The single recombinant M. tuberculosis protein DPPD provides enhanced performance of skin testing among HIV-infected tuberculosis patients.

Diagnostic testing for M. tuberculosis infection has advanced with QuantiFERON and GeneXpert, but simple cost-effective alternatives for widespread TB screening has remained elusive and purified protein derivative (PPD)-based tuberculin skin testing (TST) remains the most widely used method. PPD-based tests have reduced performance, however, in BCG vaccinees and in individuals with immune deficiencies. We compared the performance of skin testing with the recombinant DPPD protein against that of a standard PPD-based skin test. Our data indicates similar performance of DPPD and PPD (r2 = 0.7689) among HIV-negative, active TB patients, all of whom presented greater than 10 mm induration following administration. In contrast to results demonstrating that PPD induced indurations greater than 5 mm (i.e., the recommended threshold for positive results in this population) in only half (19 of 38) of the HIV positive TB patients, 89.5% (34 of 38) of these participants developed indurations greater than 5 mm when challenged with DPPD. Importantly, none of the patients that were positive following PPD administration were negative following DPPD administration, indicating markedly improved sensitivity of DPPD among HIV-infected individuals. Our data indicate that DPPD has superior performance in skin testing than the current TST standard.

Co-infection with HTLV-1 is associated with a shorter survival time for HIV-1-infected patients in Bahia, Brazil.

Co-infection with HTLV-1 reaches 20% among patients infected by HIV-1 in Bahia, Brazil. To evaluate its impact on survival, we conducted a retrospective, case-control study involving 198 patients (63 cases). Co-infection was associated with parenteral exposure (P = 0.0001) and female sex (P = 0.02). Co-infected patients had a shorter mean survival (1849 days) than controls (2430 days, P = 0.001), regardless of sex or baseline CD4 cell count. In Bahia, Brazil, co-infection with HIV-1 and HTLV-1 is associated with a shorter survival time.

Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test.

Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult particularly because sensitization with non-tuberculous mycobacteria leads to false positive tests. Using the guinea pig model of tuberculosis, we have recently described a recombinant antigen (DPPD) that could circumvent this problem. The DPPD gene is unique to the M. tuberculosis complex organisms and is absent in the organisms representative of all other members of the Mycobacterium genus. Moreover, DPPD induced strong DTH in 100% of the guinea pigs infected with M. tuberculosis and in none of the guinea pigs immunized with nine different species of Mycobacterium. Here we present results of a clinical investigation using DPPD. Mantoux test using both PPD and DPPD was initially performed in 26 patients with confirmed pulmonary tuberculosis and in 25 healthy PPD negative individuals. The results indicated that both PPD and DPPD elicited DTH in 24 out of the 26 patients. No DTH was observed in any of the PPD negative individuals. In addition, a small clinical trial was performed in a population of 270 clinically healthy and randomly selected individuals. DPPD produced a bimodal histogram of skin reaction size and PPD produced a skewed histogram. Because the DPPD gene is not present in non-tuberculous bacilli, these results suggest that this molecule can be an additional tool for a more specific diagnosis of tuberculosis.

A randomized, placebo-controlled trial of granulocyte-macrophage colony-stimulating factor and nucleoside analogue therapy in AIDS.

Preliminary preclinical and clinical data suggest that granulocyte-macrophage colony-stimulating factor (GM-CSF) may decrease viral replication. Therefore, 105 individuals with AIDS who were receiving nucleoside analogue therapy were enrolled in a placebo-controlled, double-blind study and were randomized to receive either 125 microgram/m(2) of yeast-derived, GM-CSF (sargramostim) or placebo subcutaneously twice weekly for 6 months. Subjects were evaluated for toxicity and disease progression. A significant decrease in mean virus load (VL) was observed for the GM-CSF treatment group at 6 months (-0.07 log(10) vs. -0.60 log(10); P=.02). More subjects achieved human immunodeficiency virus (HIV)-RNA levels <500 copies/mL at >/=2 evaluations (2% on placebo vs. 11% on GM-CSF; P=.04). Genotypic analysis of 46 subjects demonstrated a lower frequency of zidovudine-resistant mutations among those receiving GM-CSF (80% vs. 50%; P=.04). No difference was observed in the incidence of opportunistic infections (OIs) through 6 months or survival, despite a higher risk for OI among GM-CSF recipients. GM-CSF reduced VL and limited the evolution of zidovudine-resistant genotypes, potentially providing adjunctive therapy in HIV disease.

Safety and efficacy of reduced doses of ritonavir (RTV) plus saquinavir (SQV) in the treatment of AIDS patients in Brazil

The use of reduced doses of Ritonavir (RIT) and Saquinavir (SQV) is considered a potent alternative in treating patients infected by HIV-1. We tested a combination of 300mg of RIT plus 600mg of SQV, twice daily, in association with two reverse transcriptase inhibitors to treat AIDS patients for a period of 6 monts. Evaluation of HIV-1 RNA plasma levels, CD4+/CD8+ cell count and biochemical/hematological parameters (liver enzymes, serum electrolytes, creatinin, blood glucose, uric acid, white blood cell count, platelet count, and hemoglobin level) were performed after 30, 90 and 180 days of therapy. Clinical failure and adverse reactions were also recorded in order to assess safety and efficacy of the treatment. A total of 30 AIDS patients (25 male; 5 female) were enrolled in the study. Eight patients discontinuede the therapy due to intolerance, 2 patients presented clinical failure (onset of AIDS defining events during the study period), 2 patients were excluded due to protocol violation. Five patients tolerated only a lower dose of RIT (400mg/day). Patients who completed 6 months of therapy had a drop in viral load from 4.8ñ.7log10median4.9log) to 3.4ñ1.0log10(median 2.6log), and an increase in CD4+ count from 109ñ86 cells/ml(median 84 cells/ml) to 249ñ114 cells/ml(median 265cells/ml), compared to baseline values. However, patients who used a lower dose of RIT (400mg/day) had a less impressive drop in viral load values(mean0.6log10RNA copies/ml) when compared with those using the 600mg/day of the drug(mean 2.4log10). The percentage of patients presenting undetectable levels of HIV-1 RNA in plasma was quite different for the 2 groups: 92 percent of patients with a viral load <400 RNA copies/ml were using 600mg of RIT. The combination of reduced doses of RIT and SQV reduced viral load >1.0log10 after 6 months in 83 percent of study patients. The dose of 600mg/day of RIT was more effective in reducing viral load than 400mg/day, but was less well-tolerated. CD4+ cell counts increased in all patients regardless of the RIT dose used.

Safety and Efficacy of Reduced Doses of Ritonavir (RTV) Plus Saquinavir (SQV) in the Treatment of AIDS Patients in Brazil.

The use of reduced doses of Ritonavir (RIT) and Saquinavir (SQV) is considered a potent alternative in treating patients infected by HIV-1. We tested a combination of 300mg of RIT plus 600mg of SQV, twice daily, in association with two reverse transcriptase inhibitors to treat AIDS patients for a period of 6 months. Evaluation of HIV-1 RNA plasma levels, CD4+/CD8+ cell count and biochemical/hematological parameters (liver enzymes, serum electrolytes, creatinin, blood glucose, uric acid, white blood cell count, platelet count, and hemoglobin level) were performed after 30, 90 and 180 days of therapy. Clinical failure and adverse reactions were also recorded in order to assess safety and efficacy of the treatment. A total of 30 AIDS patients (25 male; 5 female) were enrolled in the study. Eight patients discontinued the therapy due to intolerance, 2 patients presented clinical failure (onset of AIDS-defining events during the study period), 2 patients werc excluded due to protocol violation. Five patients tolerated only a lower dose of RIT (400mg/day). Patients who completed 6 months of therapy had a drop in viral load from 4.8+/-.7 log(10) median 4.9 log) to 3.4 +/- 1.0 log(10) (median 2.6 log), and an increase in CD4+ count from 109 +/- 86cells/ml (median 84cells/ml) to 249+/- 114 cells/ml (median 265 cells/ml), compared to baseline values. However, patients who used a lower dose of RIT (400mg/day) had a less impressive drop in viral load values (mean 0.6 log(10) NA copies/ml) when compared with those using the 600mg/day of the drug (mean 2.4 log(10)). The percentage of patients presenting undetectable levels of HIV-1 RNA in plasma was quite different for the 2 groups: 92% of patients with a viral load <400 RNA copies/ ml were using 600mg of RIT. The combination of reduced doses of RIT and SQV reduced viral load >1.0 log(10) after 6 months in 83% of study patients. The dose of 600mg/day of RIT was morc effective in reducing viral load than 400mg/day, but was less well-tolerated. CD4+ cell counts increased in all patients regardless of the RIT dose used.

Comparison of the prevalence of antibodies to human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) in Brazil and Colorado.

The prevalence of human herpesvirus 8 (HHV-8; Kaposi's sarcoma [KS] herpesvirus) infection was determined by IFA in 297 persons living in Brazil and Colorado. The prevalence of antibody to HHV-8 in human immunodeficiency virus (HIV) type 1-seropositive gay men with and without KS was similar in Brazil and Colorado. In Brazil, the prevalence of HHV-8 antibody was significantly greater in HIV-1-seronegative gay men than in HIV-1-seronegative male intravenous drug users. HHV-8-seropositive Brazilian gay men who had a clinical diagnosis of KS or who were infected with HIV-1 had significantly higher titers of HHV-8 antibody than did HHV-8-seropositive, HIV-1-seronegative Brazilian gay men. These findings provide further support for the association between HHV-8 infection and KS and suggest that, as in the United States, HHV-8 infection is transmitted sexually in Brazil.

Co-Infection by HTLV-I/II is Associated With Increased Viral Load in PBMC of HIV-1 Infected Patients in Bahia, Brazil.

To evaluate the viral load in peripheral blood mononucler cells (PBMC) of patients infected by HIV-1 we performed 96 quantitative cultures in 74 patients infected by HIV-1, using the co-culture method. The viral load was expressed in tissue culture infectious doses (TCID), and the results were analyzed according to gender, age and clinical stage of patients, duration of previous antiretroviral therapy, detectable p24 antigenemia, CD4(+)/CD8(+) cell counts, co-infection by HTLV-I/II and viral subtype. We detected a statistically significant association between co-infection by HTLV-I/II and viral load higher than >50 TCID (p=0.003). We also found a significant association between co-infection by HTLV-I/II and p24 antigenemia (p=0.028). CD4(+) cell counts were significantly higher for patients presenting negative cultures, but there was no detectable association between lower CD4(+) cell counts and higher TCID. The majority of patients were infected by subtype B virus. The observation in this study that co-infection with HTLV-I/II was significantly associated with higher viral load raises the possibility that these agents act as co-factors of AIDS progression, in doubly-infected patients.

rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis.

The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease.

Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi.

The study attempted to identify immunologic markers for progression of Leishmania donovani chagasi infection to disease in children in an area endemic for visceral leishmaniasis (VL). [3H]thymidine uptake of lymphocytes stimulated with L. donovani chagasi antigen from children with asymptomatic infection (25,286 +/- 11,648) and from children with self-healing subclinical infection (15,511 +/- 4681) was greater (P = .001) than that observed with lymphocytes from children who progressed to classic VL (4811 +/- 2984). The interferon-gamma (IFN-gamma) levels from asymptomatic and subclinically infected children (74 +/- 90 units/ml) were higher (P = .02) than those observed in children who progressed to VL (7 +/- 8 units/ml). Absence of lymphocyte blastogenesis and IFN-gamma production were associated with progression of infection to classic VL. In the presence of these markers, children should be closely followed to identify signs and symptoms that would permit early initiation of therapy.

Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease.

One hundred fourteen Leishmania isolates from patients with different clinical forms of leishmaniasis in the State of Bahia, Brazil, were characterized by indirect radioimmune binding assay using specific monoclonal antibodies (serodeme analysis). Seventy-five of these isolates were also analyzed by enzyme electrophoresis, based on 11 enzyme loci; parasite species were compared, according to their characteristic zymodemes, to those of WHO Leishmania reference strains. All isolates could be classified into one of three species: Leishmania amazonensis (n = 40), L. braziliensis (n = 39) or L. chagasi (n = 35). The most interesting information obtained from this study is the realization that L. amazonensis is capable of producing a wide spectrum of disease in humans. Infection with this parasite was associated with many different clinical presentations, including cutaneous leishmaniasis [CL] (20/49 cases), mucocutaneous leishmaniasis [MCL] (5/13 cases) and, of special note, visceral leishmaniasis [VL] (11/46 cases), as well as four cases of post kalaazar dermal leishmaniasis [PKDL]. In situ tissue parasite characterization, by immunoperoxidase assay and employing anti-L. amazonensis amastigote monoclonal antibodies, confirmed the infection with this species in two cases of CL, one case of DCL, one case of MCL and one case of PKDL. Our results also demonstrate the difficulty of parasite differentiation based on clinical grounds, since at least L. amazonensis infection can be associated with all types of leishmanial diseases, and different Leishmania species may be associated with indistinguishable clinical presentations. Since leishmanial parasites may vary in their biological behavior or in their response to treatment, it is important that their identification be made by reliable methods.

Tumor necrosis factor (cachectin) in human visceral leishmaniasis.

High tumor necrosis factor-alpha (TNF alpha) levels were present in the serum of 24 of 28 active visceral leishmaniasis (VL) patients (142.9 +/- 113.9 pg/ml, mean +/- SD), whereas levels were not elevated in 26 of 30 patients with cryptic leishmanial infection (16 asymptomatic, 4 with self-healing subclinical infection, and 10 posttreatment VL cases). Serum TNF alpha levels were also not elevated in 15 normal volunteers (11.3 +/- 15.6 pg/ml) and in 10 patients with tegumentary leishmaniasis (19.1 +/- 10.8 pg/ml). Leishmanial infection of human monocyte-derived macrophages enhanced the basal TNF alpha production by these cells, and this effect was further potentiated by treatment with recombinant interferon-gamma. After effective treatment of VL patients, serum TNF alpha levels dropped rapidly (129 +/- 112 vs. 9 +/- 13 pg/ml in 10 days), even before clinical parameters such as spleen size or parasitism, white blood cell count, or levels of hemoglobin returned to normal values. On the other hand, patients unresponsive to treatment remained with elevated levels (276 +/- 69 vs. 155 +/- 71 pg/ml in 10 days). Thus, serum TNF alpha levels in VL patients are a good parameter to monitor in determining host response to therapy.

Treatment of visceral leishmaniasis with pentavalent antimony and interferon gamma.

Acute visceral leishmaniasis is associated with an antigen-specific immunosuppression of mononuclear cells as evidenced by defective in vitro production of interferon gamma. We evaluated treatment with recombinant human interferon gamma in combination with conventional pentavalent antimony therapy in patients with visceral leishmaniasis. Six of eight patients with visceral leishmaniasis (mean duration, 17 months) that had been unresponsive to multiple courses of pentavalent antimony responded to treatment with recombinant human interferon gamma (100 to 400 micrograms per square meter of body-surface area per day) in addition to pentavalent antimony (20 mg per kilogram of body weight per day) for 10 to 40 days. The other two patients improved initially but then relapsed and required treatment with amphotericin B. Eight of nine additional patients with previously untreated severe visceral leishmaniasis were also successfully treated with the combination of interferon gamma and pentavalent antimony. The 14 patients who responded to this regimen had marked improvement in symptoms and in measures of anemia and leukopenia, as well as weight gain, a decrease in spleen size, and an absence or reduction of leishmanias in splenic aspirates. These patients had no recurrence of illness after a mean (+/- SE) follow-up of 8 +/- 1 months. Fever was the only major side effect of interferon gamma. We conclude that the combination of interferon gamma and pentavalent antimony is effective in treating seriously ill patients with refractory or previously untreated visceral leishmaniasis.