Case Study MICR 454L Emerging and ReEmerging Infectious Diseases S2008

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Case StudyMICR 454L Emerging and Re-Emerging
Infectious DiseasesS2008

• Case 61
• Nadine Rodriguez
• Irving J. Phillips
• Vanessa Munoz
• Poonam Malave

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Case Summary

• 21-year-old male from North Carolina had no
  recent travel history, drank alcohol, and denied
  sexual contact
• Complaints of nausea, vomiting, diffuse body
  aches, productive cough, fever and loose, watery
  diarrhea
• Had not urinated in the prior 24 hours
  complained of dizziness on standing.
• Denied headache or abdominal pain
• Sore throat 3 weeks previously and urinary tract
  infection a month or two ago.
• Given intravenous fluids,
• Stool specimen was positive for Entamoeba
  histolytica (or Entamoeba dyspar).
• Infectious disease (ID) service was consulted
  after 1 day in hospital.
• On physical examination
• Fever of 39.1oC, a heart rate of 104 beats/min,
  and blood pressure of 134/84 mmHg.
• Enlarged tonsils, but no cervical, axillary or
  inguinal adenopathy.
• Lab results were significant for a white blood
  cell count of 2,200 with 57 polymorphonuclear
  leukocytes, 33 lymphocytes, and 6 atypical
  lymphocytes. Aspartate aminotransferase (AST) was
  650 U/liter, alanine aminotransferase (ALT) was
  830 U/liter, and lactate dehydrogenase (LDH) was
  1,000 U/liter.
• Hepatitis A, B, and C virus and HTV serologic
  test results were negative
• The etiologic agent of his primary illness was
  detected by culture, positive antigen test, and
  PCR (polymerase chain reaction).
• E. histolytica was a secondary infection

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Key Information Pointing to Diagnosis

• Young male, 21 years of age
• Fever of 39.1 oC
• Enlarged tonsils, sore throat for 3 weeks
• Low white blood cell count
• Liver enzymes were elevated
• E. histolytica as a secondary infection.

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The Diagnosis for Case 61

• We concluded that HIV is the primary infection
  after ruling out several other viruses.
• Patient denied sexual contact, which made
  diagnosis hard.
• The white blood cell count indicated immune
  compromise, pointing to a possible infection with
  HIV.

http//www.vircolab.com/content/backgrounders/www.
vircolab.com/hiv_virus.gif
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Microbiology of HIV

• 2 copies of () ssRNA
• Icosahedral, enveloped virus with glycoprotein
  spikes
• Fusion occurs at the plasma membrane
• Reverse transcription occurs within the
  pre-integration complex in the cytoplasm
• Genome is copied to cDNA which is integrated into
  the host DNA
• mRNA synthesis and viral replication take place
  in the nucleus
• Budding occurs at the plasma membrane

http//images.google.com/images?qhivndsp18um1
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www.aegis.com/topics/basics/hivandaids.html
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Pathogenesis of HIV

• Initial infection of Langerhans or Dendritic
  cells
• Both carry virus to CD4 T cells and/or
  macrophages
• Infected cells hone to lymphoid tissue
• Massive viremia leads to systemic infection
• HIV-specific immune response occurs
• Persistent infection is established despite an
  immunological response to the virus
• Partial immunological control of virus
  replication leads to accelerated production of
  virus

Fauci, 2003 Fauci AS HIV and AIDS 20 years of
science.Nat Med. 2003 9(7) 839 - 843. PubMed
12835701.
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Cell Disruption

• Infected DCs infect and induce apoptosis of
  naïve CD4 T cells
• CD4 T cells are killed by fraticide apoptosis
• ACAD pro-apoptotic proteins
• Due to CD4 T cell loss, CD8 T cells defect in
  maturation and function

• Infected macrophage produces NO
• May cause dementia
• NF?B in same cell for virus transcription
• Syncytia sites of interstrain recombination
• High rate of virus production

http//www.nature.com/nri/journal/v3/n5/fig_tab/nr
i1087_F4.html
http//www.nature.com/icb/journal/v77/n4/fig_tab/i
cb199937f4.html
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Diagnostic Tests

• Diagnostic tests for HIV are very sensitive and
  specific in nature
• Window period time during which false negative
  results are obtained (no antibodies are detected
  despite HIV being present). Most people (99)
  have a window period of three months following
  infection with HIV. During this period, an
  infected individual can transmit HIV to others
  despite HIV going undetected with an antibody
  test
• This patient tested negative because he was in
  the window period or early prodromal period,
  where CD4 cells are not producing antibodies.
  Viral load is high and therefore antigens were
  detected
• The various kinds of tests employed to detect
  HIV
• Antibody tests
• ELISA
• Western blot
• Rapid or point-of-care tests
• Antigen tests - Detect for the presence of viral
  p24 protein using monoclonal antibodies
• Nucleic acid based tests (NAT)

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Therapy

• Guidelines for starting antiviral therapy have
  been proposed by panels of experts from the U.S.
  Department of Health and Human Services
• Anti-retroviral drugs currently available
• Reverse Transcriptase Inhibitors
• Nucleoside Analogues AZT, FTC
• Non-nucleoside analogues Efavirenz, Nevirapine
• Nucleotide Analogues Tenofovir
• Protease Inhibitors - Ritonavir, Viracept ,
  Lopinavir
• Other treatments Use of fusion inhibitor T-20
  (Fuzeon), Highly Active Anti-Retro Viral Therapy
  or HAART (combination of 3 or more drugs)
• Side effects such as appearance of rash, nausea,
  fatigue, and formation of kidney stones occur by
  use of these drugs
• New drugs, which target the viral co-receptors
  CCR5 and CXCR4, are currently in development

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Prognosis

• Key tests used to assist in prognosis
• CD4 T-cell counts
• Viral Load tests
• Prognosis depends on the effectiveness of
  anti-retroviral treatment(s) received before the
  immune system has been severely compromised
• Key factors that determine the risk of
  progression to AIDS and subsequent death of
  individuals beginning a first-time treatment
  regimen
• Having a CD4 T-cell count below 200/mm3
• Having a viral load above 100,000/mL at the
  beginning of treatment
• Being 50 years of age or older
• IV drug use
• Having had a prior AIDS-defining illness
• Other factors contributing to prognosis
• Prevention of other infections
• Maintaining a healthy life style

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Prevention

• Sexual promiscuity must be avoided
• Safe sex practices must be followed
• Needles and razors should not be shared
• Counseling must be provided to infected people to
  cope with HIV infection
• Organizing education camps for families of
  individuals affected by HIV
• Blood donors must be screened for HIV

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Epidemiology
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Epidemiology

• In the U.S. alone, the number of persons
  diagnosed with AIDS is upwards of 982,498 since
  1981
• As of 2006, the number of deaths from AIDS has
  reached 545,805
• There have been 2.5 million new cases of HIV
  infection worldwide (UNAIDS, December, 2007)
• As of January 31, 2008, 25,221 total cases of HIV
  have been reported to the California Department
  of Public Health. Of these, 24,469 (97)
  correspond to living cases

http//www.cdc.gov/features/dsblackHIV/
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Populations at Risk

• African Americans
• Account for 55 of infected individuals
• Ages 13-24
• Young men who have sex with men (MSM)
• Typically minorities
• Prisons
• Heterosexual females
• Infants

http//gianmuga.blogspot.com/2007/11/011207-world-
hivaids-day.html

• People living with HIV in 2006

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Threats

• Emergence of drug-resistant strains
• Climate changes are predicted to have a negative
  impact on HIV infection rates.
• Despite educational and treatment efforts, HIV
  prevalence rates continue to increase.
• In 2007 alone, the UN reported a total of 2.1
  million deaths worldwide from AIDS.

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Primary Research Article Contributing to the
Understanding of HIV

• Janas et al. (2008) Productive infection of human
  immunodeficiency virus type 1 in dendritic cells
  requires fusion-mediated viral entry. Virology.
  375 442-451
• Experimental approach
• Cell culture
• DCs generated from purified CD14 monocytes
  stimulated with GMCSF and IL-4
• Flow Cytometry
• DCs stained with MAbs or isotype-matched IgG,
  using FACSCalibur flow cytometry
• HIV-1 Stocks
• Luciferase-reporter HIV -1 stocks generated by
  cotransfections
• HIV-1 entry, infection and transmission assays
• DCs were trypsinized, lysed and quantified by
  ELISA.
• Real-time PCR quantification of HIV-1 DNA in
  infected DCs
• Analysis of HIV-1 entry into DCs by cellular
  fractionation
• Western blotting
• Of DC lysates
• Statistical analyses
• Using Wilcoxon paired t test or Dunnetts
  multiple comparison test with Prism software
• Main findings
• HIV-1 enters DCs via endocytosis, but
  fusion-mediated viral entry is needed for
  productive infection
• HIV-VSV-G infection with DC is low pH dependent,
  while R5-tropic HIV-JRFL infection is
  pH-independent.

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Representative Figure

• Conclusion
• DCs are thought to serve as viral reservoirs for
  dissemination of HIV-1 in vivo
• Use of T-20 as a fusion inhibitor could not
  distinguish between plasma membrane fusion or
  intravesicular fusion
• Similarly, flow cytometry could not distinguish
  between these two fusion events
• cytosolic p24 fractions may serve as an
  important indicator of productive infection in
  DCs
• Relevance to the case
• If DCs serve as a reservoir for dissemination of
  HIV-1, the patient could have transmitted it to
  others before testing positive

Fig. 3. Productive HIV-1 infection in DCs
requires fusion-mediated viral entry. (A)
Blockade of fusion-mediated HIV-1 entry into DCs
diminishes viral replication. DCs were infected
with HIV-1NLAD8 for 2 h at 37 C in the presence
or absence of T-20. Supernatants of infected DCs
were measured for p24 levels at 3, 5 and 7 dpi.
(B) T-20 does not impair HIV-1NLAD8 endocytosis
into DCs. DC-associated p24 was measured after
incubation with HIV-1NLAD8 for 2 h at 37 C in
the presence or absence of T-20. HIV-1-pulsed DCs
were extensively washed, trypsinized and lysed
for p24 detection. (C) Endocytosed HIV-1 in DCs
does not generate significant amounts of late
reverse transcription (RT) products. DCs were
infected with HIV-1NLAD8, washed, trypsinized,
and cultured for 12 h before the cells were
lysed for real-time PCR detection (40 ng of
cellular DNA per sample was used). T-20 was
present during the viral incubation and the 12-h
culture. All data show the meansS.D. of
triplicate samples Data for one representative
experiment out of four are shown.
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Take Home Message

• HIV involves uptake by dendritic cells and
  subsequent infection to CD4 lymphocytes
• Typical symptoms include
• Fever, rash, diarrhea, body aches, headaches,
  lymphadenopathy and flu like symptoms
• Pathogen is HIV-1
• Diagnostics include ELISA as a screening test and
  Western blot for confirmation. RT-PCR is also
  used to determine viral load
• Therapy is based on the stage of HIV infection
  and involves anti-HIV drugs including reverse
  transcriptase inhibitors, protease inhibitors,
  fusion inhibitors and combination therapy
• Prognosis depends on the degree of immune system
  compromise. HIV has no cure, leads to full-blown
  AIDS and eventual death from recurrent
  opportunistic infections
• Prevention methods include abstinence, screening
  and education
• Transmission is via sexual contact, intravenous
  drug use, blood transfusions, and breastfeeding
• Threat is worldwide and incidence rates are still
  high

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References

• Fauci, A.S. (2003) HIV and AIDS 20 years of
  science.Nat Med. 9(7) 839 - 843. PubMed
  12835701
• http//data.unaids.org/pub/EPISlides/2007/2007_epi
  update_en.pdf
• http//www.cdc.gov/hiv/resources/factsheets/us.htm
  
• www.cdph.ca.gov/data/statistics/Pages/OAHIVAIDSSta
  tsLanding.aspx
• http//www.unsw.edu.au/news/pad/articles/2008/apr/
  HIV_forum.html
• http//www.cdc.gov/features/dsblackHIV/
• http//www.medicinenet.com/human_immunodeficiency_
  virus_hiv_aids/article.htm
• http//www.aidsmap.com/en/docs/838DDDA5-614F-488A-
  BE41-BA4DBE1AA4EC.asp
• http//www.hhs.gov/
• http//www.ucsfhealth.org/adult/medical_services/i
  nfect/hiv/conditions/hiv/signs.html
• http//www.cdc.gov/hiv/resources/Factsheets/youth.
  htm
• http//www.vircolab.com/content/backgrounders/www.
  vircolab.com/hiv_virus.gif
• http//images.google.com/images?qhivndsp18um1
  hlennewwindow1clientfirefox-achannelsrls
  org.mozillaen-USofficialstart36saN
• www.aegis.com/topics/basics/hivandaids.html
• http//www.nature.com/nri/journal/v3/n5/fig_tab/nr
  i1087_F4.html
• http//www.nature.com/icb/journal/v77/n4/fig_tab/i
  cb199937f4.html

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Thank You!
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Any Questions?