Impact of proficiency testing program for laboratories conducting early diagnosis of HIV-1 infection in infants in low- to middle-income countries.

A voluntary, cost-free external quality assessment (EQA) program established by the U.S. Centers for Disease Control and Prevention (CDC) was implemented to primarily monitor the performance of laboratories conducting HIV Early Infant Diagnosis (EID) from dried blood spots (DBS) in low- to middle-income countries since 2006. Ten blind DBS proficiency test (PT) specimens and 100 known HIV-positive and -negative DBS specimens (to be used as internal controls) were shipped triannually to participating laboratories with reports for the PT specimens due within 30 days. The participant's results and a summary of the performance of all participating laboratories and each diagnostic method were provided after each test cycle. Enrollment in the CDC PT program expanded progressively from 17 laboratories from 11 countries in 2006 to include 136 laboratories from 41 countries at the end of 2012. Despite external pressures to test and treat more children while expanding EID programs, mean PT test scores significantly improved over time as demonstrated by the upward trend from mid-2006 to the end of 2012 (P=0.001) and the increase in the percentage of laboratories scoring 100% (P=0.003). The mean test scores plateaued over the past 10 testing cycles, ranging between 98.2% and 99.7%, and discordant test results still occur but at a rate of no higher than 2.6%. Analysis of these test results suggests a positive impact of proficiency testing on the testing performance of the participating laboratories, and a continuous training program and proficiency testing participation may translate into laboratories improving their testing accuracy.

Generation of dried tube specimen for HIV-1 viral load proficiency test panels: a cost-effective alternative for external quality assessment programs.

Participation in external quality assessment programs is critical to ensure quality clinical laboratory testing. Commercially available proficiency test panels for HIV-1 virus load testing that are used commonly in external quality assessment programs remain a financial obstacle to resource-limited countries. Maintaining cold-chain transportation largely contributes to the cost of traditional liquid proficiency test panels. Therefore, we developed and evaluated a proficiency test panel using dried tube specimens that can be shipped and stored at ambient temperature. This dried tube specimens panel consisted of 20 µl aliquots of a HIV-1 stock that were added to 2 ml tubes and left uncapped for drying, as a preservation method. The stability of dried tube specimens at concentrations ranging from 10² to 106·5 RNA copies/ml was tested at different temperatures over time, showing no viral load reduction at 37 °C and a decrease in viral load smaller than 0.5 Log10 at 45 °C for up to eight weeks when compared to initial results. Eight cycles of freezing-thawing had no effect on the stability of the dried tube specimens. Comparable viral load results were observed when dried tube specimen panels were tested on Roche CAPTAQ, Abbott m2000, and Biomerieux easyMAG viral load systems. Preliminary test results of dried proficiency test panels shipped to four African countries at ambient temperature demonstrated a low inter assay variation (SD range: 0.29-0.41 Log10 RNA copies/ml). These results indicated that HIV-1 proficiency test panels generated by this methodology might be an acceptable alternative for laboratories in resource-limited countries to participate in external quality assessment programs.

Evaluation of blood collection filter papers for HIV-1 DNA PCR.

BACKGROUND: The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4-6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. OBJECTIVES: The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. STUDY DESIGN: DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37°C with high humidity, and -20°C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37°C), at weeks 4, 8, and 18 (-20°C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at -20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. RESULTS: HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. CONCLUSIONS: Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries.

Evaluation of a high-throughput diagnostic system for detection of HIV-1 in dried blood spot samples from infants in Mozambique.

We performed a comparative analysis between Roche Amplicor HIV-1 DNA test and CAPTAQ assay for the detection of HIV in 830 dried blood spot (DBS) pediatric samples collected in Mozambique. Our results demonstrated no statistical difference between these assays. The CAPTAQ assay approached nearly 100% repeatability/accuracy. The increased throughput of testing with minimal operator interference in performing the CAPTAQ assay clearly demonstrated that this method is an improvement over the Roche Amplicor HIV-1 DNA test, version 1.5.

Systemic and mucosal immunological responses during repeated mucosal SHIV(162P3) challenges prior to and following infection in pigtailed macaques.

Local and systemic immunological changes following vaginal HIV-1 exposures are poorly characterized and may influence susceptibility to infection. Therefore, we examined longitudinal mucosal, plasma cytokine profiles and viral-specific T-cell responses (vSTRs) before and during weekly repeated low-dose SHIV(SF162P3) viral challenges in six female pigtailed macaques, even in the absence of overt systemic infection. Following a single viral challenge, induction of several cytokines was detected consistently in cervico-vaginal lavages (CVL). With additional exposure and documented systemic infection, a hallmark of response profile was defined as peak levels in both CVL (MCP-1, MIP-1alpha, TNF-alpha, IL-1beta, IL-1RA and IL-8) and plasma cytokines (MCP-1, eotaxin and IL-1RA) in the macaques. In the periphery, vSTRs were observed within the first one or two viral challenges, but prior to the detection of systemic infection in 5/6 exposed pigtailed macaques. These findings provide valuable information regarding mucosal HIV-1 infection that may benefit microbicide research and development.

Direct stringency comparison of two macaque models (single-high vs. repeat-low) for mucosal HIV transmission using an identical anti-HIV chemoprophylaxis intervention.

BACKGROUND: In our previous work, oral chemoprophylaxis with tenofovir disoproxil fumarate (TDF) provided partial protection in rhesus macaques against repeated low-dose (RL) intrarectal SHIV162p3 exposure. METHODS: Here, we make a direct comparison of these previous findings with data generated using a single high (SH)-dose challenge strategy. RESULTS: All 5 (100%) control macaques were infected after a SH challenge and only three of five (60%) TDF-treated macaques became infected. The remaining two TDF-treated macaques remained virus-negative and were susceptible to virus infection upon re-challenge in the absence of oral TDF. Thus, two of five (40%) TDF-treated macaques were protected by the pre-exposure chemoprophylaxis regimen. By comparison with the RL challenge system, only one of four (25%) of TDF-treated macaques were protected from infection, whereas four of four (100%) control macaques became infected using RL challenges. CONCLUSION: Taken together, these findings indicate that the stringency of the RL challenge model for testing antiretroviral interventions is not lower and possibly greater than that of the SH challenge model.

Chemoprophylaxis with tenofovir disoproxil fumarate provided partial protection against infection with simian human immunodeficiency virus in macaques given multiple virus challenges.

We examined the efficacy of tenofovir disoproxil fumarate (TDF) in blocking simian human immunodeficiency virus (SHIV) infection in Chinese rhesus macaques. Once weekly for 14 weeks or until a macaque became infected, 12 male macaques were inoculated intrarectally with amounts of SHIV(SF162P3) (10 median tissue culture infective doses; 3.8 x 10(5) virus particles) that were approximately 5-fold higher than the human immunodeficiency virus type 1 RNA levels noted in human semen during an acute infection. Of the 12 macaques, 4 received oral TDF daily, 4 received oral TDF once weekly, and 4 (control animals) received no TDF. The control animals became infected after receiving a median of 1.5 virus inoculations; macaques receiving TDF daily (1 macaque remained uninfected after 14 inoculations) and those receiving TDF weekly became infected after a median duration of 6.0 and 7.0 weeks, respectively. Although infection was delayed in treated macaques, compared with control macaques, the differences were not statistically significant (P=.315); however, the study was limited by the small numbers of animals evaluated and the variability in blood levels of TDF that resulted from oral dosing. These data demonstrate that treatment with oral TDF provided partial protection against SHIV infection but ultimately did not protect all TDF treated animals against multiple virus challenges.

Frequency of HIV-1 dual subtype infections, including intersubtype superinfections, among injection drug users in Bangkok, Thailand.

OBJECTIVES: To estimate the frequency and incidence of dual HIV-1 subtype infections, including superinfections, among recent seroconvertors from a cohort of injection drug users (IDUs). METHODS: A total of 1209 HIV-negative IDUs were followed in a prospective cohort study at 15 methadone clinics in Bangkok, Thailand. After 2308 person-years (PY) of follow-up, 133 seroconverted to HIV-1, of which approximately 20% were subtype B and 80% were CRF01_AE (formerly called subtype E). Specimens from 126 individuals were available at time of first seropositive test and specimens from 80 of these 126 individuals were also available more than 12 months later. For each infected participant, we calculated the amount of time to superinfection, loss to follow-up, or to the closest visit more than 12 months after the time of initial seropositivity. RESULTS: Of all 126 seroconverters seen at the time of the first seropositive test result, there was no apparent case of concurrent dual subtype infection detected despite 2301 PY of observation. Overall, the incidence of superinfection was 2.2 per 100 PY [95% confidence interval (CI), 0.3-7.8]. The 1-year incidence of CRF01_AE superinfection following subtype B primary infection was 3.9 per 100 PY (95% CI, 0.1-21.9) and the incidence of subtype B superinfection following CRF01_AE primary infection was 1.5 per 100 PY (95% CI, 0.04-8.3). CONCLUSIONS: Determination of the frequency and incidence of dual HIV-1 subtype infection demonstrates that HIV-1 superinfection is not uncommon in a population with high HIV-1 incidence with more than one circulating strain.

In vitro comparison of topical microbicides for prevention of human immunodeficiency virus type 1 transmission.

A standardized protocol was used to compare cellular toxicities and anti-human immunodeficiency virus type 1 (HIV-1) activities of candidate microbicides formulated for human use. The microbicides evaluated were cellulose acetate phthalate (CAP), Carraguard, K-Y plus nonoxynol-9 (KY-N9), PRO 2000 (0.5 and 4%), SPL7013 (5%), UC781 (0.1 and 1%), and Vena Gel, along with their accompanying placebos. Products were evaluated for toxicity on cervical and colorectal epithelial cell lines, peripheral blood mononuclear cells (PBMCs), and macrophages (MPhi) by using an ATP release assay, and they were tested for their effect on transepithelial resistance (TER) of polarized epithelial monolayers. Anti-HIV-1 activity was evaluated in assays for transfer of infectious HIV-1 from epithelial cells to activated PBMCs and for PBMC and MPhi infection. CAP, Carraguard, PRO 2000, SPL7013, and UC781 along with their placebos were 20- to 50-fold less toxic than KY-N9 and Vena Gel. None of the nontoxic product concentrations disrupted the TER. Transfer of HIV-1(Ba-L) from epithelial cells to PBMCs and PBMC and MPhi infection with laboratory-adapted HIV-1(Ba-L) and HIV-1(LAI) isolates were inhibited by all products except Carraguard, KY-N9, and Vena Gel. KY-N9, Vena Gel, and Carraguard were not effective in blocking PBMC infection with primary HIV-1(A), HIV-1(C), and HIV-1(CRF01-AE) isolates. The concordance of these toxicity results with those previously reported indicates that our protocol may be useful for predicting toxicity in vivo. Moreover, our systematic anti-HIV-1 testing provides a rational basis for making better informed decisions about which products to consider for clinical trials.

New HIV type 1 CRF01_AE/B recombinants displaying unique distribution of breakpoints from incident infections among injecting drug users in Thailand.

The goals of this study were to identify and characterize recombinant human immunodeficiency virus type 1 (HIV-1) genomes among incident infections in a prospective cohort study of injecting drug users (IDUs) in Bangkok, Thailand. Through cross-sectional, comparative phylogenetic analysis of the protease and env (C2-V4) gene regions, subtype discordance was observed in HIV-1 sequences from 4 of 111 IDUs (3.5%). Near-full-length HIV-1 genome sequences of the four strains revealed that in all four, the gp120 sequences clustered with a CRF01_AE prototype, while the remainder of the genomes displayed distinct mosaic patterns, with multiple breakpoints between HIV-1 CRF01_AE and subtype B-like regions. Two of the four HIV-1 recombinant strains displayed a nearly identical mosaic structure, suggesting the possible emergence and spread of a potentially new circulating recombinant form of HIV-1. Further characterization of these and other recombinant genomes through long-term follow-up will be important in understanding the generation of viral diversity and escape from the hosts immune responses. This information will be especially important for vaccine development.

Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users.

In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.

Higher viral loads and other risk factors associated with HIV-1 seroconversion during a period of high incidence among injection drug users in Bangkok.

We analyzed data from a prospective cohort study of injection drug users (IDUs) attending methadone treatment clinics in Bangkok, Thailand, during 1995-1998 to characterize factors associated with a period of high incidence (PHI) from July 1996 through January 1997 compared with periods of lower incidence. Sociobehavioral characteristics were similar for all participants during and outside the PHI except for the following: there was more reported drug injection while IDUs were incarcerated during the PHI (odds ratio, 1.67; p =.02) and significantly higher proportions of persons reported heroin injection (91% vs. 75%, respectively; p =.02) and higher frequencies of daily injection and sharing of injection equipment (40% vs. 25%, respectively; p =.05) during the PHI than outside the PHI. Through most of the first year after seroconversion, plasma HIV-1 loads were significantly higher in persons who seroconverted during the PHI than in those who seroconverted outside the PHI. Higher viral loads may potentially contribute to faster disease progression and increased infectiousness or transmissibility to subsequent contacts. Our findings suggest that prevention efforts to reduce the effective size and turnover within IDU sharing networks may have a significant impact on the epidemic by disrupting the rapid transmission of HIV-1 from recently infected, highly infectious individuals.

Genetic analysis of incident HIV-1 strains among injection drug users in Bangkok: evidence for multiple transmission clusters during a period of high incidence.

During 1995-1996, 1,209 HIV-1-negative injection drug users (IDUs) attending methadone treatment clinics operated by the Bangkok Metropolitan Administration in Bangkok, Thailand, were enrolled in a prospective cohort study. Through 1998, 133 of these IDUs had seroconverted to HIV-1; 130 of these seroconverters were included in this study. HIV-1 CRF01_AE and subtype B strains accounted for 79% and 21% of the incident infections, respectively. To examine phylogenetic relationships among these incident HIV-1 strains, we used several phylogenetic inference methodologies to analyze the env (C2-V4) sequences in blood samples collected soon after seroconversion. These analyses consistently revealed eight phylogenetic clusters comprising 21 incident strains (bootstrap method, >80%; six CRF01_AE and two subtype B clusters). Two factors were found to be associated with the eight clusters. The first factor was temporal: seven of the eight clusters comprised 17 sequences from IDUs whose estimated dates of seroconversion were within a period of high incidence from July 1996 through January 1997. The second factor was a possible geographic association: four clusters were observed among IDUs who had attended the same methadone treatment clinics. These phylogenetic clusters likely represent subgroups within larger HIV transmission networks among IDUs in Bangkok. Despite prevention efforts, the incidence of HIV-1 infection among the Bangkok IDU population continues to be high. A better understanding of transmission networks and factors associated with such networks can help guide prevention efforts.