**16. Challenges of phenotypic methods**

Despite having high sensitivity phenotypic assays are laborious, expensive, and timeconsuming and can only be done in sophisticated laboratories by highly qualified personnel. More so the presence of CXCR4 viruses, which frequently constitute less than 5% of viral population is a challenge as phenotypic assays' sensitivity does not seem to permit this range of detection threshold. Consequently, individuals may be misdiagnosed as harboring only CCR5 coreceptor using virus, when in actual fact they may also be harbouring R4 virus in very low quantities such that once initiated on CCR5 coreceptor antagonists their CXCR4 tropic viruses may emerge. Due to the foregoing bottlenecks there is need for the development of simple, more sensitive, accurate and less expensive tests with a shorter turnaround time to replace slow and resource intensive phenotypic assays.

### **17. Genotypic coreceptor analysis methods**

HIV env gp120 is composed of about 400 amino acids which consists five relatively conserved constant (C1-C5) and five hyper-variable regions (V1-V5). The genetic determinants of HIV-1 coreceptor usage are localized in the V3 loop of gp120 which has a highly conserved crown motif and glycosylation sites. The third variable region also called the V3 loop is composed of 31-39 amino acids. . The V3 loop is closed by a disulfide bridge formed by two cysteines. Functionally it is critical in maintaining the right conformation to facilitate coreceptor interaction with the virus. This region has been shown to be the major determinant of viral tropism and accordingly, prediction of coreceptor usage based on the interpretation of V3 sequences using bioinformatics tools could be a good alternative to infer tropism in the clinical routine Sequences in the N-terminus of V3 loop have been shown to modulate the levels of infection through CCR5 coreceptor. Minor or a few sequence alterations or mutations in V3 are sufficient to switch coreceptor use from CCR5 to CXCR4 or from dual-tropic to X4-tropic virus. Additional mutations within the V1/V2 have also been observed during coreceptor switching. Studies have shown that such mutations seem to compensate for the harmful V3 mutations. Functional studies have demonstrated that the V3 loop interacts with the N-terminal extra-cellular domain of CCR5 and the extracellular loop 2. Bioinformatics tools based on V3 sequences can be used to predict HIV-1 tropism. The identification of viral genotypic changes associated with different coreceptor usage has led to the development of sequence-based algorithms to predict coreceptor usage. Different rules have been published based on the amino acid sequence of the env V3 region of HIV-gp120, which is known to be the major determinant of coreceptor usage.

#### **18. Predictive algorithms of HIV-1 coreceptor usage**

242 Immunodeficiency

assays.

Francisco, USA. The test can detect 10% of X4 variants with 100% sensitivity. More recently, an enhanced Trofile® assay with better sensitivity to improve detection of low level X4-using variants has been developed that can detect 0.3% of these variants with 100% sensitivity. Trofile® phenotypic assay continues to be the only clinically validated assay to identify coreceptor tropism and is considered the gold standard for tropism testing capable of

Another phenotypic tropism assay, Phenoscript-tropism; Eurofins is also on the market. It was specifically developed to cater for phenotypic test for the evaluation of viral tropism in HIV-1 non-B subtypes. It is worthwhile to appreciate that the HIV-1 env V3 loop is

Despite having high sensitivity phenotypic assays are laborious, expensive, and timeconsuming and can only be done in sophisticated laboratories by highly qualified personnel. More so the presence of CXCR4 viruses, which frequently constitute less than 5% of viral population is a challenge as phenotypic assays' sensitivity does not seem to permit this range of detection threshold. Consequently, individuals may be misdiagnosed as harboring only CCR5 coreceptor using virus, when in actual fact they may also be harbouring R4 virus in very low quantities such that once initiated on CCR5 coreceptor antagonists their CXCR4 tropic viruses may emerge. Due to the foregoing bottlenecks there is need for the development of simple, more sensitive, accurate and less expensive tests with a shorter turnaround time to replace slow and resource intensive phenotypic

HIV env gp120 is composed of about 400 amino acids which consists five relatively conserved constant (C1-C5) and five hyper-variable regions (V1-V5). The genetic determinants of HIV-1 coreceptor usage are localized in the V3 loop of gp120 which has a highly conserved crown motif and glycosylation sites. The third variable region also called the V3 loop is composed of 31-39 amino acids. . The V3 loop is closed by a disulfide bridge formed by two cysteines. Functionally it is critical in maintaining the right conformation to facilitate coreceptor interaction with the virus. This region has been shown to be the major determinant of viral tropism and accordingly, prediction of coreceptor usage based on the interpretation of V3 sequences using bioinformatics tools could be a good alternative to infer tropism in the clinical routine Sequences in the N-terminus of V3 loop have been shown to modulate the levels of infection through CCR5 coreceptor. Minor or a few sequence alterations or mutations in V3 are sufficient to switch coreceptor use from CCR5 to CXCR4 or from dual-tropic to X4-tropic virus. Additional mutations within the V1/V2 have also been observed during coreceptor switching. Studies have shown that such

distinguishing pure R5, D/M and pure X4 populations.

implicated in the determination of phenotypic tropisms

**17. Genotypic coreceptor analysis methods** 

**16. Challenges of phenotypic methods** 

At least eight different bioinformatics tools have been used to predict viral tropism in different HIV-1 subtypes which uses phenotypic data to predict the corresponding viral genotype. Studies of genotypic predictors have been retrospective with patient samples selected based on availability of phenotypic tropism determinations. Three of the interpretation systems namely, WetCat, WebPSSM, geno2pheno [coreceptor] are freely available on internet. All three focus on the env-V3 region and only take the amino acid sequence into account. Such genotypic systems provide the possibility for rapid screening of patients who may be administered with CCR5 blockers like maraviroc. On cloned viruses belonging to genetic subtype B, the specificity and sensitivity of most predictive methods exceed 90% and 80%, respectively. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists in populations pre-screened with a phenotypic assay. The first genotypic algorithm designed to predict HIV-1 tropism takes into account only the net charge of amino acids at two key residues located within the V3 loop, amino acids at positions 11 and 25. The most widely used is the 11/25 rule which focuses on identifying sequence patterns within the V3 loop. Predictions using the "11/25 charge rule" are relatively satisfactory.
