ADVANCES IN DIAGNOSES OF INFECTIOUS DISEASE IN DOGS IN CLINICAL PRACTICE THROUGH Molecular biology POC

DVM VITOR MÁRCIO RIBEIRO

THE CHALLENGE OF MEDICAL PRACTICE

Knowledge Diagnosis Management – Infection stage identification - Treatment Control Prevention

KNOWLEDGE X EXPERIENCE

The only source of

knowledge is

experience

ESSENTIAL ITEMS

Knowledge Diagnosis Management - Infection stage identification - Treatment Control Prevention

DIAGNOSIS

“THE INTERPRETATION OF KNOWLEDGE!”

WHAT ARE WE SEEKING TO DIAGNOSE?

The disease

Infected animal with clinical manifestations caused by the infectious agent

The infection Animal showing no manifestation of clinical signs of an infectious agent in its system

WE DIAGNOSE WE CHECK

The presence of an agent The stage of the disease

◦ Ehrlichia canis Ehrlichiosis

◦ Babesia vogeli Babesiosis

◦ Leishmania infantum Leishmaniasis

among others among others

HOW WE DIAGNOSE I - INFECTION

INDIRECT METHODS Antibody Search – "It doesn't always confirm the infection"

INDIRECT IMMUNOFLUORESCENCE ASSAY (IIFA)

Sample used BLOOD / SERUM / PLASMA

INDIRECT ELISA - Rapid Test / Plate CEREBROSPINAL FLUID

IMMUNOCHROMATOGRAPHY URINE

TISSUES

HOW WE DIAGNOSE I - INFECTION

DIRECT METHODS Antigen Search – “Confirms the infection”

DIRECT IMMUNOFLUORESCENCE ASSAY (DIFA)

DIRECT ELISA - Rapid Test / Plate

IMMUNOCHROMATOGRAPHY

CYTOLOGICAL / HISTOLOGICAL / ELECTRON MICROSCOPY EXAMINATION

CELL CULTURE AND ISOLATION

POLYMERASE CHAIN REACTION (PCR)

XENODIAGNOSIS

Sample used

BLOOD / SERUM / PLASMA

CEREBROSPINAL FLUID

URINE

STOOL

TISSUES

HOW WE DIAGNOSE II - DISEASE LINKED TO INFECTION

INFECTION STAGE HEMATOLOGICAL TESTS

BLOOD COUNT

BONE MARROW EXAMINATION

Sample used

WHOLE BLOOD

BONE MARROW

HOW WE DIAGNOSE II - DISEASE LINKED TO INFECTION

INFECTION STAGE BIOCHEMICAL ANALYSIS

KIDNEY FUNCTION - Urinalysis, urinary protein, SDMA, urea, creatinine

LIVER FUNCTION - Bile acids, ALT, GGT, FA

SERUM PROTEINS - Albumin, globulins

C-REACTIVE PROTEIN

Sample used

BLOOD PLASMA

URINE

BODY FLUIDS

HOW WE DIAGNOSE II - DISEASE LINKED TO INFECTION

INFECTION STAGE

MEDICAL IMAGING

DOPPLER ULTRASOUND

X-RAY

MEDICAL ULTRASOUND CT/RMI

NEEDS AND ADVANCES

MORE SENSITIVE ANALYSES

SPECIFIC ANALYSES FASTER METHODS AVAILABLE TECHNIQUES SUITABLE COST

MOLECULAR DIAGNOSIS

PCR

The term “molecular diagnosis” refers to tests that detect nucleic acids (DNA / RNA)

The methods most commonly used by diagnostic laboratories for detection and characterization of nucleic acids are:

◦ 1. Polymerase Chain Reaction (PCR) ◦ 2. Quantitative Real-Time PCR (qPCR) ◦ 3. Reverse Transcription PCR (RT-PCR) ◦ 4. Duplex and Multiplex Real-Time PCR ◦ 5. DNA Sequencing

IS MOLECULAR DIAGNOSTICS A SOLUTION?

Molecular diagnostic tests offer some advantages depending on the infection and the course of the disease, but it is essential to understand their limitations Clinicians must properly order molecular diagnostic tests according to the course of the disease Other diagnostic tests should not be abandoned because they can provide valuable incidental information

PCRs

Offer increased sensitivity and specificity Detect minimal levels of infectious agents (DNA/RNA) Are suitable for infectious disease diagnostic panels

Have short implementation times (Point of care (POC))

INFECTION DIAGNOSTIC PANEL POC PCRun

1 - Ehrlichia canis

2 – Anaplasma platys

3 – Babesia canis

4 – Babesia gibsoni

Are better able to diagnose subclinical infections There is a need to improve techniques that allow Lower costs

Smaller laboratory structure

Large-scale utilization

WHAT HAPPENS IN AN INFECTION

VIRAL INFECTION

The viral messenger RNA enters the ribosome causing the cell to begin producing the proteins of

viral reproduction Viruses are obligate intracellular parasites: the lack of hyaloplasm and ribosomes prevents them

from having metabolism In their life cycle, viruses need a cellular environment containing ribosomes and other necessary

substances to synthesize viral proteins, allowing multiplication of their genetic material

VIRAL INFECTION

Viruses modify the metabolism of the cell they parasitize, which can cause cell degeneration and death Viruses penetrate cells or are phagocytosed by them, thus exposing viral genetic material

Canine Distemper Virus

CLINICAL CASE

available at:

https://www.researchgate.net/publication/14641773_A_canin

e_distemper_virus_epidemic_in_Serengeti_lions_Panthera_leo

[accessed December 8, 2020].

Extreme climatic conditions can change historical relationships between host and pathogen, and synchronize temporal and spatial convergence of multiple infectious agents, triggering more deadly epidemics than those due to single pathogens.

In 1994, A CDV epidemic affected Serengeti lions (Panthera leo), killing 1/3 of the population In 2001, a second high-mortality CDV epidemic hit the lion population of Ngorongoro Crater

Serological analyses indicated that at least five “silent” CDV epidemics had

swept these two lion populations between 1976 and 2006 with no clinical signs or

measurable mortality, indicating that CDV was not necessarily fatal Clinical and pathological findings suggest that hemoparasites represent the

main contributing factor to the fatal epidemics

Using quantitative real-time PCR, we measured the magnitude of infections with hemoparasites in these populations over 22 years Significantly high levels of Babesia were observed during the 1994 and 2001 epidemics when, in addition to CDV exposure within the groups, conditions of extreme drought along with high mortality of herbivores and high outbreaks of ticks were also observed Lions were infected by an unusual number of Babesia parasites, which were more pathogenic due to the immunosuppressive effect of CDV co-infection, leading to high mortality These mass mortality events may become increasingly common if extreme weather changes disrupt the stable historical relationship between coexisting pathogens and their susceptible hosts

VIRAL AGENTS

Virus name Occurrence Disease severity

Adenovirus type 1 ? Severe

Canine coronavirus Rare? Mild

Canine Distemper Virus Common Mild to severe

Canine Herpesvirus 1 ? Mild in adults and severe in puppies

Canine infectious respiratory disease (kennel cough) Common Mild to moderate

Canine papillomavirus ? Mild

Parvovirus Common Serious

Aujeszky's disease (pseudorabies) ? Serious

Rabies Rare Serious

WHATS HAPPENS IN AN INFECTION

BACTERIAL INFECTION

INTRACELLULAR BACTERIA

Chlamydia spp., Anaplasma spp., Ehrlichia spp., Rickettsia spp., Orientia spp. and Coxiella spp. replicate exclusively within eukaryotic host cells

EXTRACELLULAR BACTERIA

Extracellular bacteria are free-living organisms

BACTERIAL AGENTS

Bacteria name Occurrence Disease severity

Anaplasma platys Common Mild to severe

Ehrlichia canis Common Mild to severe

Bordetella bronchiseptica Common Mild

Leptospira interrogans Common Severe

Brucella canis Rare? Moderate to severe

Rickettsia rickettsii Rare? Moderate to severe

Borrelia burgdorferi Rare? Moderate to severe

E

Ehrlichia sp

A single organism adheres to the plasma membrane (Figure 1), enters the cell by membrane invagination, and forms morulae after binary fission.

These elementary corpuscles can leave the cells by breaking through cell membranes or by exocytosis.

Ehrlichia sp may not destroy the cell - after its multiplication, it is released by the cell, which remains unharmed Ehrlichia sp secretes proteins that immunomodulate the infected cell as well as neighbouring cells avoiding its activation, to infect all of them successively

Pathogeny - Ehrlichiosis

After an incubation period of 8 to 20 days, three stages of Ehrlichiosis

disease follow:

* Acute phase * Subclinical stage * Chronic phase

Samples for testing

Blood (Peripheral blood / Bone Marrow - BM)

Serum

Cerebrospinal Fluid - predisposition to bleeding

Tissues

Diagnosis

• Anamnesis

• Signs and symptoms

• Laboratory

– Thrombocytopathies

]

– Serology Screening / Disease stage determination

– Serum proteins

] Confirmation

– Morula identification

– Conventional PCR / PCRun or Real-Time PCR

Diagnosis - Ehrlichiosis / Anaplasmosis

• PCR and positive IIFA suggest acute phase

• PCR positive and IIFA negative – beginning of infection

• PCR negative and IIFA positive

– Chronic phase

– Asymptomatic

– Microbiological cure

CLINICAL CASE

14 months ago

Tick disease - positive serology Ehrlichia canis / negative BM cytology Since then - non-regenerative anemia and thrombocytopenia Treatment - 2 series of doxycycline 10 mg/kg bid - 28 days Immunosuppressants (Prednisone)

On the day of the visit: Sudden blindness - intraocular hemorrhage Severe pancytopenia Treatment - azathioprine

Exames

Tempo de coagulação aumentado

Ehrlichia canis

Prescrição

Mantida a medicação

19/06/2019Controle

09/07/2020

Three drugs are indicated:

◦ Doxycycline (first-line treatment) ◦ Minocycline and Rifampicin (second-line treatments)

The treatment allows

◦ Clinical cure in most cases ◦ However, it is not always effective in clearing the infection.

Diagnosis - Ehrlichiosis / Anaplasmosis

• PCR and IIFA positive suggest Acute Phase (OCCASIONAL CHRONIC PHASE)

• PCR positive and IIFA negative – beginning of infection

• PCR negative and IIFA positive – Chronic phase – Asymptomatic – Microbiological cure

Reviews – disease stage determination

Review relapses

Possibility of maintenance of the infection - RECIDIVISM

Follow-up by quantitative serological analyses can be a guide

Blood PCR to control parasitemia

Leptospira interrogans

Leptospirose canina

Green, 4th edition, 2012

CLINICAL CASE

29/11/2020

PCRun™

29/11/2020 29/11/2020

PROTOZOAN AGENTS

Bacteria name Occurrence Disease severity

Babesia vogeli Common Mild to severe

Babesia gibsoni Common Mild to severe

Leishmania infantum Common Mild

Rangelia vittali Common Severe

Trypanosoma sp Rare? Moderate to severe

Giardia lamblia (assemblies) Rare? Moderate to severe

Hepatozoon canis Rare? Moderate to severe

WHAT HAPPENS IN AN INFECTION

Different protozoa parasitize different cells:

Two main parasites

Babesia sp - parasitize red blood cells

Leishmania sp – parasitize cells of the mononuclear phagocytic system (MPS)

◦ Others - Rangelia vitalli, Giardia lamblia, Trypanosoma sp, Hepatozoon canis

Babesia sp

Espécies encontradas no Brasil• Babesia vogeli

• Babesia gibsoni

• Babesia rossi

B. gibsoni

Imidocarb dipropionate and diminazene aceturate are considered ineffective for treating

infections with B. gibsoni. Thus, the combination of atovaquone and azithromycin is the current

choice for treating this infection. However, suppression of parasite replication by this last

combination may not be associated with parasite clearance (Jefferies et al., 2007b; Lin et al., 2012,

Kirk et al., 2017)

Conclusions

Treated dogs must be followed up and monitored by blood tests and PCR to

determine persistence of infection and parasitemia. In addition, new and

supplementary drugs and the synergistic effects of current drug combinations

against infection with Babesia should be studied to find effective treatments for

canine babesiosis.

CASO CLÍNICO

CLINICAL CASE

Name of the Animal: SHEDY

Age: 6 years, one month

Breed: American Cocker Spaniel

Sex: Male

Weight: 10.6 kg

Species: Canis

Regional Board of Veterinary Medicine No

(CRMV/MG) 18397

Medical indication: Samples collected for

laboratory tests

PROGRESS NOTES

The owner states that the pet had cystitis, liver

inflammation and gallbladder with biliary

sludge. Patient took Flagyl for 15 days, as well

as omeprazole and ursocol. The patient was

followed up by Dr. [name not legible]

The animal came for sample collection, and a

quotation was authorized yesterday. The animal

will be released after sample collection.

Today the animal is fine, eating balanced liver

dog food with selective appetite. The urine was

brownish, but now it is just concentrated. Feces

were doughy yesterday, but are more solid

today.

PCRun™

PCRun™

CLINICAL CASE

Patient Record

The animal has been in a temporary shelter for 15 days. He was a street dog. Two

doses of Vermifuge were administered, the last one 10/25/2020. The dog was

fine until then but is now showing doughy feces. Liquid feces started yesterday,

and he started having diarrhea with blood this morning. The dog is also very

lethargic and vomiting. He is dehydrated.

Babesia vogeli // Babesia gibsoni

Leishmania infantum

• Clinical disease – 13%

• Seropositives – 26%

• PCR positive – 63%

• 67% - sum of positives per serology or by PCR

IMPORTANT!

The study shows that The prevalence of infection by Leishmania in an enzootic area is greater than assumed The main tissue’s parasite reserve was the dog's skin This result demonstrates the importance of sensitive tests that detect both etiological agents and if animals have immunological protection

Distribution of canine leishmaniosis in endemic focus:

Dogs that show clinical signs of the disease (orange). Seropositive dogs without signs of illness (beige). Dogs with clinical disease, asymptomatic seropositive,

and seronegative are PCR+ (purple) The fourth subset includes dogs that are seronegative

and do not harbor parasites (blue).

INFECTION WITH L. infantum

• Half of the serological information in a study using eight different serological tests in a clinical cohort with suspicion of Leishmania canis infection proved inconclusive, offering an admixture of seropositive and seronegative data

Rapid Tests (RTs) have greater sensitivity in serum samples with higher antibody titers than the IIFA

TRs exhibited false negatives in samples with low antibody titers

The use of RTs for diagnosis of Visceral Leishmaniasis (VL) in dogs with low antibody titers, such as asymptomatic dogs, should be carefully evaluated - vaccination criteria

IIFA with the highest number of false positives

OBJECTIVE:

◦ This study aimed to assess the accuracy of ◦ TR-DPP (Biomanguinhos®) ◦ EIE Canine-Visceral-Leishmaniasis-Biomanguinhos (EIE-CVL) (Biomanguinhos®) ◦ Enzyme immunoassay (ELISA) rK39 (in-house) ◦ Direct agglutination test (DAT-Canis)

◦ Reference standard comprising parasitological and molecular techniques.

123 predominantly asymptomatic dogs

◦ Residents in an CVL enzootic area

ONE HUNDRED TWENTY-THREE DOGS – 54 NEGATIVES / 69 POSITIVES

TEST SENSITIVITY % Specificity %

95% CI

95% CI

RT-DPP 21.74 92.59

EIE-CVL 11.59 90.74

ELISA Rk39 37.68 83.33

Dat-Canis 18.84 96.3

CONCLUSION

◦ Need to improve tests’ sensitivity and specificity for asymptomatic dogs

VL diagnosis is still a challenge due to the lack of a sensitive technique, especially in different prevalence

scenarios. This study demonstrated that real-time PCR identified the presence of DNA from Leishmania in

asymptomatic dogs that tested negative in serological tests recommended by the official Brazilian protocol

for CVL Our results reinforce that the molecular method is essential for confirming the diagnosis of CVL, especially in

asymptomatic animals from non-endemic regions.

CLINICAL CASE

25/02/2014

09/04/2014 29/08/2014

Demais exames:

• Imunocromatografia de pele – negativo

• Cultura de MO – negativo

06/05/2015

11/05/2015

AVANÇO NOS DIAGNÓSTICOS MOLECULARES - PAINÉIS

CLINICAL CASE

PROGRESS RECORD

DATE OF LAST VISIT 10/15/2020 EVOLUTION

INFORMATION

ANIMAL IS CLOSE TO NORMAL, WALKING

NIMBLY, AS SEEN IN THE VIDEO. IT IS EVEN

RUNNING AWAY FROM MEDICATIONS.

STOOLS ARE IN A DOUGHY PATTERN WITH

BLOOD.

CURRENT TREATMENT AND MEDICATION

CURRENT MEDICATIONS:

TobraDex drops - apply in the right ear - 3 drops

every 8 hours (START ON 10/09/2020) //

prednisolone 3 mg/ml - 1 ml by mouth once a day -

this dose is half of what patient took in the last 30

days, it started on 10/09/2020 and will be maintained

until 10/30/2020 // Famotidine 3 mg + ondansetron /

SAM-e 180 mg sid / Penvir 125 / Acetylcysteine +

Vitamin E.

MENTAL STATE

• LEVEL OF AWARENESS (Brainstem and Cortex -

ARAS)

THE ANIMAL INTERACTS BETTER THESE

DAYS. NO CHANGES REGARDING HEARING

AND SEEING. YESTERDAY IT BEHAVED

QUIETER THAN TODAY PROBABLY BECAUSE

OF DEFECATION, UPON OWNER

CONSIDERATION.

The most used molecular tests for detection of nucleic acids are:

◦ 1. Polymerase Chain Reaction (PCR) ◦ 2. Quantitative Real-Time PCR (qPCR) ◦ 3. Reverse Transcription PCR (RT-PCR) ◦ 4. Duplex and Multiplex Real-Time PCR ◦ 5. DNA sequencing

New times

Molecular microbiology has revolutionized the field of microbiology by reducing test response time from weeks to HOURS Provided elevated SENSITIVITY - SPECIFICITY -

QUANTIFICATION Molecular assays provide improved DIAGNOSIS in suspected bacterial, viral, and protozoan infectious events

Molecular microbiology approaches such as POLYMERASE CHAIN REACTION (PCR) seek to: Detect targeted portions of genetic material (DNA or RNA) from biological samples The material is amplified - producing large numbers of copies and detecting even very small amounts of microbial genetic material

In general, these molecular methods are considered Moderately or very complex

• Require extensive training

]

This makes them unfeasible in many labs.

• Sterile technique

• Post-analytical assessments

• Take hours to execute

INNOVATION

More recent sample-to-response tests are simplified and involve minimal processing and hands-

on time. With these simplified molecular assays, more technologists and laboratories perform these tests

outside clinical microbiology laboratories, increasing their use and the number of people involved in

the process.

HENCE

The POINT OF CARE molecular diagnosis currently allows: Research on the main day-to-day etiological agents Results available in less than two hours Carried out at the clinic itself After screening with rapid tests, the possibility of confirmation by PCR POC in a short time

The prognostic implication of an adequate treatment guided by quick molecular diagnosis and

monitoring of the “cure” (QUALITATIVE / QUANTITATIVE ASSESSMENT)

POC ON CANINE INFECTIOUS AGENTS - PCRun

1. Canine molecular detection Kit of Ehrlichia canis

2. Molecular detection kit of Anaplasma platys

3. Canine molecular detection kit of Babesia canis

4. Canine molecular detection kit of Babesia gibsoni

5. Molecular detection kit of Leptospira canine pathogen

6. Molecular detection kit of Leishmania infantum

7. Canine Parvo Virus Molecular Detection Kit

8. Canine Distemper RNA Molecular Detection Kit

POC ON FELINE INFECTIUOS AGENTS - PCRun

1. Molecular detection of feline panleukopenia virus 2. Molecular detection of feline Mycoplasma haemophelis 3. Molecular detection of Feline FeLV DNA Provirus 4. Feline FeLV RNA Provirus Detection

MATERIAIS P/ ENVIO

INCLUSIVIDADE / EXCLUSIVIDADE

DON’T FORGET!

CONCLUSIONS

ADVANCES HAVE PROVIDED GREATER POSSIBILITIES FOR DIAGNOSIS TRY TO ASSOCIATE METHODS, AND ALWAYS SEEK TO INTERPRET THEM FOR DECISION MAKING CLINICS AND HOSPITALS CAN USE ADVANCED AND SIMPLE IMPLEMENTATION TECHNOLOGIES THESE METHODOLOGIES HAVE INCREASED THE SPEED AT WHICH RESULTS ARE PROVIDED - BEFORE THEM WE HAD GREAT DIFFICULTY AGILELY PROVIDING RESULTS

MOLECULAR EXAMS MUST BE INCLUDED IN THE STAGE DETERMINATION OF ALL DISEASES

ADVANCES NEVER STOP ....

ribeirovm@yahoo.com.br