Immune thrombocytopenic purpura might be an early hematologic manifestation of undiagnosed human immunodeficiency virus infection.

Little research focuses on the association between immune thrombocytopenic purpura and human immunodeficiency virus infection in Taiwan. This study investigated whether immune thrombocytopenic purpura might be an early hematologic manifestation of undiagnosed human immunodeficiency virus infection in Taiwan. We conducted a retrospective population-based cohort study using data of individuals enrolled in Taiwan National Health Insurance Program. There were 5472 subjects aged 1-84 years with a new diagnosis of immune thrombocytopenic purpura as the purpura group since 1998-2010 and 21,887 sex-matched and age-matched, randomly selected subjects without immune thrombocytopenic purpura as the non-purpura group. The incidence of human immunodeficiency virus infection at the end of 2011 was measured in both groups. We used the multivariable Cox proportional hazards regression model to measure the hazard ratio and 95 % confidence interval (CI) for the association between immune thrombocytopenic purpura and human immunodeficiency virus infection. The overall incidence of human immunodeficiency virus infection was 6.47-fold higher in the purpura group than that in the non-purpura group (3.78 vs. 0.58 per 10,000 person-years, 95 % CI 5.83-7.18). After controlling for potential confounding factors, the adjusted HR of human immunodeficiency virus infection was 6.3 (95 % CI 2.58-15.4) for the purpura group, as compared with the non-purpura group. We conclude that individuals with immune thrombocytopenic purpura are 6.47-fold more likely to have human immunodeficiency virus infection than those without immune thrombocytopenic purpura. We suggest not all patients, but only those who have risk factors for human immunodeficiency virus infection should receive testing for undiagnosed human immunodeficiency virus infection when they develop immune thrombocytopenic purpura.

The incidence of human herpes virus-8 expression in lymph node biopsies from human immunodeficiency virus-positive patients.

AIMS: Human immunodeficiency virus (HIV)-related lymphadenopathy is characterized by a wide spectrum of histological changes. Three patterns have been described which correspond to clinical stages of HIV/acquired immune deficiency syndrome (AIDS). Castleman disease is a heterogeneous group of disorders. A recently described variant, multicentric Castleman disease (MCD), of which some cases are associated with human herpes virus-8 (HHV-8), has been reported in both HIV-seropositive and -negative patients. Considerable morphological overlap occurs between one of the patterns of HIV lymphadenopathy and this variant. METHODS AND RESULTS: This retrospective histopathological study on 95 cases of HIV-reactive lymphadenopathy assessed the incidence of the different patterns and HHV-8 on immunohistochemistry (IHC). Of the 95 cases, 78 (82.1%) were HHV-8-negative, of which 46 (59.0%) were classified as pattern A, 20 (25.6%) as pattern B and 12 (15.4%) as pattern C. Nine (31.0%) of 29 cases with pattern B and 8 (40.0%) of 20 cases with pattern C were HHV-8 positive. In total 15 cases of MCD were diagnosed in this series. CONCLUSION: This study draws attention to the overlap between HIV lymphadenopathy and MCD. We recommend that cases of HHV-8-associated MCD should be investigated for HIV infection.

Human immunodeficiency virus and the central nervous system.

The pandemic of HIV/AIDS continues to grow daily. Incident cases among women, intravenous drug users and ethnic minorities comprise the fastest growing segment of the HIV-infected population, and the number of HIV-infected individuals over the age of 50 is growing rapidly. Today, the central nervous system and the immune system are seen as main targets of HIV infection. Significant progress in the knowledge and treatment of AIDS has been obtained in recent years. The neurological manifestations directly related to HIV are acute viral meningitis, chronic meningitis, HIV-associated dementia (HAD), vacuolar myelopathy, and involvement of the peripheral nervous system.

Human immunodeficiency virus and the central nervous system

The pandemic of HIV/AIDS continues to grow daily. Incident cases among women, intravenous drug users and ethnic minorities comprise the fastest growing segment of the HIV-infected population, and the number of HIV-infected individuals over the age of 50 is growing rapidly. Today, the central nervous system and the immune system are seen as main targets of HIV infection. Significant progress in the knowledge and treatment of AIDS has been obtained in recent years. The neurological manifestations directly related to HIV are acute viral meningitis, chronic meningitis, HIV-associated dementia (HAD), vacuolar myelopathy, and involvement of the peripheral nervous system.

Localisation of HHV-8 in AIDS related lymphadenopathy.

BACKGROUND: Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposi's sarcoma (KS), malignant lymphoma (Hodgkin's and non-Hodgkin's), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. AIM: To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposi's sarcoma development. METHODS: A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). RESULTS: Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. CONCLUSIONS: The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

Genetic subtypes of HIV type 1 in Republic of Congo.

To assess the molecular epidemiology of HIV-1 in Republic of Congo (Congo), we investigated 29 HIV-1s obtained from 82 Congolese AIDS and ARC patients in 1996 and 1997. Part of the env region including the V3 loop was phylogenetically analyzed. The genotypes observed were varied: of 29 specimens, 12 (41 %) were subtype A, 1 (3%) was subtype D, 6 (21%) were subtype G, 6 (21%) were subtype H, 2 (7%) were subtype J, and 2 (7%) could not be classified as any known subtypes (U, unclassified). The heterogeneous profile of HIV-1 infection was different from the profiles of neighboring Central African countries. These data show that subtypes G and H as well as subtype A were circulating with high prevalence. The fact that new genetic subtypes (J and U) are circulating indicates a need for a greater surveillance for these subtypes both in Congo as well as in other parts of the world.

Maintenance of high-avidity rubella-specific IgG antibody and titres in recent HIV seroconvertors and in patients progressing to the AIDS-related complex and AIDS.

The avidity (functional affinity) and titre of rubella-specific IgG antibodies were examined in sequential and cross-sectional sera from 38 adult HIV-infected patients, whose HIV status ranged from pre- and recent HIV seroconversion to the AIDS-related complex (ARC) and AIDS, in order to determine whether a preexisting mature antibody response to rubella is maintained or if there is a need for rubella (re)vaccination. Thirty-five patients were already rubella-seropositive and one became rubella-seropositive during the time in which sera were collected. Although the avidity of rubella-specific IgG was higher in HIV-positive patients than in their age-and sex-matched HIV-negative counterparts, the difference was not significant. The titres of this antibody, however, were significantly higher in the HIV-positive patients. No significant decrease in antibody avidity or titre were seen in sequential sera from individual HIV-positive patients except when the titres in pre-HIV-seroconversion sera were compared with the titres in sera from patients with AIDS, where a significant decrease was observed. This would suggest that preexisting humoral immunity to rubella in HIV-infected patients is not compromised with HIV disease progression and there should be no need to revaccinate.

Spectrum of clinical diseases in HIV-infected adults at the Lagos University Teaching Hospital: a five-year experience [1992-1996].

The study was conducted to document the spectrum of clinical diseases in HIV infected patients at the Lagos University Teaching Hospital (LUTH) over a period of five years: 1992-1996. Patients with symptoms suggestive of HIV infection in both in and out-patients at LUTH were studied. Their blood specimens were screened for HIV infection using enzyme immunosorbent assay (EIA) technique and positive results were confirmed by western blotting techniques. The following were documented: risk factors, clinical history and physical findings. Of the 5,010 patients screened in a five-year period, 759 (15.15%) were found to be HIV positive. Of these 759 patients, 406 (53.5%) were young adults in their third decade (20-30 years). Heterosexual intercourse was the major risk factor in these patients (76%). Progressive loss of weight occurred in 77.8%, prolonged fever in 73%, chronic cough in 50%, painless lymphadenopathy in 40%, chronic diarrhoea in 35%, Kaposi's sarcoma in 0.52% and non-Hodgkin's lymphoma in 0.65%. It appears the scourge of AIDS has eventually hit Nigeria. There is the need for reinvigoration of preventive efforts but some energy has to be channeled towards patient care.

Induction of apoptosis by primary HIV-1 isolates correlates with productive infection in peripheral blood mononuclear cells.

OBJECTIVE: To study the apoptosis-inducing capacity of HIV-1 primary isolates in human peripheral blood mononuclear cells (PBMC) in relation to the viral biological phenotype. DESIGN AND METHODS: Four HIV-1 primary isolates capable of replicating and inducing syncytia in the MT-2 cell line and two primary isolates lacking these properties were used to infect PBMC with the same infectious doses. The kinetics of virus production in the culture supernatants were followed in relation to apoptosis induction in PBMC as determined by intracellular labelling of apoptotic DNA strand breaks and flow cytometry analysis. RESULTS: When low virus dose was used (0.001 m.o.i.), productive virus infection, with peak reverse transcriptase (RT) activity at days 5-7, was followed by high numbers of apoptotic cells at day 10 post infection. Tenfold higher inoculum dose (0.01 m.o.i.) resulted in enhanced virus production with peak RT activity at day 3 followed by high numbers of apoptotic cells at day 5 after infection. The apoptosis-inducing capacity of virus isolates was independent of their capacity to induce syncytia or replicate in the MT-2 cell line. However, upon cocultivation of infected PBMC with MT-2 cells, only virus with the MT-2 tropic phenotype initiated productive infection and induced apoptosis in MT-2 cells. CONCLUSIONS: These results show that apoptosis induction in PBMC by primary HIV-1 isolates is closely related to the kinetics of virus replication but is not influenced by other biological properties of the virus such as syncytium-inducing capacity and MT-2 tropism.

Phylogenetic analysis of human immunodeficiency virus 1 in Ghana.

Eleven human immunodeficiency virus 1 (HIV-1) isolates from Ghanaian acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients obtained by our serosurvey in 1986-1994 were genomically analyzed and phylogenetically compared with other known strains. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains were circulating in Ghana and the majority of them (9 of 11 isolates) belonged to clade (subtype) A which is now furiously epidemic in Africa. Another isolate (1 of 11) belonged to clade D, and the remaining one (1 of 11) belonged to "clade G". This "clade G" virus grouped by the env analysis belonged to clade A by its pol sequence, suggesting an A/G intersubtype recombinant. The characteristic sequences in the V3 tip which have not yet been reported were observed in these Ghanaian isolates, which should be taken into account for future vaccine programs.

PIP: The molecular epidemiology of HIV-1 in Ghana was investigated through genomic and phylogenetic analysis of isolates from 11 AIDS or AIDS-related complex patients obtained in 1986-94. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains are circulating in Ghana. 9 of the isolates belonged to clade A, 1 to subtype D, and 1 to "clade G"--an A/G intersubtype recombinant. The V3 loops of all isolates were composed of 35 amino acid residues--a characteristic not previously described. These molecular data on the genetic variability of the envelope glycoprotein of HIV-1 should be useful for future vaccine studies in West Africa.

Predominance of HIV-1 among patients with AIDS and AIDS-related complex in Ghana.

We determined the prevalence of HIV among AIDS and AIDS-Related Complex (ARC) patients seen within one year in two hospitals in southern Ghana. Subjects were screened by an ELISA procedure for anti-HIV antibodies. Specific identification of the HIV type was done with a particle agglutination (PA) kit. All PA-determined dual specimens were then confirmed by Western blotting and Pepti-Lav 1/2 monoepitope kit. Virus isolation was attempted from symptomatic patients by co-culturing patient peripheral blood monocyte cells (PBMCs) and CD4+ cell lines. PBMCs and HIV isolates were characterised by PCR. By ELISA, 43.5% of the subjects (253) had anti-HIV antibodies. Of these, 61 (24%) were HIV-1 positive and 42 (18.6%) were dually reactive by PA. However, only 19% were confirmed as true dually-infected cases by western blotting and Pepti-Lav through all 42 samples were HIV-1 positive on the two tests. No subject was infected with HIV-2 alone. Three viruses were isolated. By PCR two of them had both HIV-1 and HIV-2 proviral sequences while the third virus was HIV-1 only. HIV-1 prevalence now predominates over HIV-2 implying a switch in the HIV infection pattern in Ghana. Furthermore mixed infections exist. The predominance of HIV-1 infection in Ghana may indicate a similar trend in other parts of West Africa.

PIP: Recent studies have suggested that HIV-2 infection is becoming less prevalent in Ghana, while the prevalence of HIV-1 is increasing. To confirm such a modification in the HIV infection profile in Ghana, a 1-year serologic and molecular study was conducted among 253 patients from 2 hospitals in southern Ghana (Accra and Dzodze in the Volta region) with confirmed or suspected AIDS. All 253 serum specimens were screened with enzyme-linked immunosorbent assay (ELISA) and particle agglutination (PA); the 42 dually reactive specimens were subsequently confirmed by Western blot and Pepti-Lav tests. By ELISA, 110 samples (43.5%) were positive for anti-HIV antibodies; this rate was 39.2% in Accra and 81.0% in the Volta region. Of these, 61 (24.1%) were HIV-1 positive and 42 (18.6%) were dually reactive by PA. No case of HIV-2 alone was detected. Most dually reactive cases were a cross-reaction between genetically similar regions of the 2 HIV types. Only 19% of the 42 PA-diagnosed dually reactive specimens were confirmed by Western blot and Pepti-Lav as true cases of HIV-2 only infection, and all these specimens were strongly positive for anti-HIV-1 antibodies. 3 viruses were isolated. By polymerase chain reaction, 2 had both HIV-1 and HIV-2 proviral sequences, while the third was HIV-1 only. This study's findings provide support for the hypothesis that most individuals with antibodies to both HIV-1 and HIV-2 are probably infected with HIV-1 alone. Intensified population surveillance aimed at isolating more HIV strains in West Africa could reveal the true extent of HIV genomic variation and facilitate the design of more specific diagnostic kits.

Human herpesvirus 6 in human immunodeficiency virus-infected individuals: association with early histologic phases of lymphadenopathy syndrome but not with malignant lymphoproliferative disorders.

Preliminary evidence suggested that human herpesvirus-6 (HHV-6) may act as a cofactor in acquired immunodeficiency syndrome (AIDS) and may contribute to the pathogenesis of lymphoproliferative disorders occurring in individuals infected with the human immunodeficiency virus (HIV). To understand better the biological and clinical significance of HHV-6 infection in the context of HIV-related immunosuppression, the polymerase chain reaction was used to study the frequency and variant distribution of HHV-6 in peripheral blood mononucleated cells (PBMCs) from HIV-seropositive individuals, either asymptomatic or with lymphadenopathy syndrome (LAS) or with overt AIDS. Non-neoplastic and malignant lymphoproliferative disorders from both HIV-infected and HIV-seronegative patients were also investigated using the same series of samples for the presence of Epstein-Barr virus (EBV). When compared with healthy blood donors (12/42, 29%), HHV-6 prevalence in PBMCs showed a progressive decline in HIV-seropositive individuals with asymptomatic HIV infection (3/26, 11%) and in patients with LAS (1/13, 8%) and a significant reduction in patients with overt AIDS (1/20, 20%; P = 0.02). The decrease correlated with the number of CD4+ cells at the time of examination. In addition, HHV-6 DNA sequences were significantly more prevalent in LAS biopsies (13/20, 65%) than in HIV-unrelated reactive lymphadenopathies (2/10, 20%; P = 0.02) and the presence of HHV-6 sequences correlated closely with a histologic pattern of follicular hyperplasia (13/16, 81%; P = 0.003). Strikingly, HHV-6 prevalence decreased in PBMCs of LAS patients, suggesting that the likelihood of interactions between HHV-6 and HIV varies in different body districts. In particular, the demonstration that all HHV-6-carrying LAS samples were also positive for HIV infection suggests that LAS lymph nodes constitute one of the sites where biologically relevant interactions between the two viruses might occur. Also, the prevalence of EBV was higher in LAS (14/20, 70%) than in non-neoplastic lymph nodes from HIV-seronegative individuals (4/10, 40%), although the difference was not statistically significant. EBV was associated strongly with HIV-related malignant lymphoproliferative disorders, being detected in 100% of patients with Hodgkin's disease (HD) and 53% of B-cell non-Hodgkin's lymphomas (NHL). In contrast, the prevalence of HHV-6 DNA in HD and B-cell NHL arisen in HIV-infected patients (30% and 6% respectively) was remarkably lower and similar to that observed in lymphoproliferative disorders from HIV-seronegative patients. Finally, as observed in healthy individuals, HHV-6 variant B was more prevalent than variant A in benign and malignant lymphoproliferative disorders from bot HIV-infected and HIV-seronegative patients. These results suggest that the interactions between HHV-6 and HIV could be different in the various phases of HIV disease and in different districts; HHV-6 has probably no direct role in the pathogenesis of HIV-associated B-cell NHL and HD cases, and behave differently from EBV; and HIV-related immunosuppression does not alter the distribution of HHV-6 variants in these tissues, as observed in the case of EBV.

Characterization of Epstein-Barr virus-infected cells in benign lymphadenopathy of patients seropositive for human immunodeficiency virus.

The authors investigated 25 benign lymph nodes in patients infected with the human immunodeficiency virus (HIV) by in situ hybridization (ISH) and immunohistochemistry (IHC) to detect and characterize the Epstein-Barr virus (EBV)-infected cells. After ISH, 22 lymph nodes were found to contain various numbers of Epstein-Barr-encoded RNA (EBER)-positive cells. Most of these cells were B cells. In six lymph nodes with numerous EBV-infected cells, EBNA2-positive/LMP1-positive lymphoblastoid cells were detected by IHC. Exceptional cells (in two specimens) were positively labeled with antigen-Z Epstein-Barr replicative activator (ZEBRA) antibody or BamHI Left Frame 1/Not I (BHLF1/Not I) probes, indicating that EBV replication is not enhanced in the lymphocytes. In normal conditions (healthy individuals), small lymphocytes that express a restricted pattern of viral genes do escape immune response, whereas lymphoblastoid cells do not. Thus, impaired immune system may account for the late proliferation of lymphoblastoid cells (Epstein-Barr nuclear antigen [EBNA]2positive/latent membrane protein [LMP]1 positive) in HIV-infected patients, and could explain why EBV-driven, acquired immunodeficiency syndrome (AIDS)-related, non-Hodgkin's lymphoma occur more frequently in patients with low CD4-positive T cells.

Characteristics of EBV-infected cells in HIV-related lymphadenopathy: implications for the pathogenesis of EBV-associated and EBV-unrelated lymphomas of HIV-seropositive individuals.

The present study was performed with the aim of better defining the possible role of Epstein-Barr-virus (EBV)-infected cells in the pathogenesis of HIV-related lymphadenopathy syndrome (LAS). In addition, since LAS has been considered as a pre-lymphomatous lesion, we also wished to elucidate the possible contribution of EBV-carrying cells present in LAS tissues to the development of HIV-associated malignant lymphomas. To this end, we have characterized EBV-infected cells in LAS lymph nodes in terms of EBV DNA prevalence, tissue distribution in relation to HIV-carrying cells, virus sub-type, expression of latent and replicative antigens, and presence of clonal EBV episomes. When compared with HIV-unrelated lymphadenopathies (4/10, 40%), LAS showed a higher prevalence of EBV DNA (14/20, 70%). Comparable values of EBV prevalence were detected in LAS with follicular hyperplasia (12/16, 75%) and with follicular involution (4/4, 100%). All EBV+ non-neoplastic lymph nodes from HIV-seronegative patients carried type-I EBV, whereas LAS specimens showed almost equivalent distribution of the 2 EBV sub-types. Of the 14 EBV-carrying LAS, 4 (29%) were positive by Southern-blot analysis for the BamHI-W region of the virus genome but negative for the presence of monoclonal EBV episomes. In situ hybridization revealed a remarkably higher load of EBV-infected cells in LAS than in HIV-unrelated lymphadenopathies. In LAS lymph nodes, EBV-carrying cells were identified as isolated, cytologically normal elements, sometimes with immunoblastic morphology, usually scattered throughout the interfollicular areas. By contrast, the expression of HIV p24 was restricted to germinal center cells. All the EBV+ LAS samples were negative for the expression of EBV-encoded latent (LMP-1 and EBNA-2) and replicative proteins (BZLF-1, BHLF-1, EA-D, EA-R and VCA). In addition, amplification of the immunoglobulin heavy-chain genes using 2 different polymerase-chain-reaction protocols showed evidence of B-cell clonal expansion in 2/20 (10%) LAS, one EBV- case, and one sample with low numbers of EBV-infected cells. These results suggest that (i) EBV-carrying cells are probably not involved in the development of LAS, either directly or indirectly; (ii) type-2-EBV-infected cells are present in LAS lymph nodes from the early phases of HIV infection; (iii) EBV-carrying LAS per se probably does not constitute a lesion at high risk for subsequent development of EBV+ lymphomas; (iv) it is unlikely that a high viral load or strong EBV-mediated antigenic stimulation plays a contributory role in the development of EBV-unrelated lymphomas of HIV-seropositive individuals.

Detection of southern African human immunodeficiency virus type 1 subtypes by polymerase chain reaction: evaluation of different primer pairs and conditions.

The purpose of the study was to develop a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect HIV-1 subtypes present in the Western Cape, South Africa. Twenty-two virus strains, belonging to HIV-1 subtypes B, C and D, were randomly selected for PCR evaluation. Cell lysates prepared from these virus-infected cultured cells were tested using 5 different primer pairs: gag SK38/SK39; gag 22/SK39; gag a/b, gag c/d (nested); env SK68/SK69 and LTR SK29/SK30. Eight different PCR profiles were evaluated: one profile each for the 3 gag primer pairs, 3 profiles for the env and 2 profiles for the LTR primer pairs. The number of PCR cycles, time per cycle and/or annealing temperature were changed in each profile. The optimum PCR profile for a specific primer pair was defined as that which detected one copy of proviral plasmid DNA after dot-blot hybridisation. Gag primer pairs detected HIV-1 DNA in all 22 samples. With the env primer pair, suboptimal conditions failed to detect most of the HIV-1 subtype C samples. By increasing the number of cycles and time per cycle, a 100% sensitivity was achieved. With the LTR primer pair all samples were detected by decreasing the annealing temperature and increasing the individual cycle times. This confirms that once PCR conditions are optimised, all HIV-1 subtypes in our study could be detected using different PCR primer pairs.

PIP: During 1984-92, in South Africa, virologists isolated HIV-1 from HIV/AIDS patients at hospitals in the Western Cape. Two virologists from the University of Stellenbosch Hospital in Tygerberg selected 22 virus strains, belonging to HIV-1 subtypes B, C, and D, to study in order to develop a specific and sensitive polymerase chain reaction (PCR) protocol using env, gag, and LTR primers. They used five different primer pairs to prepare cell lysates from the HIV-infected cultured cells: gag SK38/SK39, gag 22/SK39, gag a/b, gag c/d (nested), env SK68/SK69, and LTR SK29/SK30. The virologists evaluated eight different PCR profiles: one profile each for the three gag primer pairs, three profiles for the env, and two profiles for the LTR primer pairs. They changed the number of PCR cycles, time per cycle, and/or annealing temperature in each profile. The PCR profile for a specific primer pair that detected one copy of proviral plasmid DNA after dot-blot hybridization was considered the optimum PCR profile. Gag primer pairs detected HIV-1 DNA in all 22 samples. The env primer pair did not detect most HIV-1 subtype C samples. When the researchers increased the number of cycles and time per cycle, the env primer pair achieved 100% sensitivity. When they decreased the annealing temperature and increased the individual cycle times, the LTR primer pairs detected all samples. These findings support that optimization of a PCR assay is necessary to achieve high assay sensitivity, specificity, and reproducibility and that PCR sensitivity should be considered seriously when interpreting PCR results for HIV diagnosis.

Clinical evaluation of Amplicor HIV-1 test for detection of human immunodeficiency virus type 1 proviral DNA in peripheral blood mononuclear cells.

We report here the clinical evaluation of Amplicor polymerase chain reaction (PCR) assay for the detection of the human immunodeficiency virus type 1 (HIV-1) in peripheral blood mononuclear cells (PBMCs). Results obtained with Amplicor HIV-1 test were compared to serological status and a standard PCR assay using SK38/SK39 and oligomer hybridization with SK19. A panel of 208 well-characterized specimens was analyzed, including PBMC lysates from 47 antibody-negative high-risk individuals, eight antibody-negative low-risk subjects, two subjects with acute retroviral disease, 35 asymptomatic seropositive subjects (59 samples) with CD4 counts > 400/mm3, 31 patients (46 samples) with AIDS-related complex (ARC), 30 patients (40 specimens) with AIDS, and six seropositive patients with unknown clinical status. Amplicor demonstrated a specificity of 100% and a sensitivity of 98.7%. Of the two false-negative samples with Amplicor, one was negative for beta-globin amplification, whereas a dilution of the other sample turned positive for HIV-1. Inhibitors of Taq polymerase were thus believed to be responsible for the negative results. This study demonstrates that commercialized nonisotopic PCR assays reach adequate levels of sensitivity and specificity for diagnosis of HIV-1 infection and could be considered in clinical situations in which serology is not helpful.

The predictive value of changes in serologic and cell markers of HIV activity for subsequent clinical outcome in patients with asymptomatic HIV disease treated with zidovudine.

OBJECTIVE: To determine if serologic marker responses to zidovudine treatment during the first year of antiretroviral therapy could predict subsequent HIV disease progression independently of absolute CD4 lymphocyte responses. METHODS: We conducted a case-control study in patients with asymptomatic HIV disease, who were initiating zidovudine therapy in a randomized, prospective trial. A total of 102 patients who progressed to AIDS or advanced AIDS-related complex and 177 randomly selected controls matched by baseline CD4 cell count and duration of follow-up had serum samples (from prior to and at 8, 16, 32 and 48 weeks of zidovudine treatment) assayed for acid-disassociated HIV p24 antigen, beta 2-microglobulin (beta 2M), neopterin, soluble interleukin (IL)-2 receptor, soluble CD4 protein and soluble CD8 protein. RESULTS: Median time to event for cases was 20.2 months; median follow-up on study was 35.4 months for controls. After controlling for absolute CD4 count at baseline, increased baseline serum concentrations of HIV p24 antigen, beta 2M, neopterin, and soluble IL-2 receptor were highly predictive of increased risk of HIV disease progression. In a multiple logistic regression model, controlling for baseline marker values, change in beta 2M consistently added independent value to change in CD4 count in predicting subsequent risk of disease progression. CONCLUSIONS: Monitoring serum immunologic markers, in particular beta 2M, in addition to absolute CD4 lymphocyte counts prior to and within the first 4 months after initiating dideoxynucleoside therapy can increase the accuracy of estimations of subsequent long-term risk of clinical HIV disease progression. This information may be useful to clinicians and patients who are making decisions about initiating or changing antiretroviral therapy.

Human immunodeficiency virus type-1 can be detected in monocytes by polymerase chain reaction.

Lymphocytes and monocytes from 25 patients infected with human immunodeficiency virus type-1 (HIV-1)--13 asymptomatic, seven with the AIDS-related complex (ARC) and five with the acquired immunodeficiency syndrome (AIDS)--were lysed and subjected to PCR with three primer pairs: SK38/SK39 (gag), SK68/SK69 (env) and SK29/SK30 (LTR). Amplified DNA was solution-hybridised with 32P-labelled probes (SK19, SK70 and SK31, respectively) and detected by PAGE-autoradiography. HIV-1 DNA was detected as follows. Asymptomatic patients: monocytes--gag 61.5%, env 100%, LTR 0%; lymphocytes--gag 100%, env 92.3%, LTR 53.84%. ARC patients: monocytes--gag 71.4%, env 57.1%, LTR 0%; lymphocytes--gag 100%, env 71.4%, LTR 71.4%. AIDS patients: monocytes--gag 80.0%, env 100%, LTR 0%; lymphocytes--gag 100%, env 60%, LTR 60%. The presence of HIV-1 DNA was confirmed in the monocyte fraction. In this cell subset, the env gene-directed primers were the most effective for amplification, whereas the LTR gene-directed primers failed to amplify HIV-1 DNA. The different pattern of amplification found in monocytes may suggest that these cells could be infected by a genetic variant of the virus.