DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS

The incidence of EBV-associated infectious mononucleosis in the United States has been estimated to be 1.6-99 cases per 100,000 persons, whereas the prevalence of EBV infection reaches >90% of the entire population by the third decade of life. Therefore, a number of primary EBV infections are clinically silent or atypical. The classic triad of symptoms of acute infectious mononucleosis--fever, pharyngitis, and lymphadenopathy--is well known, but infectious mononucleosis often displays atypical presentations that are difficult to diagnose. Among 17 infants with serological evidence of current or recent primary EBV infection, none developed signs or symptoms that were suggestive of acute infectious mononucleosis. The initial diagnoses included respiratory tract infection (in 43% of patients), otitis media (in 29%), pharyngitis (in 21%), and gastroenteritis (in 7%).

Recently, we  and others reported the application of automated hematologic analysis to the identification of atypical and apoptotic lymphocytes in peripheral blood samples. Here, we describe 17 cases of atypical and 11 cases of typical infectious mononucleosis that were diagnosed by use of automated hematologic analysis applied to the screening of atypical and apoptotic lymphocytes in peripheral blood samples.

PATIENTS AND METHODS
From April 1996 through March 1999, all blood samples (21,553) obtained at Neagari Hospital (Ishikawa, Japan) as part of routine medical care were subjected to automated hematologic analysis for the identification of atypical and apoptotic lymphocytes. By use of this method, we identified 28 patients with atypical and apoptotic lymphocytes in peripheral blood samples. In 11 patients, 2 or 3 elements of the classic triad of infectious mononucleosis were present and the attending physician suspected infectious mononucleosis from a review of patient history and physical examination. We have defined these 11 cases as "typical infectious mononucleosis." In the remaining 17 patients, 0 or 1 of the classic triad was present and the attending physician failed to suspect infectious mononucleosis from a review of patient history and physical examination. We defined these 17 cases as "atypical infectious mononucleosis." Serological testing for viruses was performed for 15 of these 17 patients (table 1).

Peripheral blood smears, obtained by use of the wedge-pull smear technique and stained by use of the May-Grunwald-Giemsa method, were examined by means of light microscopy. Apoptotic lymphocytes were identified morphologically by loss of cell volume, chromatin condensation along the nuclear membrane with intensely basophilic staining, and/or nuclear fragmentation into spherical structures that were surrounded by nuclear membrane and that contained condensed chromatin. For visual WBC differential determinations, 500 cells were counted. Blood differential counts were obtained by use of the MAXM A/L automated hematology analyzer (Beckman-Coulter). Cell volume, conductivity, and light scatter were measured by use of low-frequency direct current and high-frequency electromagnetic probes combined with laser light. Serum antibody titers to EBV capsid antigen and nuclear antigen were determined by immunofluorescence by means of standard techniques. The presence of IgM antibodies to viral capsid antigen in the absence of measurable antibodies to EBV nuclear antigen was considered evidence for primary EBV infection. Antibody titers to CMV were detected by use of ELISA. Primary infection was diagnosed by the presence of IgM antibody to CMV. Group differences were calculated by use of the Mann-Whitney test.

RESULTS
Automated hematologic analysis of peripheral blood samples that were obtained as part of routine medical care identified atypical and apoptotic lymphocytes in 28 patients. Among these 28 patients, 17 had sought medical attention for an acute illness, and the attending physician had not suspected infectious mononucleosis. Clinical presentations and laboratory test results for these 17 patients, defined here as "atypical infectious mononucleosis," are summarized in table 1. Eleven of these 17 patients had one of the symptoms of infectious mononucleosis from the classic triad of fever, pharyngitis, and lymphadenopathy, whereas 6 patients had none of the symptoms. One patient, who had initially presented to the hospital with fever alone, returned to the hospital with lymphadenopathy 3 days after discharge from the clinic, at which time the clinical diagnosis of typical infectious mononucleosis was made. The clinical diagnoses made at presentation included upper respiratory tract infection in 8 patients, fever of unknown origin in 3 patients, gastroenteritis in 2 patients, and tonsillitis, drug allergy, hepatitis, and asthma in 1 patient each. Serological testing revealed primary EBV infection in 8 and primary CMV infection in 3 of the 17 patients (table 1). Two patients had varicella-zoster virus or rotavirus infections diagnosed on the basis of clinical observations. In the other 4 patients, the etiologies remained undefined: each of 3 patients tested serologically negative for EBV, CMV, and hepatitis B virus, and 1 patient was not tested for these agents. Selected clinical and laboratory data regarding these 17 cases, defined here as atypical infectious mononucleosis, are summarized in table 2. All patients recovered rapidly from their illness.

The remaining 11 patients, who had evidence of atypical and apoptotic lymphocytes determined by means of automated hematologic analysis, had sought medical attention for an acute illness, and the attending physician had made a clinical diagnosis of infectious mononucleosis. Selected clinical and laboratory data regarding these 11 cases, defined here as "typical infectious mononucleosis," are summarized in table 2. Nine of the 11 patients had 3 of the symptoms of infectious mononucleosis from the classic triad, whereas 2 patients had 2 of the symptoms. For 9 of these 11 patients, there was serological evidence of primary EBV infection with IgM antibody to viral capsid antigen but no antibody to EBV nuclear antigen. For 1 patient, there was serological evidence for convalescent primary infectious mononucleosis with IgG antibodies to viral capsid antigen and EBV nuclear antigen, but no IgM antibodies to viral capsid antigen and no antibodies to CMV or hepatitis B or C viruses. Serological studies were not performed for 1 patient.

All patients with typical or atypical infectious mononucleosis had abnormal elevations in levels of aspartate and alanine aminotransferase and lactate dehydrogenase, WBC count, and percentages of lymphocytes, atypical lymphocytes, CD8+ lymphocytes, and HLA-DR-positive CD3+ lymphocytes, and they had decreases in percentages of CD4+ lymphocytes (table 2). In general, these laboratory abnormalities did not significantly differ (P > .05) between patients with typical infectious mononucleosis and those with atypical infectious mononucleosis. However, the percentages of CD8+ lymphocytes were significantly higher among patients with typical infectious mononucleosis than they were among those with atypical infectious mononucleosis (P < .05). Also, visual inspection of atypical lymphocytes in peripheral blood smears failed to show morphological differences between patients with atypical and patients with typical infectious mononucleosis (figure 1).

A striking feature of the CD45RO+ atypical T lymphocytes in the peripheral blood of patients with infectious mononucleosis is that they spontaneously and rapidly died of apoptosis when removed from the patients and incubated in vitro. Immediately after the blood was drawn, peripheral blood smears revealed that atypical lymphocytes constituted 9.5%, 10.7%, and 1.2% of the WBC in patients with atypical infectious mononucleosis, patients with typical infectious mononucleosis, and healthy adults, respectively (table 2). After the blood was held for 4 h at 37 Celsius, apoptotic lymphocytes constituted 8.2%, 8.3%, and 0.9% of the WBC in patients with atypical infectious mononucleosis, patients with typical infectious mononucleosis, and healthy adults, respectively (table 2). The percentage of apoptotic lymphocytes in patients with either atypical or typical infectious mononucleosis was, on the average, significantly higher than that in healthy adults. However, percentages of apoptotic cells in patients with atypical infectious mononucleosis were not significantly different from those in patients with typical infectious mononucleosis. By means of visual inspection, apoptotic lymphocytes in blood smears from patients with atypical and patients with typical cases displayed no morphological differences (figure 1).

On the MAXM analyzer, the atypical lymphocytes appeared as an upward-spreading lymphocyte cluster, whereas the apoptotic lymphocytes appeared as a new cluster under the lymphocyte cluster in VOLUME/DF1 scattergrams. By visual inspection, atypical and apoptotic lymphocyte dusters from patients with atypical infectious mononucleosis were indistinguishable in the scattergrams from those from patients with typical infectious mononucleosis (figure 2).

DISCUSSION
In this study, we report the diagnosis, clinical presentation, and clinical course of atypical cases of infectious mononucleosis that were not suspected to be infectious mononucleosis by the attending physicians. During a 3-year period, we diagnosed 17 cases of atypical infectious mononucleosis and 11 cases of typical infectious mononucleosis through the screening of peripheral blood samples for the presence of atypical and apoptotic lymphocytes. This screening was performed by use of automated hematologic analysis applied to the detection of atypical and apoptotic lymphocytes. Atypical lymphocytosis was reported to have a high degree of sensitivity and an even greater degree of specificity for the diagnosis of acute infectious mononucleosis. Therefore, the application of automated hematologic analysis to the detection of atypical and apoptotic lymphocytes may represent an effective screening test for the diagnosis of atypical cases of infectious mononucleosis.

On the basis of the relationship between the incidence of EBV-associated acute infectious mononucleosis and the prevalence of EBV infection [1,2], the number of patients with atypical cases of infectious mononucleosis could be greater than the number of patients with typical infectious mononucleosis. However, it has often been difficult to diagnose infectious mononucleosis or even suspect infectious mononucleosis from a review of patient history and physical examination when infectious mononucleosis has atypical presentations. Often no symptoms or only individual symptoms from the classic triad of fever, lymphadenopathy, and pharyngitis are present. Among the 17 patients with atypical infectious mononucleosis reported here, 6 patients had no symptoms whereas 11 patients had 1 symptom from the classic triad. The clinical diagnosis at presentation was upper respiratory tract infection in 53% of patients with atypical infectious mononucleosis described here. This presentation was reported at a similar frequency in a previous retrospective analysis of primary EBV infection in infants. Of interest, only 1 of the 17 cases of atypical infectious mononucleosis was later diagnosed as typical infectious mononucleosis by the attending physician on the basis of the development of new symptoms. Therefore, we suspect that only a few of the cases of atypical infectious mononucleosis became typical infectious mononucleosis during the course of the illness.

Laboratory findings often prove critical to the screening of atypical infectious mononucleosis, which is consistent with the observation that infectious mononucleosis was originally described as "mononuclear leukocytosis" in children . In this study, we found that laboratory abnormalities, including hematology and chemistry, were similar in nature and magnitude among patients with atypical and those with typical infectious mononucleosis. Therefore, in the context of atypical clinical presentations, laboratory abnormalities should be carefully evaluated for evidence of infectious mononucleosis.

Prompt communication between the hematology laboratory and the attending physicians resulted in the diagnosis of atypical infectious mononucleosis for 17 patients for whom the diagnosis of infectious mononucleosis was not suspected. As a consequence, no additional tests and no therapy were prescribed. However, retrospective analysis of patients with primary EBV infection that had been proven by means of serological testing has revealed that unnecessary diagnostic procedures and medical treatments were entertained and great medical costs ensued . On average, Is similar to $12,000 was spent in health costs per patient, and 33% of the patients were hospitalized. Tests performed for diagnostic workups included rheumatologic profiles, tumor markers, bacteriologic cultures, chest radiographs, abdominal sonograms, radionuclide scans, and biopsies. In 53% of the patients, antibiotic treatments were prescribed. In 12% of the patients treated with beta-lactam antibiotics, a maculopapular rash developed.

This study was not designed to evaluate the potential cost-saving benefits from automated hematologic analysis to the diagnosis of infectious mononucleosis, nor was it designed to evaluate the specificity and sensitivity of this technology. However, the current results document the potential utility of automated hematologic analysis to the screening of infectious mononucleosis and suggest that additional studies should be considered. Early screening for infectious mononucleosis can result not only in the reduction of health care-related costs but also in prompt relief being provided to the patients.

Appropriate informed consent was obtained from the patients and their patients or guardians, and clinical research was conducted in accordance with guidelines for human experimentation as specified by the US Department of Health and Human Services.

Table 1. Clinical and laboratory parameters for 17 patients with atypical infectious mononucleosis (IM).
Legend for Chart:

A - Patient no.
B - Sex, age
C - Clinical days[a]
D - Symptoms
E - Clinical diagnosis
F - Serum antibodies (titer)
G - Final diagnosis

A     B                                     C
       D                                     E
       F                                     G

1   M, 10 months                             7

       Fever                                 URTI

       VCA IgM (40); VCA IgG (40)            EBV IM

2   M, 2 years                               9

       Cough                                 URTI

       VCA IgM (>40); VCA IgG (1280)         EBV IM

3   F, 2 years                               6

       Fever, cough                          URTI

       VCA IgM (40); VCA IgG (320)           EBV IM

4   M, 3 years                               5

       Fever, cough                          URTI

       VCA IgM (160); VCA IgG (430);         EBV IM
       EBNA-negative

5   M, 4 years                               5

       Fever, diarrhea                       Enteritis

       VCA IgM (>40); VCA IgG (1280);        EBV IM
       EBNA-negative

6   F, 6 years                               6

       Low-grade fever                       FUO

       VCA IgM-positive; VCA IgG-positive    EBV IM

7   M, 10 years                              2

       Skin eruption                         Drug allergy

       VCA IgM (40); VCA IgG (320);          EBV IM
       EBNA-negative

8   E 32 years                               30

       Cough                                 Asthma

       VCA IgM (40); VCA IgG (160)           EBV IM

9   M, 22 years                              28

       Fatigue, headache                     URTI

       VCA IgM-negative; VCA                 CMV IM
       IgG-positive; CMV IgM-positive;
       CMV IgG-positive

10  M, 27 years                              30

       Epigastric discomfort                 Acute hepatitis

       VCA IgM (10); VCA IgG (1280);         CMV IM
       EBNA-positive; CMV IgM-positive;
       CMV IgG-positive; HBsAg-negative;
       HCV antibody-negative

11  M, 39 years                              4

       Malaise, pharyngitis,                 URTI
       abdominal pain

       VCA IgM (<10); VCA IgG (1280);        CMV IM
       EBNA-positive; CMV IgM-positive;
       CMV IgG-positive

12  F, 8 months                              6

       Fever, cough, vomiting,               Rotavirus enteritis
       diarrhea

       ND                                    Rotavirus IM

13  M, 1 year                                5

       Fever                                 FUO

       ND                                    VZV IM

14  F, 8 months                              6

       Fever, cough, skin                    URTI
       eruption

       VCA IgM (<10); VCA IgG (<10)          Unknown IM

15  F, 1 year                                1

       Cough, pharyngitis                    URTI

       VCA IgM (<10); VCA IgG (160);         Unknown IM
       EBNA-positive; CMV
       IgM-negative; CMV IgG-negative

16  F, 4 years                               4

       Lymphadenopathy,                      Tonsillitis
       cough, rhinorrhea

       VCA IgM (<10); VCA IgG (320);         Unknown IM
       EBNA-positive

17  M, 5 years                               7

       Fever                                 FUO

       VCA IgM (<10); VCA IgG (320);         Unknown IM
       EBNA-positive

NOTE. CMV, cytomegalovirus; EBNA, Epstein-Barr virus nuclear
antigen; EBV, Epstein-Barr virus; FUO, fever of unknown origin;
HBsAg, hepatitis B virus surface antigen; HCV, hepatitis C
virus; ND, not determined; URTI, upper respiratory tract
infection; VCA, Epstein-Barr virus capsid antigen; VZV,
varicella-zoster virus.

[a] Days that the patient was symptomatic.

Table 2. Comparison of demographic and laboratory data for patients with typical and atypical infectious mononucleosis and healthy subjects.
Legend for Chart:

A - Parameter
B - Healthy subjects (n = 22)
C - Subjects with typical infectious mononucleosis (n = 11)
D - Subjects with acute illness (n = 17)

A                                                  B
                          C                        D

Age                                       22 years to 49 years
                 21 months to 33 years    8 months to 39 years

Sex, no. male/no. female                  17/5
                 7/4                      10/7

Clinical days[a]                          --
                 4.0 +/- 1.6              9.5 +/- 9.7

Fever                                     0
                 11                       9

Pharyngitis                               0
                 9                        2

Lymphadenopathy                           0
                 10                       1

Hepatomegaly                              0
                 2                        1

Splenomegaly                              0
                 1                        1

Aspartate aminotransferase level, IU/L    19 +/- 5
                 116 +/- 112              130 +/- 265

Alanine aminotransferase level, IU/L      20 +/- 9
                 178 +/- 211              110 +/- 205

Lactate dehydrogenase level, IU/L         361 +/- 48
                 1029 +/- 3.8             994 +/- 389

WBC count, cells/mm3                5520 +/- 1440
                 14,231 +/- 7495          11,118 +/- 3635

Lymphocytes, %                            34.4 +/- 6.9
                 52.5 +/- 9.1             55.0 +/- 13.0

Atypical lymphocytes, % of WBC            1.2 +/- 1.3
                 10.7 +/- 3.9             9.5 +/- 5.3

Apoptotic cells,[b] % of WBC              0.9 +/- 0.4
                 8.3 +/- 3.6              8.2 +/- 3.9

CD4 cells, %                              41.2 +/- 6.7
                 13.3 +/- 4.2             22.7 +/- 11.2

CD8 cells, %                              22.0 +/- 4.4
                 64.5 +/- 11.6            50.2 +/- 13.3

HLA-DR+ cells in CD3 cells, %       26.6 +/- 11.4
                 64.4 +/- 28.3            31.2 +/- 18.9

Final diagnosis                           Normal

                 Typical infectious       Atypical infectious
                 mononucleosis            mononucleosis

NOTE. Data are no. of patients or mean value +/- SD, unless
otherwise indicated.

[a] Days that the patient was symptomatic.

[b] Percentage of apoptotic cells in blood smear after 4 h of
incubation at 37 Celsius.

Figure 1. Representative peripheral blood smears from a healthy subject, a patient with typical infectious mononucleosis (IM), and a patient with atypical IM associated with primary Epstein-Barr viru s infection immediately after blood was taken or after 4 h of incubation at 37 Celsius. May-Grunwald-Giemsa stain; original magnification, x 1000.

Figure 2. Representative blood scattergrams of anticoagulated peripheral blood samples obtained from a healthy subject, a patient with Epstein-Barr virus--associated acute infectious mononucleosis (typical IM), and a patient with upper respiratory tract infection and serological evidence of primary Epstein-Barr virus infection (atypical IM), subjected to automated hematologic analysis with the MAXM analyzer (Beckman-Coulter) either immediately after blood was drawn or after 4 h of incubation at 37 Celsius. Ly, lymphocytes; Mo, monocytes; Ne, neutrophils; Eo, eosinophils; Apop, apoptotic cell cluster.

Last updated Jan 4/07