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CASE REPORT
MICROSCOPY ‘AIDS’ IN DIAGNOSING A FEBRILE INFANT
Annelize Crous, MB ChB, DA (SA)
Department of Haematology, University of Pretoria, and National Health Laboratory Services, Tshwane Academic Division
Adele Visser, MB ChB, PG (Dip) TM, DTM&H, MMed (Clin Path), FCPath (SA) (Clin Path)
Department of Microbiology, Division of Clinical
Pathology, University of Pretoria, and National Health Laboratory
Services, Tshwane
Academic Division
A 5-month-old South African girl presented to a casualty department
with a short history of fever. General examination did not reveal
organomegaly or neck stiffness. In keeping with local guidelines,
malaria was excluded on antigen testing and microscopy (thick and thin
smear with Giemsa stain).1
Rickettsia typhi, R. conorii and Coxiella burnetti were also excluded on the basis of serological testing.
The full blood count revealed pancytopenia with a haemoglobin (Hb)
level of 7.9 g/dl (normal 10 - 15 g/dl), a white cell count (WCC) of
1.17×109 /l (normal 5.50 - 18.00×109 /l) with an absolute lymphocyte count of 0.80×109 /l, and a platelet count of 47×109 /l (normal 140 - 350×109 /l).
Biochemical values were suggestive of renal dysfunction, with a serum
urea level of 9.6 mmol/l (normal 1.4 - 5.0 mmol/l) and a creatinine
level of 180 μmol/l (normal 14 - 34 μmol/l), as well as a
markedly elevated C-reactive protein level of 243.1 mg/l (normal 0.1 -
7.5 mg/l).
On investigation of the patient’s immune status, it was
established that she was HIV-1-positive as confirmed by HIV-1 DNA
Amplicor assay version 1.5 (Roche Diagnostics, Mannheim, Germany), with
an HIV-1 viral load of 2 738 930 RNA copies/ml by Abbott
m2000 Real Time HIV-1 Assay (Abbott Diagnostics, Johannesburg) and a
CD4+ lymphocyte percentage of 28%.
Initial standard paediatric blood cultures did not yield any growth
after 7 days of incubation. A follow-up full blood count, performed 2
days after the initial investigations and assayed on the ADVIA 2120
(Bayer HealthCare, Diagnostic Division, Isando, Gauteng), differed
markedly from the previous results, showing a WCC of 13.83×109 /l with an absolute lymphocyte count of 10.99×109
/l. On microscopic examination of the peripheral blood smear (Fig. 1,
a) these counts could not be verified and the leucocytes appeared
markedly reduced. In addition, fungal elements could be demonstrated
(Fig. 1, a and b).
Subsequent blood cultures, performed in paediatric bacterial blood culture bottles, confirmed the aetiological agent to be Candida albicans.
Fungal morphology clearly showed the presence of oval yeast forms,
slightly smaller than a lymphocyte nucleus (Fig. 1, a), with
blastoconidia and pseudohyphae (Fig. 1, b). The fungus was identified
by demonstrating formation of a germ tube on 4-hour incubation in horse
serum.2
Subsequent antifungal susceptibility, performed on the Vitek 2 system
(BioMerieux, Randburg, Gauteng), demonstrated sensitivity to all
antifungal agents tested (amphotericin B, fluconazole and
voriconazole).
Discussion
HIV-1 infection has been established as a risk factor for fungaemia in both children3 and adults.4 The routine use of fungal blood cultures remains controversial, being advocated by some authors6 and discouraged by others.7
Microscopic examination of the peripheral blood smear revealed a
significant leucopenia, despite contradicting automated counts. WCCs as
well as differential leucocyte counts have been described to be
influenced by high fungal loads of Candida spp.8 The ADVIA 2120 uses both a lobularity and a peroxidise channel for leucocyte identification and quantification.8
In this case a comparison error had been flagged, indicating the
probability of falsely elevated counts. This error, however, does not
reflect on the laboratory report issued to the clinician, and is an
analytical issue that needs to be addressed by the technologist and
pathologist in the laboratory setting. Owing to their small size as
well as their peroxidise-negative properties, the yeast cells had been
identified as lymphocytes by the automated analyser (Fig. 1, c).
Considering the increased prevalence of fungal infections among HIV-1-positive patients,3
full blood count evaluation is best accompanied by morphological
evaluation to detect any discrepancies between automated counts and
actual observation.8
In most laboratory settings, morphology is evaluated once the clinician
requests a differential count. Clinicians should further request
differential counts once any full blood count parameters show
significant abnormalities, or when there is a clinical suspicion of
disease affecting the haematological system, including bleeding
tendencies, infection, hepatomegaly, splenomegaly, lymphadenopathy, etc.10
In this clinical case, the rapid change in results over a 2-day
period should have prompted the clinician to suspect an analytical
issue with the samples in question.
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in three automated hematology counters. Ann Hematol 2008;87:557-562.
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10. Bain B. Diagnosis from the blood smear. N Engl J Med 2005;353(5):498-507.
Fig. 1.
Peripheral blood smear from a 5-year-old HIV-1-positive girl, showing
marked fungaemia: a – size of fungal element in relation to lymphocyte
(arrow); b – closer view of fungal elements, showing oval yeast forms
with blastoconidia and pseudohyphae; c – peroxidase channel on ADIVA
2120 analyser. The encircled area depicts the region where lymphocytes
are typically noted based on both size and peroxidase negativity. In
this instance, these events are produced by yeast cells rather than
lymphocytes.