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Original article

Epidemiology, clinical and laboratory characteristics of currently alive HIV-1 infected former blood donors naive to antiretroviral therapy in Anhui Province, China

XU, Jian-qing; WANG, Jian-jun; HAN, Li-feng; XU, Chen; RUAN, Yu-hua; XU, Zhen-hou; CHEN, Xi; LIU, Zhen-dong; WANG, Jun; SU, Bing; DING, Xin-ping; GAO, Bing; GU, Yong-bin; CAO, Xiao-yun; XING, Hui; HONG, Kun-xue; PENG, Hong; ZHAO, Quan-bi; YUAN, Lin; FENG, Yi; ZHANG, Gui-yun; MA, Li-ying; WU, Lan; SHAO, Yi-ming

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Abstract

One hundred and thirty-five thousand six hundred and thirty immunodeficiency virus type 1 (HIV-1)-infected cases had been reported in China by the end of September, 2005. Injecting drug usage, unregulated blood/plasma donation and sexual contact constituted the major spreading pathways and accounted for 40.8%, 23.0% and 9.0% of HIV-1 prevalence, respectively.1 It was estimated that 650 000 people are currently living with HIV/AIDS in China and this number could zoom to 10 million or even more by 2010 if no effective measures will be taken. In order to restrain the HIV/AIDS pandemic and to strength the care of HIV/AIDS affected individuals, Chinese government had initiated National Free Antiretroviral Therapy (ART) Program firstly among HIV-1 infected former blood/plasma donors (FBDs) and thereafter among other high-risk groups since the end of 2003, which highlighted the urgency and importance to collect both clinical and laboratory characteristics from different HIV-1 infected populations, specially from FBDs.

Unregulated commercial blood/plasma collection among farmers occurred between 1992 and 1995 in several provinces in central China, including Henan, Anhui and Shanxi provinces, and caused the second major epidemic of HIV-1 infection in China. Although this was eradicated by Chinese government by the end of 1995,2 the practice of using contaminated blood collection equipment or re-infusing pooled buffy coats back to donors caused rapid HIV-1 and hepatitis C virus (HCV) spreading among those FBDs.3,4 Up to now, a number of HIV-infected FBDs remain alive, however, their disease status are largely unknown.

HIV-1-infected FBDs represent a unique population to study HIV/AIDS disease progression because the outbreak of HIV-1 infection occurred within a narrowed period and the majority of FBDs were infected with HIV from a common-source exposure to contaminated blood. Current survivors are comprised of typical progressors and long-term survivors (long-term non progressors) since they have lived for more than 10 years after HIV-1 infection. To characterize this population and thereafter to study viral and host factors associated with disease progression will be likely to add important information to the body of current knowledge. Meanwhile, the collected data will be served to China National Free ART Treatment Program. Under the support of China Integrated Programs for Research on AIDS (CIPRA), we conducted a cross-sectional study among HIV-1-infected FBDs in Fuyang prefecture city in Anhui Province, one of the provinces with the high prevalence of unregulated blood collection in early 1990s and thereby severely devastated by HIV/AIDS. Their demographic information, clinical manifestations and several key laboratory indicators are described and analyzed here.

METHODS

Subjects

The cross-sectional study was initiated after the study protocol was sequentially approved by the National Institute of Health, USA, the Institutional Review Board (IRB) of China Center for AIDS/STD Control and Prevention and the IRB of Anhui Provincial Center for Disease Control and Prevention, respectively. Participants were recruited from Funan County and Yingzhou District in Fuyang City through two clinics, one clinic is located in Wanhua Township Hospital in Funan County, and the other resides in Wandian Township Hospital in Yingzhou District. An administrated-interviewer screening questionnaire was used to collect information about participants' demographics and high-risk behaviors, and then information on medical and medication history, and recent clinical manifestations was collected by physicians. Thereafter, a detailed physical examination was conducted and participants were staged according to WHO Staging System5 by physicians. All the above procedures were completed within day 1 at visit and the blood was collected in the morning of day 2 to reconcile with the set time of the blood shipping airplane. Each clinic averagely enrolled 10 participants every two days, which resulted in enrolling 20 participants every two days for two clinics. A subject was eligible for HIV-1 seropositive FBD only if all the following criteria apply: age > 18 years, self-reported history of blood donation in 1995 or before, documentation of HIV-1 infection by ELISA and confirmed by Western blot test, ART naive by self report, negative pregnancy testing for females, willing to provide adequate locator information for study retention purpose, willing and able to provide informed consent. HIV-1 negative control satisfied following criteria: age > 18 years old, HIV-seronegative by ELISA, negative pregnancy testing for females, willing to provide adequate locator information, willing to receive repeat HIV testing, and willing and able to provide informed consent. Participant's HIV-1 infections were previously identified through an ELISA screening and a subsequent Western blot assay confirmation by China CARE Program (launched in early 2003 by Chinese government. This is a comprehensive community-based HIV/AIDS program; its activities include advocacy, communication, preventive services, voluntary counseling and testing, free treatment of opportunistic infections, free antiretroviral therapy, and community-based care and support). All participants signed the written informed consents.

Laboratory tests

Blood from all participants was collected into EDTA Vacutainer® (BD Company, USA) by venipuncture, shipped to the laboratory at China National Center for AIDS/STD Control and Prevention (NCAIDS) within 10 hours by air, and then aliquoted and distributed to different laboratories for following assays: hematology testing, CD4+ and CD8+ T cell counts, HIV antibody testing, hepatitis C antibody testing, hepatitis B surface antigen testing, and plasma HIV-1 RNA (viral load). All assays were conducted according to their corresponding standard operation procedures (SOP).

The hematology parameters, including hematocrit, hemoglobin and white blood cell (WBC) differentials, were generated by ADVIA 70 Bayer Hematology System in the Clinical Laboratory at Beijing Youan Hospital. HIV antibody testing [Anti-HIV(1+2) ELISA kit, Wantai, Beijing; Anti-HIV-1 Western blot kit, Aoya, Hangzhou, China], hepatitis C antibody testing (Anti-HCV antibody testing kit, Lizhu Diagnostics, Zhujiang, China) and hepatitis B surface antigen testing (HBsAg ELISA kit, Wantai, Beijing, China) were performed in National Reference Laboratory at NCAIDS. CD4+/CD8+ T cell counts were quantified by a single-platform flow cytometry (EPICS-XL, Coulter) with TruCount package (BD, USA). Viral loads were quantified by COBAS AMPLICOR™ HIV-1 MONITOR Test, v1.5 (Roche Diagnostics, USA). Both assays were performed in the Division of Research on Virology and Immunology Laboratory at NCAIDS in Beijing. The reliability of those assays was proved by international standard external quality assurance programs.

Data collection and statistical analysis

All data were collected on case report forms (CRFs) and transferred to database in Beijing via Datafax System. Database manager had been monitoring data transferring and reported data errors to study site or lab once he identified; he continued to do so until the error had been corrected. The data from database were then converted into statistical analysis (SAS 9.1 for Windows, SAS Institute Inc., NC, USA) for analysis. All data were analyzed either according to HIV sero-positive/negative or to stratified CD4+ T cell counts. ANOVA and t test were used to calculate statistical values for continuous variables whereas chi-squared test or Fisher exact test was used for categorical variables. Finally, univariate and multivariable linear regression analysis were employed to examine the association of CD4+ T cell count with all other criteria. A P value less than 0.05 was considered statistically significant.

RESULTS

Demographic and epidemiologic characteristics

Three hundred and fifty-three subjects were enrolled in this study from June 1 to August 26, 2005, including 294 HIV-positive FBDs and 59 HIV-seronegative individuals (Table 1). Their ages ranged from 27 to 65 years old with a mean of (42 ± 8) for HIV-positive participants and from 27 to 62 years old with a mean of (44±8) for HIV seronegative participants. The majority of HIV-1-infected FBDs were either illiterate (44.6%) or only received primary school education (42.1%). No injecting drug usage was identified in HIV-positive participants. All HIV-positive participants are naive to ART. Both HIV-positive and seronegative groups were comprised of about a half male and a half female study subjects. While all HIV-positive participants had blood/plasma donation history, 31 out of 59 seronegative subjects (52.5%) also had the practices of blood/plasma donation, however, those HIV-seronegative subjects donated significantly less frequent than that in HIV-positive subjects (16 times in seronegative subjects versus 49 times in HIV-positive subjects), indicating that the risk to HIV-1 infection is positively associated with more frequent to donate blood/plasma. Blood donation vastly occurred 10.8–12.8 year ago. No differences were observed on the history of smoking among HIV-positive and -negative groups. All detailed data are shown in Table 1.

Table 1
Table 1:
Demographic and epidemiologic characteristics of participants

Clinical and laboratory characteristics

Clinical manifestations, such as fatigue, poor appetency, fever, diarrhea, weight loss, dermatosis, etc appeared more frequent in HIV-positive group than in sero-negative group (all P<0.05), indicating those symptoms might be associated with HIV-1 infection (Table 2). Hemoglobin, hematocrit and lymphocytes profoundly decreased in HIV-positive group from sero-negative group (all P<0.001). Furthermore, HIV-positive group has much less CD4+ T-cell counts and percentages than that in HIV sero-negative group (334 cells/μl vs 665 cells/μl for absolute counts, 20% vs 37% for percentages). In contrast, CD8+ T-cell counts and percentages are significant higher in HIV-positive group than in HIV sero-negative group. Overall, the ratio of CD4/CD8 in HIV-infected population reduced to 0.45 from 1.35 in sero-negative population. HIV-HCV co-infection is more prevalent in HIV positive participants (89.5%) than in HIV sero-negative participants (50.8%, P<0.001), however, both are highly above what has been reported in general population.6 Considering only 31 out of 59 HIV sero-negative subjects have the plasma donation history and HCV mainly transmitted through blood, overall 97% HIV sero-negative FBDs were infected by HCV. In contrast, the prevalence of HIV-HBV co-infection was much lower in HIV-positive participants (3.7%) than in HIV sero-negative group (13.6%). All data are detailed in Table 2.

Table 2
Table 2:
Clinical and laboratory characteristics of participants

Clinical manifestations and laboratory criteria among different CD4+ T-cell count groups

Out of 294 HIV-positive participants, 77 (26.2%) had CD4 <200 cells/μl, 85 (28.9%) and 83 (28.2%) had CD4+ T-cell ranges between 200–349 and 350–499, respectively. Forty-nine (16.7%) remained relatively healthy with CD4+ T-cell counts ≥500 cell/μl. Since FBDs on highly active anti-retroviral therapy (HAART) and cases died of HIV/AIDS before excluded in our study, our results may significantly underestimate the fraction of subjects on advanced HIV/AIDS, which in turn causes the overestimation of the fraction of population with high CD4+ T-cell counts. While the majority of laboratory parameters decreased in parallel with the CD4+ T-cell counts, clinical manifestations appeared more frequently in low CD4+ T-cell subgroups. A steady increase of cases with advanced disease stages according to WHO staging system was observed with the decrease of CD4+ T-cell counts; this reverse drift noticeably reached the peak in the subgroup with CD4+ T-cell counts <200 cells/μl (occupied 64.3% cases in all). A similar pattern was also identified for infectious dermatosis. Low CD4+ T-cell counts inversely associated with high viral loads, which is clearly exemplified with the percentage increase in the fraction with viral loads ≥30000 copies/ml plasma. All date are detailed in Table 3.

Table 3
Table 3:
Demographic, clinical and laboratory features among subgroups of HIV-1-positive FBDs with stratified CD4+ T-cell counts

Association of clinical and laboratory characteristics with CD4+ T cell counts

When unadjusted univariate linear regression analysis between CD4+ T-cell counts and demographic, clinical or laboratory factors was performed, more than 20 variables might be associated with CD4+ T-cell change (Table 3, P>0.10). However, when adjusted multivariate linear regression analysis was employed, only five factors, including total lymphocyte counts, viral loads, hemoglobin level, CD8+ T-cell counts and dermatosis, were identified to be significantly associated with CD4+ T-cell counts in the final model (Table 4). Total lymphocyte counts displayed the strongest positive association with CD4+ T-cell counts (standardized regression coefficient=0.5). Moreover, a decreased hemoglobin level was observed over disease progression and dermatosis appeared more frequent in subjects with decreased CD4+ T-cell counts. In contrast, viral load and CD8+ T-cell counts were inversely associated with CD4+ T-cell counts. The all five factors together accounted for 43.9% of CD4+ T-cell count changes (Table 4).

Table 4
Table 4:
Adjusted relationship of CD4+ T-cell counts with demographic, clinical and laboratory characteristics

DISCUSSION

A comprehensive cross-sectional study on FBDs was carried out under the support of CIPRA grant. This is the first large-scale study on this unique population. It was reported that this population had a compelling high prevalence at 9.1%-12.5% of HIV-1 infection,7,8 which resulted in 2.1% HIV-1 infection in their descendants,9 therefore, FBDs is a population severely devastated by HIV-1 infection and accounts for 23% prevalence of HIV-1 infection in China. However, more detailed study on the status of disease progression in this population has been largely unavailable so far. After more than a decade of HIV-1 infection, the majority of FBDs have progressed into AIDS stage whereas a small fraction remained relatively healthy as long-term survivors/non-progressors, it is critical to collect information on both host and viral factors associated with disease progression from this unique population before the loss of entire typical progressor population, which will be likely to contribute to the understanding of protective and pathogenic factors during HIV/AIDS disease progression.

We have examined demographic, clinical and key laboratory characteristics among 294 HIV-1 infected FBDs. Our data showed that the practice of unregulated blood/plasma donation in Fuyang prefecture city occurred more than a decade ago in less educated farmers. More frequent blood/plasma donation was clearly associated with the risk of HIV-1 infection, which is in accordance with previous observation in this population.7 Similar to injecting drug users10 and other study on FBDs,11 an extremely high prevalence of HCV infection was observed in FBDs, which indirectly corroborated the practice of re-infuse of pooled buffy coat back to donors since this practice is similar to the needle sharing and could cause HIV and HCV spreading. HCV co-infection could cause hepatitis and may partially account for the appearance of clinical manifestations, such as fatigue and weight loss. The influence of HCV co-infection on disease progression will be further analyzed between subpopulations with HIV-1 infection alone and HCV-HIV dual infection. Albeit in small number, there is a trend of less HBV co-infection (3.7%) with HIV-1 in FBDs comparing to general Chinese population (reported as between 8.0%-15.3%12,13), which may be explained by that HBV co-infection could facilitate disease progression14,15 and thereby leads to a shortened life expectancy in dual infected subjects.

Importantly, when we examined data by stratified CD4+ T-cell counts, a deterioration in both clinical manifestation and laboratory parameters and an increase of viral loads were observed in parallel with the decrease of CD4+ T-cell counts, suggesting that CD4+ T-cell counts is a reliable indicator for disease progression among FBDs. Noticeably, the majority of cases at WHO Stage III emerged after CD4+ T-cell counts dropped below 200 cells/μl, indicating that 200 cells/μl of CD4+ T-cell counts is a reliable dichotomous line for AIDS disease progression and it is beneficial to patients to initiate antiretroviral therapy if their CD4+ T-cell counts is <200 cells/μl. In addition, total lymphocyte counts decreased in parallel with CD4+ T-cell counts, which is in accordance with previous reports16–18 and total lymphocyte counts may be used as a surrogate for CD4+ T-cell counts in resource-poor regions.

Due to the fact that skin and mucosa are the interfaces interacting with outside pathogens, immunodeficiency may firstly cause repeated or persistent infection on those surfaces and thereby skin or mucosal infection could predict the disease progression to AIDS. In accordance with this hypothesis, previous report demonstrated that persistent thrush, an infection caused by fungus, was a predictor for progression to AIDS;19 Similarly, dermatosis was observed to be associated with disease progression in our study. The discrepancy between our study and previous report may be caused by the fact that thrush could cause worse suffering than dermatosis and thereby stimulate HIV-infected individuals to approach the free ART program. Since free ART program as an important component in China Cares Program had been started since the end of 2003, the majority of HIV-1 infected FBDs at advanced WHO stage may have been recruited for treatment and our study only enrolled subjects naive to ART. Therefore, the data from our study are likely to underestimate the fraction of subpopulation with CD4+ T-cell counts <200 cells/μl, the appearance of clinical symptoms which could cause worse suffering and to overestimate the fraction of subpopulation with CD4+ T-cell counts >500 cells/μl. We also failed to identify significant difference of Karnofsky Performance Scores among groups with different CD4+ T-cell counts and none of study participants was diagnosed at WHO stage IV, which may attribute to the fact that less active individuals were either unable to take a long walk to participate in our study or have been enrolled into the free ART program.

Overall, coinfection with HCV but not HBV is highly prevalent among HIV-1-infected FBDs, a quarter of them naive to HAART have progressed into AIDS phase (CD4+ T-cell counts <200 cells/μl). Five factors, including total lymphocyte counts, viral load, hemoglobin level, CD8+ T-cell counts and dermatosis, related to CD4+ T-cell count changes. Decreased total lymphocyte counts, lower level of hemoglobin and appearance of dermatosis were observed to be associated with the decreased CD4+ T-cell counts whereas viral loads and CD8+ T-cell counts were inversely associated with CD4+ T-cell counts. It is known that high viral loads is the driving force to cause faster depletion of CD4+ T cells20,21 and stronger CD8+ T-cell activation; HIV-1 infection also damage other arms of immune system, including innate immunity,22,23 thereby resulting in the emergence of opportunistic infection, such as dermatosis. In accordance with previous reports,16–18 both total lymphocyte counts and hemoglobin are predictive for progression of HIV-1 infection. A cohort study on FBDs has been ongoing; all factors observed in this cross-sectional study will be further tested for association with disease progression in the cohort study.

Acknowledgements:

We would like to thank CHANG Jason (Clinical Protocol Development Associate), JIAO Jin-hua (WESTAT Corporation, USA) and Ray Chen (National Institute of Health, USA) for their helpful comments and suggestions, and also to acknowledge all study participants for their contribution.

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Keywords:

human immunodeficiency virus; blood donors; disease progression; co-infection; hepatitis C

© 2006 Chinese Medical Association