material. Viral DNA suitable for PCR amplification was obtained by reverse transcription from RNA recovered from 200 μl of serum, as described . PCR fragments were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, California, USA) and sequenced. To determine virus subtype and investigate evolutionary relationships between the two isolates, maximum likelihood phylogenetic analyses was performed using the GTR+G model of nucleotide substitution, as described previously . Tree searches were conducted in PAUP v4.0b10 using a nearest-neighbor interchange heuristic search strategy and bootstrap. The sequences have been assigned GenBank accession numbers GQ387120-GQ387157.
Rare cases of HIV-1 infected patients with clinical symptoms of AIDS but repeatedly negative for HIV antibody screening have been described [1–3]. There may be several reasons for negative HIV screening results in patients who are HIV-infected. Among host factors, the most well recognized is the window period, generally 20–25 days after infection, depending on the specific enzyme immunoassay reagents used . Late seroconversions of up to 40 months after infection have been described [13,14]. Other host factors that could explain seronegative infection include profound hypoglobulinemia, B-cell functional defects and seroreversion [12,15]. This does not appear to be the case in patient 98PTHEM103 because he had negative results from antibody tests in all occasions and his immunological parameters were otherwise normal.
In conclusion, patient 98PTHEM103, a previously healthy individual, exhibited a fatal infection with the absence of an HIV-specific humoral response. The results suggest a massive infection with a highly aggressive CRF14_BG-like strain and/or the presence of an unidentified immunological deficiency that has prevented the formation of HIV-1-specific antibodies.
The clinical and laboratorial contributions of Manuela Mafra and Sandra Bento for the early identification and study of these patients are gratefully acknowledged.
Author's contributions: N.T., R.C., and V.B. designed the research. N.T., R.C., V.B., I.B. and H.B. performed the research, interpreted the data and wrote the paper. All authors reviewed and accepted the final manuscript.
Financial Support: This work was supported by grant CRIA-CR3751 from Comissão Nacional de Luta Contra a SIDA, Portugal.
1. Cardoso AR, Goncalves C, Pascoalinho D, Gil C, Ferreira AF, Bartolo I, Taveira N. Seronegative infection and AIDS caused by an A2 subsubtype HIV-1. AIDS 2004; 18:1071–1074.
2. Baldrich-Rubio E, Anagonou S, Stirrups K, Lafia E, Candotti D, Lee H, Allain JP. A complex human immunodeficiency virus type 1 A/G/J recombinant virus isolated from a seronegative patient with AIDS from Benin, West Africa. J Gen Virol 2001; 82:1095–1106.
3. Candotti D, Adu-Sarkodie Y, Davies F, Baldrich-Rubio E, Stirrups K, Lee H, Allain JP. AIDS in an HIV-seronegative Ghanaian woman with intersubtype A/G recombinant HIV-1 infection. J Med Virol 2000; 62:1–8.
4. Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, Saragosti S, et al
. Identification of a new human immunodeficiency virus type 1 distinct from group M and group O. Nat Med 1998; 4:1032–1037.
5. Bartolo I, Casanovas J, Bastos R, Rocha C, Abecasis AB, Folgosa E, et al
. HIV-1 genetic diversity and transmitted drug resistance in healthcare settings in Maputo, Mozambique. J Acquir Immune Defic Syndr 2009; 51:323–331.
6. Sandhu GS, Kline BC, Stockman L, Roberts GD. Molecular probes for diagnosis of fungal infections. J Clin Microbiol 1995; 33:2913–2919.
7. Thomson MM, Delgado E, Manjon N, Ocampo A, Villahermosa ML, Marino A, et al
. HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users. AIDS 2001; 15:509–516.
8. Esteves A, Parreira R, Piedade J, Venenno T, Franco M, Germano de Sousa J, et al
. Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal. AIDS Res Hum Retroviruses 2003; 19:511–517.
9. Sing T, Low AJ, Beerenwinkel N, Sander O, Cheung PK, Domingues FS, et al
. Predicting HIV coreceptor usage on the basis of genetic and clinical covariates. Antivir Ther 2007; 12:1097–1106.
10. Saksena NK, Wang B, Dyer WB. Biological and molecular mechanisms in progression and nonprogression of HIV disease. AIDS Rev 2001; 3:133–144.
11. Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci USA 2003; 100:4144–4149.
12. Sullivan PS, Schable C, Koch W, Do AN, Spira T, Lansky A, et al
. Persistently negative HIV-1 antibody enzyme immunoassay screening results for patients with HIV-1 infection and AIDS: serologic, clinical, and virologic results. Seronegative AIDS Clinical Study Group. AIDS 1999; 13:89–96.
13. Cho YK, Sung H, Bae IG, Oh HB, Kim NJ, Woo JH, Kim YB. Full sequence of HIV type 1 Korean subtype B in an AIDS case with atypical seroconversion: TAAAA at TATA box. AIDS Res Hum Retroviruses 2005; 21:961–964.
14. Dalmau J, Puertas MC, Azuara M, Marino A, Frahm N, Mothe B, et al
. Contribution of immunological and virological factors to extremely severe primary HIV type 1 infection. Clin Infect Dis 2009; 48:229–238.
15. Jurriaans S, Sankatsing SU, Prins JM, Schuitemaker H, Lange J, Van Der Kuyl AC, Cornelissen M. HIV-1 seroreversion in an HIV-1-seropositive patient treated during acute infection with highly active antiretroviral therapy and mycophenolate mofetil. AIDS 2004; 18:1607–1608.