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A Seronegative Case of HIV-1 Subtype C Infection in Botswana

  1. Vladimir Novitsky1,3,
  2. Tendani Gaolathe2,
  3. Elias Woldegabriel1,
  4. Joseph Makhema1,3, and
  5. M. Essex1,3
  1. 1Botswana-Harvard School of Public Health AIDS Initiative Partnership for HIV Research and Education, Gaborone, Botswana
  2. 2Infectious Disease Care Clinic, Princess Marina Hospital, Gaborone, Botswana
  3. 3Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts
  1. Reprints or correspondence: Dr. M. Essex, Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave., Boston MA 02115 (messex{at}hsph.harvard.edu).
 
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Abstract

We report the first case, to our knowledge, of antibody-negative human immunodeficiency virus type 1 (HIV-1) subtype C infection, which was identified during screening for acute HIV-1 infection in Botswana. Results of tests for HIV-1 antibodies were consistently negative, including rapid and regular enzyme-linked immunosorbent assay and Western blot. The nonrecombinant HIV-1 subtype C infection was confirmed by viral genotyping within the gag, pol, and env genes. The period between referral of the patient in a clinically stable condition and AIDS-related death was ∼3 months. The reported case indicates the importance of studying the prevalence of seronegative HIV-1 infection in southern Africa, where subtype C predominates.

Routine HIV-1 testing by rapid or standard ELISA has become a commonly used health care practice. The high sensitivity of currently used licensed ELISA kits results in a relatively low frequency of false-negative results during HIV-1 testing. Testing during the seroconversion “window period” is the most recognized reason for a temporarily negative ELISA result; other reasons may include atypical virus or agammaglobulinemia. Although false-negative ELISA results can be confirmed by alternative methods that target viral RNA in plasma and/or cell-associated proviral DNA, the nucleic acid—based methods are not commonly used for routine HIV-1 testing.

Some patients may not develop antibodies after primary HIV infection, and a lack of adequate immune response may facilitate rapid progression of disease [1]. Rare cases of seronegative HIV-1 subtype B infection have been reported in Europe and North America [1,2,3,4,5–6]. Among non-B clades, HIV-1 subtype A infection was found in a persistently HIV-1-seronegative woman from Ivory Coast [7], subtype A2 virus was identified in an HIV-1-seronegative woman in Portugal [8], HIV-1 CRF01_AE (a circulating recombinant form of HIV-1 that is composed predominantly of subtype A and a unique envelope designated lineage E) was documented in 4 HIV-1-seronegative drug users in Thailand [9, 10], and a rare A/G recombinant was found in an HIV-1-seronegative woman from Ghana [11]. To date, no studies, to our knowledge, have reported cases of seronegative HIV-1 subtype C infection, which is the predominant HIV-1 subtype in the worldwide epidemic.

Here, we report a case of persistently antibody-negative HIV-1 subtype C infection in Botswana. A 46-year-old woman was referred to our study of primary HIV-1 subtype C infection in Botswana for HIV screening with 2 documented antibody-negative rapid ELISA tests performed elsewhere during the previous year. At the time of referral, she did not present with any opportunistic or chronic illnesses but had complaints of recent deterioration in general health and weight loss. The patient had received antifungal treatment for oral candidiasis and nonspecific oral ulcers at the local city-council clinic. No behavioral risk for HIV infection was identified by interview of the patient or review of medical records. She denied having any sexual relationships for >6 months prior to study entrance and reported no blood transfusions or injection drug use.

After receiving counseling and signing an informed consent form, the patient was tested for HIV-1 antibodies and viral RNA. The antibody tests, including rapid ELISA (Determine HIV-1/2, Abbott, and Uni-Gold HIV kit, Trinity Biotech) and regular ELISA (Murex HIV 1.2.0, Abbott, and Ortho HIV-1/2 Ab Capture, Ortho Diagnostics), yielded negative results. Viral RNA was detected in plasma by RT-PCR at >750,000 copies/mL. The tests were repeated, and the results remained the same. Western blot (HIV 2.2 Western Blot assay, Gene Labs Diagnostics) did not reveal any reactive bands. IgG and IgM levels were within the normal range (1410 mg/dL and 179 mg/dL, respectively), whereas the IgA level was elevated to 588 mg/dL. Her CD4+ T cell count was 38 cells/mm3, and her CD8+ T cell count was 138 cells/mm3. The patient was offered antiretroviral therapy but declined. Her viral load fluctuated from 451,000 copies/mL to >750,000 copies/mL over 2 months. Her CD4+ cell count decreased to 19 cells/mm3. The CD4+/CCR5+ T cell percentage was detected within the range of 10.3%–17.3%, and the CD4+/CXCR4+ T cell percentage dominated at ∼95%.

Esophageal candidiasis and severe oral herpes were treated with fluconazole and oral acyclovir. Eight days after the initiation of HAART (600 mg of efavirenz once daily and 150 mg of lamivudine plus 300 mg of zidovudine twice daily), the patient presented with inability to communicate and unusual behavior. Altered mental status and HIV encephalopathy were listed as probable clinical conditions. Cryptococcal meningitis and intracranial space—occupying lesions or infections were ruled out by analysis of CSF specimens and CT of the brain. The biochemical laboratory results are summarized in table 1. The patient died 4 days after admission. Encephalopathy or meningitis, HIV infection, and pulmonary tuberculosis were listed as the diseases or conditions leading to death. Immune reconstitution syndrome [12] or herpetic etiology of encephalopathy could not be ruled out. Postmortem examination was not available, and therefore, the cause of death could not be ascertained.

Figure 1
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Figure 1

Analysis of phylogenetic relationships between HIV-1 sequences, providing evidence that the sequence from Botswana (indicated by a filled diamond) clusters with HIV-1 subtype C references in gag (A), pol (B), and env (C) with high bootstrap values. Neighbor-joining trees are presented. Bootstrap values ⩾80 are shown.

Table 1
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Table 1

Clinical values for a patient presenting with HIV-1 antibody—seronegative AIDS.

Previous reports of seronegative HIV-1 infections described patients who progressed to AIDS rapidly [1]. Rapid progression was also observed in the current case. Routine identification of antibody-negative HIV infections may be associated with substantial logistical challenges. Nevertheless, initiation of HAART in patients identified to be seronegative who have high plasma viral loads apparently should not be deferred.

It is known that high levels of circulating immune complexes can be found during the late stage of HIV infection. Formation of immune complexes in the sera of patients with AIDS and AIDS-related complex may be responsible for failure to detect free HIV antibodies in some patients [13, 14]. Therefore, we cannot exclude formation of immune complexes as a potential reason for negative results of tests for HIV antibodies in the study patient. However, patients with detectable circulating immune complexes usually have increased levels of each immunoglobulin class [15]. Consequently, the normal levels of IgG and IgM, as well as the results of seronegative tests performed within the year prior to referral, might argue against immune complexes being the main cause of the HIV antibody—negative status of the patient in the current study.

To address the potential uniqueness of the patient's virus, we performed sequence analysis across structural viral proteins to determine the HIV-1 subtype and to confirm or exclude recombination across the viral genome. The gag, pol, and env genes were sequenced from the earliest available specimens from the time of referral. A total of 1358 bp was analyzed in gag; 1302 bp in pol, encoding the entire protease and 435 amino acids of the N-terminal reverse transcriptase; and 1182 bp in env, encoding the C2-V5 region of gp120. Phylogenetic analysis demonstrated that the query sequence clustered with HIV-1 subtype C reference sequences within gag, pol, and env (figure 1). The patient's virus was a typical subtype C virus, which diverged within the expected range from the subtype C consensus and/or ancestral sequence. Detailed sequence analysis did not suggest lack of cross-reactivity between the antigens in the ELISA or Western blot kits. The phylogenetic data provided evidence that a potential mismatch between the HIV-1 antigens in diagnostic kits and in patient's virus is unlikely to account for the observed seronegativity in the clinical case described here.

Recombinant analysis using the RIP 3 package [16] did not reveal any breakpoints across the analyzed sequences in gag, pol, and env (data not shown). Viral quasispecies were analyzed in gag and env by single genome sequencing, a new method developed in our laboratory based on a recent report by Palmer et al. [17]. The mean value of gag quasispecies nucleotide distance was 6.1% to both HIV-1 subtype C consensus and ancestral sequences. The mean value of env quasispecies nucleotide distance was 9.6% to HIV-1 subtype C consensus sequence and 9.9% to HIV-1 subtype C ancestral sequence. Analysis of intrapatient viral diversity revealed a mean value of 0.25% and a median diversity of 0.15% in gag and a mean value of 0.6% and a median diversity of 0.55% in env. The data obtained strongly suggest that the patient had nonrecombinant HIV-1 subtype C infection with a relatively low level of viral intrapatient diversity at the time of analysis.

In summary, we report the first case, to our knowledge, of antibody-negative HIV-1 subtype C infection. Although the exact time and source of HIV-1 subtype C infection is unknown, it is likely that infection occurred >1 year before death. Repeated tests for HIV-1 antibodies consistently yielded negative results, including rapid and regular ELISA and Western blot. The levels of IgG and IgM were within the normal range, whereas the IgA level was slightly elevated, which excludes agammaglobulinemia as a cause of antibody-negative HIV infection. The nonrecombinant HIV-1 subtype C infection was confirmed by viral genotyping within the gag, pol, and env genes. The period between referral of the patient in a clinically stable condition and AIDS-related death was ∼3 months.

This is the first documented case of seronegative HIV-1 subtype C infection. Our findings indicate the importance of future studies to identify the prevalence of seronegative HIV infection in the subtype C epidemic in southern Africa to determine whether viral nucleic acid testing should be introduced in the algorithm of routine HIV-1 screening.

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Acknowledgments

We thank the Tebelopele staff at Gaborone, Botswana, for their ongoing support and collaboration; Erin McDonald, Melissa Ketunuti, and Gaseboloke Mothowaeng, for dedicated clinical support; Busisiwe Mlotshwa, Lemme Kebaabetswe, Simani Gaseitsiwe, Caitlin Bonney, and Michaela Herzig, for excellent laboratory assistance; and Lendsey Melton, for outstanding editorial help.

Financial support. National Institutes of Health (AI057027-02).

Potential conflicts of interest. All authors: no conflicts.

  • Received March 26, 2007.
  • Accepted May 8, 2007.
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  1. Clin Infect Dis. (2007) 45 (5): e68-e71. doi: 10.1086/520683
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