Anticipating future emerging infectious diseases through collaborative research: a reflection on

the Zika outbreak

Constância Ayrestans@cpqam.fiocruz.br

Roundtable Meeting on Zika Virus: Improving Detection, Preparedness, and Response, Through Surveillance and

Research

Vector control

Vector control

Integrated control;Sustainable strategies;Permanent;Monitoring through surveillance;Choice of a good tool (sensitivity);

CICLO BIOLÓGICO

15 to 30 days

Qu

iesc

ent

eggs

Dis

per

sed

Ovi

po

siti

on

Passive Dispersion

Biological traits of Aedes aegypti

• Highly antropophilic females;

• Monogamy (breeding can occur in 24 hours after emergence);

• Adapted to the urban environment (preference for premises);

• Prefer temporary breeding sites;

• Colonize reservoirs of potable water (>80% of breeding sites are inside houses).

National Program for Dengue Control(PNCD)

PNCD components

1. Epidemiological surveillance

2. Vector control

3. Patients assistance

4. Integration of Agents (PACS/PSF)

5. Sanitation

6. Social mobilization and comunication

7. Training

8. Legislatioin

9. Social and political approach

10. Evaluation

Methods of control

• Focal – treatment of breeding sites.

• Perifocal – residual treatment of

surfaces.

• Space spray -Ultra Low Volume spray.

Focal treatment (larvicides)

Larvae

Pupae

Adult

Egg Temephos Bti Diflubenzuron Pyriproxifen

Perifocal: Insecticide Residual Spraying (adulticides)

“strategic points”

PNCD guidelines: use of larvicide only when necessary

Powder

Use in the field - aplication

IPE

Space- Spray

Rede Nacional de Monitoramento

- Morena

Regiões

N

NE

SE

S

CO

Laboratório de

Entomologia/CE

Laboratório de

Entomologia CPqAM

(Fiocruz/RJ)

Laboratório de criação de

Aedes aegypti Geraldo

Magella Buralli/SP

(*) Rede Nacional de Monitoramento de Aedes aegypti a Inseticidas

Processo de avaliação para ingresso

de dois novos laboratórios:

1. Medicina Tropical – UnB

2. Universidade Federal do Paraná

Temephos resistance in Brazil

Chediak, M. et al., 2016

2002 - Programa Nacional de Controle da Dengue- PNCD

Aedes aegypti – Histórico de Controle no Brasil

•PNCD measures have not been enough to reduce Ae. aegypti populations, andconsequently, to minimize the endemic-epidemic transmission of dengue,chikungunya and zika;

• Natural Ae. aegypti populations are highly resistant to chemical insecticide;

• Low coverage of larvicide control associated to: high percentage of closedpremises/or refusal to inspection by the Health Agents (pendencies) and a highnumber of vacant lands;

Aedes aegypti control in Brazil

SMCP-Aedes

Pernambuco - Northeastern

Sites – Real-Scale trials

3,000 inhabitants

Area: 17 km2 (15 villages)

Fernando de Noronha Island

.Archipelago 500 Km

87,538 inhabitants

Area: 335.5 km²

Santa Cruz do Capibaribe

Arid region 200 Km

Ipojuca

76,000 inhabitants

Area: 527.3 km²*

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Mosquito surveillance

Sentinel - Ovitrap Network

* Santos et al 2003. Field Evaluation of Ovitraps Consociated with Grass Infusion and Bacillus thuringiensis var. israelensis to determine Oviposition

Rates of Aedes aegypti. Dengue Bulletin, 27: 156-161.

SMCP-Aedes

Fixed sampling stations

262 – Santa Cruz do Capibaribe

76 – Ipojuca Centro

103 – Fernando de Noronha

Period in the field: 30 days

Containing Bti*

The sentinel-ovitrap (S-Ovt)

Araújo et al., 2007; Melo-Santos et al 2009. Long lasting persistence of Bacillus thuringiensis israelensis larvicidal activity in Aedes aegypti breeding sites.

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Social Mobilization - Information

Public health authorities

SMCP-Aedes

Technical staff of health services

Vídeo FIOCRUZ

Community mobilization

Information Flow

Egg collection Counting Data analysis

Intervention Actions Planning Kernel Maps

SMCP-Aedes

21

Control-ovitrap (C-Ovt) Production

Non-Govermental Organization - ICRIAC

collective ovitraps

manufactoring

SMCP-Aedes

* Regis et al 2013. Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian

Cities. PLoS ONE 8(7): e67682. doi:10.1371/journal.pone.0067682.

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Areas with high concentration of mosquito (hot spots).

SMCP-AedesKernel-Maps as decision support

Eg

g D

en

sit

y

Lo

w

Hig

h

Site infestation SMCP-Aedes

Infestation found after >10 years of temephos** or Bti treatments (6 cycles/year) – PNCD.

May 2008

Santa Cruz**

POI = 100%

EDI = 1,597 eggs/trap/month

Ipojuca**

POI = 92.6%

EDI = 561.2 eggs/trap/month

Fernando de Noronha

POI = 84%

EDI = 180,6 eggs/trap/month

• POI: Positive Ovitrap Index• EDI: Egg Density Index (Mean number of eggs/OVT-S/ 30 days)

Jan 2011

Araujo et al 2013. The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for

management. Parasites & Vectors, v. 6, p. 297-305, 2013.

SMCP-Aedes - Sensitivity to detect the A. aegypti presence

Egg laid = presence of blood fed Aedes females

The system detects continuous mosquito oviposition in most sites.

POI = 63 % – 100 %

SMCP-Aedes

* Regis et al 2013. Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian

Cities. PLoS ONE 8(7): e67682. doi:10.1371/journal.pone.0067682.

House Index

Santa Cruz = 3.8 to 12%

Ipojuca = 0 to 0.8%

Fernando de Noronha = 0 to 0.5%

Index based on larval surveys are not sensible enough to revel the true level of infestation by A. aegypti

25

Befo

re

Co

ntr

ol

SMCP-Aedes

* Regis et al 2013. Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian

Cities. PLoS ONE 8(7): e67682. doi:10.1371/journal.pone.0067682.

90% in Santa Cruz

77% in Ipojuca

Egg density

Egg densities decreased progressively during the control intervention period, however the POI remained higher than 80%.

Aft

er

C

on

tro

l

Egg suppression >7,500,000

The tools and strategies used in SMCP-Aedes allowedquantification of the active population of A. aegypti, indicatingperiods and places with high virus transmission risk.

Final Considerations

The methods and tools used motivated the assimilation ofknowledge and encouraged social participation.

SMCP-Aedes16

The strategy using control-ovitrap is cost-effective and compatiblewith other control measures.

Surveillance will enable control programs to:

Identify vectors;

Evaluate the impact of vector control strategies;

Management of insecticide resistance;

Generate risk maps for exposure to arboviruses;

Define priority areas for vector control;

Predict outbreaks.

Why mosquito surveillance is important in SE Asia?

Zika virus has been present in areas of Southeast Asia for manyyears, and several countries have reported occasional cases orsmall outbreaks of Zika virus infections. Zika virus isconsidered endemic in some countries, and a large number oflocal residents are likely to be immune;

infections have occurred among travelers to Southeast Asia; Recent variations have been observed in the number of cases

reported in Southeast Asia. This can reflect changes inawareness of Zika virus, surveillance and testing for Zika virus,or changes in intensity of Zika virus transmission.

CDC

The surveillance efforts are focused on identifyingarbovirus infections in mosquitoes as predictors of thepotential risk of transmission to humans;

to monitor the further expansion of the viruses; To provide base-line information for increment surveillance

programs and initiating control activities in the event that ZIKV is introduced into the region.

Aims:

May 2013 May 2014

Monitoring of Ae. aegypti in the City of Praia, Cape Verde by Positive Ovitrap Index (POI) for 2013 and 2014.

• Demonstration that members of the suspected populationcommonly feed upon vertebrate hosts of the pathogen;

• Demonstration of efficient transmission of the identifiablepathogen by the suspected vectors under controlledexperimental conditions;

• Repeated demonstration that suspected vector collectedunder natural conditions, harbor the identifiable, infectivestage of the pathogen;

• Demonstration of a convincing biological association intime and space between the suspected vectors and clinicalor subclinical infections in vertebrate hosts.

(Barnett, 1962)The incrimination of arthropods as vectors of disease

RT-qPCR results

Electronic Microscopy

ZIKV in salivary gland cell of Culex

ZIKV detected by FTA cards

Ae. aegypti 106

Cx. quinquefasciatus 106

Ae. aegypti 104

Cx. quinquefasciatus 104

Surveillance

Field work

Diagnostic

Analysis and Outcomes

Collection Transportation Storage

Surveillance

Field work material

Aspiration

• indoor

• outdoor

FTA cards

• Inactivate the virus

• RNA extraction

Collection

RNAlater

alive

Transportation

Registration

Sampling proportionality

Need to reduce the error

Need to increase the chance of detection

Vector infection is a rare event

MIR Density

Feedingbehavior

• Number of bites

• mosquito/person

Collection

• Time period

• Samplingmethods

• area

Phase of outbreak

• Few humaninfections

• peak

• post-peak

We need to take into account:

the sampling process has to be adjusted to the most rare event

• MIR

Considering ecological and biological factors

• Time of outbreak, habits

Vector ZIKV Infection Rate

Local Aedes Culex MIR

N Pools ZIKV+ Total Pools ZIKV+ Aedes Culex

Recife-PE (2016) 408 02 1496 03 4.9 2,0

Vitória-ES 14 01 286 04 71.4 13.98

Rio de Janeiro 315 03 385 0 9.5 0

Vector ZIKV Infection Rate

Local Aedes Culex MIR

N Pools ZIKV+ Total Pools ZIKV+ Aedes Culex

Cabo Verde 100 0 20 0 0 0

Mexico 279 15 151 0 53,7 0

Malaysia 1277 01 - - 0,78 -

French Polynesia 2039 01 286 0 0,49 0

Micronesia (Yap) 41.2% 0 28.1% 0 0 0

Gabon 853 0 690 0 - -

Senegal* 250 1 22* 1 4.08 45.45

ZIKV Diagnostic

Nature Reviews Microbiology , S30-S37

Direct and Indirect Laboratory Diagnostic Methods

Detection of ZIKV by RT-qPCR and RT-PCR

Bad!

RT-qPCR

Lanciotti, 2016

Primary x Secondary Flavivirus Infection

ZIKV Serology Summary

Other Samples?

• N=6 patients• ZIKV RNA is detectable in urine at a higher load and with a longer duration than in serum

Serum

Urine

Other Samples?

• 182 patients• ZIKV was more frequently detected in saliva compared to blood.• The use of saliva sample increased the rate of molecular detection of ZIKV at the acute phase of the disease but did NOT enlarge the window of detection of ZIKV RNA.

ZIKV in Semen

•In December 2013, during a Zika virus (ZIKV) outbreak in French Polynesia, a patient in Tahiti sought treatment for hematospermia.

•Eight weeks after first signs of Zika, he described a second episode of symptoms compatible with ZIKV infection

• 2 weeks after second episode: RNA loads were: 2.9 × 107 copies/mL and 1.1 ×107 copies/mL in the first and second semen samples, respectively, and 3.8 ×103 copies/mL in the urine sample. • ZIKV was isolated from his semen.

Sample Collection and Shipment

Refrigerate biological fluids and tissues at 4 °C immediately after collection; Tissue fragments should be fixed in 10% formalin (1 part of tissue + 9 of formalin)

Transport • 4-8oC if transport within 3 days • Freeze at -80°C if storage for weeks or months before processing and shipment to reference laboratory

How to Destroy Your Sample

Keep samples without refrigeration Repeated freeze-thaw cycles

- Destroys IgM- Kills the virus

To avoid hemolysis: do not freeze unseparated blood

Obrigada!

Thanks!