17 Diagnostics

17.1 The ideal test

  • Cheap
  • Quick
  • Sensitive
  • Specific
  • Well tolerated
  • Easy
  • Non invasive
  • Prognosticates
  • Low tech
  • Safe
  • “Green”

But tests that identify parasites are less useful for immune patients, identifying patients with parasites doesn’t mean they necessarily have disease!

The history is that up until 2010, the diagnostic “test” was whether you had fever or not (according to WHO). Microscopy wasn’t incentivised, just treat everyone anyway. This resulted with massive over-diagnosis. In tanzania, half of the admissions with “severe malaria”, were slide negative! These slide negative patients did worse, because they didn’t have malaria! The new treatments were effective but costly.

17.2 Microscopy

17.2.1 The thick film

No red cells seen as you lyse the red cells, leaving the parasites. This means you can’t tell species apart as that relies on red cell changes.

Good for identifying parasitaemia. Quick to read.

17.2.2 The thin film

You can see the red cells, the trophozoites inside, and the changes to the red cells.

The thin film is less sensitive, but improved speciation

17.2.3 Thick and thin film

Both on the same slide

17.2.4 Interpreting a film

  • What’s the species? - Thin film
  • What’s the density of infection? - Thin film
  • Number of parasites per high power field - Thick Film
  • Staging of parasites (trophozoites vs schizonts)

17.2.4.1 Problems

need expertise, electricity, microscopes, maintenance, stocks

17.3 Other approaches

17.3.1 Rapid Diagnostic Tests

They detect plasmodia antigens (not antibodies)

Immunochromatographic (they use an antibody, to test for the antigen)

Tests for 3 antigens in common use

  • HRP2
  • LDH
  • Aldolase

17.3.2 RDT Problems

HRP2 is only produced by falciparum so only useful for that

Pf LDH, not as heat stable

These RDTS are about as good as microscopy for infection or not

These RDTS are not as good as speciation as a microbiologist, nor do they allow you to prognosticate.

HRP-2 sticks around for several weeks (2-6 weeks), so you can’t use it to monitor for infection. So in a high transmission settings, practically everyone will be positive

The real problem is in terms of resistance, what happens when the parasites start developing deletions of these antigens, so the RDTs don’t pick up infection. This first started in the amazon, with reported false positives. Now it’s being seen increasingly elsewhere. In general, rates in africa are low-ish (1.5-2%), but there’s not that much data. In eritrea this year, there was a study showing 60% rates of slide positive, RDT negative malaria. This a potentially major major problem.

So how to respond. You need a couple of negative tests (of different types of tests) before saying that someone doesn’t have malaria. We need some national programmes to screen for PfHRP2 deletions (to determine the level of the problem). We need to improve performance of LDH tests (but these are currently not nearly as good as the HRP2 tests)

17.4 Malaria Control - Entomology, Vector Control, Vaccines

Janneke Snetselaar, Matt Kirby, Baz 04/09/18

  • Malaria - Mosquitoes (Anopheles)
  • Lymphatic filiarisis - Mosquitoes (Anopheles,Culex,Aedes)
  • Dengue - Mosquitoes (Aedes)
  • Yellow fever - Mosquitoes (Aedes)
  • Human African Trypanosomiasis - Tsetse fly

There are >3000 species of mosquitoes

Most significant mosquito families as vectors of disease:

  • Anopholes
  • Culex
  • Aedes

17.5 Life Cycle

Starts with feeding, only females take blood meal (blood meal is just for egg development) on day one, rest day one and two, lay egges day three.

They are larva then pupa, emerge then fertilise once.

The mosquito can live approxiametly a month. Depending on temperature and humidity.

Males survive on nectar.

Mosquitos are atrracted to:

  • CO2 (Furthest distance)
  • Body Odour (Sweat)
  • Heat (closest distance)

17.6 Identification

17.6.1 Eggs

Anopheles: Laid as single eggs that float

Culex: lay egges in raft (approx 60 eggs)

17.6.2 Larvae

  • Anopholes rest parallel to water surface

  • Aedes and culex rest at an angle to water surgace as they have a breathing siphon

  • Anopheles feed at surface water

  • Culex feed throughout water

17.6.3 Adults

Anophelines female from male:

  • Male have feathery palps next to antenna (females dont)
  • Culex have same male female divide with palps

Anopheles versus culex:

  • Anopheles sit butt up in the air (45)
  • Aedes will rest parallel to wall

17.7 Behaviour

Culex:

  • Feeds primarily at night
  • They are a nuisance biter (biting in large densities)
  • Vector of filiarisis
  • Very common
  • Breed in urban areas, in polluted water (soap, sewage, canal)

Aedes:

  • Feeds primarily during day
  • Vector of dengue, zika, yellow fever, chikungunya
  • Very aggressive
  • Very difficult to control
  • Lay eggs in water, but can survive dessication (Drying)
  • Eggs often laid in puddles in car tyres, other man made (originally forest dwelling)

Anopheles:

  • Feeds at night
  • Vector of malaria, filiarisis
  • Breeds in open, unpolluted, sun exposed, often small temporary puddles
  • Breeds in habitats often created by man
  • Very large and rapid reproductive output
  • Prefers to bite humans (over cattle)
  • Attracted to human odours, likes to go indoor homes
  • Great flexibility to changing environments
  • Has multiple resistance mechanisms

Tsetse fly

  • Both genders take blood meal
  • Prefers animals over men
  • Long puparal output
  • Prefers v vegetation output

17.8 Plasmodium falciparum

Distribution: This realy overlaps with An. gambiae (needs lots of rainfal, lower temp), and An. arabiensis (exists in more arid areas, higher temperatures)

Malaria in tanzania more common in coastal areas (sea and lakes). This is overlapping with the high humidity, and high prevelance of An. gambiae species

17.9 Malaria Control through Vector Control

Janneke Snetselaar, Matt Kirby, Baz 04/09/18

Key Factors:

  • Insecticide Treated Nets
  • Indoor Residual Spraying

The global strategic plan was for 80% to have protection with one of the above factors.

  • Coverage = access to protection
  • Usage = use of protection

The importance of vector control. Bhatt et al. 2015 Nature 526, 207-2011

68% of cases averted from 2005-2015 can be attributed to bed nets

The remainder is through spraying and ACTs

  • LLIM (long lasting insecticidal net)
  • ITN (insectacide treated net)

The 80% target has been met for nets, it hasn’t been with IRS (for sub saharan africa). It hasn’t been for IRS due to mosquito resistance, and new effective agents being more expensive.

17.9.1 In Tanzania

Main areas for transmission around the coast. There has been some rebound in the most recent years. You need to be putting more effort in when close to elimination, than when you are further away.

17.9.2 Vector Control

A mosquito has to live around 10 days to transmit malaria. It has to bite twice!

How can you control malaria through vectors?

So if you can m (mosquito density) in the mcdonalad equation affect P (the daily survival rate) If you can affect a you affect anthropophily (tendency of mosquito to bite humans)

17.9.2.1 Indoor residual spraying

Spray on the walls of the house, once per year. About a month or two before the season starts, and it will last the whole season.

Insecticides last less well on mud than on concrete. Last around 3 months on mud, 6 months on concrete.

Insecticides are tested for mammalian toxicity, and don’t really have any.

We have agents that last longer, don’t bioaccumulate, are safer, have less resistance, than DDT. No one should be using DDT anymore.

Has a big impact on m (mosquito density) and p (survival rates)

  • Kills mosquitos when they first seek a blood meal

  • Targets mosquitos that feed and rest indoor

  • You measure the age of mosquito population by whether they’ve laid eggs or not.

  • IRS will result in younger populations of mosquitoes

IRS allows you to supress population

The average reduction of malaria prevalence from IRS is 62%

17.9.2.1.1 Problems with IRS
  • The timing (For duration)
  • Community fatigue (big impact on the community, to perform this)
  • High level of disturbance
  • Needs proper training, difficulties with logistics, planning, scale up, infrastructure
  • Safety for those administering
  • Insecticide resistance
  • Cost, one common IRS is around 20-30 dollars per house per year (~5 dollars per house)

17.9.2.2 Bed Nets

They are toxic to mosquitoes, they are a physical barrier, they have repellant to mosquitoes

They are personal protection rather than community protection.

Have an impact on a and p if high coverage of nets

Long-lasting nets (coated + binder, or incorporated - which is a plastic net were the insecticide is within the fibre of a net), giving 3+ years bioefficacy and still viable after 20 washes.

Nets dominate vector control sales, initially targetting vulnerable groups (children and pregnant women). THere are annual distributions at schools, antenatal care. They aim is for universal coverage.

For every 1000 children you prevent 6 deaths.

In stable malaria transmission area, you reduce incidence of uncomplicated malaria by 50% compared to no nets.

RBM recommends nets scale up in high transmission and stable malaria endemic areas.

17.9.2.2.1 Problems with nets
  • Nets require owners to use them correctly
  • Mosqquitoes must be biting indoors late at night
  • For full potential of nets a coverage of 35-65% of the population is necessary
  • Nets must be treated with safe insecticides, resistance is a big threat - you have more physical contact with a net than you do with IRS, so we’ve been using pyrethroids for the last 30 years as it’s the only one we’re certain is safe enough
  • Insecticide is really important, without insecticide, as soon as you get a few holes, the net would be completely ineffective

17.9.3 Larval control

Targets mosquito density

You aren’t breaking human vector control

So it is a more targetted intervention only good for certain scenarios.

Larval control only really works when breeding sites are: Few, Fixed, Findable

17.9.4 Insecticide Resistance

Insecticide resistance is spreading, looking at the last 15 years compared to the previous 50. There has been a strong development of resistance to pyrethroids.

There are multiple resistance mechanisms, some mechanisms will protect against more than one insecticide/method of administration.

There are 13 forms of compounds that we have available for IRS. We can rotate through these for insecticide resistance strategey.

For nets we have very few options in comparison. All our options are part of one class, they’re all pyrethroids

17.9.4.1 How to beat resistance?

A strategy with less selection pressure for mutations of resistance? Our current strategies kill of 99% of mozzies, the only ones that survive are the ones that are resistant, they will rapidly become the majority in a population.

Alternative strategies would let mosquitoes survive long enough to pass on genes, but not long enough to pass on malaria. An example would be an infective fungus that kills off mosquito after a few days.

An other option would be physical barriers such as house screening. Mosquitos here though well develop “behavioural resistance” and would learn to bite outside.

New strategies are new chemicals for in combination with pyrethroids. These are coming soon.

Or chemicals that target mosquito population through sterilisation through damaging the ovaries of the mosquito.

Plus multiple strategies in combination.

17.9.5 Conclusion

Vector control is critical to control diseases

We need to apply the strategies properly, to be effective and stay effective

We need new tools!

17.10 Vaccines

Phil Gothard 4/9/18

Does the elimination of malaria depend on having a vaccine?

If R0 is > 1 then number of infected people will grow until everyone is infected, or natural immunity develops.

We think there is immunity to malaria, we aren’t too sure what it is!

We saw in 1961 when people were given pooled serum with antibiotics from Gambia, that people were able to clear malaria infection faster, protecting from severe infection. So maybe there’s an antibody thing, but malaria is an intracellular parasite, so that’s a bit odd. It’s still not clear what immune compartment is used for protection!

We know that natural immunity is slowly acquired (several years), and quickly lost (several months)

What if a vaccine is short acting, just means they get malaria later in life, and then get severe malaria then? This is a real concern in development of vaccines. Especially as you get less fatal syndromes as an infant (infants still die though!)

Malaria is a complicated parasite with huge strain variation, plus it’s very polymorphic.

If you inject irradiated sporozoites into mice, you can protect them from malaria. And a similar thing is seen in american soldiers, but you need 100,000 irradiated sporozoites and it is less than 10 month protections

17.10.1 Ideal vaccine

  • Complete protection
  • Safe
  • Long lasting
  • Cheap
  • Easy to deliver

17.10.2 Good Enough Vaccine

  • Protects 1/2 population
  • Target key populations
  • Needs to last a whole season
  • Can incorporate in an existing health system

17.10.3 Difficulty in developing a vaccine

The candidate vaccine we currently have targets a pre-erythrocytic stage, giving short term immunity, a sterile immunity

  • Which lifecycle stage?
  • Which antigen?
  • Which immune compartment?
  • What logistics of vaccine?
  • How do you clinically validate it

17.10.4 RTS,S

It is currently in late stage trials

It targets the circumsporozoite protein (CSP) on sporozoites. First identified in 1984

RTS,s was formulated in 1989 (CSP fused with hepatitis B surface antigen, suspended with free Hep B surface antigen)

First evidence of protection from RTS,S in 1997

First phase 2 trial in adults in the gambia in 2001 This gave 34% protection for 8 weeks. This trail was sponsered by GSK, monitored by GSK, WHO, everyone! The drug company sponsoring the trial insisted on storing the data, and doing the analysis! This was later switched to the clinicians studying the trial also doing the analysis.

So then trialed again with an adjuvant ASO2A, with children mozambigue in 2005

Then a new adjuvant AS01E, in children in Tanzania in 2008

Then a large phase 3 trial across Africa in 2015, compared against the rabies vaccine. 11 centres in 7 countries. Several doses up to one year. Followed up 3-4 years. Outcome was “clinical malaria” rather than parasitaemia. The efficacy was greater in children than infants. But still poor 20-30%. But does avoid a lot of cases. Reduced 65 cases per 100 children over four years. More marked in low transmission areas.Effect wanes over 4 years. Associated with a redicution in anti-CSP antibodies.

Still not licensed as a vaccine

17.10.5 Sporozoite vaccine

Small vaccine trial.

135,0000 sporozoites per injection. 4 or 5 vaccinations. Effective.

But an IV vaccine for african children? Not really faesible

17.10.6 Current position

The WHO in 2017

RTS,s/AS01 has had a positive regard. For 4 doses between 5-17 months. In areas for moderate to high transmission.

A large RCT over 4 studies.

We won’t get results until 2020