Human Immunodeficiency VirusType-1 (HIV-1) Disease Progression and Viral Activity: A Seroepidemiological and Molecular Study

Syed Zahid Bukhari1, Javed Iqbal Qazi2, Ahmad Mohammad Ashshi3and Naheed Zia4


ABSTRACT

Objective:To determine the frequency and epidemiological characterization of human immunodeficiency virustype-1

(HIV-1) infection, HIV disease progression, immune status and viral activity.

Study Design:Descriptive study.

Place and Duration of Study: Department of Microbiology, University of the Punjab and Institute of Public Health, Lahore,

from September 2005 to August 2008.

Methodology:The study enrolled samples from general population, high risk groups and spouses of HIV+ deport workers

with criteria; positive double enzyme linked immunosorbent assay (ELISA) and positive western blot. Immune status and

viral activity was determined by cluster determinants (CD4+ and CD8+) cell count, ratio of CD4+/CD8+ on flow cytometer,

and HIV RNAviral load on polymerase chain reaction (PCR).

 

 

Results: Atotal of 116 HIV+ untreated subjects enrolled after screening of 2260 blood samples. The seroprevalence rate

 

in general population, high risk individuals and spouses of HIV+ deport workers was found 0%, 0.4% and 26%

 

respectively. The CD4+ cell count was found 533/mm3(range 12-1800/mm3)

 

   and plasma viral load 27,122 copies/ml

 

(range 00-40,621). The CD4+/CD8+ ratios < 0.5, < 1, < 1.5 and < 2 appeared as 17.2%, 30.2%, 51.7% and 0.9% respectively.

 

Significant correlation was observed between plasma viral load, CD4+ count and CD4+/CD8+ ratio (p = 0.001). CD4+

 

T-cell counts < 200 cells/mm3was found in 23 HIV+ patients.

 

Conclusion:There was a low frequency of HIV in the general population and high risks groups as compared to very high

 

frequency in spouses of HIV+ deport workers with significant correlation of viral activity and immune status.

 

INTRODUCTION

 

The first human immunodeficiency virus(HIV)- positivePakistani was identified in 1987.1Till March 2010, 3325patients were registered at National AIDS ControlCentre, NIH, Islamabad.2Human immunodeficiencyvirustype 1 (HIV-1) specific CD8+ T-cells play a key rolein the control of viral replication during HIV-1 infection.The cytotoxic T-lymphocyte (CTL) response is mainlymeasured at the early stage of infection and itsappearance coincides with a rapid fall in plasma viremiaduring the early stage of infection with HIV-1.3Among allHIV high risk individuals in Pakistan, female sexworkers (FSWs) formed the largest group reported, withestimates of 79127 and five different sub-typologies.

 

1Department of Microbiology and Infection Control, Hera General

 

Hospital, Makkah, Saudi Arabia.

 

2Department of Microbiology, Faculty of Live Sciences,

 

University of the Punjab, Lahore.

 

3Department of Laboratory Medicine, Faculty of Applied Medical

 

Sciences, Umm Al-Qura University, Makkah, KSA.

 

4Department of Obstetrics and Gynecology, Sir Ganga Ram

 

Hospital, Lahore.

 

Correspondence:Dr. Syed Zahid Bukhari, P.O. Box.  20865,

 

Makkah, Saudi Arabia.

 

E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

 

Received December 13, 2010;  accepted June 18, 2012.

 

Injection drug users were the second largest groupfollowed by male sex workers and hijra sex workers withestimates of 31555, 19320 and 14702, respectively.4The rationale behind the study was that in humanimmunodeficiency virustype-1 (HIV-1) infection there isa decline in viral replication that has been attributed tohost immunity, but the components of this responsevaries among individuals particularly the ability ofcytotoxic T-lymphocytes to control viral burden andinfluence the outcome of the disease.

 

The objectives of the study were to determine HIVfrequency in general population, high risk individuals forHIV and spouses of HIV+ deport Pakistani workers andto find out the immune status and plasma viremia withHIV disease progression.

 

METHODOLOGY

 

It was a descriptive study with repeated cross-sectionalsurveys at fixed intervals on HIV+ patients followedlongitudinally from September 2005 to August 2008.This study was conducted at Department ofBacteriology, Institute of Public Health and MicrobiologySection of Faculty of Live Sciences University of thePunjab, Lahore. The technical assistance was gainedfrom Armed Forces Institute of Pathology, Rawalpindi,and National Institute of Health, Islamabad. The study was approved by the Ethical Committee of the Universityof the Punjab and principles of Helsinki Declaration werefollowed. The consent form was prepared in Urdu. Thosepersons who did not sign the consent form wereexcluded from the study population in general as wellas high risk individuals. The clinico-immunologicalprogression of HIV disease was observed

 

 

 

The subjects representing general population consistedof both sexes and all age groups residing in thesurrounding area of Institute of Public Health, Lahore.Simple random sampling procedure was adopted by thestudy team comprised of one Doctor and two LaboratoryTechnicians of either gender. The questionnaireinformation related to HIV was solicited and the bloodsamples were collected according to format preparedprior to the sample collection. The standard precautionswere followed during the blood extraction and clinicalexamination of the patients. The high risk individualsincluded were commercial sex workers (CSW) orprostitutes residing in Shahi Mohalla, Lahore. Asimplerandom sampling was done on the basis of sampleframe provided by the leader of the singing and dancinggirl’s union. Homosexual workers (HSW) were individualswho were reputed for indulging in homosexuality aspassive partner,6contacted through their administrativechief (Guru). The third group was patients receivingmultiple transfusions for haemoglobinopathies mainlythalassemia major and those leukemia contactedthrough Mayo and Services Hospitals, Lahore. The othergroups were professional donors from various privateclinics and volunteer donors registered in differentprivate blood transfusion associations as well asintravenous drug users (IDUs) admitted in drug addictioncontrol centres of Psychiatry units of Mayo and ServicesHospitals and patients having generalized lymphadeno-pathy for more than 3 months in two or more extrainguinal sites were included. The spouses of Pakistaniworkers with HIV/AIDS deported from Gulf countrieswere included in the study through the Provincial AIDSPrevention and Control Centres. This group representsall provinces of Pakistan; Sindh, Punjab, Balochistan,Khyber Pakhtunkhwa including Federal Capital areas-Islamabad, and Azad Jammu and Kashmir (AJK).The criterion for inclusion in the study population was;positive HIV on double ELISAand confirmed on Westernblot. The manufacturer instructions (Abbott Murex) werefollowed for ELISAprocedure. Lav. Blot 1 Ac- Ab-Ak kit(Diagnostic Pasteur, France) was used to detect HIV-antibodies in serum by immunoblotting (Western blot) inorder to confirm the status of ELISApositive serumsamples. The criteria of exclusion was those HIV+patients who had received anti-retroviral treatment.

 

CD4+ and CD8+ T-lymphocytes count was performed onflow cytometer; FAC Scan Becton Dickinson Immuno-cytrometry System 2350 Qune Drive San Jose, California95131, (bench top).5

 

Blood samples were drawn aseptically by venepunctureinto a sterile K3 EDTA(lavender top) vacutainer bloodcollection tubes. Aminimum of 1 ml of whole blood wasrequired. White blood cell count (WBC) and a differentialcell count was obtained from the same sample of wholeblood before staining. Sysmex KX-21N AutomatedHaematology Analyzer was used for white blood cell(WBC) count.  An acceptable WBC concentration rangefor the method used was described to be ranging from3.5 x 103to 9.4 x 103

 

WBC/µl. Samples with countsgreater than 9.4 x 103WBC/µl were diluted with IXphosphate-buffered saline (PBS) containing 0.1%sodium azide, or cell wash. For the samples with lessthan 3.5 x 103WBC/µl, more blood was required. Bloodwas stained with reagent Athrough F within 6 hours afterdrawn (Becton Dickinson catalog No. 349524). For thepercent lymphocyte, conversion software option wasselected, which automatically calculated each reportedlymphocyte subset as a percentage of lymphocytes inthe lymphocyte analysis gate.

 

Viral load was determined by quantification of viral RNAin peripheral blood by RT-PCR (reverse-transcriptionPCR) on Roche Diagnostics System which comprisedof the following four equipments; COBAS AmplicorPCR analyzer-Roche Diagnostic; COBAS AmpliPrepTManalyzer-Roche Diagnostic;  Amplilink PC; and Barcodescanner.7The results were categorized in four groups as< 500, 500 to 10,000, 10,001 to 100,000 and > 100,000copies/ml.

 

Statistical Package for Social Sciences (SPSS) statisticalsoftware version 10 was used. Descriptive statisticsincluding 95% confidence interval between upper andlower limit-Confidence Interval (CI) were determined.Pearson correlation was considered significant atthe 0.01 level (2-tailed). Continuous variables weresummarized using median, mean and standarddeviation (SD) and categorical variables using frequen-cies and percentages. The chi-square test was used forcomparing proportions.

 

RESULTS

 

Of the 2260 total individuals tested for HIV, 1050(46.46%) belonged to the general population, 1010(44.69%) represented high-risk groups and 200 (8.85%)spouse of HIV+ deported workers. The seroprevalencerate of HIV in general population, high-risk individualsand spouses of deported workers was found 0%, 0.4%and 26% respectively. The mode of transmission amongHIV+ individuals was as follows; heterosexual 75(64.7%), homosexual transmission 2 (1.75%), IDUs 4(3.4%), blood transfusion 12 (10.3%) and unknown23 (19.8%). The area wise distribution of HIV+/AIDScases was as follows. Federal/Islamabad in 14 (12.1%),Punjab in 33 (28.4%), Sindh in 47 (40.5%), KhyberPakhtunkhwa in 16 (13.79%), Balochistan in 5 (4.3%)and AJK in one (0.9%) case.

 

Table I:Clinical positive findings in HIV/AIDS cases.

 

Symptoms / Signs / Diagnostic

Number of positive HIV+ cases (%)


 


Federal area

Punjab

Sindh

Khyber

Balochistan

AJK

Total

     

Pakhtunkhwa

     


 


Kaposi sarcoma

0

0

0

0

0

0

0

Disseminated/ extrapulmonary / non-cavity

             

pulmonary tuberculosis

0

0

0

0

0

0

0

Oral candidiasis / hairy leukoplasia

1 (0.86)

3 (2.59)

8 (6.90)

1 (0.86)

0

0

13 (11.2)

Pulmonary tuberculosis with cavitations,

             

or unspecified

0

0

4 (3.4)

0

0

0

4 (3.4)

Herpes zoster in a person of 60 years or less

0

0

3 (2.6)

0

0

0

3 (2.6)

Central nervous system dysfunction

0

0

0

0

0

0

0

Diarrhea one month or more

1 (0.86)

3 (2.59)

6 (5.17)

0

0

0

10 (8.6)

Fever of at least 38°C for at least a month

1 (0.86)

6 (5.17)

4 (3.45)

1 (0.86)

0

0

12 (10.3)

Cachexia or weight loss of more than 10%

1 (0.86)

4 (3.45)

6 (5.17)

1 (0.86)

0

0

12 (10.3)

Asthenia of at least a month

0

0

0

0

0

0

0

Persistent dermatitis

0

3 (2.59)

7 (6.03)

0

0

0

10 (8.6)

Anaemia, lymphopenia, and/or thrombocytopenia

0

4 (3.45)

10 (8.62)

1 (0.86)

0

0

15 (12.9)

Persistent cough or any pneumonia, and/or

             

thrombocytopenia

0

0

0

0

0

0

0

Lymphadenopathy of at least two non-inguinal sites

0

2 (1.72)

7 (6.03)

0

0

0

9 (7.8)


                                                            

                              


Acute retroviral syndrome like mild fever, lethargy,malaise and headache was found in 37 cases (31.87%);56 (48.3%) were asymptomatic. The clinical findingsamong the 116 HIV/AIDS cases are given in Table I.Immunologically HIV disease progression was catego-rized into five stages; asymptomatic primary in 2 (1.7%),symptomatic primary in one (0.9%), asymptomatic latentin 49 (42.2%), symptomatic latent in 41 (35.3%) andAIDS in 23 cases (19.8%). During the follow-upexaminations 20 cases (17.24%) of the 116 HIV/AIDScases developed opportunistic infections. Theseincluded pulmonary tuberculosis in 4 (3.4%), oralcandidiasis in 13 (11.2%), and herpes zoster in 3 (2.6%)cases. Patients characterized with CD4+ lymphocytecounts < 200/ml and CD4+ percentages < 14% werepresented with opportunistic infections. Patients with aviral load over 10,000 copies/ml also had a morefrequent incidence of opportunistic infections. In general,the high viral load was associated with more frequentopportunistic infections, regardless of respective CD4+lymphocytes' level. Four of the HIV/AIDS patients (3.4%)had associated infection (co-infection). Three subjectswere found hepatitis B (HbsAg) positive, while anti HCVappeared in one case. Six HIV/AIDS patients (5.2%)showed sexually transmitted diseases other than HIV.Syphilis was diagnosed in 4 of the cases followingscreening test, haemagglutination (TPHA) for Tryponemapallidumtest. Similarly, gonorrhea was diagnosed on thebasis of typical history of urethral discharges in two ofthe seropositive HIV individuals, which was confirmed byexamining direct Gram's stained smears.

 

The distribution of CD4+ cell count among 116HIV+/AIDS patients was as follows; patients presented

 

33and < 200/mm3with cell counts < 500/mm, < 350/mm

 

were 63 (54.3%), 33 (28.4%) and 20 (17.2%) respec-

 

Table II:CD4+/CD8+ ratios amongst the HIV positive and AIDS patients.CD4+/CD8+ HIV disease status

 

ratio categoryHIV positive casesAIDS casesp-value

 

No.%No.%value< 0.5002017.2< 0.0001< 1.03429.31010.860.0019< 1.55850.00021.72< 0.0001< 2.0010.86000.617Total9380.172319.78-

 

Figure 1: Viral load (copies/ml) and CD4+ (cells/cmm) count among

 

HIV+/AIDS patients.

 

tively. All above patients were tested again for CD4+ cellcount after 6 months and the count remained the samein three groups mentioned above. Of total 116HIV+/AIDS patients, 56 (48.3%) showed viral load < 500copies/ml; 24 (20.7%) 500-10,000 copies/ml; 24 (20.7%)10,001-100,000 copies/ml and 20 (17.2%) > 100,000copies/ml. Figure 1 shows the description of viralload (copies/ml) and CD4+ count (cells/cmm) amongHIV+/AIDS patients. The distribution of CD4+/CD8+ratios amongst the HIV positive and AIDS patients isgiven in Table II.

 

DISCUSSION

CONCLUSION


 


International labour migration, or the movement ofpeople across national borders for employment, is agrowing phenomenon and an increasingly importantaspect of global, regional and national economies.However, HIV has become a key issue of concern withcross border and overseas migration. In the presentstudy highest prevalence rate (26%) of HIV infection wasfound among the spouses of deported Pakistaniworkers; a similar figure was reported in other study.8Pakistan has high rate of external migration. Mobility andmigration themselves are not risk factors for HIV, butthey may lead to situation in which people may becomevulnerable to the infection; separation from spouse,family, society and traditions, together with isolation,loneliness and a sense of anonymity can lead tounsocial and sexual practices, which may increase theexposure to HIV. This group may be a major threat ofHIV epidemic in Pakistan and this can indirectly beproved that all clients of HIV clinics in Pakistan are theexpatriated migrant workers and their immediaterelations.9We found that a large number of deport HIV+Pakistani workers (82%) were returned from the Gulfcountries similar to other studies.10The above workerswere screened there on routine investigations, which aremandatory for renewal of contract and resident permit.In the current study, HIV frequency in general populationand high risk population was found low which may bedue to social, cultural and religious impact on the riskfor contracting HIV disease in Pakistan. Very lowprevalence of HIV among CSW, HSW and other highrisk group was comparable to other reports.11-13Thecharacteristics of HIV-specific immune responses andthe parameters of HIV infection in Pakistani population isnot fully understood because of the non-availability ofdata. The relationship between HIV-specific T-lymphocyteresponse and viral replication was studied amongPakistanis who are infected with HIV-1. High viral loadwas found in a significant number of HIV+ patients whichlater developed symptoms of AIDS and was consideredas an indicator of the disease's progression.14,15Therewere 23 HIV+ symptomatic patients in this study whoshowed high viral load and rapidly decreasing CD4+count < 200/mm3

 

and urgently seeking antiretroviraltreatment,16but unfortunately none was received duringthe study period. HIV+ patients had elevated numbers ofCD8+ cells during early infection, which was consideredas a viral set point determination on HIV diseaseprogression.17The influence of CD8+ T-lymphocytefunction on HIV disease progression is of considerableinterest as cytotoxic T-lymphocytes (CTLs) are the maineffecter cells of the specific cellular immune response.Activated by CD4+ T-helper cells, anti-HIV specificCD8+ T-cells have a crucial role to play in the control ofviremia,18-20and increased in response to ongoing viralreplication.21

 

Alow frequency of HIV was found among the generalpopulation and the high-risk groups while it was higheramong the spouse group of HIV+ deport workers. Themajority of deport workers returned from the Gulf countriesand appeared a major threat of HIV disease in Pakistan.High viral load and low total CD4+ T-lymphocyte countwere considered to be one of the main indicators of theprogression of HIV-induced immunodepression in patients.

 

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