CHAPTER FIVE: - DISCUSSION
5.1 Introduction
This chapter offers the discussion, conclusion, and recommendations. This is in line with the
specific research objectives: to profile the Nucleoside Reverse Transcriptase Inhibitor drug-
resistant and susceptibility patterns of naive HIV-positive patients from Machakos Level 5
hospital to profile Nucleoside Reverse Transcriptase Inhibitor drug-resistant and
susceptibility patterns of naive HIV positive patients from Machakos Level 5 hospital.
5.2 Summary and Findings
Research shows that in East Africa, most infections are caused by subtype A, plus also
subtypes C and D, plus some URFs. Subtype A is also distributed in Russia and the former
Soviet Union, Central Asia (Akhome, 2021; Bbosa et al., 2019). In Kenya, research studies
carried out throughout the country in various regions from the coast, western and central
Kenya indicate the detection and, in some areas, the predominance of the subtype A plus also
the presence of subtypes C and D. This study's sequences on analysis had shown genetic
similarities of between 85 – 97% alignment with HIV subtype A as shown by the
representative sequence BLAST and HIV BLAST results. This is similar to what other
researchers have shown from previous studies carried out here in Kenya, which show the
predominance of subtype A, where they range from 44% to 74% detection (Adhiambo, et al.,
2021; Adungo, et al., 2014; Gounder, et al., 2017; Hassan, et al., 2018; Kageha, et al., 2012;
Khamadi, et al., 2009; Kinyua, et al., 2018; Kitawi, et al., 2015; Koigi, et al., 2014; Luvai, et
al., 2015; Mabeya, 2021; Onywera, et al., 2017; Yang, et al., 2003). At the time of the study,
the amplified and sequenced samples data indicated the presence of only subtype A and no
other subtype was detected from our samples, which is a difference from other studies carried
out in Kenya.
When the Recombinant Identification Program (RIP) hosted by the Los Alamos Database
was used to analyze for and determine recombinations in this study's sequences, from the
results, the most dominant curve was that of the A1 sub-subtype. This was also confirmed by
the REGA HIV subtyping tool, which also confirmed the genotyping as sub-subtype A1 with
100% support. When the phylogenetic tree was drawn using similar sequences obtained
from the Genbank Database, this study's sequences aligned with other Kenyan sub-subtype
A1 sequences. A unique observation is the clustering of this study's samples on the inferred
phylogenetic tree after 2000 replicates. Previous Kenyan studies carried out on various
groups, including expectant mothers, intravenous drug users, and HIV infected individuals,
1
, have majorly the sub-subtype A1, which is the most predominant strain detected with
detection rates ranging from 44% to 86% (Adhiambo, et al., 2021; Adungo, et al., 2014;
Gounder, et al., 2017; Hassan, et al., 2018; Kageha, et al., 2012; Khamadi, et al., 2009;
Kinyua, et al., 2018; Kitawi, et al., 2015; Koigi, et al., 2014; Lihana, et al., 2009; Luvai, et
al., 2015; Mabeya, 2021; Nduva, et al., 2020; Nyamache, et al., 2013; Onywera, et al., 2017;
Yang, et al., 2003). The neighbor-joining method for constructing phylogenetic trees uses
pairs of operational taxonomic units (OTU) or neighbors to minimize branch lengths, starting
with a starlike tree that is repeatedly run at each replication stage to ensure the integrity of the
tree structure (Saitou & Nei, 1987). The Kimura 2 parameter model used in this analysis has
the role of estimating genetic distances between different nucleotide sequences during an
evolutionary process (Kimura, 1980; Nishimaki & Sato, 2019).
5.2.1 Profile of Resistance to Nucleoside Reverse Transcriptase Inhibitor (NRTI) Drugs
The nucleoside reverse transcriptase inhibitors (NRTIs) are drugs that inhibit the reverse
transcription of viral DNA within cells. They contain nucleotide base analogs that will cause
the chain termination or non-extension of the DNA during reverse transcription of the HIV
viral DNA while inside an infected cell using the HIV reverse transcriptase (immunopedia,
2021). Their mode of action either follows a discriminatory pathway, leading to the decrease
of the binding affinity of NRTI-triphosphate over the natural nucleotide on the reverse
transcriptase binding site. Again, this may decrease the NRTI-phosphate over the natural
nucleotide rate of incorporation of the reverse binding site. The second pathway is an
excision pathway dependent on adenosine triphosphate (ATP) or pyrophosphate; therefore,
mutations that increase the affinity of reverse transcriptase for ATP or increase the analog
complex's removal rate are favored. Also, changes in the translocation ability of the residues
from the active site (N-site) to the post-translocation site (P-site) as well as the rate of
dissociation of the template/primer from the enzyme can enhance the excision pathway
(immunopedia, 2021).
Generally, NRTIs are drugs that include abacavir (ABC), zidovudine (AZT), emtricitabine
(FTC), lamivudine (3TC), tenofovir (TDF), stavudine (d4T), didanosine (ddl), and zalcitabine
(ddC) (immunopedia, 2021, NASCOP, 2018). All the sequenced samples had a D67E
mutation in this study, which is a change of amino acid aspartic acid (D) to glutamic acid (E).
Aspartic acid (D) is an important building block used in making proteins in the body, while
glutamic acid (E) is used to form proteins in the body (Immunopedia, 2021). According to the
HIV research, this is a non-polymorphic NRTI selected mutation at position 67 that is a
2
5.1 Introduction
This chapter offers the discussion, conclusion, and recommendations. This is in line with the
specific research objectives: to profile the Nucleoside Reverse Transcriptase Inhibitor drug-
resistant and susceptibility patterns of naive HIV-positive patients from Machakos Level 5
hospital to profile Nucleoside Reverse Transcriptase Inhibitor drug-resistant and
susceptibility patterns of naive HIV positive patients from Machakos Level 5 hospital.
5.2 Summary and Findings
Research shows that in East Africa, most infections are caused by subtype A, plus also
subtypes C and D, plus some URFs. Subtype A is also distributed in Russia and the former
Soviet Union, Central Asia (Akhome, 2021; Bbosa et al., 2019). In Kenya, research studies
carried out throughout the country in various regions from the coast, western and central
Kenya indicate the detection and, in some areas, the predominance of the subtype A plus also
the presence of subtypes C and D. This study's sequences on analysis had shown genetic
similarities of between 85 – 97% alignment with HIV subtype A as shown by the
representative sequence BLAST and HIV BLAST results. This is similar to what other
researchers have shown from previous studies carried out here in Kenya, which show the
predominance of subtype A, where they range from 44% to 74% detection (Adhiambo, et al.,
2021; Adungo, et al., 2014; Gounder, et al., 2017; Hassan, et al., 2018; Kageha, et al., 2012;
Khamadi, et al., 2009; Kinyua, et al., 2018; Kitawi, et al., 2015; Koigi, et al., 2014; Luvai, et
al., 2015; Mabeya, 2021; Onywera, et al., 2017; Yang, et al., 2003). At the time of the study,
the amplified and sequenced samples data indicated the presence of only subtype A and no
other subtype was detected from our samples, which is a difference from other studies carried
out in Kenya.
When the Recombinant Identification Program (RIP) hosted by the Los Alamos Database
was used to analyze for and determine recombinations in this study's sequences, from the
results, the most dominant curve was that of the A1 sub-subtype. This was also confirmed by
the REGA HIV subtyping tool, which also confirmed the genotyping as sub-subtype A1 with
100% support. When the phylogenetic tree was drawn using similar sequences obtained
from the Genbank Database, this study's sequences aligned with other Kenyan sub-subtype
A1 sequences. A unique observation is the clustering of this study's samples on the inferred
phylogenetic tree after 2000 replicates. Previous Kenyan studies carried out on various
groups, including expectant mothers, intravenous drug users, and HIV infected individuals,
1
, have majorly the sub-subtype A1, which is the most predominant strain detected with
detection rates ranging from 44% to 86% (Adhiambo, et al., 2021; Adungo, et al., 2014;
Gounder, et al., 2017; Hassan, et al., 2018; Kageha, et al., 2012; Khamadi, et al., 2009;
Kinyua, et al., 2018; Kitawi, et al., 2015; Koigi, et al., 2014; Lihana, et al., 2009; Luvai, et
al., 2015; Mabeya, 2021; Nduva, et al., 2020; Nyamache, et al., 2013; Onywera, et al., 2017;
Yang, et al., 2003). The neighbor-joining method for constructing phylogenetic trees uses
pairs of operational taxonomic units (OTU) or neighbors to minimize branch lengths, starting
with a starlike tree that is repeatedly run at each replication stage to ensure the integrity of the
tree structure (Saitou & Nei, 1987). The Kimura 2 parameter model used in this analysis has
the role of estimating genetic distances between different nucleotide sequences during an
evolutionary process (Kimura, 1980; Nishimaki & Sato, 2019).
5.2.1 Profile of Resistance to Nucleoside Reverse Transcriptase Inhibitor (NRTI) Drugs
The nucleoside reverse transcriptase inhibitors (NRTIs) are drugs that inhibit the reverse
transcription of viral DNA within cells. They contain nucleotide base analogs that will cause
the chain termination or non-extension of the DNA during reverse transcription of the HIV
viral DNA while inside an infected cell using the HIV reverse transcriptase (immunopedia,
2021). Their mode of action either follows a discriminatory pathway, leading to the decrease
of the binding affinity of NRTI-triphosphate over the natural nucleotide on the reverse
transcriptase binding site. Again, this may decrease the NRTI-phosphate over the natural
nucleotide rate of incorporation of the reverse binding site. The second pathway is an
excision pathway dependent on adenosine triphosphate (ATP) or pyrophosphate; therefore,
mutations that increase the affinity of reverse transcriptase for ATP or increase the analog
complex's removal rate are favored. Also, changes in the translocation ability of the residues
from the active site (N-site) to the post-translocation site (P-site) as well as the rate of
dissociation of the template/primer from the enzyme can enhance the excision pathway
(immunopedia, 2021).
Generally, NRTIs are drugs that include abacavir (ABC), zidovudine (AZT), emtricitabine
(FTC), lamivudine (3TC), tenofovir (TDF), stavudine (d4T), didanosine (ddl), and zalcitabine
(ddC) (immunopedia, 2021, NASCOP, 2018). All the sequenced samples had a D67E
mutation in this study, which is a change of amino acid aspartic acid (D) to glutamic acid (E).
Aspartic acid (D) is an important building block used in making proteins in the body, while
glutamic acid (E) is used to form proteins in the body (Immunopedia, 2021). According to the
HIV research, this is a non-polymorphic NRTI selected mutation at position 67 that is a
2
