Many clinics in metropolitan cities across Europe now routinely report 25 to 40% of their patient profile as having non-B clade virus, including London-based hospitals.

It is currently estimated that one quarter of HIV-infected people in the United Kingdom have contracted a non-B subtype. Such infections are found almost exclusively among people who have contracted HIV through heterosexual sex. A British study of over 600 people born in the United Kingdom or Europe found that 14% of the men and 35% of the women were infected with non-B subtypes (Parry 2001).

Due to the preponderance of the B subtype in western countries, resistance research has focused on the evolution and mutations associated with the B subtype. Little is known about how drug resistance may evolve differently in people infected with non-B subtypes, although this is a growing area of interest.

There is increasing recognition of the impact sub-type has on pathways to resistance during anti-HIV treatment. There is also recognition of the need for further research in non-B sub-types to inform the treatment decisions of people with non-B sub-types and the analysis of their resistance assays.

Sub-types and resistance mutations

There is increasing evidence that the HIV subtypes each have distinct pathways to resistance, although many key resistance mutations occur in both non-B and B sub-types which are exposed to antiretroviral drugs (Palmer 2001; Weidle 2001). The prevalence of protease and reverse transcriptase mutations differ among treated patients according to sub-type. However, understanding pathways to resistance in terms of B and non-B sub-types may be unhelpful given that there may be significant distinctions between the resistance pathways of non-B clades.

All HIV subtypes feature naturally occurring genetic variations known as polymorphisms. In some cases, natural polymorphisms can be associated with drug resistance.

There is some evidence that some natural polymorphisms may be more common in people infected with some non-B subtypes (Perez-Alvarez 2001). Generally these natural variations are not primary mutations which confer significant resistance, but secondary mutations which may contribute to resistance or improve the fitness of resistant virus.

In some instances, however, polymorphisms which confer significant resistance have been detected in people with non-B sub-types. For instance, K103N associated with high level resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been reported three untreated people infected with non-B sub-types (Akinsete 2004). Other identified mutations associated with reduced susceptibility to NNRTIs, such as the 135 mutation, have also been identified as natural variations in non-B sub-types (Florance 2003).

Sub-type C and mutations

There is a growing body of research into polymorphisms and resistance mutations seen in sub-type C.

Studies have reported a higher prevalence of certain secondary protease substitutions, such as K20R, M36I, M46 and I93L among people with sub-type C who have never taken antiretroviral drugs (Cane 2001; Grossman 2005).

A study of 40 blood samples containing HIV sub-type C which had not been exposed to anti-HIV therapy showed that 90% contained polymorphisms M36I and I93L which have been identified as secondary protease mutations. Nearly half showed the K20R mutation, the L63P/T/V mutation and 5 of 40 samples had the V77I polymorphism. A quarter exhibited both K20R and M36I, associated with resistance to indinavir (Crixivan) and ritonavir (Norvir). Three samples showed the V82I polymorphism which has been linked to low level resistance to nelfinavir (Viracept; Besslong 2005).

An analysis of sub-type C virus from 28 untreated Zambians found a high prevalence of particular polymorphisms (Handema 2003). The most frequent secondary mutations in the protease and reverse transcriptase genes were I93L, L89M, M361I, M361V, R211K and S48T. Other polymorphisms found in the reverse transcriptase gene were M41N and D67A.

Another study found that efavirenz (Sustiva) can trigger a V106M mutation in people with sub-type C which leads to cross-resistance to all the NNRTIs (Brenner 2003). V106A has long been recognised as a nevirapine (Viramune) mutation in people with subtype B. However, V106M has not previously been identified. This study suggests that V106M may be a signature NNRTI mutation in people with sub-type C.

A study of people infected with either sub-type B or C and treated with ritonavir-boosted lopinavir (Kaletra) showed L63P occurred more commonly in sub-type B than C. Other lopinavir-associated mutations such as L10I/V/F, M46I, F53L, I54V, A71V, V82A, I84V, L90M had roughly similar prevalence rates in the two sub-types (Grossman 2005).

Key protease inhibitor (PI)-associated mutations such as D30N and L90M may be significantly less common in sub-type C than in sub-type B virus following treatment, although there may be some diversity within C sub-types (Doualla-Bell 2005). It has also been reported that key reverse transcriptase-associated mutations including D67N, K103N and T215Y are less common in sub-type C compared to sub-type B (Cane 2001).

A study of 164 people who were failing first-line therapy found that 77% had resistance to NNRTIs most commonly with M184V. All people taking efavirenz developed the K103N mutation but it was more common in B compared to A or C sub-types. The G190 mutation was most common for sub-type C and A but not for B (Cane 2005). The V106M mutation occurs in sub-type C in response to efavirenz and confers 100- to 1000-fold resistance to all NNRTIs (Cane 2005; Turner 2003).

Other sub-types

There is also evidence of resistance-related polymorphisms in other HIV sub-types. For example, two reverse transcriptase mutations (A98S and R211S) and two protease inhibitor-related mutations (M46L and 19P) have been identified in sub-type G virus among a Spanish cohort.

A study comparing resistance mutations in sub-type B and G which developed during treatment with nelfinavir. In sub-type B, 66% of virus showed the D30N mutation (strongly associated with N88D), 28% had the L90M and 6% had N88S. In contrast, 74% of sub-type G virus showed the L90M mutation, 12% D30N, 10% N88S, 5% I54V/L and 2% L46I (Camacho 2005).

There is also evidence of a difference in the 82 mutation in sub-types B and G seen during tipranavir therapy. Mutation 82A was more common in sub-type B but 82T and 82S predominate in sub-type G (Camacho 2005).

HIV sub-type D in infants exposed to nevirapine displayed a high level of resistance to nevirapine. Interestingly, the resistance profile was I135L, T139V and V245T. This is quite distinct from the usual NNRTI resistance mutations of Y181C, K103N and G190A (Baird 2004).

A comparison of resistance mutations in individuals with sub-type B and G infections showed that whilst sub-type B patients had all developed nelfinavir resistance characterised by the D30N mutation, which does not cause protease inhibitor cross-resistance, sub-type G patients all developed nelfinavir resistance characterised by the L90M mutation (associated with broad cross-resistance), together with the I54M mutation in six of ten cases (not previously associated with nelfinavir resistance; Gomes 2002).

Analysis of sub-type CRF02_AG in Ghana found no key PI resistance mutation but phenotypic testing showed that the virus was much less susceptible to nelfinavir and lopinavir and moderately less susceptible to indinavir and saquinavir than B sub-types (Kinmoto 2005).

Impact on response to therapy

There has been considerable debate about the effect non-B subtype polymorphisms and resistance pathways may have on response to therapy and the likelihood of resistance.

In February 2003, Dr S Spira and colleagues from McGill University in Canada wrote: Increasing evidence suggests that all clades of HIV probably display similar sensitivity to antiviral drugs. However, viruses from some subtypes and / or geographical regions may have a greater propensity to develop resistance against certain drugs than do other viral variants. They added that variations in viral fitness may also impact on efficacy of treatment and the consequent pace of HIV disease progression.

The evidence published to date supports this equivocal finding.

Several studies have found that different resistance pathways do not undermine the benefits of antiretroviral therapy. For example, a study of 79 treatment-naive Africans found that viral subtype had no impact on response to therapy and that no specific polymorphism impacted on clinical outcome (Frater 2001). A randomised study found no significant difference in virologic response at weeks 24 or 48 when comparing children with B and non-B subtypes (Pillay 2002). Thirdly, an in vitro study has found that susceptibility was not significantly reduced in non-B viral isolates with secondary mutations (Schapiro 2001).

However, there is some evidence that subtype may affect response to therapy. A retrospective case-control study evaluating 42 consecutive matched pairs of patients with subtype C and subtype B HIV infection found that people with subtype C had an inferior viral load response to treatment at 24 and 48 weeks, with high rates of secondary mutational changes in the protease and reverse transcriptase enzyme (Loveday 2002).

A study of six Ethiopians has suggested that NNRTI-related resistance mutations may also develop more rapidly in the sub-type C virus and that people infected with this virus may have a greater chance of being naturally resistant to the NNRTIs (Loemba 2002). This confirms several reports of the rapid development of drug resistance among women given nevirapine monotherapy to prevent mother-to-child HIV transmission but certainly needs to be tested in larger trials. There have also been reports of previously unreported mutations in the subtype C virus (103E, 106M, 62V, 75E) in response to efavirenz treatment (Brenner 2003; Loemba 2002). The clinical implications of these findings are unknown.

There is evidence that D30N may impact more significantly on subtype C than subtype B (Gonzalez 2004).

As this summary suggests, little is known about the baseline resistance profiles of non-B subtypes. Even less is known about how the non-B subtypes will evolve in the presence of antiretroviral drugs and the impact this will have on response to therapy. The implications for second-line and salvage therapy choices are yet to be established.

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