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ORIGINAL ARTICLE
Year : 2018  |  Volume : 32  |  Issue : 2  |  Page : 98-102

Quantification of human immunodeficiency virus-1 viral load and its correlation with CD4 cell count in antiretroviral therapy naïve patients attending regional institute of medical sciences hospital, Imphal


Department of Microbiology, Regional Institute of Medical Sciences, Imphal, Manipur, India

Date of Web Publication25-Oct-2018

Correspondence Address:
Dr. Paotinlal Haokip
Department of Microbiology, Regional Institute of Medical Sciences, Imphal - 795 004, Manipur
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jms.jms_107_16

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  Abstract 

Background: While the CD4 cell count plays a pivotal role for antiretroviral therapy (ART) initiation, plasma viral load (PVL) provides additional guiding information, especially in patients with a relatively high CD4 cell count. This study was carried out to quantify PVL and CD4 cell count in ART naïve cases and determine their correlation.
Materials and Methods: A cross-sectional study was conducted on 82 ART naïve patients of ≥15 years of age attending the Fluorescent Activated Cell Sorter Count (FACSCount) center, Department of Microbiology at a tertiary care center after the Institutional Ethics Committee approval, from August 2014 to November 2015. Blood samples were collected after obtaining written informed consent. PVL was quantified by COBAS® TaqMan® human immunodeficiency virus type 1 (HIV-1) version 2.0 Test and CD4 cell count was measured by FACSCountTM System. The correlation analysis was performed by Pearson's correlation test (r) using SPSS 16.0 software.
Results: In this study, mean PVL and CD4 cell counts were 108,000 ± 206,200 copies/mL and 348.2 ± 296 cells/μL, respectively. Among those with PVL ≥50,000 copies/mL, CD4 cell count was >350 cells/μL in 10.4%. PVL was not detectable in 8.5%. There was significant negative correlation (r = −0.54, P < 0.00) between PVL and CD4 cell count in ART naïve patients.
Conclusion: Consideration of PVL in ART initiation guidelines for those with CD4 cell count >350 cells/μL will maximize ART coverage of highly infectious ART naïve patients, which in turn will reduce the risk of HIV transmission at individual and population level.

Keywords: Antiretroviral therapy, antiretroviral therapy naïve, CD4 cell count, plasma viral load


How to cite this article:
Haokip P, Singh HR, Laldinmawii G, Marak EK, Roy A. Quantification of human immunodeficiency virus-1 viral load and its correlation with CD4 cell count in antiretroviral therapy naïve patients attending regional institute of medical sciences hospital, Imphal. J Med Soc 2018;32:98-102

How to cite this URL:
Haokip P, Singh HR, Laldinmawii G, Marak EK, Roy A. Quantification of human immunodeficiency virus-1 viral load and its correlation with CD4 cell count in antiretroviral therapy naïve patients attending regional institute of medical sciences hospital, Imphal. J Med Soc [serial online] 2018 [cited 2018 Nov 17];32:98-102. Available from: http://www.jmedsoc.org/text.asp?2018/32/2/98/207669


  Introduction Top


Quantification of human immunodeficiency virus type 1 (HIV-1) ribonucleic acid (RNA) copies per milliliter of plasma (i.e., viral load) is used as a marker of risk of disease progression,[1] to decide when to initiate antiretroviral therapy (ART) in ART naïve patients and to monitor response to ART.[1],[2] Plasma viral load (PVL) indicates the risk of HIV transmission at the individual and population levels[3],[4] and is a useful technique to diagnose HIV infection in infants.[5]

While the CD4 cell count plays a pivotal role for initiation of ART, PVL provides additional guiding information, especially in patients with a relatively high CD4 cell count.[6] Except for patients failing first-line ART, PVL monitoring is not recommended under the national program, in India.[7] However, some guideline recommends viral load measurement at entry into care (baseline), at the initiation of ART, and on a regular basis thereafter.[8]

In an asymptomatic HIV-1 infected individual, there is lower CD4 cell count and higher viral load.[9],[10] However, some individuals with a high CD4 cell count may have a high viral load and vice versa.[11]

Hence, this study was carried out to explore baseline PVL and its correlation with baseline CD4 cell count in newly diagnosed HIV seropositive ART naïve patients attending Fluorescent Activated Cell Sorter Count (FACSCount) Centre, Department of Microbiology at a tertiary care center in North Eastern India.


  Materials and Methods Top


A cross-sectional study was conducted on 82 HIV-seropositive ART naïve patients of ≥15 years of age attending the FACSCount Centre, Department of Microbiology at a tertiary care center in North Eastern India from August 2014 to November 2015. The study was carried out after obtaining approval from the Institutional Ethics Committee. For the purpose of this study, we defined “ART naïve” as those individual who have been diagnosed as HIV-seropositive recently, but not yet taking ART.

Blood samples were collected after proper counseling and after obtaining written informed consent from each patient. About 3–4 ml of blood samples were collected in vacutainer tube containing ethylenediamine tetraacetic acid as anticoagulant. Fifty microliters whole blood for CD4 cell count estimation was immediately separated by reverse pipetting. Plasma was separated from the remaining whole blood sample within 6 h of collection and transfer to a sterile, 2.0 mL polypropylene screw cap tube. A volume of 500 μL of plasma was used for viral load quantification on the same day. For few patients where the test could not be performed on the day of sample collection, the plasma obtained was stored at 4°C and processed within 5 days. All the samples were processed according to the manufacturer's instructions.

The CD4 cell counts were estimated by Becton Dickinson (BD) FACSCount System (BD, San Jose, CA, USA) using BD FACSCount CD4/CD3 Reagent Kit.

Baseline PVL quantification was carried out at the Molecular Diagnostic Laboratory, Department of Microbiology by The COBAS® TaqMan® HIV-1 Test, version 2.0 for use with the high pure system, using the high pure system viral nucleic acid kit for manual specimen preparation and the COBAS® TaqMan® 48 Analyzer (Roche Diagnostics Ltd., Switzerland) for automated amplification and detection. The test can quantitate HIV-1 RNA over the range of 34–10,000,000 copies/mL.[12] The controls (negative control, low- and high-positive control) used were those provided in the kit.

Participants were stratified according to PVL (<10,000; 10,000–49,999; ≥50,000 copies/mL) and CD4 cell count (≤350 and >350 cells/μL or ≤500 and >500 cells/μL). The CD4 cell count threshold was based on current national and international guidelines; viral load thresholds were based on prior HIV transmission related studies that there is a low risk of transmission with PVLs <10,000 copies/mL[13] and high risk of transmission at PVLs ≥50,000 copies/mL.[4]

Data on demographic variables and other baseline characteristics of the study population were also taken from each patient. Among married patients, HIV status of the spouse was confirmed based on the HIV test report they produced.

The correlation between PVL and CD4 cell count was analyzed by Pearson correlation test (r) using Statistical Package for the Social Sciences (SPSS) software (SPSS Inc. Released 2007. SPSS for Windows, Version 16.0. Chicago, IL, USA). For all analyses, P < 0.05 was considered statistically significant.

Quality control

The FACSCount Centre, Department of Microbiology have been participating in the external quality assurance system (EQAS) conducted by the National AIDS Research Institute, Pune, Maharashtra, India.

Our center is one of the selected 11 National AIDS Control Organization (NACO) laboratories that have been identified to initiate and perform HIV-1 PVL testing[14] and as per NACO recommendation, our center have been participating in EQAS conducted by the Royal College of Pathologists Australasia Quality Assurance Programme, St. Leonards, New South Wales (Australia).


  Results Top


The age ranged in the study population was 16–60 years (mean age 37.1 ± 8.6 years). Of the 82 patients, 44 (53.7%) were females and 38 (46.3%) were males. Majority of the cases (i.e., 94%) belonged to the age group of 15–49 years. Based on the history of the participants, the most common route of acquiring HIV-infection was heterosexual intercourse (75.7%) and injecting drug use (19.5%).

Of 77 (93.9%) married patients, 59 (76.6%) had HIV seropositive spouse and 18 (23.4%) had HIV seronegative spouse. PVL was higher among the patients with seropositive spouse; 42 (71.2%) had viral load of ≥10,000 copies/mL. On the contrary, 15 (83.3%) patients with HIV seronegative spouse had PVL of <10,000 copies/mL.

In this study, baseline PVL and CD4 cell count ranged from target not detected, that is, <34–1,080,000 copies/mL and 9–1566 cells/μL, respectively.

Overall mean and standard deviation of PVL and CD4 cell count in the study population were 108,000 ± 206,200 copies/mL and 348.2 ± 296 cells/μL, respectively. The overall median and interquartile range (IQR) of PVL and CD4 cell count were 14,900 copies/mL (IQR: 3662–88,700) and 273.5 cells/μL (IQR: 138.5–464.2), respectively.

The median PVL was 27,600 copies/mL in the males but only 10,600 copies/mL in the females (P < 0.030). The mean PVL was 157,870 copies/mL in the males but only 67,057 copies/mL in the females (P < 0.046). The CD4 cell counts did not differ according to sex [Table 1] and [Table 2].
Table 1: Mean plasma viral load and mean cluster of differentiation 4 cell count of antiretroviral therapy naïve patients (n=82)

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Table 2: Median plasma viral load and cluster of differentiation 4 cell count of antiretroviral therapy naïve patients (n=82)

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[Table 3] shows ART naïve patients stratified by PVL and CD4 cell count. Of 82 patients studied, 27 (32.9%) patients had PVL <10,000 copies/mL and among them 18 (66.7%) had CD4 cell count >350 cell/μL. Forty-eight (58.5%) patients had viral load ≥10,000 copies/mL and among them 48 (85.4%) had CD4 count ≤350 cell/μL. Fifty (61%) patients had CD4 cell count ≤350 cell/μL and 64 (78%) patients had CD4 cell count ≤500 cell/μL. Among those with PVL ≥50,000 copies/mL, the proportions with CD4 cell count >350 cells/μL and CD4 cell count >500 cells/μL were 10.4% and 3.5%, respectively.
Table 3: Stratification of antiretroviral therapy naïve patients by plasma viral load and cluster of differentiation 4 cell count

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Viral load was not detectable in seven patients (8.5%) in whom the CD4 cell count ranged from 453 to 1225 cells/μL (mean 740 ± 297 cells/μL). Among this group, 100% and 71.4% of patients had CD4 cell count >350 cells/μL and >500 cells/μL, respectively.

There was a significant negative correlation between the PVL and CD4 cell count in HIV-1 seropositive ART naïve patients with “r” of −0.54, (P ≤ 0.00).


  Discussion Top


After searching the literature online, to the best of our knowledge, this study provides the first estimates of baseline PVL in HIV seropositive ART naïve individuals in the North-Eastern part of the country.

In this study, the mean CD4 cell count was 348.2 cells/μL which is in agreement with studies among similar patient groups from African country (266 cells/μL)[15] but higher than values reported from Northern India (112 cells/μL).[16] This difference may be due to variation in mean CD4 cell count among healthy HIV-seronegative adults of different parts of India.[17] Only 22% had CD4 cell count >500 cells/μL, which is lower to a similar study from Africa (58.2%).[2]

The mean PVL of 108,000 copies/mL obtained among the ART naïve individuals with detectable PVL is similar to a report by another Indian study from New Delhi (192,686 copies/mL).[16] Our finding is lower to a report from African study (450,370 copies/mL)[15] but a little higher to a report from Spain (35,730 copies/mL).[11] The high mean viral load in African countries may be the result of recurrent infections in HIV-infected individuals in Africa, especially malaria and some bacterial infections.[15]

Among those with PVL ≥50,000 copies/mL, the proportions with CD4 cell count >350 cells/μL and >500 cells/μL were 10.4% and 3.5%, respectively, which is similar to a cross-sectional study from African countries.[2]

Our study showed that HIV-1 RNA was not detectable in 8.5% of the ART naïve patients which is in concordance with another similar studies from Spain (16%)[11] and Nigeria (9.5%).[15] The exact reason for undetectable PVL is not clear. This could be due to the strong immune status of these patients as reflected by their high CD4 cell counts (mean 740 ± 297 cells/μL). Another reason may be due to the presence of variants of HIV-1 that the COBAS® TaqMan® HIV-1 Test (version 2.0) kit for viral load assay is unable to detect.[12] 71.4% of the individuals with undetectable HIV-1 RNA in plasma had CD4 cell count >500 cells/μL which is similar to report from African country (56.3%).[15]

In this study, the median PVL was significantly lower in women than in men (10,600 vs. 27,600 copies of HIV-1 RNA per milliliter, P < 0.030), but there was no significant gender difference in mean CD4 cell count (P < 0.131) and median CD4 cell count (P < 0.090). Our study confirms earlier reports that PVL levels are significantly lower in women than in men.[11],[18] However, another similar study from India[19] negates recent reports that PVL levels are higher and CD4 cell counts are lower for HIV-1 infected men than HIV-1 infected women.[4],[10]

Our study showed a statistically significant negative correlation (r = −0.54, P < 0.00) between PVL and CD4 cell count in HIV-seropositive ART naïve patients, that is, majority of the individuals (89.6%) with low CD4 cell count (≤350 cells/μL) had higher viral load (≥50,000 copies/mL) and vice versa (66.7% with high CD4 cell count >350 cells/μL had lower viral load <10,000 copies/mL). Our study confirms earlier reports from India[10] and Spain.[11] However, this feature is not always absolute, as some individuals with a high CD4 cell count may have high PVL and vice versa.[11] In our study too, a 25-year-old female had both high CD4 cell counts (1566 cells/μL) and high PVL (80,300 copies/mL).

In India, as per NACO guidelines,[7],[14] CD4 cell count ≤350 cells/μL is being used as the basis for initiation of ART in adults and adolescents. ART initiation based on current NACO guidelines will avert treatment of 39% of ART naïve patients, including 10.4% of ART naïve patients with viral load ≥50,000 copies/mL with CD4 cell count >350 cells/μL, who represents a group of ART naïve patients with an increased risk of HIV-1 transmission and disease progression.[4],[9] The higher the viral load at initiation of therapy, the longer it takes to drop below the level of detection. In one study, the range was between 15 days with a baseline PVL of 1000 copies/mL and 113 days with a baseline of 1 million viral copies/mL.[20] PVL in ART naïve patients provides additional important information for clinical decisions on when to initiate first-line ART especially, in patients with a relatively high CD4 cell count.[7] Therefore, consideration of PVL in ART initiation guidelines for those with CD4 cell count >350 cells/μL or with CD4 cell count above the recommended threshold for ART initiation will maximize ART coverage of highly infectious ART naïve patients, which in turn will reduce the risk of HIV transmission and disease progression at individual and population level.

The World Health Organization (WHO) guidelines[21] recommended earlier initiation of ART for clinical benefit, at CD4 count ≤500 cells/μL. In our study, we observed that if this guideline is followed 78% of ART naïve patients will qualify for ART initiation including, 96.5% of patients with high PVL (≥50,000 copies/mL) who are highly infectious. However, based on recent studies, the WHO 2016 guidelines[22] recommended that ART should be initiated in all people living with HIV, irrespective of CD4 cell count and WHO clinical stage. This will substantially reduce sexual transmission, HIV morbidity, improve the quality of HIV treatment and bring us one step closer in achieving universal access to HIV treatment.

Limitations

In this study, information on ART naïve status was obtained by self-report, thus there is potential for some patients on ART for a variable period. Further investigations or follow-up of one patient with both high CD4 cell count and high PVL was not done and therefore, the reasons remain obscure.


  Conclusion Top


Our study provides baseline PVL and CD4 cell count in HIV-seropositive ART naïve patients of this part of the country. In this study, we found that there is statistically significant negative correlation between PVL and CD4 cell count in HIV-seropositive ART naïve patients. Our study confirms earlier reports that PVL levels are significantly lower in women than in men, but no statistically significant gender differences in the CD4 cell count. Our results strongly support the WHO recommendation to start ART with CD4 count ≤500 cells/μL. Consideration of PVL in ART initiation guidelines for those with CD4 cell count >350 cells/μL or with CD4 cell count above the recommended threshold for ART initiation will maximize ART coverage of highly infectious ART naïve patients, which in turn will reduce the risk of HIV transmission and disease progression at individual and population level.

Acknowledgments

The first author is thankful to the Department of Biotechnology (DBT) Nodal Centre, Tezpur University, Assam, India, Ministry of Science and Technology, Government of India for funding this study in part under “MD/MS Thesis Grant phase 7 and 8 (2014–2015)” vide Sanction No. BT/Med/15/Vision-NER/2011, dated November 2, 2011 and Memo No. TU/DBT-NC/MD/MS-P7&8/14-15/32, dated August 27, 2015. We thank all the individual who participated in this study. We also expressed our gratitude to all the staff of CD4 count center and DBT Molecular Diagnostic Laboratory, Department of Microbiology, Regional Institute of Medical Sciences, Imphal, Manipur, India for their cooperation and technical assistance during the study.

Financial support and sponsorship

Financial assistance received under “MD/MS Thesis Grant phase 7 and 8 (2014–2015)” from the Department of Biotechnology Nodal Cell, Tezpur University vide Sanction Order No. BT/Med/15/Vision-NER/2011, dated November 2, 2011 and Memo No. TU/DBT-NC/MD/MS-P7 and 8/14-15/32, dated August 27, 2015.

Conflicts of interest

There are no conflicts of interest.

 
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