Conceived and designed the experiments: AN JvdW. Performed the experiments: AN ZM JS PK DA. Analyzed the data: JW JvdW NvN. Wrote the paper: JvdW. Critically reviewed the manuscript: AN JvdW ZM JS PK DA NvN.
Jian Wu is a consultant (JW consulting is a one person business) and was paid to conduct the statistical analyses for this paper. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
HIV prevalence and incidence among sexually active women in peri-urban areas of Ladysmith, Edendale, and Pinetown, KwaZulu-Natal, South Africa, were assessed between October 2007 and February 2010 in preparation for vaginal microbicide trials.
Sexually active women 18–35 years, not known to be HIV-positive or pregnant were tested cross-sectionally to determine HIV and pregnancy prevalence (798 in Ladysmith, 1,084 in Edendale, and 891 in Pinetown). Out of these, approximately 300 confirmed non-pregnant, HIV-negative women were subsequently enrolled at each clinical research center (CRC) in a 12-month cohort study with quarterly study visits. Women in the cohort studies were required to use a condom plus a hormonal contraceptive method. HIV prevalence rates in the baseline cross-sectional surveys were high: 42% in Ladysmith, 46% in Edendale and 41% in Pinetown. Around 90% of study participants at each CRC reported one sex partner in the last 3 months, but only 14–30% stated that they were sure that none of their sex partners were HIV-positive. HIV incidence rates based on seroconversions over 12 months were 14.8/100 person-years (PY) (95% CI 9.7, 19.8) in Ladysmith, 6.3/100 PY (95% CI 3.2, 9.4) in Edendale, and 7.2/100 PY (95% CI 3.7, 10.7) in Pinetown. The 12-month pregnancy incidence rates (in the context of high reported contraceptive use) were: 5.7/100 PY (95% CI 2.6, 8.7) in Ladysmith, 3.1/100 PY (95% CI 0.9, 5.2) in Edendale and 6.3/100 PY (95% CI 3.0, 9.6) in Pinetown.
HIV prevalence and incidence remain high in peri-urban areas of KwaZulu-Natal.
The South African province of KwaZulu-Natal is experiencing one of the worst HIV epidemics worldwide. The epidemic has been described as hyperendemic, generalized and mature, with HIV prevalence rates in the general population of over 15%
KwaZulu-Natal is divided into 11 districts. The HIV epidemics in two of these districts have been extensively studied: the urban district of eThekwini (Durban Metropolitan Area and surrounding area) and the rural district of uMkhanyakude (where the Africa Centre of the University of KwaZulu-Natal is located)
We conducted HIV prevalence and incidence studies in sexually active women in the above-mentioned peri-urban areas of KwaZulu-Natal to better understand how many and where new HIV infections are occurring and to assess the feasibility of undertaking vaginal microbicide trials for HIV prevention in these populations.
In preparation for future vaginal microbicide trials for HIV prevention in KwaZulu-Natal, cross-sectional studies (targeting 800–1,000 women each) were conducted at three clinical research centers (CRC) to determine HIV prevalence and to identify HIV-negative, non-pregnant women for enrollment in subsequent cohort studies (targeting 300 women each). The main aim of the cohort studies was to determine HIV incidence in seroconversions per 100 PY. The CRCs were located in Ladysmith, Edendale, and Pinetown. Each CRC established a Community Advisory Group (CAG) to provide community input in study procedures and to assist the researchers with community education and mobilization. CRC staff, with the assistance of CAG members, organized meetings in public spaces (at public meetings, in shopping centers and in waiting areas of clinics), where the study was presented. Women who expressed an interest in study participation were then invited to visit the CRC for screening and possible enrollment. In addition, door-to-door or family visits were conducted by study staff. While the recruitment strategies were CRC-specific, the same study procedures were followed at each CRC from the moment women were screened for study participation.
Women were eligible for the cross-sectional studies if they were 18–35 years, not HIV-positive or pregnant by self-report, not breastfeeding, and sexually active (defined as at least one penetrative vaginal coital act per month for the previous three months). Women who tested HIV- and pregnancy-negative in the cross-sectional studies, still met the entry criteria described above, and met additional entry criteria for the cohort studies, were subsequently offered enrollment into the cohort studies. These additional entry criteria included using a condom plus a hormonal (oral or injectable) contraceptive method
At all study visits, women were interviewed regarding demographics, sexual behavior, vaginal hygiene practices, and medical history; and received HIV risk reduction and contraceptive counseling, condoms, and syndromic management of sexually transmitted infections (STI) free of charge
An HIV testing algorithm was used to determine the presence of prevalent and incident HIV infections. Women were first tested by OraQuick ADVANCE Rapid HIV-1/2 Antibody Test using oral swabs (OraSure Technologies Inc., Bethlehem, PA, USA) or by Uni-Gold Recombigen HIV test using blood (Trinity Biotech, Bray, Wicklow, Ireland). When this first HIV test was positive, blood samples were tested by Determine HIV-1/2 rapid test (Inverness Medical Professional Diagnostics, Princeton, NJ, USA), and by enzyme-linked immunosorbant assay (ELISA) if a tiebreaker was needed. Blood samples from women who were confirmed HIV-positive were also tested by BED assay (Calypte Biomedical Corporation, Portland, OR, USA) according to the manufacturer's instructions. A specimen with a final normalized optical density value of less than or equal to 0.8 was considered to be from a participant who was infected less than 155 days before
Case report forms were processed using the DataFax data management system (Clinical DataFax Systems Inc., Hamilton, Ontario, Canada) and analyzed using SAS version 9.2 (SAS Institute, Cary, NC, USA). Descriptive statistics were used to summarize baseline demographic, behavioral and clinical characteristics. Categorical variables were expressed as percentages, and continuous data as medians with inter-quartile ranges.
Incidence rates in the cohort studies were calculated based on a Poisson distribution with PY at risk in the denominator. A person's time at risk began at the enrollment visit and ended at the last study visit attended (usually the Month 12 visit) or when HIV infection or pregnancy occurred. HIV infection and pregnancy were assumed to have occurred at the mid-point between the last available negative test and first positive test. A woman who reached an HIV endpoint was no longer considered at risk for HIV but was still considered at risk for pregnancy, and vice versa. HIV incidence rates and 95% confidence intervals based on BED results in the cross-sectional studies were calculated using the formula, and accompanying spreadsheet, provided by McWalter and Welte
Age-adjusted and multivariable logistic regression models were used to assess predictors of prevalent HIV infection and pregnancy, with p-values from the Wilcoxon-Mann-Whitney test for continuous variables and the Chi-square and Fisher's exact tests for categorical variables. Age-adjusted Cox proportional hazards regression models were used to assess predictors of HIV seroconversion and incident pregnancy.
Women were enrolled in the cross-sectional studies between 2007 and 2009 as follows: 798 women in Ladysmith, 1,084 women in Edendale, and 891 women in Pinetown. The Ladysmith and Edendale CRCs subsequently enrolled 300 women in their cohort studies and the Pinetown CRC 297 women, accumulating 223, 254, and 223 PY respectively. In Ladysmith, 129 of 300 (43%) participants completed all scheduled visits; 53 women withdrew early from the cohort study, 32 were lost to follow-up, and none died. In Edendale, 210 of 300 (70%) participants completed all scheduled visits; 6 women withdrew early from the cohort study, 24 were lost to follow-up, and none died. In Pinetown, 167 of 297 (56%) participants completed all scheduled visits; 5 women withdrew early from the cohort study, 74 were lost to follow-up, and none died.
In the cross-sectional studies, the median age of study participants was 23 or 24 years (
Ladysmith | Edendale | Pinetown | |
Characteristic n (%) | N = 798 | N = 1,084 | N = 891 |
Age in years (median; range) | 24 (18–35) | 24 (18–35) | 23 (18–35) |
Race: Black African | 792 (99.2) | 1,081 (99.7) | 890 (99.9) |
Marital status | |||
Single | 723 (90.6) | 988 (91.1) | 799 (89.7) |
Married/or living together | 75 (9.4) | 90 (8.3) | 91 (10.2) |
Separated/divorced/widowed | 0 | 6 (0.6) | 1 (0.1) |
Education | |||
No school | 2 (0.3) | 5 (0.5) | 3 (0.3) |
Some/completed primary school | 26 (3.3) | 40 (3.6) | 81 (9.1) |
Some/completed high school | 651 (81.6) | 1,002 (92.5) | 748 (84.3) |
Some/completed tertiary school | 119 (14.9) | 36 (3.3) | 55 (6.2) |
Source of income |
|||
Formal/informal work | 193 (24.2) | 56 (5.2) | 123 (13.8) |
Government grants | 321 (40.2) | 639 (59.1) | 509 (57.2) |
Husband/partner | 95 (11.9) | 25 (2.3) | 78 (8.8) |
Other | 471 (59.0) | 397 (36.7) | 234 (26.3) |
Average monthly income | |||
0-R500 | 542 (67.9) | 1034 (95.7) | 772 (86.6) |
>R500 | 256 (32.1) | 47 (4.3) | 119 (13.4) |
Male sex partners in last 3 months | |||
1 | 718 (90.0) | 998 (92.1) | 780 (87.5) |
2 or more | 80 (10.0) | 86 (7.9) | 111 (12.5) |
Male sex partners in last 7 days | |||
0 | 152 (19.9) | 215 (19.9) | 135 (15.2) |
1 | 608 (79.5) | 859 (79.3) | 750 (84.4) |
2 or more | 5 (0.7) | 9 (0.8) | 4 (0.4) |
Condom used during last sex act | 374 (46.9) | 577 (53.2) | 550 (62.0) |
Condom use in last 7 day |
|||
Always | 228 (35.5) | 404 (42.2) | 322 (42.2) |
Inconsistent | 197 (30.7) | 137 (14.3) | 182 (23.9) |
Never | 217 (33.8) | 416 (43.5) | 259 (33.9) |
Any chance that any current sex partner is HIV-positive | |||
Yes | 210 (26.3) | 178 (16.7) | 182 (20.5) |
No | 235 (29.5) | 187 (17.5) | 125 (14.1) |
Don't know | 352 (44.2) | 702 (65.8) | 579 (65.3) |
Ever had anal sex | 4 (0.5) | 28 (2.6) | 3 (0.3) |
Ever had oral sex | 115 (14.4) | 169 (15.6) | 145 (16.3) |
Ever vaginal cleansing before or after sex | 8 (1.0) | 20 (1.8) | 67 (7.6) |
Ever vaginal drying before or after sex | 1 (0.1) | 16 (1.5) | 37 (4.1) |
Self assessment of HIV risk |
|||
No/low risk | 392 (51.4) | 449 (42.2) | 407 (50.1) |
Moderate risk | 58 (7.6) | 188 (17.7) | 20 (2.5) |
High risk | 312 (40.9) | 426 (40.1) | 385 (47.4) |
Reported genital symptom at baseline |
34 (4.3) | 15 (1.4) | 27 (3.0) |
Multiple responses allowed.
Women who reported any sexual intercourse in the last 7 days only.
Women who said ‘don't know’ were excluded.
Includes lower abdominal pain, genital discharge, odor, ulcers, sores, itching or swelling, burning pain on urination.
More than 80% of women at all three CRCs reported that they themselves, or they and their partner together, decided about condom use (data not shown). About one third of women (28% in Ladysmith, 16% in Edendale, and 39% in Pinetown) reported to have refused sex in the last 7 days due to lack of a condom. The most common reasons for using a condom were ‘to protect myself from HIV’ (49% in Ladysmith, 74% in Edendale, and 70% in Pinetown), followed by ‘to prevent pregnancy’ (41% in Ladysmith, 66% in Edendale, and 52% in Pinetown), and ‘to protect myself from STIs’ (29% in Ladysmith, 38% in Edendale, and 58% in Pinetown). Protecting sexual partners from HIV or STIs was less often mentioned in Ladysmith and Edendale, and rarely mentioned in Pinetown (data not shown). The most common reason for not using a condom was partner refusal (40% in Ladysmith, 28% in Edendale, and 33% in Pinetown).
HIV prevalence was higher than 40% at all three CRCs: 42.0% (95% CI 38.5, 45.5) in Ladysmith, 46.1% in Edendale (95% CI 43.1, 49.1), and 41.3% (95% CI 38.0, 44.6) in Pinetown. Factors positively associated with prevalent HIV infection at all three CRCs in age-adjusted and multivariable models were: age, lower educational level, self-assessment of HIV risk as moderate or high (compared to no or low risk), and suspected positive or unknown HIV serostatus of a current sexual partner; no or inconsistent condom use was associated with HIV infection in all age-adjusted models but not in all multivariable models (
Determinant | Ladysmith (N = 798) | Edendale (N = 1,084) | Pinetown (N = 891) | |||
% HIV+ | Age-adjusted OR (95% CI) | % HIV+ | Age-adjusted OR (95% CI) | % HIV+ | Age-adjusted OR (95% CI) | |
Marital status: | ||||||
Married/living together | 48.0 | 0.8 (0.5, 1.3) | 60.0 | 1.0 (0.6, 1.5) | 53.3 | 1.2 (0.8, 2.0) |
Single, separated or divorced (reference) | 41.4 | 44.8 | 39.9 | |||
Highest level of education achieved: | ||||||
Some/completed primary education | 50.0 | 2.9 (1.2, 7.4) | 70.0 | 9.1 (2.9, 28.9) | 54.3 | 6.2 (2.5, 15.1) |
Some/completed high school | 45.3 | 3.0 (1.9, 4.9) | 46.2 | 4.3 (1.7, 10.7) | 41.8 | 3.7 (1.7, 8.1) |
Some/completed tertiary education (reference) | 21.8 | 16.7 | 14.8 | |||
Source of income: | ||||||
Formal/informal work (reference) | 53.9 | 48.2 | 44.2 | |||
Government grants | 36.4 | 0.6 (0.4, 0.9) | 52.1 | 1.2 (0.7, 2.1) | 46.8 | 1.1 (0.7, 1.7) |
Husband/Other | 39.4 | 0.9 (0.6, 1.3) | 35.9 | 1.0 (0.5, 1.8) | 30.7 | 0.9 (0.5, 1.4) |
Average monthly income5 | ||||||
0-R500 (reference) | 40.2 | 45.8 | 42.5 | |||
>R500 | 45.7 | 0.8 (0.6, 1.1) | 51.1 | 1.1 (0.6, 2.0) | 33.3 | 0.5 (0.3, 0.8) |
Number of sex partners in last 3 months | ||||||
1 (reference) | 40.7 | 46.3 | 40.1 | |||
More than 1 | 53.8 | 1.9 (1.2, 3.2) | 43.0 | 1.2 (0.7, 1.9) | 49.5 | 1.7 (1.1, 2.6) |
Condom use in last 7 days | ||||||
Always (reference) | 27.2 | 32.4 | 32.5 | |||
Inconsistent | 50.3 | 2.5 (1.7, 3.8) | 58.1 | 2.6 (1.7, 4.0) | 48.6 | 1.9 (1.3, 2.8) |
Never | 47.5 | 2.1 (1.4, 3.1) | 56.7 | 2.4 (1.8, 3.3) | 50.8 | 1.8 (1.3. 2.6) |
Ever had oral sex |
||||||
Yes | 43.5 | 1.0 (0.7, 1.6) | 43.8 | 1.0 (0.7, 1.4) | 41.7 | 1.1 (0.7, 1.5) |
No (reference) | 41.7 | 46.4 | 41.2 | |||
Self assessment of HIV risk | ||||||
No/low risk (reference) | 24.7 | 25.2 | 26.8 | |||
Moderate risk | 53.4 | 3.6 (2.0, 6.4) | 58.3 | 3.8 (2.6, 5.6) | 60.0 | 3.0 (1.2, 7.7) |
High risk | 58.7 | 4.1 (3.0, 5.8) | 61.5 | 3.9 (2.9, 5.3) | 53.3 | 2.9 (2.1, 3.9) |
Any chance that any current sex partner is HIV+ | ||||||
Yes | 51.0 | 3.2 (2.1, 4.9) | 62.4 | 4.7 (2.9, 7.8) | 51.1 | 2.6 (1.6, 4.3) |
No (reference) | 23.4 | 20.9 | 27.2 | |||
Don't know | 49.1 | 3.0 (2.0, 4.3) | 48.2 | 3.1 (2.0, 4.6) | 41.6 | 1.8 (1.2, 2.8) |
Reported genital symptom at baseline | ||||||
Yes | 55.9 | 1.9 (0.9, 4.0) | 46.7 | 1.1 (0.4, 3.2) | 70.4 | 4.2 (1.7, 10.2) |
No (reference) | 41.4 | 46.1 | 40.4 |
Each row represents one bivariable model including age and the predictor of interest.
Anal sex, vaginal cleansing and vaginal drying were too infrequently reported to be assessed as a predictor of HIV prevalence (see
Determinant | Ladysmith (N = 798) | Edendale (N = 1,084) | Pinetown (N = 891) |
Adjusted OR (95% CI) | Adjusted OR (95% CI) | Adjusted OR (95% CI) | |
Age (year) | 1.12 (1.07, 1.17) | 1.16 (1.12, 1.20) | 1.13 (1.09, 1.18) |
Highest level of education achieved: | |||
Some/completed primary education | 1.87 (0.59, 5.94) | 12.77 (2.93, 55.66) | 4.57 (1.50, 13.95) |
Some/completed high school | 1.92 (1.09, 3.40) | 6.72 (2.02, 22.40) | 3.21 (1.21, 8.53) |
Some/completed tertiary education (reference) | |||
Average monthly income | |||
0-R500 (reference) | |||
>R500 | 0.90 (0.59, 1.37) | 1.46 (0.62, 3.41) | 0.45 (0.26, 0.76) |
Number of sex partners in last 3 months | |||
1 (reference) | |||
More than 1 | 1.95 (1.09, 3.50) | 1.02 (0.58, 1.79) | 1.27 (0.76, 2.12) |
Condom use in last 7 days | |||
Always (reference) | |||
Inconsistent | 1.89 (1.16, 3.07) | 2.27 (1.39, 3.72) | 1.24 (0.71, 2.16) |
Never | 1.27 (0.78, 2.06) | 1.40 (0.96, 2.03) | 1.07 (0.65, 1.76) |
Self assessment of HIV risk | |||
No/low risk (reference) | |||
Moderate risk | 2.72 (1.35, 5.46) | 3.54 (2.29, 5.47) | 2.29 (0.65, 8.04) |
High risk | 3.16 (2.10, 4.75) | 3.15 (2.12, 4.67) | 2.12 (1.32, 3.40) |
Any chance that any current sex partner is HIV+ | |||
Yes | 2.61 (1.55, 4.40) | 2.90 (1.62, 5.21) | 2.58 (1.33, 5.03) |
Don't know | 2.36 (1.48, 3.78) | 2.34 (1.46, 3.76) | 1.56 (0.89, 2.72) |
No (reference) |
Overall HIV incidence rates based on seroconversions during the 12-month follow-up period in the cohort studies were 14.8/100 PY (95% CI 9.7, 19.8) in Ladysmith, 6.3/100 PY (95% CI 3.2, 9.4) in Edendale, and 7.2/100 PY (95% CI 3.7, 10.7) in Pinetown (
Women enrolled in the 12-month cohort studies visited the CRC at 3, 6, 9, and 12 months after enrollment for HIV testing. HIV incidence rates were calculated based on a Poisson distribution with PY at risk in the denominator. They are expressed as number of cases per 100 PY with 95% confidence intervals. HIV infection was assumed to have occurred at the mid-point between the last available negative test and first positive test.
Ladysmith | Edendale | Pinetown | |
HIV incidence after 12 months | 14.8 (9.7, 19.8) | 6.3 (3.2, 9.4) | 7.2 (3.7, 10.7) |
HIV incidence in first 6 months | 17.4 (10.3, 24.5) | 5.5 (1.7, 9.3) | 8.6 (3.5, 13.7) |
HIV incidence in second 6 months | 11.0 (4.2, 17.8) | 7.3 (2.3, 12.4) | 5.2 (0.6, 9.8) |
HIV incidence BED adjusted (155 days; 1.7%) |
15.0 (10.1, 19.9) | 10.2 (6.8, 13.7) | 11.6 (7.6, 15.7) |
HIV incidence BED adjusted (187 days; 1.7%) |
12.5 (8.4, 16.6) | 8.5 (5.6, 11.4) | 9.7 (6.3, 13.0) |
Pregnancy incidence after 12 months | 5.7 (2.6, 8.7) | 3.1 (0.9, 5.2) | 6.3 (3.0, 9.6) |
Pregnancy incidence in first 6 months | 7.4 (2.8, 12.0) | 2.0 (0, 4.4) | 7.0 (2.4, 11.6) |
Pregnancy incidence in second 6 months | 3.2 (0, 6.7) | 4.4 (0.5, 8.3) | 5.3 (0.7, 9.9) |
The pregnancy prevalence rates in the cross-sectional studies were low at all three CRCs in accordance with the recruitment strategy (only women reporting not to be pregnant were eligible for study participation): 2.6% (95% CI 1.6, 4.0) in Ladysmith, 4.1% (95% CI 3.0, 5.4) in Edendale, and 1.5% (95% CI 0.8, 2.5) in Pinetown. Pregnancy was associated with inconsistent condom use (age-adjusted OR 3.5, 95% CI 1.1, 11.4) and self-reported genital symptoms (age-adjusted OR 4.2, 95% CI 1.2, 15.2) in Ladysmith, and with ‘never used condoms’ (age-adjusted OR 4.1 (95% CI 1.8, 9.7) and self-reported moderate or high HIV risk (age-adjusted OR 3.7 (95% CI 1.5, 9.6) and 3.8 (95% CI 1.7, 8.8), respectively) in Edendale. In the cohort studies, overall pregnancy incidence for the 12-month period was 5.7 (95% CI 2.6, 8.7) in Ladysmith, 3.1 (95% CI 0.9, 5.2) in Edendale, and 6.3 (95% CI 3.0, 9.6) in Pinetown. Again, no trends were observed over time (
Urine pregnancy tests were done at each study visit (screening, enrollment, and 3, 6, 9, and 12 months after enrollment in the cohort study). If test result was positive, the participant was to continue on study for follow-up per protocol. Estimated date of conception and estimated due date were to be recorded. If possible, follow-up was to continue for pregnancy outcome. Contraceptive counseling was provided and condoms were dispensed at each study visit.
Our data confirm that HIV prevalence and incidence continue to be high in sexually active women aged 18–35 years living in peri-urban areas of KwaZulu-Natal. Our prevalence rates are similar to those reported in the 2009 and 2010 national antenatal surveys but higher than those reported in the 2008 HSRC population-based household survey (26% for women and men combined and for all districts of KwaZulu-Natal combined)
Our HIV incidence rates of 6.3 to 14.8 per 100 PY suggest that HIV transmission is still rampant in KwaZulu-Natal. Our incidence rates for Edendale (6.3/100 PY; uMgungundlovu district) and Pinetown (7.2/100 PY; eThekwini district) fall within the range of rates recently reported for urban and rural women in eThekwini and uMkhanyakude districts (6.4/100 PY among urban women and 6.5/100 PY among rural women aged 14–30 years
As expected, HIV incidence rates based on the adjusted BED-CEIA results were higher than those based on seroconversions per 100 PY for the two sites with lower HIV incidence (Edendale and Pinetown;
While HIV incidence at the three study sites seems sufficiently high for implementation of HIV microbicide efficacy trials, retention rates would have to be improved (currently 43–70%) and pregnancy incidence would have to be reduced. Women in our studies were required to use a condom and a hormonal method of contraception but the high pregnancy incidence rates indicate that these methods were not used correctly and consistently.
A few limitations of our data should be noted. The eligibility criteria for study participation may have limited generalizability of our results. The HIV prevalence rates apply only to young, sexually active women who were not known to be HIV-infected or pregnant, and who agreed to be tested regularly for HIV. The total number of seroconversions in each prospective cohort study were low (16–33 cases) and the 95% confidence intervals were therefore wide. The low retention rates of our cohort studies (43–70%) may have biased our HIV incidence estimates based on seroconversions. We do not have any indications that the women who left the cohort studies early were at higher or lower risk of HIV acquisition than the women who remained in the study but we cannot be certain. The 95% confidence intervals of the cross-sectional BED-based HIV incidence estimates were also wide. Furthermore, we did not measure local false-recent rates or window periods and could therefore not adjust our BED estimates as recommended by WHO
In conclusion, HIV prevalence and incidence remain very high in sexually active women living in peri-urban areas of KwaZulu-Natal. HIV prevention interventions in these populations should be strengthened.
The authors gratefully acknowledge the study teams at the CRCs in Ladysmith, Edendale, and Pinetown and the study team at the International Partnership for Microbicides, in particular Dr. Mercy Kamupira (Clinical Safety Physician) and Karen Bester (Project Manager). The authors would also like to thank Dr. Sarah Braunstein for conducting preliminary data analyses.