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��ࡱ�>��	NP����IJKLM���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������a	��m[bjbj��	�ex�Ebx�EbƳ,����������""2$2$2$T�����$�$�$�%<Z&D�$�b�'H�(�(�(�(W��U�4���T�V�V�V�V�V�V�$�����z��2$%����W�%�%�z�""�(�(�\�0-�-�-�%�	"��(2$�(.�&-�%�T�-�-���� �#����(�����`:�I�����;���<�C�<�V�x9�Zx�����2$���%�%�-�%�%�%�%�%�z�z����%�%�%��%�%�%�%���������������������������������������������������������������������%�%�%�%�%�%�%�%�%��B� :	Immunophenotypic Characterization of ART-Na�ve, HIV Infected Subjects in Kombewa Sub-county, Kisumu: a Cross Sectional Study of Rural Population in Western Kenya
Jew Ochola1, 2, David H. Mulama2, Jackson Cheruiyot2, 
1KEMRI/USAMRD-K, Walter Reed Project, Kisumu County, Kenya 
2Department of Biological Sciences, Masinde Muliro University of Science and Technology, Kakamega County Kenya 
Abstract 
Reliable information on clinical immunophenotypic biomarkers is critical in the management of Hepatitis B and C and mycobacterium tuberculosis infections especially in ART Na�ve HIV positive adults. HIV plasma viral load levels, CD4 cell counts, and the level of liver function bio-molecules can be monitored to assess the body status and treatment response to interventions. This was a cross sectional laboratory investigation study aimed at investigating the immunological phenotypes in HIV positive adults from Kisumu West Sub-county, so that overall patient care, participation of individuals in clinical trials and evaluation of adverse events can be improved. Median ALT values in Hepatitis C positive HIV positive ART Naive participants were higher than in the Hepatitis C negative participants (p=0.0494). The study underscores the need for monitoring liver enzymes levels in ART naive HIV positive patients as well, screening for viral hepatitis before initiation of Anti-Retro Viral therapy as these diseases pathologically alters the body�s immune biomarkers. 
Introduction
Proper management of common public health issues more so in developing countries where medical infrastructure is undeveloped is urgently needed. This calls for availability and proper understanding of appropriate biomarkers that can be readily accessed for monitoring disease progression, therapeutic intervention as well as prognosis that can be applied in treatment and clinical studies for reference purposes.  These values are urgently needed in sub-Saharan African that is ravaged by both parasitic diseases such as malaria and schistosomiasis, bacterial diseases as well as viral diseases such as HIV Aids (Naylor et al., 2008). Human Immunodeficiency Virus (HIV) infection and Acquired Immunodeficiency Syndrome (AIDS) are a twin global health challenges (UNAIDS Global report, 2013). Infected subjects are usually susceptible to multiple infections. Mycobacterium tuberculosis and viral hepatitis are two of the commonest opportunistic co-infections seen among HIV-positive patients worldwide. The common hepatitis infection in HIV infected subjects is due to hepatitis B (HBV) and Hepatitis C (HCV) (Verucchi et al., 2004). The complications associated with HBV and HCV are the leading cause of chronic liver diseases especially in and immunological compromised patients (WHO, 2002). Global HIV prevalence is estimated at 40 million while, chronic HBV and HCV infection accounts for an estimated 370 million, and 130 million respectively (Alter, 2005). It is well known that infections with HBV and/or HCV have a severe and invasive impact on the health of millions of people around the world, as these infections are often asymptomatic (Wondimeneh et al., 2013).
Reliable information on clinical immunophenotypic biomarkers is critical in the management of these diseases. HIV plasma viral load levels, CD4 cell counts, and the level of liver function bio-molecules can be monitored to assess the body status and treatment response to interventions. World Health Organization (WHO) advises that appropriate mechanisms be applied when certain virologic, immunologic, or clinical features are observed (Ekouevi et al. 2014). For instance, the WHO recommends initiation of HAART in HIV patients co-infected with Hepatitis B or Hepatitis C irrespective of value of CD4 count (WHO, 2009). The implication in HIV patients who have CD4 counts above 350cells/�l but who may be positive for Hepatitis B and/or Hepatitis C is not factored in the treatment regimen of Highly Active Anti-Retroviral Therapy (HAART) until they get to end-stage liver damage (National Institutes of Health, 1990). There is need to establish reference values for liver enzymes to provide a guideline in monitoring of patients who may be prone to liver toxicity. 
The WHO estimates that one third of the world's population is infected with Mycobacterium tuberculosis with nine million new infections reported annually (WHO 2011). Globally, an estimated 13% of TB patients are co-infected with HIV (Naidoo et al., 2013).  Although data on the epidemiology of these infections in the general population in Kenya exists, there is limited information on Clinical biomarkers and immunophenophytic characteristics that may be putative in their management (Ballah et al., 2013). There is relatively scanty information regarding liver enzyme levels (hepatic biomarker characteristics) and immuno-phenotypic characteristics among ART na�ve HIV positive adults with or without Viral Hepatitis and Mycobacterium tuberculosis. Proper determination of the levels of clinical biomarker analytes in HIV positive ART na�ve adults with or without Hepatitis B, C and Mycobacterium tuberculosis is important in clinical management. 
To date no study has been done to characterize the level of clinical markers and immunophenotypes in the vulnerable population within Kombewa. The aims of this study were to investigate the immunological phenotypes in HIV positive adults from Kisumu West Sub-county so that overall patient care, participation of individuals in clinical trials and evaluation of adverse events can be improved.
Methods
Study Site
The study was conducted in Kombewa Sub-Country, which is part of the Kisumu County, Kenya. The study site lies at 34�45� E 0�10� S, average elevation 1,289 m above sea level, near the northeast shore of Lake Victoria and 40 km northwest of the County capital of Kisumu town. This is a predominantly a rural population and mainly of the Luo ethnic group. The main economic activity of the inhabitants is fishing on Lake Victoria, the second fresh water lake in the world as well as other fresh rivers that drain into it. The stable diet is maize meal, sorghum, millet, cassava, fish and local vegetables. Farming, mostly of food crops, is done during the two rainy seasons; the �long rainy season� between March and May and the �short rainy season� between October and December, in a tropical climate, which are usually accompanied by spikes in malaria intensity. Basically, this region is a malaria holo-endemic region as well as other parasitic diseases such as schistosomiasis. 
Study population and participant recruitment
The Kombewa Center operates a Health and Demographic Surveillance System which conducts biannual demographic and syndromic surveillance surveys in addition to collecting population data (including in- and out-migrations, births, deaths and verbal autopsies to assign cause of death) for participating households. The HDSS study area population of approximately 150,000 (a 369 square km area); serves as the catchment area for most epidemiologic and research studies conducted at the CRC in Kombewa. Every building within the HDSS area has been marked and, therefore has an address. The KHDSS is a longitudinal population registration system designed to track the evolving demographic and health status of the study populations over time. Various studies nested on this platform take advantage of the sampling frame inherent in the HDSS, whether at individual, household/compound or regional levels. This study comprised of adults aged 18 years and above who were enrolled in a larger cohort study to assess the impact of clinical practices, biological factors and socio-behavioral issues on HIV infection and disease progression in an African context. 
Ethical considerations and approval
This study was approved by the KEMRI Ethics Review Committee (ERC) and the Walter Reed Army Institute of Research (WRAIR) Institutional Review Boards. The study was conducted in accordance with Good Clinical Practice (GCP), the Declaration of Helsinki and local rules and regulations of the Kenya Government Expert Committee on Clinical Trials of the Pharmacy and Poisons Board. The researcher requested an approval for the use of the data for this sub-study from the sponsor�s protocol chair and the study Principal Investigator. Written and signed informed consent was sought from study participants before samples were drawn. 
Disclaimer 
The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70�25.
Blood collection, immunological analysis and Quality control
Blood samples were collected by trained phlebotomist, by use of veni-puncture procedures from participants who obtained a written consent. Blood samples for the Hepatitis B, C and biochemical analysis were collected aseptically into 10 ml vacutainer tubes (Becton Dickson, New Jersey USA). Blood samples for viral load and CD4 analyses were collected in EDTA anticoagulated tube. Samples for Viral load were centrifuged to obtain plasma. Serum samples for serological assays for hepatitis B and C markers were stored at "20�C until testing time. The procedure for Hepatitis B Surface antigen ELISA summarily involved addition of 100�l of the controls or specimens to the appropriate wells of the microwell plate.  Two Positive Controls, two Low Positive Controls, and three Negative Controls were assayed on each plate. The plate was then covered with a plate sealer to minimize evaporation and then incubated for 60 to 65 minutes at 37�1�C using a dry-heat static incubator. At the end of the incubation period, the plate cover was removed and the fluid aspirated from each well into a biohazard container. The plate was then washed a minimum of five times with a Wash Solution (at least 400 �l/well/wash), with a soaking duration of 30 to 60 seconds between each wash. The wash solution was aspirated after each wash and after the last wash, the inverted plate was blotted on clean, absorbent paper towels. 100�l of Working Conjugate Solution was then added to each well containing a specimen or control. The plate was then covered with a plate sealer to minimize evaporation and then incubated for 60 to 65 minutes at 37�1�C using a dry-heat static incubator. At the end of the incubation period, the plate was then washed as in the steps above. 100�l of the Working TMB Solution was then added to each well containing a specimen or control and incubated for 30 to 33 minutes at room temperature (15-30�C) in the dark. 100�l of Stopping Solution was then added to each well to terminate the reaction and then the results of the absorbance read within 30 minutes after adding the Stopping Solution, using the 450 nm filter with 615 nm to 630 nm as the reference and results evaluated for acceptability (Appendix 5 shows a full procedure for the ELISA test for Hepatitis B Surface Antigen).
ELISA procedure for determination of anti- HCV antibodies summarily involved addition of 200ul of specimen diluents to all wells. 20ul of the controls, calibrators or specimens were then added to the appropriate wells. The microwell strip holder was then covered with a plate sealer and incubated at 37C�1C for 60min�5min.  The sample solutions were then aspirated from the microwells then filled completely with wash buffer and washed for 5 times. The plate was inverted and firmly tapped on a clean paper towel to remove excess wash buffer, if necessary. 200ul of conjugate was then added to all wells and then the micro-well strip holder was covered with a new plate sealer and then incubated at 37C for 60min�5min. The plate was then washed as above. The substrate solution, that contains OPD tablets, was then added to the plate and then incubated at room temperature in the dark for 30min�1min. 50ul of 4N sulfuric acid was then added to all wells and the plate was read at a wavelength of 490nm with a reference wavelength of 620nm or 630nm within 60min following the addition of sulfuric acid.  The results were then evaluated for acceptability (Appendix 6 shows a full procedure for the ELISA test for anti-Hepatitis C antibody).
CD4 determinations were done by use of FacsCount flow cytometer analyzer. These samples were analyzed in whole blood using the standard operating procedures and as per the manufacture instructions. Blood samples for interferon Gamma test were collected in special blood collection tubes (Mitogen, TB antigen and Nil blood collection tubes). The samples were incubated at 37�C for 16-24 hours after collection and then analyzed using ELISA test as per the kit instructions and standard operating procedures. The results were then evaluated via a software for positivity or negativity for active or exposure to Mycobacterium Tuberculosis infection (Appendix 7 shows full procedure for the ELISA test for the Interferon Gamma Release Assay).
Serum Biochemistry levels were assessed the same day of blood collection by use of C111 biochemistry analyzer. The serum biochemistries were evaluated using the standard procedures and as per the instrument manufacturer instructions to determine the serum levels for the Clinical Biomarker characteristics. 

Data management and Statistical Analysis 
The collected data was entered and organized into excel spreadsheet and analyzed using Graphpad prism V5 software. Both descriptive and inferential statistics were used in the analysis. Non-parametric methods were used for data that is not normally distributed. Categorical variables were analysed by use of fisher exact test. Spearman�s rank correlations were used in bi-variate analysis for non-normally distributed data. Due to data distribution and/or sample size, Mann Whitney test were used in comparison of categorical variables. P value of < 0.05 was considered statistically significant under two tailed tests. 
Results 
The study investigated 57 (n=57) adult study participants from Kisumu West Sub-County who were ART Na�ve HIV positive having or not the Hepatitis B and Hepatitis C. Residents of this locality are mainly of the Luo ethnic group and their economic mainstay is subsistence farming and fishing (Waruiru et al., 2014). The study had data set of 57 participants from Kisumu West Sub-County (n=57) that was above the calculated minimum sample size of 45 study participants. Majority (45.6%) of the study participants were from the age group 21 to 30 years, while 21.0% of the study participants were illiterate. More than 96% of the population was from the Luo ethnic group (see Table 1)


Table 1: Socio-demographic characteristics of the study participants in the immunophenotypic arm of the study
Characteristics
Age bracket (years)Gender
Total (%)Males n (%)Females n (%)21 to 307(26.9)19(73.1)26(45.6)31 to 4012(54.5)10(45.5)22(38.6)41 to 502(33.3)4(67.7)6(10.5)51 to 60
1(33.3)
2(67.7)
3(5.3)
Marital statusSingle8(32)17(68)25(43.9)Married9(64.3)5(35.7)14(24.6)Divorced2(67.7)1(33.3)3(5.3)Widowed
 3(60)
2(40)
5(8.8)
ReligionChristian22(39.3)34(60.7)56(98.2)Muslims0(0.0)1(100.0)1(1.8)
Tribe/ethnicityLuo21(38.2)34(61.8)55(96.5)Luhya1(50.0)1(50.0)2(3.5)Other0(0.0)0(0.0)0(0.0)
EducationCertificate and above5(45.5)6(54.5)11(19.3)High school6(33.3)12(67.7)18(31.6)Primary school7(43.8)9(56.2)16(28.1)Illiterate4(33.3)8(67.7)12(21.0)Socio-geodemographic characteristics of study participants showing age, marital status, religion and tribe. The percentages are shown in the parenthesis.
Elevated Liver Enzymes Clinical Biomarker in HIV positive ART na�ve adults Co-infected with Hepatitis
Initiation of anti-retroviral therapy requires a proper understanding of the health status of HIV infected individuals, as these drugs have been known to lead to liver toxicity and pathology. The study examined the levels of liver enzymes (ALT and AST) in HIV sero positive individuals who were either infected with various types of hepatitis (Hepatitis B, Hepatitis C) as well as  and Mycobacterium tuberculosis.
Effect of Hepatitis B on Liver Enzymes
To investigate if Hepatitis B infection affected Liver enzymes in the study participants form Kisumu West, the levels of AST and ALT were compared in HIV patients. It was observed that there were no significant differences in the median levels of ALT and AST. The median ALT value in hepatitis B positive individuals was 20.35 IU/L compared to the median value in hepatitis B negative individuals which was 20.3 IU/L (p=0.6282).   For AST values, the median AST value in hepatitis B positive individuals was 30.0 IU/L compared to the median values in hepatitis B negative individuals which was 26.2 IU/L (p=0.7786).   Figure 1 shows the levels of clinical biomarkers in hepatitis B positive adults compared to hepatitis B negative adults. 
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	Fig 1: The levels of Liver Enzymes in ART Na�ve adults with and without Hepatitis B 
Effect of Hepatitis C on Liver Enzymes
The study found that there were significantly high differences in the levels of liver enzymes in the HIV positive participants who were hepatitis C positive compared with those who were negative. The median ALT value in hepatitis C positive individuals was 30.8 IU/L compared to the median values in Hepatitis C negative individuals which was 19.8 IU/L (p=0.0494).   For AST values, the median AST value in hepatitis C positive individuals was 42.4 IU/L compared to the median values in Hepatitis C negative individuals which was 29.4 IU/L (p=0.3630).   Fig 2)
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	Figure 2-Box and whisker plots showing the levels of clinical biomarker 	characteristics in adults with and without Hepatitis C from Kisumu West 
Effect of Mycobacterium infection on Liver Enzymes
To investigate whether Mycobacterium tuberculosis infection affected liver activity, liver enzymes in those who were positive and negative were compared. It was observed that there were no significant differences in the median ALT and AST values in Mycobacterium tuberculosis negative individuals compared to the Mycobacterium tuberculosis positive individuals. The median ALT value in Mycobacterium tuberculosis positive individuals was 18.1 IU/L compared to the median values in Mycobacterium tuberculosis negative individuals which was 22.2 IU/L (p=0.0956).   For AST values, the median AST value in Mycobacterium tuberculosis positive individuals was 27.3 IU/L compared to the median values in Mycobacterium tuberculosis individuals, which was 33.2 IU/L (p=0.1626). Figure 3 shows the results for the levels of clinical biomarker characteristics in IGRA negative versus IGRA positive participants (see fig 3)
	
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	Figure 3: Box and whisker plots showing the levels of clinical biomarker 	characteristics 	in adults with and without Mycobacterium tuberculosis from Kisumu 	


Table 2 below shows a summary of the clinical biomarker levels in the different groups of the participants in the study 




Table 2-Summary of the median levels of the Clinical Biomarker characteristics in HIV positive ART Na�ve individuals in the study.
�Median Levels of clinical Biomarker Characteristics (IU/L)���Hepatitis B statusHepatitis C statusIGRA status�HBV Neg HBV PosP valuesHCV NegHCV PosP valuesIGRA Neg IGRA Posp valuesALT20.320.350.628219.830.80.049422.218.10.0956AST26.2300.778629.442.40.36333.227.30.1626
This table shows the summary of median levels of clinical biomarker characteristics in HIV positive ART Na�ve individuals from Kisumu West
Immunophenotypic characterization of HIV infected ART na�ve adults with Hepatitis and Mycobacterium tuberculosis Co-infections
The study also examined the levels of CD4 and viral load immune-phenotypes in individuals with and without Hepatitis B, Hepatitis C and Mycobacterium tuberculosis co-infections in HIV positive ART na�ve adults. 
Hepatitis infection and Immunophenotypes
The study revealed that there were no significant differences in the median levels of CD4 in the HIV positive ART na�ve adults who had hepatitis B positive against those who didn�t have hepatitis B. the median CD4 value in hepatitis B positive individuals was 519.5 cells/ul compared to the median values in Hepatitis B negative individuals which was 378 cells/ul (p=0.6732).   
Similarly for hepatitis C, the study revealed that there were no significant differences in the median levels of CD4 in hepatitis C positive individuals compared to the median levels in hepatitis C negative individuals (p=0.2918). (Figure 4) 
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	Fig 4.30: Box and whisker plots for the CD4 immunophenotype levels in Hepatitis B and 	C.
Effect of Mycobacterium tuberculosis infection on CD4 Immuno-phenotypes
As well in comparing the median CD4 values in Mycobacterium tuberculosis positive individuals against Mycobacterium tuberculosis negative individuals, there were no significant differences in the median value of these. The median CD4 value in Mycobacterium tuberculosis negative individuals was 377 cells/ul compared to 447.5 cells/ul in Mycobacterium tuberculosis positive individuals (p=0.8664). Figure 4 (c) shows the box and whisker plots for the CD4 immunophenotype levels for the study participants in Kisumu West Sub-county.	
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	Fig 4 (c): Box and whisker plots for the CD4 immunophenotype levels in 	Mycobacterium tuberculosis.
The effect of hepatitis and Mycobacterium tuberculosis co-infection on HIV viral load 
The study further investigated if hepatitis B, hepatitis C and Mycobacterium tuberculosis had an impact on HIV viral loads. .It was observed that there were no significant differences in the median levels of viral load the HIV positive ART na�ve adults who had hepatitis B positive against those who didn�t have hepatitis B. The median viral load levels in hepatitis B positive individuals was 29603 copies/ml compared to the median level in Hepatitis B negative individuals which was 30430 copies/ml (p=0.9779).   
For hepatitis C, the study also revealed that there were no significant differences in the median viral load levels in hepatitis C positive individuals compared to the median levels in hepatitis C negative individuals. The median viral load levels in hepatitis C positive individuals was 30017 copies/ml compared to the median level in Hepatitis C negative individuals which was 38885 copies/ml (p=0.7240).    Similarly, in comparing the median viral load levels in Mycobacterium tuberculosis positive individuals against Mycobacterium tuberculosis negative individuals, there were no significant differences in these. The median viral load value in Mycobacterium tuberculosis negative individuals was 30123 copies/ml compared to 30883 copies/ml in Mycobacterium tuberculosis positive individuals (p=0.7729). The researcher did a log transformation of the viral load values due to its distribution as well and presented the findings. Figure 5 shows the box and whisker plots for the viral load levels in ART na�ve adults from Kisumu west.
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Figure 5-The levels of the viral load immunophenotype in HIV positive ART na�ve adults with and without Hepatitis B, Hepatitis C and IGRA from Kisumu West Sub-county (log transformed). 
Table 3 shows the summary of the median levels of the immunophenotypes in HIV positive ART Na�ve individuals in the study
The relationship between the immunophenotypes and Liver Enzyme biomarker characteristics	
To investigate the relationship between HIV viral load levels and serum levels of ALT, among other variables, spearman�s correlation analysis was used to do this. The results of the study shows that a positive correlation (spearman�s correlation coefficient, r = 0.609, p= 0.069) between HIV viral load and ALT was observed. The rationale for studying this is that for example, liver dysfunction is a real challenge in the management of HIV infected patients. To investigate whether there was any relationship between a rise in alanine amino transferase and aspartate amino transferase, the values were compared using spearman�s correlation. It is important to monitor trend of these liver functions especially before introduction of anti-retrovirals in order to evaluate effects that could result. It was observed that there was a positive correlation between where a rise in ALT was correlated with a rise in AST. Figure 6 shows the correlation of the clinical biomarkers and the immunophenotypes. .
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	Fig 6-Spearman�s correlation showing the relationship between the 				immunophenotypes and clinical biomarker analytes in HIV positive ART na�ve 			adults 
 Discussion 
For the immunophenotypic characteristics, the study looked at the immuno-phenotypic and hepato-biomarker characteristics of HIV positive adults not on anti-retroviral drugs who were infected or not with viral hepatitis and exposed to or not to Mycobacterium tuberculosis in Kisumu West Sub-County. Most studies have focused on different aspects of HIV positive group on ART since they are the majority and they are mostly accessible. As some modes of exposure/transmission to HIV virus are almost the same as in viral hepatitis relating blood infections, as well those who are HIV positive are susceptible to Mycobacterium tuberculosis. This necessitates studying the immune-phenotypic and hepato-biomarker characteristics in this group of ART Na�ve individuals. The study also assessed the relationship between the immunophenotypic and hepato-biomarker characteristics as well gender disparity in regard to exposure of Mycobacterium tuberculosis in HIV ART Na�ve adults in Kisumu West Sub-county.
Characterization of HIV positive ART na�ve adults with Hepatitis and Mycobacterium Co-infections 
In this study, regarding liver enzymes, there was a significant increase in the serum liver enzymes ALT in HIV-HCV co-infected people as compared to hepatitis C negative individuals. Although the study participants who were co-infected with the viral hepatitis and HIV were few, the study supports other studies conducted by Zhou et al., (2007) who reported that many people with chronic hepatitis have elevated liver enzymes levels. 
ALT is produced in hepatocytes, the major cell type in the liver. ALT is often inaccurately referred to as a liver function test. The level of ALT in the blood may be elevated in conditions in which hepatocytes are damaged or die. As cells are damaged, ALT leaks out into the bloodstream. All types of hepatitis (viral, alcoholic, drug-induced, etc.) cause hepatocyte damage that can lead to elevations in the serum ALT activity. The ALT level may also be elevated in cases of liver cell death resulting from other causes, such as shock or drug toxicity. The level of ALT may correlate roughly with the degree of cell death or inflammation, however, this is not always the case. An accurate estimate of inflammatory activity or the amount cell death can only be made by liver biopsy. (Jens et al., web page).
AST is an enzyme similar to ALT but less specific for liver disease as it is also produced in muscle and can be elevated in other conditions (for example, early in the course of a heart attack). AST is also inaccurately referred to as a liver function test by many physicians. In many cases of liver inflammation, the ALT and AST activities are elevated roughly in a 1:1 ratio. In some conditions, such as alcoholic hepatitis or shock liver, the elevation in the serum AST level may higher than the elevation in the serum ALT level. (Jens et al., web page). In a similar research in Northern Nigeria, Adewole et al., (2009) reported a high mean level of ALT among HIV patients co-infected. Increased levels of liver enzymes observed among HIV patients co-infected with HCV in this present research may be because of the asymptomatic nature of HCV that leads to late diagnosis. At this stage, the patient might have developed most symptoms and thus, in chronic stage of the infection since 70-80% of people infected with HCV develop chronic infections (AIDS Info Net, 2010). Also, Boyer, (2001) reported that HCV is the leading cause of chronic liver disease in the United States. Even though the normal levels of AST and ALT vary depending on the individual, the range for normal AST is reported between 10-40 U/L and ALT between 7 - 56 U/L. More specifically, the recommended AST values are 14-20 U/L for males, and 10-36 U/L for females (Jens et al., web page). 44 (77.19%) participants had AST values within the recommended range while 52 (91.23%) of the participants had ALT values within the recommended range.
Liver enzyme elevations are a frequent finding in HIV-infected patients as a consequence of several risk factors, however the analysis of these events is limited as precise etiology is rarely clearly defined. Abnormalities in liver function tests could be produced exclusively by direct inflammation in hepatocytes, caused by the HIV virus. Although the mechanisms by which HIV causes hepatic damage are still unknown, studies have shown that it may be as result of apoptosis (induced by caspases 2, 7 and 8) and mitochondrial dysfunction with decreasing mitochondrial DNA in several tissues. Another injury mechanism is permeability alteration in mitochondrial membrane by HIV proteins which stimulate an inflammatory response (Pol et al., 2004). Alanine aminotransferase (ALT) is a hepatic enzyme that may be used as a marker of hepatocellular injury. (Zechini et al., 2008). In this study, the results showed insignificant differences in the levels of clinical biomarker analytes in those who were IGRA negative versus those who IGRA positive with an implication that the status of Mycobacterium tuberculosis may not per se affect the hepato biomarker analytes.  However, the impact of viral hepatitis on the immune system and liver enzymes needs further studies in both on HAART and HAART naive HIV positive patients.
The results for this study showed an overall median CD4 count of 433 cells/mm3 and median of 407cells/mm3 for the 57 adult participants studied. CD4 and Viral load levels were studied in those who were Hepatitis B and C positive adults against those who were negative for Hepatitis B and C. In this study there were no significant differences in the median CD4 and HIV viral load levels in the HIV positive ART Na�ve adults from Kisumu West who are either co-infected or not with Hepatitis disease. This was inconsistent with a study done by Mohammad, that reported that within a NVS cohort, individuals without chronic HCV had a statistically significant elevation in mean CD4 count compared to those NVS with chronic HCV. The correlation of CD4 count with HCV status in the study was statistically significant for earliest recorded CD4 count and there was a trend toward significance with the most recent CD4 count as well (Mohammad et al., 2013). In this study there was a similarly statistically significant elevation in the mean CD4% and mean CD4/CD8 ratio in those without chronic HCV compared to the chronic HCV group. (Mohammad et al., 2013). 
However, in the current study from Kisumu West, the researcher acknowledges a limitation of low numbers of Hepatitis positive cases partly due to low prevalence of Hepatitis in the area thus limiting adequate conclusions to be drawn from this. 
In this study of 22 males and 35 females, there was also difference on the mean CD4 values in relation to gender. The median CD4 value in the females was slightly higher than males (437 cells/mm3 in females compared to 427 cells/mm3 in males). Similar findings as for this were observed in studies that have been conducted in. Kenya (Erick et al., 2013), Uganda (Tugume et al., 2008) and Nigeria (Akinsegun et al., 2012). The higher CD4 values in females compared to males could probably due to biological factors. It has been speculated that gender and age-related variations within the immune system parameters may contribute to the pathogenesis of several gender and age-related diseases such as autoimmune disorders in female patients (Ngowi et al., 2009, Kibaya et al., 2008). Further, it has been shown that there are gender differences in the generation of CD8 cells during HIV-1 infection, due to increased immune activation compared to men (Meier et al., 2009). Since the number of subjects with Hepatitis B and C were few, comparison of the positive Hepatitis B and C individuals in terms of gender could not be done to effectively yield statistical significant results. 
Globally, the recommended CD4 range is 400 � 1600 cells/mm3. Some people would be initiated to medication when their CD4 counts reaches 350 cells/mm3.  On the other hand, according to AIDS.gov, one of the qualifications for an AIDS diagnosis is CD4 count less than 200 cells/mm3.  Almost half of the patients (49.1%) had CD4 count less than 400 cells/mm3. Those who required to be initiated on medication were 42.10%, while 28.07% had CD4 counts less than 200 cells/mm3.
Median viral load levels were statistically significantly higher in males as compared to females. In this study, again it was not possible to characterize the immune and biomarker characteristics in the viral hepatitis positive individuals. This is because even in the general population prevalence of the viral hepatitis is low and so getting higher numbers to be studied sometimes is hard. This study only had four individuals who were positive for hepatitis C and four who were positive for Hepatitis B, one of these who had Hepatitis B and C co-infection. Characterizing these on their own would not provide statistical significant results, and thus causality could not be inferred.
The relationship between the phenotypic and clinical biomarkers in HIV positive ART na�ve adults with or without Hepatitis B, C and Mycobacterium tuberculosis
To investigate the relationship between HIV viral load levels and serum levels of ALT, among other variables, spearman�s correlation analysis was used to do this. Rationale for this is that for example, liver dysfunction is a real challenge in the management of HIV infected patients, thus evaluation of the liver enzymes and CD4 levels in relation to viral load levels is necessary. Especially a proper understanding on how this is depicted in HIV positive ART Na�ve patients is required. This is due to the fact that the use of ART has completely modified the pattern of hepatic events in HIV infection resulting in significant decrease in morbidity and mortality among HIV infected patients (cooper et al., 2007. The results of the study shows that a positive correlation between HIV viral load and ALT was observed. This is consistent with findings for another study (Ballah et al., 2013). As seen above, ALT could be used as markers of hepatocellular injury. Liver enzyme elevations are frequent in human immune deficiency virus (HIV)-infected patients which may be caused by the HIV virus in those without other risk factors for liver damage.  The finding of positive correlation between HIV viral load and ALT levels in HIV infected naive patients suggests some relation between ALT level and HIV-1 viral load in HIV patients. It is thus recommended evaluating patients with high ALT for early anti-retroviral therapy (ART) in those without risk factor for liver damage regardless of the CD4+ cell count, especially where facility for estimating viral load is not available. ALT should be monitored given the observed positive correlation between the HIV viral load and serum levels of ALT enzyme in HIV infected naive patients. Though not statistically significant, the results for this study would show that patients with elevated ALT level may also have high HIV-1 viral load. Thus liver function tests should be monitored closely in these patients. Thus such patients with high ALT should be evaluated for early start of antiretroviral treatment in those without risk factor for liver damage regardless of the CD4+ cell count, especially where facility for estimating viral load is not available.
Limitations
Further, similar study of clinical characterization and immunophenotypes that would utilize higher number of Hepatitis B and C positive individuals and with higher number of individuals in general could be warranted for proper characterization of the immune-phenotypic and hepato-biomarker characteristics. The sample size could have been small for this study due to the criteria of limited data that was only accessible in ART Na�ve individuals.
Conclusion
There were significant differences in regard to the median levels of the ALT in hepatitis C infected group compared to hepatitis C non infected participants. For the other immuno-phenotypes (CD4, Viral load) and clinical biomarker analytes (ALT and AST enzyme levels) there were no significant differences in both the HIV positive ART Naive adults with or without Hepatitis B, C and Mycobacterium tuberculosis infection. However, before arriving at a conclusion, a study with more respondent who tested positive for Hepatitis B or C would give a correct picture. Screening for viral hepatitis should also be performed alongside HIV testing in order to detect it early for effective therapy and good prognosis. 
Competing interests
The authors declare no financial or personal relationships that would influence them in developing the article.
Author contributions
Author JOc conceived the project, designed the study, conducted the laboratory aspects of the study, performed data analysis and drafted the manuscript. Author JC and DHM helped in study design, professional guidance, data analysis and drafting the manuscript. All authors read and approved the final manuscript.
Acknowledgements 
The authors would like to thank the study participants who participated in the study thus allowing data generation. We thank the sponsor protocol chair for granting permission to use the data.   
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Supporting information
S1 File-Approval letter for use of data








 PAGE   \* MERGEFORMAT 26



Fig 1 (a)

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Fig 2 (a)

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Fig 3 (a)

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Fig 4 (a)

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Fig 5 (b)

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Fig 6 (b)

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