Use of Serum Amyloid A to Differentiate between Infectious and Non-Infectious Diseases in Horses

CENTER FOR EQUINE HEALTH EQUINE RESIDENT RESEARCH FINAL REPORT; 2016

Resident Name: Dr. Fiona Wensley Department: Equine Internal Medicine

Faculty Advisor:

Name: Dr. Nicola Pusterla Department: Equine Internal Medicine 

Collaborators 

Name: Danielle Carrade-Holt 

Department: Central Lab Receiving

Name: Julie Burges

Department: Central Lab Receiving

Name: Dr. Phil Kass

Department: Veterinary Epidemiology

Name: Kaitlyn James

Department: Veterinary Epidemiology

EQUINE RESIDENT RESEARCH FUNDING PROGRAM; FINAL REPORT 2016

  1. Project Title:                                                            .                              .         .            .

Use of Serum Amyloid A to Differentiate between Infectious and Non-Infectious Diseases in Horses

  1. Investigators:

Dr. Fiona M Wensley BVM&S; Dr. Nicola Pusterla, DVM, PhD, Diplomate ACVIM;  Dr. Danielle

Carrade-Holt, PhD; Julie Burges, MS; Phil Kass, DVM, MPVM, MS, PhD; Kaitlyn James, MPH, PhD

  1. Research Hypothesis:

Serum Amyloid A (SAA) levels alone. o SAA in c:ombinatio.n with the haeatological paameers, including white blood cell count and f1bnnogen , will better d1fferent ate 1nfect1ou from non-infect1 us inflammatory disease . Serum Amy loid A will also help better define the seventy of the underlying disease , as well as detecting a level of subclinical disease prior to the development of abnormal clinical signs and conventional hematological parameters .

  1. Study  Objectives:

The goals of this study are to improve correlations of Serum Amyloid A with plasma fibrinogen, and neutrophil count and to assess the use of SAA as a screening tool to identify infectious disease. The information gained from this study will help the equine practitioner and laboratory diagnostician build trust in the SAA testing offered by Stablelab.

  1. Materials and Methods:

Case selection

314 equine patients presented to the VMTH with the inclusion criteria of a Complete Blood Count (CBC) submission . SAA samples were run on whole blood using the Stablelab Lateral Flow Immunoassay . The horses were classified into 5 groups using clinical and diagnostic modalities from the VMTH Medical records . No reference to the complete blood count or SAA results were used for aid in classification . The 5 groups included healthy (n=36) ; healthy recently vaccinated (n=1O) ; inflammatory , non-infectious (n=106) ; inflammatory , infectious (n=121); and non-healthy , non­ inflammatory  (n=41).

Stastistical Analysis

Non-parametric tests were used for data analysis , including Kruskal-Wallis one-way analysis of variance . Sensitivity , specificity , and positive and negative predictive values were determined. Multivariate logistic regression analysis was used to determine odd ratios. A P value  of <0.05 was considered  significant.

Healthy (n=36)

Healthy recently vaccinated (n=10)

Inflammatory,  Non-infectious (n=106)

Inflammatory, Infectious (n=121)

Non-healthy,

Non-infectious (n=41)

  • Normal physical exam
    • Preoperative castration
    • CEM exam
    • • Within prior 5 days
      • • Osteoarthritis
        • Inflammatory airway disease
        • • Gastric ulceration
          • • Inflammatory bowel disease
          • • Colic; gastric impaction; abnormal abdominocentesis ; torsion; strangulating or ischemic lesions
            • Post-operative
            • Renal injury
            • Non-infected keratitis
            • Uveitis
            • Neonatal maladjustment
            • Hypersensitivity reactions
            • Laminitis
              • • Abscess
              • • Cellulitis
              • • Sepsis
                • • Peritonitis
                • • Septic arthritis
                • • fncisionaf infection
                  • • Enteritis/enterocolitis
                  • • Clostridial myonecrosis
                  • • Thrombophlebitis
                  • • Omphalophlebitis
                    • • Placentitis
                    • • Sinusitis
                    • • Cystitis
                      • • Pyelonephritis
                      • • Pneumonia
                      • • Parasitic infection
                  • • Colic ; enterolithiasis ; mild colon impaction ; spasmodic
                    • • Neoplasia
                      • • PPID
                      • • Routine dentistry
 

Ta. ble 1  Examp es of Group c ass1 tea ton

f. Results:

Demographics

The majority of breeds were warmbloods, thoroughbreds and quarter horses, with no significant difference seen between the groups. Differences in sex and age were seen between the groups , however this was likely due to study design so was not further statistically analyzed . Ages ranged from 0-40 years with 30 foals under 3 weeks of age.

T,able 2 DemographJ.CS

 

Healthy (n=36)

Recently Vaccinated Healthy (n=10)

Inflammatory Non-Infectious (n=106)

 

Inflammatory Infectious (n=121)

Non-inflammatory, non-infectious,  non- healthy (n=41)

Breed

Quarter horse (n=69)

4 (11%)

0 (0%)

21 (20%)

32 (26%)

12 (30%)

Thoroughbred (n=44)

1 (3%)

0 (0%)

19 (17%)

20 (17%)

4 (10%)

Warm blood (n=84)

18 (50%)

9 (90%)

23 (22%)

23 (20%)

11 (26%)

Sex

Mare (n=161)

24 (66%)

9 (90%)

55 (52%)

57 (47%)

16 (39%)

Gelding (n=112)

0 (0%)

0 (0%)

39 (37%)

48 (40%)

25 (60%)

Stallion (n=41)

12 (34%)

1 (10%)

12 (11%)

16 (13%)

0 (0%)

Age

Age range (median)

0-12 (6)

4-11 (8)

0-29 (10)

0-40 (10)

1-31 (14)

Hematological  Variables

The median SAA value for inflammatory non-infectious and inflammatory infectious were significantly different at 9.5µg/ml and 181µg/ml, respectively. Significant  SAA elevations  were  seen  in  the recently vaccinated horses (median of 550µg/mL). Neutrophil count medians were similar between groups, although large ranges were seen in the three unhealthy groups. Fibrinogen values showed increased ranges within both of the inflammatory groups with an increased range up to 1200mg/dl in the inflammatory infectious group.

Table 3. Hematological  Variables. Absolute ranges for SAA, neutrophils, and fibrinogen with the

medJ" an vaIue m. paren thesJ.s, fior aII 5 qrou

S.

 

 

Healthy (n=36)

 

Healthy Vaccinated (n=10)

 

Inflammatory Non-Infectious (n=106)

 

 

Inflammatory Infectious (n=121)

Non-inflammatory Non-infectious  Non- healthy (n=41)

 

SAA

0 - 685

1 - 1046

0 - 1180

0 - 3000

0 - 954

(0-20ug/ml)

(7)

(550.5)

(9.5)

(181)

(8)

                                                  -  -                                 -- -                                              -    

Neutrophil

 

 

2611 - 7054

 

 

3368 - 6814

 

 

627 - 14071

 

 

175 - 22716

 

 

1342 - 12145

s

(2600-

(4784.5)

(4166.5)

(5042)

(4914)

(4793)

6800/µ )

Fibrinogen (S400

mg/dl)

 

200 - 500

(300)

 

200 - 500

(300)

 

100 - 600

(300)

 

100 - 1200

(400)

 

100 - 400

(300)

SAA Distribution

To refine the model, the SAA values were categorized into normal, low, medium and high ranges. SAA was normal for 86% of the healthy group and for 80% of the non-healthy , non-inflammatory group . The recently vaccinated, apparently healthy horses, had a moderate increase in SAA values. High elevations between 1000 and 3000µg/ml were mainly distributed in the inflammatory infectious group (78%), with none in the healthy group . SAA values in the medium or high range correlated well with changes to the white blood cell count and fibrinogen.

 

 

Healthy (n=36)

 

Healthy Vacci nated (n=10)

 

Inflammatory Non-Infectious (n=106)

 

Inflammatory Infectious (n=121)

Non-inflammatory Non-infectious Non- healthy (n=41)

Normal

0-20 µg /ml

31 (86%)

3 (30%)

65 (61%)

48 (40%)

33 (80%)

Low

20-200 µg /ml

3 (8%)

1 (10%)

17 (16%)

15 (12%)

3 (7%)

Med

200-1000 µg /ml

2 (6%)

5 (50%)

16 (15%)

27 (22%)

 

5 (12%)

High

1000-3000 µg /ml

 

0 (0%)

 

1 (10%)

 

8 (8%)

 

31 (26%)

 

0 (0%)

 

Table 4 SAA Distribution 

Neutrophil and Fibrinogen Distribution

The infectious group had the highest level of neutropenic patients at 14%. Unhealthy status was perfectly predicted by a neutropenia with 0% in the healthy group. The healthy and healthy recently vaccinated groups had 89% and 90% of horses within the normal range. Neutrophil count was insensitive as an inflammatory marker shown by the high percentage of unhealthy horses within the normal range. A neutophilia was seen in 31% of the infectious group. An abnormal neutrophil count of the healthy horses versus the unhealthy horses had a significant odds ratio of 0.17. A normal fibrinogen was seen in the majority of groups , with a hyperfibrinogenemia in 44% of the infectious horses. A hyperfibrinogenemia had a statistically significant odds ratio of 0.22 of predicting healthy versus unhealthy horses.

Infectious versus Non-Infectious

The  data  set  was  further  stastically  analyzed  amongst  only the  three  unhealthy groups.  The inflammatory infectious (n=121) compared with the two groups of non-infectious (n=147). When comparing the infectious inflammatory group to the two non-infectious groups , the SAA value was 2.6 times more likely to be within the medium range and 8 times more likely to be within the high range of 1000-3000µ9 /ml (Table 5). A neutropenia is 4.2 times more likely to be of infectious origin with a neutrophilia        at                         2.2   times  more      likely,     in            comparison              to     the     non-infectious groups . Hyperfibrinogenemia is 7.6 times more likely to have an infectious component when compared to the non-infectious groups . The sensitivity of SAA alone was 60% with an overall accuracy of 46% (Table 6). The specificity of the neutrophil count and fibrinogen were higher than the SAA specificity . The combination of an abnormal neutrophil count and hyperfibrinogenemia, improves the sensitivity and

overall accuracy of displaying an underlying infectious process. With the addition of elevated SAA values along with these abnormalities further increases the amount of correctly identified infectious inflammatory disorders, seen by the increased sensitivity of 70%, with the accuracy improved to 59% in comparison to each of these values alone.

 

Infectious (n=121)

 

Non-Infectious Groups (n=147)

 

 

Odds Ratio

 

Confidence Interval

Normal

0-20 µg /ml

 

48 (40%)

 

98 (67%)

1.0

(reference)

Low

20-200 µg /m l

 

15 (12%)

 

20 (13%)

 

1.53

.72,

3.25

Med

200-1000 µg /m l

 

27 (22%)

 

21 (15%)

 

2.65**

1.35,

5.11

High

1000-3000 µg /m l

 

31 (25%)

 

8 (5%)

 

7.9**

3.38,

18.52

 
T.able 5 SAA va ues Infiecf10us vs Non-Infiecf10us Groups

 

 

Sensitivity

 

 

Specificity

 

Positive Predicative Value

Negative Predictive Value

 

Overall Accuracy

SAA

60%

67%

60%

67%

46%

Neutrophil Count

45%

76%

61%

63%

34%

Fibrinogen

45%

90%

79%

67%

25%

Neutropenia, Neutrophilia and/or Hyperfibrinogenemia

 

 

56%

 

 

74%

 

 

64%

 

 

67%

 

 

40%

Neutropenia, Neutrophilia, Hyperfibrinogenemiaa nd/or Elevated SAA

 

 

70%

 

 

50%

 

 

54%

 

 

67%

 

 

59%

 
* =P value <0.05;   ** = P value <0.01 Table 6.  Infectious  versus  Non-infectious
 
 
g. Conclusions:
Serum Amyloid A performance held a greater overall accuracy when compared to neutrophil count and fibrinogen, both individually and combined, for defining infectious versus non-infectious diseases. When used in conjunction with neutrophil count and fibrinogen, serum amyloid A further enhances the accuracy of differentiating disease status. The stall side assay was found to be an excellent screening tool; if an SAA value is moderately (>200µg/ml) to markedly (>1 OOOµg/ml) elevated, abnormalities in the white blood cell count and fibrinogen can be expected. A normal SAA does not rule out inflammation, nor is a high SAA not definitive of infection, as with all hematological parameters, there is high variability among individuals.