Rapid Multiplexed Immunoassay for Detection of Antibodies to Kaposi's Sarcoma-Associated Herpesvirus.

Diagnosis of KSHV-infected individuals remains a challenge. KSHV prevalence is high in several populations with high prevalence of HIV, leading to increased risk of development of Kaposi's sarcoma (KS). While current assays are reliable for detecting antibodies to KSHV, none are routinely utilized to identify individuals with KSHV infection and thus at increased risk for KS due to assay complexity, lack of access to testing, and cost, particularly in resource-limited settings. Here we describe the addition of KSHV proteins LANA and K8.1 to a previously evaluated HIV/co-infection multiplexed fluorescence immunoassay system. This study demonstrates assay performance by measuring antibody reactivity for KSHV and HIV-1 in a collection of clinical specimens from patients with biopsy-proven KS and sourced negative controls. The KSHV assay correctly identified 155 of 164 plasma samples from patients with biopsy-proven KS and 85 of 93 KSHV antibody (Ab)-negative samples for a sensitivity of 95.1% and specificity of 91.4%. Assay performance for HIV-1 detection was also assessed with 100% agreement with independently verified HIV-1 Ab-positive and Ab-negative samples. These results demonstrate good sensitivity and specificity for detection of antibody to KSHV antigens, and demonstrate the potential for multiplexed co-infection testing in resource-limited settings to identify those at increased risk for HIV-1-related complications.

Relationship of vitamin D insufficiency to AIDS-associated Kaposi's sarcoma outcomes: retrospective analysis of a prospective clinical trial in Zimbabwe.

OBJECTIVES: The prevalence of vitamin D insufficiency in Africans with AIDS-associated Kaposi sarcoma (AIDS-KS) and the role of vitamin D in AIDS-KS progression are unknown. We hypothesized that a high prevalence of vitamin D deficiency would be found in Zimbabweans with AIDS-KS and that low baseline vitamin D would correlate with progression of AIDS-KS. METHODS: Ninety subjects were enrolled in a prospective pilot study investigation of the effect of antiretroviral therapy in the treatment of AIDS-KS in Harare, Zimbabwe. Co-formulated abacavir, lamivudine, and zidovudine was initiated; chemotherapy was provided at the discretion of the provider. Participants were followed for 96 weeks. 25-Hydroxyvitamin D was measured in stored specimens collected at study entry. The relationship between vitamin D and clinical response was described by odds ratio and 95% confidence interval. RESULTS: Samples were available for 85 participants; 45 (53%) subjects had inadequate (<75 nmol/l) 25-hydroxyvitamin D. HIV-1 RNA was significantly higher among those with insufficient vitamin D (4.7 vs. 4.5 log, p = 0.04). Tumor response, survival, and KS-IRIS were not associated with vitamin D (p ≥ 0.3). CONCLUSIONS: Vitamin D insufficiency was common among Zimbabweans with AIDS-KS but not associated with outcomes after initiation of antiretroviral therapy.

Evaluation of plasma human herpesvirus 8 DNA as a marker of clinical outcomes during antiretroviral therapy for AIDS-related Kaposi sarcoma in Zimbabwe.

BACKGROUND: The usefulness of plasma human herpesvirus 8 (HHV-8) DNA as a marker of response to treatment for acquired immunodeficiency syndrome-associated Kaposi sarcoma (AIDS-KS) in an African setting is unknown. METHODS: We conducted a prospective pilot study at the Parirenyatwa Hospital Kaposi Sarcoma Clinic (Harare, Zimbabwe) to investigate the hypothesis that the clinical response of AIDS-KS is associated with suppression of HHV-8 DNA. Antiretroviral therapy (ART) was provided as coformulation of abacavir, lamivudine, and zidovudine. Clinical response was defined as survival to week 96 with either complete or partial resolution of KS disease. RESULTS: Ninety ART-naive participants (62 men and 28 women) aged >18 years who had human immunodeficiency virus type 1 (HIV-1) infection and biopsy-confirmed KS were studied; 82% had stage T1 disease. Fifty participants received adjunctive chemotherapy. The median CD4(+) lymphocyte count increased from 124 cells/microL at baseline to 281 cells/microL, the plasma HIV-1 RNA level decreased from 4.69 to <2.60 log(10) copies/mL, the plasma HHV-8 DNA level decreased from 660 to <25 copies/mL, and HHV-8 DNA level in peripheral blood mononuclear cells decreased from 2790 to 37 copies/10(6) cells (P < .001 for each comparison). There were 14 deaths (16%) and 13 patients (15%) lost to follow-up. The most common cause of death was infection. Clinical response of KS occurred in 17 participants (19%). Pretreatment plasma HHV-8 DNA levels of <660 copies/mL were associated with greater survival (odds ratio, 2.83; 95% confidence interval, 1.07-7.53; P = .04) and a better clinical response (odds ratio, 6.38; 95% confidence interval, 1.68-24.19; P = .006). CONCLUSIONS: AIDS-KS tumor responses after ART initiation were limited. Pretreatment plasma HHV-8 DNA level may be a surrogate for KS disease that is in need of intensive clinical management.

Lack of evidence for frequent heterosexual transmission of human herpesvirus 8 in Zimbabwe.

BACKGROUND: There is conflicting evidence about the contribution of heterosexual transmission to the spread of human herpesvirus 8 (HHV-8) in southern Africa. This study evaluated the hypothesis that HHV-8 infection is associated with risk factors for human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted infections among Zimbabwean men. METHODS: HHV-8 seroprevalence was determined for 2750 participants in the Zimbabwe AIDS Prevention Project cohort of male factory workers in Harare, Zimbabwe. Potential associations of HHV-8 antibody detection with risk factors for HIV-1 infection were examined by univariate analysis. Variables with P < .1 in the univariate analysis were included in a multivariate logistic regression model. HHV-8 seroprevalence was also determined among 297 heterosexual couples. RESULTS: Prevalence of HHV-8, HIV-1, and HHV-8 and HIV-1 coinfection was 28.5% (95% confidence interval [CI], 26.8%-30.2%), 19.5% (95% CI, 18.0%-20.9%), and 6.5% (95% CI, 5.6%-7.5%), respectively. Detection of HHV-8 antibodies was independently associated with older age and HIV-1 infection but not with number of recent sex partners, marital status, education, condom use, prior sexually transmitted infections, payment for sex, chronic hepatitis B infection, or incident HIV-1 infection. HHV-8 seroprevalence was 31.7% (95% CI, 26.3-37.0) among wives in the couples tested, but HHV-8 infection of wives was not associated with HHV-8 infection of husbands (odds ratio, 1.08; 95% CI, 0.62-1.88; P = .8). CONCLUSIONS: HHV-8 and HIV-1 infection did not have common sexual risk factors among urban Zimbabwean men. Sexual transmission does not explain the high prevalence of HHV-8 in this population.

Genetic diversity of the Kaposi's sarcoma herpesvirus K1 protein in AIDS-KS in Zimbabwe.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes genetically diverse K1 alleles which have unique geographic distributions. Little is known about K1 genetic diversity in Zimbabwe where acquired immunodeficiency syndrome-associated KS (AIDS-KS) is epidemic. OBJECTIVE: Evaluate K1 diversity in Zimbabwe and compare Zimbabwean K1 diversity to other areas in Africa. STUDY DESIGN: K1 nucleotide sequence was determined for AIDS-KS cases in Zimbabwe. K1 references sequences were obtained from Genbank. RESULTS: Among 65 Zimbabwean AIDS-KS cases, 26 (40%) were K1 subtype A and 39 (60%) were subtype B. Zimbabwean subtype A sequences grouped only with African intratype A5 variants. Zimbabwean subtype B sequences grouped with multiple intratype African variants: 26 B1 (26%), four B3 (6%) and nine highly divergent B4 (14%). Zimbabwean subtype B had a lower synonymous to nonsynonymous mutation ratio (median 0.59 versus 0.66; P=0.008) and greater distance to the most recent common ancestor (median 0.03 versus 0.009; P<0.001) compared to subtype A. Within the B subgroup, the distribution of intratype B variants differed in Zimbabwe and Uganda (P=0.004). CONCLUSIONS: Greater positive selection and genetic diversity in K1 subtype B compared to subtype A5 exist in Zimbabwe. However, there were no significant associations between K1 subtype and the clinical or demographic characteristics of AIDS-KS cases.

Gender differences in AIDS-associated Kaposi sarcoma in Harare, Zimbabwe.

Reasons for gender-related differences in the risk of AIDS-related Kaposi sarcoma (AIDS-KS) are unknown. Four hundred thirty-eight male and 166 female AIDS-KS patients were evaluated in Harare, Zimbabwe. Female patients were younger than male patients in this study (median of 33 vs. 38 years; P < 0.001), mirroring the epidemiology of AIDS in Zimbabwe. In a multivariate model adjusted for CD4 T-cell count, age, prior radiation treatment, and chemotherapy, women were more likely to report fever, diaphoresis, or weight loss (odds ratio = 1.7, 95% confidence interval: 1.1 to 2.7; P = 0.009). These findings suggest an increased severity of KS or other unidentified infections among women with AIDS-KS in Zimbabwe.

Relationship of Kaposi sarcoma (KS)-associated herpesvirus viremia and KS disease in Zimbabwe.

The relationship between Kaposi sarcoma-associated herpesvirus (KSHV) viremia and KS disease was investigated in 500 subjects who received treatment in Harare, Zimbabwe. Subjects were grouped by results of human immunodeficiency virus (HIV) type 1 serological tests, KS diagnosis, and KS clinical stage. The plasma KSHV DNA concentration was associated with concomitant KS and HIV-1 infection (AIDS-KS; P<.001) and AIDS-KS clinical stage (P=.01). Plasma KSHV DNA levels were greater in AIDS-KS than in matched HIV-1-seronegative KS (P=.04). The plasma KSHV DNA level was not associated with age, sex, systemic symptoms, or CD4+ lymphocyte count. Plasma and peripheral blood mononuclear cell KSHV DNA concentrations were linearly related (r2=.44; P<.001), and the nucleotide sequence of the K1 gene highly variable region was identical in both compartments. These findings provide evidence that KSHV viremia is common in advanced AIDS-KS in Zimbabwe and suggest a relationship between KSHV lytic replication and untreated HIV-1 infection.