HIV-1/HIV-2 co-infection among voluntary counselling and testing subjects at a regional hospital in Cameroon

Background : HIV/AIDS is a major public health problem in Cameroon which had a prevalence of 5.1% in 2010 with 141 new infections per day. The fear of voluntary counseling and testing (VCT) is an obstacle to HIV prevention. Objectives : To determine the prevalence of HIV-1, HIV-2 and HIV-1/HIV-2 co-infection among people attending a health facility for VCT. Methods : Venous blood was collected from participants using aseptic techniques in a descriptive observational cross-sectional study. DETERMINE HIV-1/2 and SD BIOLINE HIV-1/2 3.0 qualitative tests were used for the detection of HIV-1 and HIV-2 in their sera. Range and consistency checks were carried out on the data and analysed using Epi-Info. Results : Of 290 individuals tested, 78(26.9%) were positive for HIV-1 and HIV-2. Among the 78 HIV positive individuals, 62 (79.5%) had HIV-1, 1(1.3%) had HIV-2 and 15(19.2%) had concurrent HIV-1/ HIV-2. Among those infected, 57(73.1%) were females including 21(26.9%) males. Conclusion : HIV-1 is the major cause of AIDS and VCT is well accepted. Co-infection with HIV-1/HIV-2 may lead to anti-retroviral drug resistance. VCT should be encouraged so that positive cases can initiate therapy on time to stay ahead of anti-retroviral drug resistance.


Introduction
HIV is a global health problem.In 2005, WHO and UNAIDS estimated that 40 million people were infected with HIV in the world; with more than 60% of them in sub-Saharan Africa.The pandemic is dominated by HIV-1, which was discovered in 1983.In 1987, HIV-2 was discovered which is very common in West Africa 1 and has not shown any significant spread from there.HIV-2 is less easily transmitted than HIV-1 and the period between initial infection and illness is longer than with HIV-1 2 .
The prevalence of HIV in Cameroon was 5.1% in 2010 with significant regional variations 3 , 141 new infections per day 4 and transmission is mainly heterosexual (90%) 4 .Part of the strategic response to HIV in Cameroon, has been the institutionalization of VCT for HIV services across the country 5 .
HIV-1/HIV-2 co-infection studies revealed a difference in the progression to AIDS among HIV-1, HIV-2 or co-infection and their response to antiretrovirals 2 .There are three categories of antiretrovirals 2 : nucleoside reverse transcriptase inhibitors (NsRTIs) (zidovudine, didanosine, stavudine, lamivudine, abacavir, zalcitabine, and emtricitabine); non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, efavirenz, delavirdine) and nucleotide reverse transcriptase inhibitors like tenofovir 2 .The protease inhibitors include saquinavir, ritonavir, indinavir, nelfinavir, lopinavir/ritonavir, amprenavir, and atazanavir.A combination of at least three antiretrovirals, usually two NsRTIs and a NNRTI are recommended for continued effectiveness and to prevent the emergence of drug-resistant HIV strains 2 .
In many countries, HIV remains a highly stigmatizing condition and there are many anecdotal reports of people being stigmatized for being seropositive.This is common when people are tested mandatorily or without adequate counseling 11,12 .As the HIV/AIDS pandemic advances, VCT should be provided along with sensitisation 13 .VCT is important in improving care and support 14 and many developing countries are instituting it as part of the primary health care package 15 .Developments in cotrimoxazole prophylaxis 16,17 and tuberculosis preventive therapies 18 for HIV people, antiretroviral (ARV) therapy 19 and PMTCT 20 , have expanded to VCT.This study was conducted to determine the HIV prevalence and its major strains (HIV-1 andHIV-2) in people coming for VCT in a regional hospital setting.

Study Area
This study took place in Buea, the headquarters of the South West Region of Cameroon.There are many ethnic groups in Buea including the Bakweri (indigenes), Bamileke, Bafou, Balondu, Metta and Bayangi among others.Buea has a population of about 200.000 inhabitants and is located 15kms from the Atlantic Ocean, and 60 kilometres from Douala, the economic capital of Cameroon.Most inhabitants practice agriculture as the main economic activity.Buea has two seasons: the dry season from October to March and the wet season from March to September.Almost all ethnic groups in Cameroon are represented in Buea, attracted by the fertile volcanic soil and the Cameroon Development Corporation, a giant agricultural corporation that seconds the state of Cameroon in employment.

Study design and setting
The design was a descriptive observational crosssectional hospital-based study in a tertiary health care facility in Buea, Cameroon.The Buea Regional Hospital Annex(BRHA) has thirteen services: administration, paediatric, surgery, maternity, theatre, x-ray, laboratory, dental, diabetes unit, tuberculosis unit, eye unit, family planning, outpatient, HIV/AIDS unit, the male and female medical units.

Study population and sample size
The study population involved males and females of different age groups who came to the BRHA for VCT from October 27 th -December 16 th 2009.Samples were collected after due counselling and informed consent from all individuals at the HIV/ AIDS counselling unit of the hospital.Only those that consented to VCT were included in the study.290 samples were collected from all individuals presented for VCT during the study period.
The authorization to carry out this study was obtained from the Faculty of Health Sciences, University of Buea.Ethical clearance was obtained from the Regional Delegation of Public Health, Buea.Informed consent was obtained from all persons before any information and blood sample were collected.

Specimen collection and laboratory analysis
Venous blood was collected by venepuncture using aseptic techniques.About 3-4ml blood samples was transferred into a labelled dry tube, centrifuged at 3000rpm for 5minutes to separate serum from red cells, labelled and stored between 2-8 o C. Serum samples were analysed using two qualitative tests: DETERMINE HIV-1/2 and SDBIOLINE-1/2 3.0.

Laborator y procedures for the immunochromatographic tests
We used DETERMINE HIV-1/2 to detect all the positive samples and SD BIOLINE HIV-1/2 3.0 to identify HIV-1, HIV-2, and HIV-1/HIV-2.The tests were performed using the WHO Bulk Procurement Scheme for HIV Assays 21 .
For DETERMINE, 10-test cards were removed and the strips placed on a table.Using a clean micropipette, 50μl serum was applied to the pad.All samples were tested with new strips from the same lot number.Results were read between 15-60 minutes.To insure assay validity, a procedural control was incorporated.When the control bar did not turn red upon assay completion, the test result was invalid and the sample retested.Red bars appearing in both the control and patient windows were interpreted as positive.Any visible red colour in the patient window was interpreted as positive.One red bar in the control window with no red bar in the patient window was interpreted as negative.When there was no red bar in the control window, even if a red bar appeared in the patient window, the result was invalid and repeated.
For BIOLINE, the test kit was removed, and placed on a table.Using a micropipette, 20μl of serum was added into the well.Four drops of assay diluent were added and results read in 5-20 minutes.The presence of only a control line within the result window indicated a negative test.The presence of two lines as control line and test line 1 within the result window indicated a positive HIV-1.The presence of two lines as control line and test line 2 within the result window indicated a positive HIV-2.The presence of three lines as control, test line 1 and test line 2 within the result window indicated a positive HIV-1/HIV-2.When the colour intensity of test line 1 was markedly darker than that of test line 2 in the result window, it was interpreted as HIV-1.When the colour intensity of test line 2 was markedly darker than that of test line 1 in the result window, it was interpreted as HIV-2.When colour intensities of test lines 1 and 2 in the result window were the same, it was interpreted as HIV-1/HIV-2.The results were entered into a log book and analyzed using Epi-Info after a double entry by two data clerks.

Discussion
The HIV-1/HIV-2 co-infection has treatment implications because nonnucleoside reverse transcriptase inhibitors, crucial in standard first-line regimens for HIV-1 in many low-income settings [like Cameroon], has no effect on HIV-2 22 .Nucleoside analogues alone are not sufficiently potent enough to achieve durable virologic control.Some protease inhibitors without ritonavir boosting are not sufficiently effective against HIV-2 22 .
The most crucial difference between HIV-1 and HIV-2 when considering suitable antiretroviral regimes is the lack of susceptibility of the firstgeneration NNRTIs, nevirapine, and efavirenz 22 .The natural resistance of HIV-2 to these drugs is due to differences in the amino acid residues that make contact with the NNRTI in the binding pocket of HIV-1 and HIV-2, particularly the Y181I and Y188L natural polymorphisms seen in HIV-2, which significantly reduce NNRTI binding 22 .HIV-1 mutations at these positions result in complete resistance to NNRTIs.Although etravirine has more activity against HIV-2 than previous NNRTIs, the presence of L181 and other structural differences in the HIV-2 NNRTI-pocket make HIV-2 naturally resistant to etravirine 22 The lack of susceptibility of HIV-2 to NNRTIs indicate that patients with co-infection need to use an adapted combination to suit their condition, adding a second-line treatment regime to the standard first-line for HIV-1 infection 22 .Second line therapy ideally include a minimum of three new drugs, with at least one from a new class, in order to increase treatment success.The implications are that patients have to spend more on testing and buying drugs 22 .This means that government policy on HIV treatment has to change occasionally whenever there is any documented resistance to treatment regimes when HIV-2 is diagnosed due to viral natural polymorphism 22 .
In this study, there were more HIV-1 than HIV-2 which contrasts the findings of a Malian study 23 where there were more HIV-2 than HIV-1.This study agrees with the work of Abdulazeez et al 10 , who found 12.1%, 0.5% and 1.6% for HIV-1, HIV-2 and HIV-1/HIV-2 respectively but different from that of Ndiaye et al 9 .The increase in HIV-1/ HIV-2 co-infection in our study and that of Abdulazeez et al 10 , suggests that there may be more co-infection with the two virus strains.
The 26.9% HIV seroprevalence was significantly high in the population as compared to the 5.3% prevalence in the Cameroonian population 24 .The presence of many higher institutions of learning in Buea increases the youth population who are more vulnerable because of their sexual habits.
VCT constitute a major component of the UNAIDS 2011-2015 strategic plan of "three-zeros" -zero new infections, zero stigma and zero deaths 25 .VCT is important for HIV prevention in communities with generalised epidemics like Cameroon.It allows adolescents to know their HIV status and to evaluate their behaviour and its consequences while a negative test reinforces the importance of safe and risk-reducing behaviours.VCT benefits include improved health status, emotional support, better coping ability, PMTCT feeding awareness options, motivation to initiate or maintain safer sexual and drug-related behaviors, and safer blood donation 26 .Stigma may actively prevent people accessing care, gaining support, and preventing onward transmission.Hence, UNAIDS 24 stipulated that testing should be voluntary, and VCT should take place in collaboration with stigmareducing activities 26 .
The International Guidelines on HIV/AIDS and Human Rights 27 advises against mandatory HIV testing for both public health and human rights reasons.The World Health Assembly 28 resolved that there was no public health rationale for measures establishing mandatory HIV screening 26 .
Our findings showed that the most infected were in the 20-29 and 30-39 age brackets which agrees with results of UNAIDS 29 , that the HIV prevalence in Cameroon is more amongst people aged 15-49 years.There was no significant difference in HIV infection with age and sex (p>0.05);hence both males and females of all age groups have the same likelihood of being infected with HIV.
During the early years of HIV infection in Cameroon, social stigma was very high and treatment was not easily available 30 .With the availability of free HIV/AIDS drugs 26 , many people are willing to go for VCT and treatment.
We could not obtain some demographic information from participants because of logistic reasons.Cost problems of conducting VCT, psychological trauma and stigma are inherent in our environment.