Microbiology /Parasitology HIV 13 December 07
Human Immunodeficienc Immunodeficiency y Virus •
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Schematic Diagram of HIV Replication
Acquired Immunodeficiency Syndrome first described in 1981 HIV-1 isolated in 1984, and HIV-2 in 1986 Belong to the Lentivirus subfamily of the Retroviridae Enveloped RNA virus, 120nm in diameter HIV-2 shares 40% nucleotide homology with HIV-1 Gag core proteins – p15, p17, p24 Pol - p16 (protease), p31 (integrase/endonuclease) Env – gp 160 (gp 120, outer membrane; gp41, transmembrane) Other regulatory proteins, fe. Tat, Rev, Vif, Nef, Vpr and Vpu
HIV Particles
HIV Genome
Replication •
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The first step of infection is the binding of gp120 to the CD4 receptor of the cell, which is followed by penetration and uncoating. The RNA genome is then reverse transcribed into a DNA provirus which is integrated into the cell genome. This is followed by the synthesis and maturation of virus progeny.
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Clinical Features 1. Seroconversion illness - seen in 10% of individuals a few weeks after exposure and coincides with seroconversion. Presents with an infectious mononucleosis like illness. 2. Incubation period - this is the period when the patient is completely asymptomatic and may vary from a few months to a more than 10 years. The median incubation period is 8-10 years. 3. AIDS-r AIDS-rela elated ted compl complex ex or persist persistent ent gener generali alized zed lymphadenopathy. 4. Ful Full-bl l-blow own n AIDS AIDS.. Stages of Infection Exposure ↓ Seroconversion ↓ Asymptomatic ↓ PGL ↓ Persists
↓ Remain Asymtpmatic ↓ AIDS
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Microbiology/Parasitology Microbiology/Parasitology – HIV by Dra Dra de Castro Classification Group I Seroconversion Illness Group II Asymptomatic Group III Persistent Generalized Lymphadenopathy Group IV A – Constitutional Disease B – Neurological Disease C – 2 Infectious Diseases D – 2 Cancers E – Other Conditions Acute seroconversion illness resembles glandular fever adenopathy flu-like symptoms 5-10% • • •
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Pneumocystis carinii Pneumonia Leading cause of morbidity and mortality • Cause infections in immunocompromised • Diagnosis in young men with no explanation for their • immunosuppression •
Was the 1st clue to the recognition of AIDS
Toxoplasma gondii • Always associated with compromised patients •
Brain – important site
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Persistent generalized lymphadenopathy (PGL) (+) 25-30% Enlarged LN: Painless o Symmetrical o • •
Opportunistic Infections Protoz Protozoal oal pneumo pneumocyst cystis is carini cariniii (now (now though thoughtt to to be be a fungi) fungi),, toxoplasmosis, crytosporidosis Fung Fungal al cand candid idia iasi sis, s, cryt crytoc ococ occo cosi sis, s, hist histop opla lasm smos osis is,, coccidiodomycosis Bact Bacter eria iall Myco Mycoba bact cter eriu ium m aviu avium m com compl plex ex,, MTB, MTB, atypi atypica call mycobacterial disease, salmonella septicaemia, multiple or recurrent pyogenic bacterial infection Viral CMV, HSV, VZV, JCV Opportunistic Tumors •
The The most most freq freque uent nt oppo opport rtun unis isti tic c tumo tumorr, Kaposi's sarcoma, sarcoma, is observed in 20% of patients with AIDS.
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KS is observed mostly in homosexuals and its relative incidence is declining. It is now associated with a human herpes virus 8 (HHV-8 (HHV-8). ).
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Malignant l ymphomas ymphomas are also frequently seen in AIDS patients.
Kaposi’s Sarcoma (HHV-8) One of the earliest diseases Arises in many sites: skin, mouth, gut, eye Arise from endothelial cells of blood vessels Bluish purple, raised irregular lesions
HIV Dementia 25% of patients with AIDS Gradual loss of cognition, progressing to overt dementia CT Scan: Loss of tissue o Widening of sulci and ventricles o • • •
Developing Countries: Local problems MDRTB MAI – BM, liver, sleen, LN • • •
Pediatric AIDS Recurrent bacterial infections Lymphoid interstitial pneumonia Pulmonary lymphoid hyperplasia • • •
Epidemiology Sexual transmiss transmission ion - male ale hom homosex osexua uals ls and and 1. Sexual consti constitut tute e the largest largest risk risk group group in N. America America and Western Europe. Europe. In developing developing countries, heterosexual spre spread ad cons consti titu tute te the the most most impo import rtan antt mean means s of transmission.
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Blood/blood products - IV drug abusers represent the seco second nd larg larges estt AIDS AIDS pati patien entt grou groups ps in the the US and and Europe. Haemophiliacs were one of the first risk groups to be identified: they were infected through contaminated factor VIII.
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Vertical transmission - the the tran transm smis issi sion on rate rate from from moth mother er to the the newbo newborn rn vari varies es from from arou around nd 15% 15% in West Wester ern n Euro Europe pe to up to 50% 50% in Afri Africa ca.. Verti ertica call tran transm smis issi sion on may may occu occurr tran transp spla lace cent ntal ally ly rout route, e, perina perinatal tally ly during during the birth birth proces process, s, or postna postnatal tally ly through breast milk.
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Other Manifestations It is now recogni recognized zed that that HIV-in HIV-infec fected ted patien patients ts may deve develo lop p a numb number er of mani manife fest stat atio ions ns that that are are not not explained by opportunistic infections or tumors. •
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The The most most freq freque uent nt neur neurol olog ogic ical al diso disord rder er is AIDS encephalopathy which is seen in two thirds of cases. Other manifestations include characteristic skin eruptions and persistent diarrhea.
Oral Hairy Leukoplakia Unique to HIV-infected HIV-infected patients Margins of tongue White ridges of fronds on the epithelium o (+) association with EBV and papilloma virus • •
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5 million people infected with HIV globally Due to the rampant rampant epidemi epidemics cs seen seen in subsubo Saharan Africa Spikes Spikes in Soviet Union and Eastern Europe, o Central Asia and East Asia 3.1 M deaths including 570,000 children o UN Data, 2005 Disease Progression Untreated form initial infection: 5% within 3 years •
Microbiology/Parasitology Microbiology/Parasitology – HIV by Dra Dra de Castro 20-25% by 6-7 years 5-10% each year <5% asymptomatic for >10 years 2% asymptomatic for > 12 years 13% of patient with viral RNA copy number of <1500/mL will develop AIDS within 9 years 93% progression in 9 years with RNA copy number > 55,000/mL Pathogenesis
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sensit sensitive ive wherea whereas s confir confirmat matory ory assays assays should should be as specific as possible.
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Screening assays - EIAs are the most frequently used screening assays. The sensitivity and specificity of the presently available commercial systems now approaches 100% but false positive and negative reactions occur. Some assays have problems in detecting HIV-1 subtype O.
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Confirmatory assays - Western blot is regarded as the gold standard standard for serological serological diagnosis. diagnosis. However, However, its sens sensit itiv ivit ity y is low lower than than scre screen eniing EIAs. IAs. Line Line immun immunoas oassay says s incorp incorpora orate te variou various s HIV antige antigens ns on nitroc nitrocell ellulo ulose se strips strips.. The interp interpret retati ation on of results results is similar to Western blot it is more sensitive and specific.
The profound immunosuppression seen in AIDS is due to the depletion of T4 helper lymphocytes. lymphocytes. In the immed immediat iate e period period follow following ing exposu exposure, re, HIV is present present at a high level in the blood (as detected by HIV Antigen and HIV-RNA HIV-RNA assays). It then settles down to a certain low level ( set-point) set-point) during during the incuba incubatio tion n period period.. During During the incuba incubatio tion n peri period od,, ther there e is a massiv massive e turnov turnover er of CD4 cells cells,, whereby CD4 cells killed by HIV are replaced efficiently. efficiently. Eventually Eventually,, the immune immune system succumbs and AIDS develop when killed CD4 cells can no longer be replaced (witnessed by high HIV-RNA, HIV-antigen, and low CD4 counts).
HIV Half-lives Activated cells that become infected with HIV produce virus immediately and die within one to two days. Prod Produc ucti tion on of viru virus s by shor shortt-li live ved, d, acti activa vate ted d cell cells s accounts for the vast majority of virus present in the plasma.
ELISA for HIV antibody
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The time required to complete a single HIV life-cycle is approximately 1.5 days. days.
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Resting cells that become infected produce virus only after immune stimulation; these cells have a half-life of at least 5-6 months. months.
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Some cells are infected with defective virus that cannot complete the virus life-cycle. Such cells are very long lived, and have an estimated half-life of approximately three to six months. months. Such Such long long-l -liv ived ed cell cell popu popula lati tion ons s pres presen entt a majo major r challenge for anti-retroviral therapy. therapy.
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Microplate ELISA for HIV antibody: coloured wells indicate reactivity
Western blot for HIV antibody There are different criteria for the interpretation of HIV Wester Western n blot blot result results s e.g. e.g. CDC, CDC, WHO, WHO, Ameri American can Red Cross. •
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The most important important antibodies antibodies are those against the envelope glycoproteins gp120, gp120, gp160, gp160, and gp41
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p24 antibody is usually present but may be absent in the later stages of HIV infection
Other diagnostic assays
Laboratory Diagnosis •
Serology is the the usua usuall meth method od for for diag diagno nosi sing ng HIV HIV infection. Serological tests can be divided into screening and confirmatory assays. Screening assays should be as
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It normally takes 4-6 weeks before HIV-antibody appears following exposure.
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A diagnosis of HIV infection made be made earlier by the detection of HIV of HIV antigen, antigen, pro-DNA, pro-DNA, and RNA. RNA.
Microbiology/Parasitology Microbiology/Parasitology – HIV by Dra Dra de Castro •
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overlapping toxicity, toxicity, it may be possible to reduce toxicity, improve efficacy and prevent resistance from arising.
However, there are very few circumstances when this is justified e.g. diagnosis of HIV infection in babies born to HIV-infected HIV-infected mothers.
Prognostic tests Once a diagnosis of HIV infection had been made, it is important to monitor the patient at regularly for signs of dise diseas ase e prog progre ress ssio ion n and and resp respon onse se to anti antivi vira rall chemotherapy. •
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HIV viral load load - HIV HIV vira virall load load in seru serum m may may be measured measured by assays which detect detect HIV-RNA HIV-RNA e.g. RTPCR, NASBA, NASBA, or bDNA. bDNA. HIV viral viral load has now been establ establish ished ed as having having good good progno prognosti stic c value, value, and in monitoring response to antiviral chemotherapy.
Anti-Retroviral Agents •
Nucleoside analogue reverse transcriptase inhibitors e.g. AZT, ddI, lamivudine
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NonNon-nu nucl cleo eosi side de anal analoq oque ue inhibitors e.g. Nevirapine
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Protease Inhibitors e.g. Indinavir, Ritonavir
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Fusion inhibitors e.g. Fuzeon (IM only)
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HAART (highly active anti-retroviral therapy) regimens normally normally comprise comprise 2 nucleoside nucleoside reverse transcript transcriptase ase inhibitors and a protease inhibitor. inhibitor. e.g. AZT, lamivudine and indinavir . Since the use of HAART, mortality from HIV has declined dramatically in the developed world.
HIV Antigen tests - they were widely used as prognostic assays. It was soon apparent that detection of HIV p24 antigen was not as good as serial CD4 counts. The use of HIV p24 antigen assays for prognosis has now been superseded by HIV-RNA HIV-RNA assays.
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Treatment Treatment (cont.) Assess effectiveness: Measure plasma viral load + CD4 count (estimate state of immune system) Start of treatment, 1 month, 3-4 months interval (+) Response: ↓ in RNA load within a few days by 1 log 10 at o 2-8 weeks < 50% by 4-6 months o Cont Contin inue ue prop prophy hyla laxi xis s and and prom prompt pt trea treatm tmen entt of opportunistic infections Pneumocystis carinii o Toxoplasma gondii o •
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Treatment Aim •
Produce the maximum lasting reduction in viral load Preserve or improve immune function o Reduce clinical problems o Prolng life o Reduce infectivity o Basis for selection Clinical state deteriorating o Plasma viral load high or rising o CD4 count falling or < 200 o Acute stage of initial infection to reduce early o viral replication replication,, achieve achieve a lower stable RNA load, preserve immune function and reduce risk of viral mutation o
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Zidovudine (AZT) AZT) was the first anti-viral agent shown to have beneficial beneficial effect against against HIV infection. infection. However, However, afte afterr prol prolon onge ged d use, use, AZT AZT-res -resis ista tant nt stra strain ins s rapi rapidl dly y appeared which limits the effect of AZT. Combinat Combination ion therapy therapy has has now now been been show shown n to be effectiv effective, e, especially especially for trials trials involving involving multiple multiple agents agents including protease inhibitors. (HAART (HAART - highly active anti-retroviral therapy) therapy) The ration rationale ale for this this approa approach ch is that that by combin combining ing drugs that are synergistic, synergistic, non-cross-re non-cross-resista sistant nt and no
Prevention •
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The risk of contracting HIV increases with the number of sexual partners. A change in the lifestyle would obviously reduce the risk. The spread of HIV through blood transfusion transfusion and blood prod produc ucts ts had had virt virtua uall lly y been been elim elimin inat ated ed sinc since e the the introduction of blood donor screening in many countries. AZT AZT had had been been shown shown to be effe effect ctiv ive e in prev preven enti ting ng transmission of HIV from the mother to the fetus. The incidence of HIV infection in the baby was reduced by two-thirds. The management of health care workers exposed to HIV through inoculation accidents is controversial. Anti-viral prophylaxis had been shown to be of some benefit but it is uncertain what is the optimal regimen. Vaccines are being developed at present but progress is hampered hampered by the high variabili variability ty of HIV. Since 1987, more than 30 HIV candidate vaccines have been tested in approximately 60 Phase I/II trails, involving more than 10,000 healthy volunteers. A phase III trial involving a recombinant gp120 of HIV subtype B was reported in Feb 2005 to be ineffective in preventing HIV infection.
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“There is no short cut, no vaccines, no magic bullet, because sexual transmission remains the main route of
Microbiology/Parasitology Microbiology/Parasitology – HIV by Dra Dra de Castro infection and the focus must therefore be on empowering people to AVOID UNSAFE SEX.” Dr. Mevron
“AIDS is no longer a death sentence for those who can get the medicines. Now it's up to the politicians to create the "comprehensive "comprehensive strategies" to better treat the disease.” Bill Clinton quotes “The subject no longer has to be mentioned by name. Someone is sick. Someone else is feeling better now. A friend has just gone back into the hospital. Another has died. The unspoken name, of course, is AIDS.” David W. Dunlap “It is impossible to maintain civilization with 12-year-olds having babies, with 15-year-olds killing each other, with 17-year-olds dying of AIDS and with 18-year-olds getting diplomas they can't read” Newt Gingrich
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