JAIDS Journal of Acquired Immune Deficiency Syndromes:
Epidemiology and Social Science
Low Sensitivity of Total Lymphocyte Count as a Surrogate Marker to Identify Antepartum and Postpartum Indian Women Who Require Antiretroviral Therapy
Gupta, Amita MD, MHS*; Gupte, Nikhil PhD†; Bhosale, Ramesh MD‡; Kakrani, Arjun MD‡; Kulkarni, Vandana MSc†; Nayak, Uma PhD§; Thakar, Madhuri PhD∥; Sastry, Jayagowri MD, MPH†; Bollinger, Robert C MD, MPH*§; for the Byramji Jeejeebhoy Medical College-Johns Hopkins University Study Group
From the *Johns Hopkins University School of Medicine, Baltimore, MD; †Byramji Jeejeebhoy Medical College-Johns Hopkins University Maternal Infant Transmission Study, Pune, India; ‡Byramji Jeejeebhoy Medical College, Pune, India; §Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; and the ∥National AIDS Research Institute, Pune, India.
Received for publication January 17, 2007; accepted August 3, 2007.
Supported by grant R01 AI 45462 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), and grant 2 D43 TW00010-20-AITRP from the Fogarty International Center, NIH, Program of International Training Grants in Epidemiology Related to AIDS.
The views expressed do not necessarily reflect the view of the Johns Hopkins University, Byramji Jeejeebhoy Medical College, or the NIH.
Correspondence to: Amita Gupta, MD, MHS, Division of infectious Diseases Center for Clinical Global Health Education, Johns Hopkins School of Medicine, 600 N. Wolfe Street, Phipps 540B, Baltimore, MD 21287 (e-mail: firstname.lastname@example.org).
Background: Some studies support the use of total lymphocyte count (TLC) as a surrogate marker for CD4 cell count to guide antiretroviral therapy (ART) initiation. However, most of these studies have focused on nonpregnant adults. In light of expanding ART access through prevention of mother-to-child transmission (PMTCT)-plus programs in resource-limited settings, we assessed the sensitivity, specificity, and positive predictive value (PPV) of TLC for predicting low CD4 counts in antepartum and postpartum women in Pune, India.
Methods: CD4, TLC, and hemoglobin were measured at third trimester, delivery, and 6, 9, and 12 months postpartum (PP) in a cohort of 779 HIV-infected women. Optimal TLC cutoff for predicting CD4 <200 cells/mm3 was determined via logistic regression where sensitivity, specificity, PPV, and an area under the receiver operating characteristic (ROC) curve were calculated.
Results: Among the 779 women enrolled, 16% had WHO clinical stage 2 or higher and 7.9% had CD4 <200 cells/mm3. Using 2689 TLC-CD4 pairs, the sensitivity, specificity, and PPV of TLC <1200 cells/mm3 for predicting CD4 <200 cells/mm3 was 59%, 94%, and 47%, respectively. The sensitivity of TLC <1200 cells/mm3 cutoff ranged between 57% and 62% for time points evaluated. Addition of hemoglobin <12 g/dL or <11 g/dL increased the sensitivity of TLC to 74% to 92% for predicting CD4 <200 cells/mm3 but decreased the specificity to 33% to 69% compared to TLC alone. A combination of TLC, hemoglobin, and WHO clinical staging had the highest sensitivity but lowest specificity compared to other possible combinations or use of TLC alone. The sensitivity and specificity of TLC <1200 cells/mm3 to predict a CD4 <350 cells/mm3 was 31% and 99%, respectively.
Conclusions: Our data suggest that antepartum and PP women with TLC <1200 cells/mm3 are likely to have CD4 <200 cells/mm3. However, the sensitivity of this TLC cutoff was low. Between 45% and 64% of antepartum and PP women requiring initiation of ART may not be identified by using TLC alone as a surrogate marker for CD4 <200 cells/mm3. The WHO-recommended TLC cutoff of <1200 cells/mm3 is not optimal for identifying antepartum and PP Indian women who require ART.
Prevention of mother-to-child transmission of HIV (PMTCT) programs linked to antiretroviral therapy (ART) provision are now being scaled up in resource-limited settings. Identifying women who require ART remains a challenge, as CD4 cell counts are still relatively expensive and may not be available in many settings. Total lymphocyte counts (TLC) have been proposed as a surrogate marker for low CD4 cell counts. However, some studies reveal that TLC may lack sufficient sensitivity and positive predictive value.1-5 Algorithms that include the combination of TLC, clinical staging, and hemoglobin may be more useful in predicting low CD4 cell counts.6 In the absence of CD4 cell count, the WHO in 2002 recommended initiating ART if the patient has TLC ≤1200 cells/mm3 and a WHO stage 2 or higher. In 2006, these guidelines were revised, and use of TLC now is suggested only if a patient has a WHO stage 2 and a TLC <1200 cells/mm3 and there is no CD4 available.5 For HIV-infected pregnant women in resource-limited settings where CD4 testing is not available, the 2006 WHO PMTCT guidelines recommend only using WHO clinical stage as criteria for initiation of ART.7 Nevertheless, in many resource-limited settings, clinicians continue to use TLC as a surrogate for CD4 cell counts, because CD4 counts are still not easily available, affordable, or standardized, and clinical stage alone is felt to be insufficient.
Pregnancy and the postpartum period are a key time when women access care and antiretroviral therapy. The WHO TLC guidelines, however, were developed using data primarily from nonpregnant adults. Furthermore, TLC may vary naturally during pregnancy and afterward.8,9 Therefore, we assessed the sensitivity, specificity, and predictive value of the optimal TLC cutoff for predicting CD4 cell counts <200 and <350 in antepartum and postpartum (PP) women in a resource-limited setting. We also assessed whether the addition of hemoglobin or WHO clinical staging to TLC in this female population improves the sensitivity, specificity, and positive predictive value (PPV) beyond TLC alone.
Our study was conducted at Sassoon General Hospital, a large urban public hospital in Pune, India. Between August 2002 and May 2006, pregnant women in the third trimester or in labor were screened for HIV and enrolled in a National Institutes of Health (NIH)-funded PMTCT clinical trial. HIV was diagnosed using 2 previously validated rapid assays.10 WHO clinical staging was assessed at enrollment and at delivery, 6, and 12 months postpartum.5
We performed complete blood counts using Sysmex K4500 cell counters (Sysmex, Hamburg, Germany). CD4 cell counts were measured by single-platform methodology using FACSCount (Becton Dickinson, Franklin Lakes, NJ) during the third trimester, at delivery, and at 6, 9, and 12 months postpartum at a certified Indian laboratory according to manufacturer's instructions.
The sensitivity, specificity, and area under the receiver operating characteristic (ROC) curve were calculated. The area under the curve varies between 0 and 1, with 1 being the value where there is 100% sensitivity and specificity.11 Optimal TLC cutoff for predicting CD4 cell counts <200 cells/mm3 was determined by logistic regression. We also assessed whether the addition of hemoglobin or WHO clinical staging5 to TLC improved the sensitivity and specificity of detecting a CD4 <200 cells/mm3. We used 3 hemoglobin cutoffs based on the quartile distribution: 10 g/dL (which is also the WHO cutoff for mild-to-moderate anemia in women), 11 g/dL, and <12 g/dL (suggested criteria by Spacek and colleagues,6 who studied a US HIV-infected population). We also assessed whether a 2-stage algorithm consisting of hemoglobin and TLC cutoffs would be useful to identify which WHO stage 1 or 2 women would meet criteria for ART initiation in the absence of CD4 cell count.
Lastly, we assessed the sensitivity, specificity, and predictive value of TLC <1200 cells/mm3 for predicting a CD4 <350 cells/mm3. CD4-based guidelines for initiating ART may evolve to a higher threshold, as starting ART at a CD4 cell count between 200 and 350 cells/mm3 may be of benefit.
A total of 779 women were enrolled into the study. The median age was 23 years and the median hemoglobin was 11.1 g/dL (Table 1). Sixteen percent of participants had a WHO clinical stage of 2 or higher. Using 2689 TLC-CD4 pairs of observations obtained from the third trimester, delivery, and 6, 9, and 12 months postpartum, the overall median TLC was 1900 cells/mm3 and the median CD4 count was 472 cells/mm3. A scatter plot illustrating the relationship between TLC and CD4 count pairs is shown in Figure 1. The overall sensitivity of TLC <1200 cells/mm3 to predict CD4 <200 cells/mm3 was 59% with a PPV of 47%. Looking at specific time points-for example, in the third trimester, when many women in PMTCT programs would be presenting for care and treatment-a TLC cutoff of 1200 cells/mm3 had a sensitivity of 57%, a specificity of 91%, a PPV of 23%, and a negative predictive value (NPV) of 98% to predict a CD4 <200 cells/mm3 (Table 2). We found similar sensitivities of the TLC <1200 cells/mm3 cutoff at delivery and at 6, 9, and 12 months postpartum. Calculating the area under the curve for several cutoffs between TLC <800 and TLC <2500 (data not shown), we found that a TLC cutoff of <1400 cells/mm3 would maximize the sensitivity and specificity to predict a CD4 <200 cells/mm3 in the third trimester, and a TLC cutoff of <1500 cells/mm3 would maximize the sensitivity and specificity at the other time points. These higher TLC cutoffs increased the sensitivity to between 74% and 80%, but decreased the specificity to between 82% and 86%.
Based on calculated sensitivities and specificities, the use of TLC <1200 cells/mm3 to predict CD4 <200 cells/mm3 would result in a 25% increase in the number of women eligible for ART compared to using the CD4 <200 cells/mm3 criteria alone. Using a higher cutoff of TLC <1500 cells/mm3 at delivery increased the percentage of women detected with a CD4 <200 cells/mm3 to 74%. With a lower specificity of 82%, TLC <1500 cells/mm3 would result in a 180% increase of women eligible for ART compared to using the CD4 <200 cells/mm3 criteria alone. Furthermore, use of TLC <1200 or <1500 cells/mm3 criteria alone would result in 35% or 57%, respectively, of women being treated with ART who would have a CD4 >250 cells/mm3, the threshold above which the risk of nevirapine toxicity is increased in some female populations.
We assessed whether the use of hemoglobin <12 g/dL in addition to TLC <1200 cells/mm3 could improve the sensitivity and specificity of CD4 <200 cells/mm3 detection. We found that this hemoglobin cutoff increased the sensitivity to 74% to 92%, depending on the time point, but decreased the specificity to 33% to 69% compared to TLC alone (Table 3). Using other hemoglobin cutoffs (<11 g/dL or <10 g/dL) did not improve the sensitivity or specificity much (data not shown for Hgb <10 g/dL). Compared to TLC alone, combining the WHO clinical stage and TLC to predict CD4 <200 cells/mm3 increased the sensitivity to 70% to 81%, but decreased the specificity to 78% to 83%. WHO stage and TLC, however, remained more specific to predict CD4 <200 cells/mm3 than the combination of TLC and hemoglobin. A combination of TLC, hemoglobin, and WHO clinical staging had the highest sensitivity but lowest specificity compared to other possible combinations or use of TLC alone.
We also assessed whether a 2-stage algorithm would be useful to identify which WHO stage 1 or 2 women would meet criteria for ART therapy in the absence of CD4 cell testing, as current WHO PMTCT guidelines do not recommend ART initiation for these stages unless CD4 cell count can be documented to be <200 cells/mm3. We determined that use of hemoglobin cutoff of <12 g/dL as the first screening test (sensitivity 86% at delivery) followed by TLC <1200 cells/mm3 (specificity of 93%) had a net sensitivity of 49%, a net specificity of 95%, and a PPV of 39% based on our CD4 <200 cells/mm3 prevalence of 6%.
We investigated the clinical utility of first screening women with WHO clinical staging and complete blood count and referring women with WHO stage 1 or 2 that either had hemoglobin <12 g/dL or TLC <1200 cells/mm3 for CD4 testing. In such a strategy, 464 women would be tested with CD4 to identify 36 of 40 women with a CD4 <200 cells/mm3 (sensitivity 90%, specificity 29%, PPV 8%). Criteria of having both hemoglobin <12 g/dL and TLC <1200 cells/mm3 would lead to CD4 testing of 43 women, with identification of 18 of the 40 women with CD4 <200 (sensitivity 45%, specificity 96%, PPV 42%).
We also assessed the sensitivity and specificity of TLC <1200 cells/mm3 to predict a CD4 <350 cells/mm3. The overall sensitivity was 31%, and the specificity was 99%. The sensitivity was improved to 64% to 76% by combining TLC <1200 cells/mm3 with hemoglobin <11 g/dL, and to 72% to 83% with the addition of WHO staging, but the specificity decreased to 38% to 53%. Compared to a TLC <1200 cells/mm3, increasing the TLC cutoff to <1500 cells/mm3 increased the sensitivity to 50% to 67% while reducing the specificity somewhat to 86% to 93%. Combining the TLC <1500 mm3 with hemoglobin and WHO staging resulted in a sensitivity of 82% to 88% and a specificity of 37% to 43%, similar to what was seen using the TLC <1200 cells/mm3 cutoff.
Whereas several studies have examined TLC as a proposed surrogate marker for CD4 cell count, the question of whether pregnancy and the postpartum period affect this relationship has not been studied. To our knowledge, our study is the first to focus on pregnant and postpartum women, a time when TLC and CD4 cell count may naturally vary. The antepartum and postpartum period is a key access point for HIV diagnosis, care, and initiation of ART for women. Although the WHO recommends initiation of ART to be guided by CD4 cell count along with clinical staging, many clinicians in resource-limited settings such as India still use TLC as a surrogate marker for CD4 due to cost, availability, and belief in its clinical utility. In our study of Indian antepartum and postpartum women, we found that the WHO-recommended TLC cutoff of <1200 cells/mm3 as a surrogate marker for CD4 <200 cells/mm3 has a low sensitivity of 59% and low PPV. Our findings are consistent with several studies in other resource limited settings.1,4,12-14 This low sensitivity and PPV did not differ substantially between third trimester, delivery, and within the first year postpartum. We determined that approximately 40% of women requiring initiation of ART would not be identified by the use of TLC cutoff of <1200 cells/mm3 alone. Furthermore, 35% of women identified by TLC <1200 cells/mm3 alone had a CD4 >250 cells/mm3. Similar to what has been previously reported, the addition of hemoglobin to TLC to predict CD4 <200 cells/mm3 in this population increases the sensitivity by ∼20% but decreases the specificity by ∼40%.6 Using a combination of WHO staging (WHO stage 2 or higher), TLC, and hemoglobin yielded the highest sensitivity, but specificity remained low, suggesting that even this combination is not a suitable surrogate marker for low CD4 cell counts.
Some studies have shown that increasing the TLC cutoff improves the sensitivity to detect a CD4 count <200 cells/mm3.4,15 We also found this to be the case; however, although increasing the TLC cutoff increases the number of women detected with CD4 <200 cells/mm3, it also has the consequence of substantially increasing the proportion of women who would have a CD4 >250 cells/mm3. Because some female populations, including pregnant women, have been documented to have a greater risk of nevirapine toxicity at CD4 >250 cells/mm3, and nevirapine remains the most commonly used drug in the WHO first-line regimen for women of reproductive age, increasing the TLC cutoff to capture more women eligible for treatment may be problematic.16-19
We had a relatively small proportion of persons who had CD4 <200 cells/mm3, but this reflects the HIV-infected antenatal clinic population in India. Because the PPV of TLC as a surrogate marker depends on the prevalence of CD4 <200 cells/mm3, it is not surprising that we found a low PPV of TLC. In our antenatal clinic, approximately 8% of women had a CD4 <200 cells/mm3, whereas in some sub-Saharan settings, antenatal women have a much higher CD4 <200 cells/mm3 prevalence and thus would be expected to see an increased PPV. For example, a CD4 <200 cells/mm3 prevalence of 20% would increase the PPV from 27% to 67%. Our data therefore may not be generalizable to all resource-limited settings, but nevertheless are consistent with several other studies that have evaluated TLC and CD4 in other HIV-infected populations and settings.2,6,13-15,20
The WHO is now emphasizing the need to perform CD4 cell counts due to concerns about insufficient sensitivity and PPV of TLC as a surrogate marker.5 Our data of antenatal and postpartum women in a resource-limited setting support this concern. When CD4 cell counts are unavailable, practitioners need to be aware of the limitations of TLC as a surrogate marker, and ideally TLC should be combined with WHO staging. The addition of hemoglobin as a marker in this antepartum and postpartum female population can improve the sensitivity but reduces the specificity, so addition of hemoglobin does not seem to be sufficiently clinically useful in improving the identification of women in need of ART in our population. Continued efforts to develop and expand access to low-cost, valid CD4 testing technologies are needed.
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