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��ࡱ�>��	UX����RST������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������	����bjbj���	�����������������"""""4����E"E"E"h�"�1%�E"�W��&��,�,�,�,�.�.�._VaVaVaVaVaVaV$dZ�]`�V�"�.N.@�.�.�.�V""�,�,�rWLILILI�.."�,"�,_VLI�._VLILIr�QT}R�,���������W�E"�@b=RKV�W0�WKR2f]I.f]}R}R�f]"3S�.�.LI�.�.�.�.�.�V�VLI�.�.�.�W�.�.�.�.��������������������������������������������������������������������f]�.�.�.�.�.�.�.�.�.�	� :	Original Research Article

Biochemical and Haematological Changes In HIV Subjects Receiving Winniecure Antiretroviral Drug In Nigeria

Bartholomew Okecuhukwu Ibeh Ph.D1, Omodamiro Olushola D M.Sc 2,  Urenna Ibeh  OD3
1Department of Medical Biotechnology, National Biotechnology Development Agency.
2Department of Biochemistry, College of Natural and Applied Sciences, Michael Okpara University of Agriculture P.M.B.7267 Umudike Abia State Nigeria.
3HIV/AIDS Unit, Emerging Health and Environment Initiative, Abuja.
Correspondence: Dr. Bartholomew. O. Ibeh1.(Email:barthokeyibeh@yahoo.com), Ph:+2348068767253 
N0.of pages: 24, Abract:250, Main Text:4,013





















ABSTRACT
BACKGROUND: Hematological and biochemical abnormalities are among the most common clinicopathological manifestations of HIV patients on ART therefore, assessment and development of indigenous ART for these abnormalities are a necessity. Our objective is to assess haematological and biochemical abnormalities in HIV patients administered with Winiecure ART,  an ethnomedicinal herb in a Nigerian setting  .
METHODS:  Fifty(50) confirmed HIV positive, ART na�ve patients aged 36�10  were accessed for haematological and biochemical responses  between 1st week (initial) and  12th week treatment period. Haematological responses  were assessed using coulter Ac-T differential analyser taken on repeated visits (1st, 2nd 3rd) and biochemical indicators (bilirubin, creatine, urea, amylase, ALT, ALP,  AST, albumin)  assayed spectrophotometrically. 
 RESULTS: The biochemical parameters ALP (P<0.05), ALT(P<0.0001), AST(P<0.001), and amylase(P>0.05) were slightly  increased at the  12th week, no change occurred in plasma creatinine and urea concentrations while albumin levels decreased insignificantly(P<0.002).
Our haematological results showed  consistent reductions in  ESR, eosinophil, absolute and differential lymphocytes and granulocytes and total WBC among the patients from 1st to 12th week  assessment period, while  haemoglobin, platelet and PCV increased(P<0.05) significantly. Thrombocytopenia (10.30%) and anaemia(76%) were reduced at the 12th week to 2% and 31% respectively while neutropenia(4.2 to 8%), leucopenia(26.8 to 30%) and lymphopenia(1to10%) increased. No cases of neutrophilia, lymphocytosis, eosinophilia and leukocytosis were observed. CONCLUSIONS:  The drug has a reduced haematological abnormalities while  normal kidney function was unaffected though there were signs of  possible  abnormal hepatic enzyme elevation.
Key words: Haematological, HIV, Winne-cure, ART, biochemical, Nigeria

Background

The human immunodeficiency virus (HIV) is one of the most important emerging infections of this century. It is probably one of the diseases with multiple impacts on persons, families, communities and the entire society. HIV also threatens human existence especially in sub-Saharan African countries where the infection is endemic. The epidemic in Nigeria, a sub-Saharan African country for instance, increased tremendously with a prevalence rate of 3.8% in 1993, to 5.8% in 2001[1]. Over the years significant progress has been made towards understanding the viral pathogenic proteins that causes the acquired immunodeficiency syndrome (AIDS) which was first isolated in 1983 from patient with AIDS related complex (ARC) and AIDS [2]. The wide spread and use of HIV antibody  screening techniques [3,4,5,6] over the decades has provided a more complex understanding of the epidemiology and clinical features of the epidemic [7,8].

It is however observed that the emergence of drug development programmes by government agencies and industries has led to many potent anti-HIV drugs [9]. Medical practitioners at present have learnt to use various combinations of these drugs to keep HIV at bay. Currently, combination of anti-HIV drugs are now highly recommended for used in the management of HIV disease; highly active anti-retroviral therapy (HAART)).  The advent of HAART, a cocktail of nucleoside and non-nucleoside analogues with potential to inhibit HIV reverse transcriptases and proteases  [10] in the scientific world has over the years led to  a series of documented reductions in the risk of  acquiring the resultant illness AIDS from the virus [I1], thus improving the  quality of life of people living with HIV/AIDS [12]. 

It is undoubtedly observed that the HIV drugs are without some adverse biological  effect thus having safety limitations. Certain documented side effects of these drugs ranges from diarrhea, nausea, abnormal distribution of body fats, anemia or neutropenia to cytopenia [13]. It is however, necessary to consider the safety and efficacy of any novel antiretroviral drug. This is also in accordance with centre for disease control and prevention (CDC) stand point which, advocated that before the use of any anti-retroviral therapy, the safety and efficiency of the drug should be considered. Thus the rationale for evaluating   possible  hematological and biochemical abnormalities in Winnie-cure antiretroviral drug.

The hematological consequences of HIV infection are dominated by peripheral blood cytopenias. These have become more common with the advent of anti-retroviral therapy and related treatments for HIV-associated infections and malignancies [14, 15]. Anaemia may occur in approximately 60-70 percent of AIDS patients; neutropenia 50 percent, and the thrombocytopenia in 40 percent of patients undergoing ART [16]. In general, the incidence and severity of low cell counts increase with advancing HIV disease. An exception is thrombocytopenia, which may be an early manifestation of HIV infection, occurring in 3 to 12 percent of asymptomatic patient [17, 18]. Myelo suppressive therapies or marno-infiltrating opportunistic diseases may further contribute to the development of cytopenia similar to the anaemia of HIV infection. The major clinical significance of the HIV�induced neutropenia is that it often produces therapy with Zidovudine (ZDV)  and some drugs used for the treatment of opportunistic diseases. Though macrocytosis occurs in more than 90% of patients receiving ZDV this does not correlate with the development of anaemia [19].

In view of the various questions raised by end users of anti-retroviral therapy such as the safety and efficacy of the drugs, a careful study of  some possible abnormalities  that may be associated with Winnie-cure an antiretroviral drug used  in Nigeria was investigated.

Materials and Methods
Study population and Ethics
Fifty (50) confirmed HIV positive patients who were previously ART na�ve  volunteered to take Winnie-cure anti-retroviral therapy at a prescribed dose of 5ml (1 tea spoonful) containing 250mg Winnie-cure extract taken 3 times daily for 5 days and then 2 times daily for the period of this study (12 weeks). The study had also 100 ART na�ve subjects attending clinic for the first time as the study reference subjects or control group. All consenting subjects were recruited consecutively for the work. Blood samples were taken  in accordance with the Nigerian National Ethics and Operational Guidelines for Research on Human subjects (NNEOGRHS).Part of the blood was collected into EDTA container to give blood-anti-coagulant concentration of 2mg/ml of blood with sterile needle and syringe (aseptic precautions were taken). The samples were collected at intervals of 6 weeks for the period of this study.

The inclusion criteria for enrolment of the study subjects were specified as follows; subjects must either be males or females aged 17 years and above, must have confirmed laboratory evidence of HIV infection, must accept to abide by the prescribed dosage of drug, have history of no previous antiretroviral therapy (ART na�ve) and have CD4 cell counts between 100 and 350 cells/�l. Exclusion criteria include those with recent blood transfusion.

A structured questionnaire to obtain demographic data   and clinical history was
completed for each patient with assistance given on request. Prior to initiation of treatment, the HIV serostatus of  the patients were reconfirmed. Plasma samples from each of the patients were tested using double rapid tests  kits  (Cappillus and Unigold: Trinity BioTech, Ireland), a rapid (Cappillus) and an  ELISA (Genie II HIV-1 & 2 kit). The tests used have different antigenic compositions.
 
Haematological analysis
The following parameters (Hb, PVC, Total WBC, Differential WBC and platelet count and ESR) were evaluated each time the samples were collected. Samples for ESR were analyzed using Westergren method [20]. Blood (2ml) was diluted in 0.5ml Trisodium Citrate solution  using the Westergren pipette  that was filled to a zero mark and mounted on a stand with the time adjusted to exactly 1 hour for the red cell to sediment. The column of the sedimented red cells was read at exactly 1 hour. Results were reported in mm/hour. Other haematological parameters (Hb,PCV), total WBC, differential WBC and platelet counts were analyzed with a haematology analyzer coulter AC.T diff. analyzer. A suspension of blood cells was passed through a small orifice simultaneously with an electric current which introduces an impedence change in the orifice determined by the size of the cell. The system counts the individual cells and provides cell size distribution. The number of cells counted per sample is approximately 100 times greater than the usual microscope count which reduces the statistical error by a factor of approximately 10 times



Abnormalities
Certain abnormalities were determined in the subjects undergoing antiretroviral treatment. Anemia was defined as haemoglobin <13 g/dl (Men) and <12 g/dl (women) while Leucopenia as total WBC count less than 4000 cells/�l. Total platelet count <150�103/�l�  was considered as Thrombocytopenia. However, Neutropenia was judged as absolute neutrophils/granulocytes count <1000 cells/�l and Lymphocytopenia considered at absolute lymphocyte count of <800 cells/�l while Lymphocytosis an increase in the number or proportion of HYPERLINK "http://en.wikipedia.org/wiki/Lymphocyte" \o "Lymphocyte"lymphocytes were considered when the absolute lymphocyte count was greater than 4000 cells/�l. HYPERLINK "http://en.wikipedia.org/wiki/Neutrophilia" \o "Neutrophilia"Neutrophilia indicates an elevated absolute granulocyte count above 7000 cells/�l while Leukocytosis is defined as a total WBC more than two standard deviations above the mean, i.e a value of greater than 11,000 cells/�l in adults. Also eosinophilia was evaluated at eosinophil count above 450 cells/ �l. Absolute CD4 cell count determination was done by flowcytometric analysis.


Biochemical Analysis
Biochemical indicators were assessed at the first and last visits only. Commercially available kits were used to assay for the following serum enzymes: ALT, AST and amylase. Urine creatine (RANDOX).  and urea (BioVision) were  also determined using test kits. Bilirubin was measured directly by use of bilirubin oxidase [21] while albumin concentration was obtained by BCG (QuantichromTm BCG Albumin Assay kit [DIAg-250]) at  620nm. This method is used daily in clinical laboratory measurement in Nigeria and the results   are comparable to those of electrophoresis. Manufacturer�s procedure for each test  were followed accordingly. Absorbance was measured with the spectrophotometer and results calculated accordingly. Normal ranges were reported based on the test kits.

  Statistical Analysis
 All  data calculations/analysis were done using the SPSS version 17 statistical software package. The data were presented as Means � Standard deviation (SD), and statistical analysis carried out using the Student�s t-test and ANOVA. Differences were considered to be of statistical significance at an error probability of less than 0.05 (P<0.05).



Results and Observation

Of the 150 subjects recruited for the study, 50 confirmed HIV positive individuals on  the antiretroviral drug with an average age of 36�10 was used. There was no significant difference between the ages of the HIV positive group and that of the reference group (40�52). Observed in the HIV positive subjects on ART were an insignificantly higher number of   HIV positive females (63%) and a significant increase of married couples (66.7%). However, fifty three percent (53%) of the ART subjects had CD4+ cells within the range of 200-350 cells/�l while 46.9% were between 50-200 cells/�l (Table 1).

Table 2 summaries the mean�SD of some haematological parameters in HIV patients on the anti-HIV drug, Winnie-cure herbal preparation before treatment (1st assessment) and subsequent appointments at 6 weeks interval otherwise referred to as visits in this study.
A comparative statistical analysis was made between the 1st assessment visit and subsequent visits  (treatment period) using student�s T-test at a 5% level of significance as shown in table 2. It was observed that there were a   consistent reduction of ESR, eosinophil, total white blood cell (WBC), absolute and differential lymphocyte, absolute & differential granulocyte  in the patients from the first to the  3rd assessment visits  with a lower value when compared with the obtained reference values. An exception was noted on ESR and eosinophil which significantly increased on comparison with the reference values. Conversely, there was statistically significant (P<0.05) increase between the 1st and  3rd  visits in haemoglobin, platelet and packed cell volume. All values obtained here were lower than the reference value.

Our biochemical data shows that the concentrations of these biochemical parameters; ALP (HYPERLINK "http://en.wikipedia.org/wiki/EC_number" \o "EC number"EC HYPERLINK "http://enzyme.expasy.org/EC/3.1.3.1"3.1.3.1;P<0.05), ALT (HYPERLINK "http://en.wikipedia.org/wiki/EC_number" \o "EC number"EC HYPERLINK "http://enzyme.expasy.org/EC/2.6.1.2"2.6.1.2; P<0.0001), AST (HYPERLINK "http://en.wikipedia.org/wiki/EC_number" \o "EC number"EC HYPERLINK "http://enzyme.expasy.org/EC/2.6.1.1"2.6.1.1;P<0.001), amylase (HYPERLINK "http://en.wikipedia.org/wiki/EC_number" \o "EC number"EC 3.2.1;P>0.05) and direct bilirubin (table 3) were statistically increased (upper range) at the third visit though still within the acceptable normal range. We observed no remarkable change in plasma creatinine and urea concentrations on comparism with the reference value however, the data obtained here remained within the acceptable normal range. Our albumin levels decreased insignificantly (P>0.002) when compared with the reference value. Reference values are values obtained from the HIV negative healthy control group. 

Various haematological abnormalities may be associated with administration of anti-HIV drugs, however our study reveals a higher percentage of Thrombocytopenia (10.30%), and Anaemia (76%) in those subjects on ART at the first visit and lower values (2% and 31% respectively) at  3rd assessment  visit /12th week (Fig. 1). Conversely, neutropenia (4.2%), leucopenia (26.8) and lymphopenia (1%) were low at the first visit  but had a respective  higher values of 8%, 30% and 10% (fig.1) at the 12th week/3rd  visit. No cases of neutrophilia, lymphocytosis , eosinophilia and leukocytosis was observed or  associated with the drug administration. 

Discussion
The study seeks to evaluate some haematological and biochemical indicators of abnormalities in HIV infected patients administered with Winnie-cure a chemotherapeutic agent used in the  treatment  of HIV/AIDS in some parts of  Nigeria. 

HIV infection has been reported to cause diverse degree of immunopathogenesis in man [22] and this has enormous haematologic and biochemical consequences. The haematologic consequences of HIV infection are dominated by peripheral blood cytopenias which, have become more common with the advent of antiretroviral therapy for HIV infection and other treatments for HIV related infections and malignancies. Winnie-cure has been shown to inhibit viral replication and could restore human immunity necessitating its use in the region as an anti-HIV medication. As an anti-retroviral therapy, it may trigger haematologic consequence as have been reported for some other anti-retroviral drugs [23]. Winniecure is a  herbal composition of  Fiscus exaspirata, Fiscus sur, Sida acuta/Sida corymbosa  with a strong hydrophilic nature, moisture content of 0.1%, density 1.936�0.2 and having the following phytochemicals; tannins,saponins and carbohydrates [24]. Previous studies have indicated its safety in humans and animals [25].

The baseline characteristics shows that there were no significant  age difference between the reference or control group with that of the test subjects  used in the study. This thus reduces the variance that may emanate from the pharmacological effect of age on chemotherapeutic agent. The number of HIV infected subjects are generally reported to be higher in  the female gender than in the male counterpart [26]. Our work reported higher number of females for those infected subjects recruited for the ART administration when compared with the reference (HIV seronegative subjects) group that has higher number of males. However, HIV/AIDS has so far, severely affected more women  mostly in sub-Saharan Africa. Here women and girls account for almost 57% of adults living with HIV/AIDS. Of  all the HIV infected youths in the world (5.4 million), over three million are female [27]. It is known that the virus is more easily passed on to young women owing to their immature vaginal tracts and easily torn tissues, also gender inequalities in many tribes especially in African prevent young women from negotiating safer sexual practices which may include use of contraceptives such as condom  may be responsible for the increase in the number of infected females rather than the sheer believe on  socio-cultural aspects of feminine disposition to attending  HIV clinic.
 
It is generally believed that single unmarried young individuals are more likely to acquire the HIV than married partners [28] but our study indicates a higher incidence of  HIV infection in married partners to  singles as also reported by some other researchers [29,30]. In as much as unmarried single individuals are likely to engage in multiple sex partners we suggest that this group may likely have higher propensity to use protection mechanism against HIV infection than in married sexual partners that may have limited sexual partners. However, Haque and Soonthorndhada [31],   reported increased STI risk perception and reason for use of condom in unmarried young individuals than their married counterpart.

HIV patients assessing ART have been reported to have an increased CD4 cell count, normally  initiating treatment from 100-260 cells/�l  baseline [32,33] conversely, starting ART at CD4 cell counts below 50 cells/�l increased the relative risk of death by approximately 60% compared to starting at 150-249 cells/ul CD4 [34]. Majority (88.9%) of our study subjects have CD4 count between 200-350 cell/ul (category II based on CDC classification).This inadvertently, indicates that the immune status of the subjects fall within the recommended CD4 count for ART initiation (Table 1).

In the course of ART, clinically significant hematologic abnormalities may be common in persons with HIV infection. Impaired haematopoiesis, immune-mediated cytopenias, and altered coagulation mechanisms have all been described in HIV-infected individuals. Here we evaluated the haematological and biochemical efficacy of Winnie-cure an antiretroviral drug found in Nigeria. In individuals abnormalities may occur as a result of HIV infection itself,  or as  a sequel of HIV-related opportunistic infections or malignancies, or as a consequence of therapies used for HIV infection and associated conditions.  In our findings a significant variation was observed in some of the haematological parameters of the HIV positive subjects on treatment with Winnie-cure compared with values obtained at baseline (Table 2). Similarly, reference values obtained from control subjects were compared as well. Consequently, the following haematological abnormalities (Figure 1) were observed; thrombocytopenia, anaemia, neutropenia, leucopenia  and lymphopenia.

Generally, the overall White Blood Cell (WBC) count is important to monitor as a significant elevation in WBC may indicate infection, lack of response to treatment, or an abnormality. � The total WBC count shows a consistent significant reduction at all the visits although the reduction at the 3rd visit was insignificant (P>0.05). The reductions observed in absolute lymphocyte and total WBC may indicate suppressive activity of  the antiretroviral drug on the virus with the resultant increase in the percentage of leucopenia (mild leucopenia; 26.8% to 30%) and lymphocytopenia (moderate lymphocytopenia; 1% to 10%) from  the 1st to 3rd  visit. Leucopenia (a decrease in the number of  HYPERLINK "http://en.wikipedia.org/wiki/White_blood_cell" \o "White blood cell" white blood cells�) and Lymphopenia (decrease in lymphocyts) a hallmark of HIV infection  is thought to be mediated by infection of the virus and subsequent killing of CD4+ T cells,  can  also be caused by certain medications including ART [35-37] and infection  [38,39]. The significant reduction found in the lymphocyte count at the 2nd visit follows the pattern already reported by  Stein  and his colleagues [40],  thus the transient lymphopenia is common and one-third of the patients may have a typical lymphocytes in the peripheral blood smear. Moreso, the gradual rise in the lymphocyte at the 3rd   visit may contribute to the fall in granulocyte count observed. The mechanism responsible for leucopenia and �lymphopenia�might be an accumulation of Winnie-cure metabolites. We would not deemphasize the possibility of a pseudoleucopenia since our result showed a mild leucopenia and an increase in Hb as the treatment progresses. Though further investigation is needed to ascertain this since our study is devoid of CD4 and viral load assessments hence its limitation.
Several research works have shown that administration of HAART especially Zidovudine (AZT) therapy causes anaemia with a significant reduction in Hb in HIV patients [41] even at 6 weeks of administration [42]. The PCV as well  increased at the 2nd and 3rd  visits, this result tend to have similar pattern with the values obtained from Hb which shows an effective therapeutic effect of the drug at the 12th  week since a decreased PCV indicates anaemia. Feiterna-Sperling and his colleagues [43] reported anaemia and neutropenia as some common side effects in Zidovudine (ZDV) on haematological parameters. However, at the 12th  week, we observed  a significant increase in the value of the Hb at P<0.05. This rise at the 3rd   visit may indicate a peak active period of the Winnie-cure treatment within the observation time frame with the resultant reduction of anaemia form 76% to 31%  seen in the treatment group. Recent research works have shown that mean haemoglobin increases significantly in patients who receives ART thus reversing HIV associated anaemia [44].The consistent reduction of ESR (49.56+51 to 37.13+35.59) and eosinophil (197.01+1.42 to 132 + 1.42) from 1st week to 12th week attest to the reduced anaemic condition found in the HIV subjects on Winnie-cure therapy. Since raised ESR  and esonophil can indicate acute anaemic condition [45]. ESR and eosinoophil increased significantly when compared with their controls, this often may rise significantly in individuals due to infection or medication and may reflect the anaemic condition seen in the subjects. The high eosinophil may suggest a  secondary or reactive rather than primary increase in blood eosinophils which, may be  associated with the drug administration. Ndakotsu et al. [46] have also reported high  ESR in HIV patients of Nigerian origin.� 
 We observed a significant reduction in both differential and absolute granulocyte count between the 1st   and 3rd    visits Using the National Cancer Institute-Common Toxicity Criteria (NCI-CTC) Version 2.0 we found the subjects undergoing treatment to be at NCI risk category 1, implying a mild neutropenia. It is of note that abnormal granulopoiesis and anti-granulocyte antibodies have been noted and described in 30-70% of HIV infected patients [47] this is believed to contribute to the observed increase in neutropenia (4.2 to 8%). Also, the major clinical significance of the HIV induced neutropenia is that it often precludes therapy with ART and some drugs used for the treatment of opportunistic diseases [48]. Neutropenia or Granulocytopenia is a problem commonly encountered in patients with HIV infection. The low granulocyte counts seen in the 3rd  visit  may reflect the  action of Winnie-cure on  HIV infection or associated conditions, however the incidence of neutropenia at the first visit (untreated patients) is consistent with other reports  [49] which have also shown a high incidence of granulocytopenia, particularly in patients with more profound immunodeficiency. 

An escalated and uncontrolled platelet count may indicate disease progression and is cause for concern in which the condition is sometimes associated with abnormal bleeding. Thrombocytopenia has been defined as any disorder in which there is an abnormally low amount of platelets and may result due to immune system malfunction
[50]. Our result here showed a consistent significant increase in platelet count from the 1st to the 3rd visit this indicates a corresponding reduction in thrombocytopenia. It may be possible that the activity of the anti-retroviral therapy led to the reduction pattern seen in the platelet count count.  
 
Numerous studies have found that liver problems are the most common non-AIDS cause of death among people with HIV, and that liver-related death is one of the major causes of mortality accounting for 14-18% of all deaths in the effective antiretroviral therapy (ART) era [51-53]. Our findings shows an elevated liver transaminase enzymes (AST- 34.1U/L, ALT-38.47U/L and ALP-97.31U/L) from  baseline (1st visit) to the 12 th week (3rd  visit).Though the transaminases increased  but when  compared with  our reference values they  are  still within the normal laboratory reference range, also as reported by Abdullahi et al. [24]. Therefore, we suggest that long term administration of the drug should be guarded  to avoid drug induced injury as Sulkowski [54]  reported that in all the PI regimens studied, the greatest risk of developing drug induced liver injury  has been observed among patients on long term therapy  receiving full-dose  of ritonavir with elevated hepatic enzymes. Furthermore, birirubin a substance  created by the activity of�  HYPERLINK "http://en.wikipedia.org/wiki/Biliverdin_reductase" \o "Biliverdin reductase" biliverdin reductase�on� HYPERLINK "http://en.wikipedia.org/wiki/Biliverdin" \o "Biliverdin" biliverdin, a green tetrapyrrolic bile pigment that is also a product of haeme catabolism is also cleared by the liver. The increased direct  bilirubin  value obtained shows that the  liver is conjugating bilirubin normally similarly,  HYPERLINK "http://en.wikipedia.org/wiki/Albumin" \o "Albumin" Albumin� a protein made specifically by the liver, is slightly decreased (3.02�2.8U/I) at the 12th week thus indicating a non-significant change.

The pancreatic enzyme amylase (71.21 � 41.26U/I)  and direct bilirubin (0.21 �0.11 mg/dl) were mildly elevated at the 12th week  when compared with the reference values.  However, increased amounts of amylase may be released into the blood when the pancreas is injured, inflamed, or blocked. This may signify possibility of pancreatic dysfunction in this case hyperamylasaemia, conversely, creatinine and urea an excellent indicator of kidney function did not  show any significant change in their plasma  levels between 1st and 3rd visits and also with the reference value thus may indicate a normal functional and intact kidney.
Conclusions
In summary the efficacy of the drug has a reduced haematological abnormalities and  normal kidney function, however, there are early signs of increasing tendency for abnormal hepatic enzyme concentrations. Also the drug  seems to may have  worked optimally at about  12 weeks of treatment with the period of test thereby indicating appreciative positive changes in the haematological  and biochemical indices. These results show that the therapeutic efficacy measured on the haematological and biochemical responses are accompanied by a reduction of these indicators of  abnormalities. They also suggest possible  tendency of the administered ART to drive a shift from the physiological normal ranges of  biochemical (mostly liver damage) indices of healthy status  to an elevated  range. 

Admittedly, the study did not consider the intriguing possibility  of  symptomatic improvement, palliative benefit, and CD4 count improvement  of the administered drug nor its comparison with any other known antiretroviral drugs thus its limitation. 

Disclaimer: We the authors wish to state that normal reference laboratory values may differ from one population to another.


Funding Received: None
Ethical Approval: Ethical clearance obtained from Emerging Health and environment Initiative reference N0;Ibeh-2010-25
Conflict of Interest: There is no potential conflict of interest 

Authors� Contribution to the Work
Dr Bartholomew O. Ibeh: Designed the work,  interpreted the results. Laboratory analysis and drafting of the original manuscripts and final approval of the version. Omodamiro Olushola: Involved in the project design,  result interpretation and analysis laboratory analysis. Urenna Ibeh: Critical revision of draft article for suitability and intellectual content, statistical analysis and recruitment of the study population


Acknowledgement: Appreciation goes to  Emerging Health and Environment Imitative (EMHEI) a non-governmental organization based in Nigeria for  their  assistance.  

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Table 1: The baseline Characteristics of the study population indicating  CD4 count grouping, ART duration, marital status, age and gender.

Characteristics               ART  
   (n=81)    Reference/
Control 
(n=100)    P-Value  Age (years; mean � SD)  

    
Gender
(Female/Male; n {%})
   
Marital Status
(Married/Single; n{%})

Length of HAART
(Weeks)  36�10



51{63}/30{37}


54{66.7)/30{37)


 12
40�52



49{49}/51{51}


48{49}/52{52}


NA

0.084


0.092


0.039 aCD4 Count
  subjects (%),n
  1.50-200 cells/ �l, 
  11.200-350 cells/ �l
  



(11.1), 9
(88.9), 72


1100�82 cells/ �l


Comparison of data were done  at P<0.05, data on gender, marital status and CD4  (measured as cells/ �l) were presented as absolute numbers (n) and percentages (%) while age +CIJQS�����������������������*	+	�	�	



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Table 2: Haematological parameters of  HIV patients on the anti-HIV drug, Winnie-cure   from the 1st assessment visit (before treatment)  to the last visit ( 12th week). 




Parameters 1st visit
(baseline)2nd visit3rd visit    Reference         
           ValueESR (mm/1hr)41.46+31.01 a                              40.33+35.36 a32.12+15.49b19.6�40Total WBC(x103 cells/�L )6.23+1.96 a                              5.70+0.06 b5.65+0.73 b8.40�1.5Absolute Lymphocytes
(x103 cells/�L )
3.04+1.44 a                              2.48+0.68b2.30+0.51b3.88�2.5Absolute Granulocytes
(x103 cells/�L )
3.03+1.46 a                              2.90+0.64 a2.70+0.72 b4.21+ 1.5Differential Granulocytes (%)

Haemoglobin (g/dl)               

Packed cell Volume (%) 

Platelet (x103/�l) 


Differential Lymphocytes (%) 


Eosinophil (�l)                           51.54+11.58 a                              
10.37+1.15 a                              
34.66+5.56a                              
211+68.11a

61.26+16.58a

197.01+1.42a49.50+9.70b
10.64+1.55 a
32.63+5.14a
305.9+56.25 b

45.50+11.90b


177 +1.44b48+ 3.76b
11.7+ 1.01 b                              
36.75+1.53b                              
310+ 49.54b
49.00+5.66b132 +1.42c
57+ 5.48
14�0.9

51+3.23

320�42

59 +2.53

242+1.42

Comparison    was made between the 1st visit and subsequent visits (treatment period) using student�s T-test at a 5% level of significance. Reference values are values obtained from the HIV negative healthy control group. Data with different superscript indicates statistical difference at P<0.05




Table 3: Significant levels of  some Biochemical parameters   between  baseline and 3rd visit in the subjects undergoing ART. 

                       Parameters1st Visit P-value  3rd  VisitP-valueReference valueAST (U/l)   
    31.66�25.20 a
P<0.00134.31 �52.21 bP<0.00125�80cALT/ (U/l)
  
ALP(U/l)21.38 � 14.26 a
  
88.4 �21.31a                     P<0.0001

P<0.0538.47 � 22.66 b

97.31�29.1bP<0.0001

P<0.05 20�12.2a
62�11cAmylase (U/l)
                                             59.43�42.62 a
P>0.05
71.21 � 41.26 b P>0.05
80�25.31bPlasma Urea (mmol/l)
   4.17�8.22 a                                    
P<0.0024.21 � 13.64 aP<0.0024.3�10bPlasma Creatinine (mmol/l)
  
Direct Bilirubin (mg/dl)

Albumin(U/l)
1.20 � 0.99 a

0.19�0.10a

3.3�2.3a                P<0.629

P<0.001

P<0.0021.10 � 1.01 a 

0.21 �0.11b

 3.02 �2.8a                           P<0.629

P<0.001

P<0.0021.23�0.9a
0.18�0.07a
4.2�1.1b

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