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 Table of Contents  
CASE REPORT
Year : 2019  |  Volume : 21  |  Issue : 1  |  Page : 44-46

Gram's stain report of bone marrow aspirate that proved to be a lifesaver in a case of fever of unknown origin


Department of Microbiology, JSS Medical College, JSS Academy of Higher Education and Research, Mysore, Karnataka, India

Date of Web Publication12-Aug-2019

Correspondence Address:
Dr. Sowmya Govindanahalli Shivappa
764, 8th Main, B-Block, Vijaynagar 3rd Stage, Mysore - 570 030, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_32_18

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  Abstract 


Gram's stain is an invaluable, simple and inexpensive staining technique which gives a clue to the aetiological bacterial agent, thus helping in choosing the appropriate antibiotic. The significance of Gram's stain in bone marrow aspirate has not been described in previous literature. This is the case report of a child who presented with fever of unknown origin, where all high-end investigations proved inconclusive, but Gram's stain of bone marrow showed both extra-cellular and intra-cellular gram-positive cocci in clusters morphologically resembling Staphylococcus. This report helped the paediatricians choose the right antibiotic that proved to be a lifesaver of the child.

Keywords: Bone marrow aspirate, fever of unknown origin, Gram's stain


How to cite this article:
Neelambike SM, Shivappa SG, Basavaraj CK. Gram's stain report of bone marrow aspirate that proved to be a lifesaver in a case of fever of unknown origin. J Acad Clin Microbiol 2019;21:44-6

How to cite this URL:
Neelambike SM, Shivappa SG, Basavaraj CK. Gram's stain report of bone marrow aspirate that proved to be a lifesaver in a case of fever of unknown origin. J Acad Clin Microbiol [serial online] 2019 [cited 2019 Aug 20];21:44-6. Available from: http://www.jacmjournal.org/text.asp?2019/21/1/44/264253




  Introduction Top


Diagnosis of infectious diseases including  Brucellosis More Details, histoplasmosis, blastomycosis, tuberculosis and leishmaniasis can sometimes be made either by detection of the organism in the bone marrow or by culture. In immunocompromised patients, disseminated organisms such as Cryptococcus neoformans and Mycobacterium avium complex can also be visualised in bone marrow smear.[1]

This is the case report of a child who presented with a fever of unknown origin (FUO), where all high-end investigations proved inconclusive, but Gram's stain of bone marrow showed both extra-cellular and intra-cellular staphylococci. This report helped the paediatricians choose the right antibiotic that proved to be a lifesaver of the child.


  Case Report Top


A 3 ½-year-old male child presented with a history of fever, cold and headache of 7-day duration. The child was treated with Azithromycin for 5 days. The child was afebrile for 2 days followed by recurrence of fever from day 7.

There was no history of vomiting, cough, pain abdomen, burning micturition, rashes, altered sensorium and convulsions. Birth, developmental and immunisation history were normal. On examination, the child weighed 14.5 kg, pulse rate 110/min, respiratory rate 22/min and BP – 100/70 mmHg. Lymph nodes were palpable in the neck, axilla, epitrochlear and inguinal regions, 1–2 in number, <0.5 cm in diameter, non-tender and mobile. No further enlargement of lymph nodes was observed during the child's stay in the hospital.

Cardiovascular system and respiratory system examination was normal. Abdominal examination showed palpable liver, 3 cm below the costal margin, soft and non-tender. There was no splenomegaly. Central nervous system (CNS) examination showed Glasgow coma scale (GCS) of 15/15; there was no neurological deficit and no signs of meningeal irritation.

Laboratory investigations

The comparison of the blood counts with the number of fever days is mentioned in [Table 1].
Table 1: Comparison of the blood counts with the number of fever days

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Peripheral smear examination showed neutrophilic leucocytosis with thrombocytosis and absence of malarial parasite. Erythrocyte sedimentation rate – 90 mm in 1 h, blood urea – 18 mg/dl, serum creatinine – 0.7 mg/dl, serum glutamic–oxaloacetic transaminase – 36 units/L, serum glutamic–-pyruvic transaminase – 12 units/L, serum ferritin – 125 ng/ml, Lactate Dehydrogenase (LDH)-808 U/L and alkaline phosphatase – 486u/L.

Blood culture and sensitivity showed no growth after 7 days of incubation. Urine routine was normal and culture and sensitivity showed no growth. Immunoglobulin M (IgM) antibodies for Leptospira and  Brucella More Details were negative by enzyme-linked immunosorbent assay, Widal test was negative, malaria antigen detection test for plasmodium Lactate Dehydrogenase (pLDH) was negative and viral capsid antigen IgM for Epstein–Barr virus was also negative. Anti-nuclear antibodies for the diagnosis of autoimmune diseases were negative, and D-dimer concentration was normal.

The radiological investigations such as chest X-ray, magnetic resonance imaging (MRI) brain and two-dimensional (2D) echocardiography (ECHO) were normal. Ultrasonography (USG) of abdomen showed normal results, which was done twice during the course of the child's stay in the hospital. Cerebrospinal fluid (CSF) analysis showed total count – 15 cells/dl, neutrophils – 40%, lymphocytes – 60%, protein –28 mg/dl, glucose – 59 mg/dl, chloride – 116 mmol/L, Gram and Ziehl–Neelsen stain were negative and culture and sensitivity – no growth.

Multiplex polymerase chain reaction (PCR) (XCyton Diagnostics) for sepsis panel was negative (Staphylococcus aureus, Group B Streptococcus, Streptococcus pyogenes, Streptococcus pneumonia, Enterococcus species,  Escherichia More Details coli, Klebsiella pneumoniae,  Salmonella More Details species, Enterobacter aerogenes, Proteus mirabilis,  Neisseria More Details meningitidis, Haemophilus influenzae, Acinetobacter baumannii, Pseudomonas aeruginosa, Leptospira, Bacteroides fragilis, Aspergillus species and Candida species).

Multiplex PCR (XCyton Diagnostics) on CSF for meningoencephalitis panel was negative (for all the organisms tested in blood PCR along with Cryptococcus neoformans, herpes simplex 1 and 2, cytomegalovirus, varicella zoster, HHV-6 and Toxoplasma gondii).

The bone marrow aspirate was sent on the 8th day of admission to microbiology laboratory. Gram's stain of bone marrow aspirate showed few Gram-positive cocci in singles, pairs and clusters, both intra-cellular [Figure 1] and [Figure 2] and extra-cellular [Figure 3] suggestive of staphylococcal infection. Bone marrow aspirate culture yielded no growth after aerobic incubation for 48 h on blood agar and MacConkey agar and in Bact Alert Blood Culture System (BioMerieux).
Figure 1: Intra-cellular Gram-positive cocci seen in bone marrow aspirate

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Figure 2: Intra-cellular Gram-positive cocci seen in bone marrow aspirate

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Figure 3: Extra-cellular Gram-positive cocci, few abutting the cell margin

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In view of this, Ceftriaxone, Amikacin and Doxycycline were stopped and Linezolid was started (intravenously for 6 days and orally for the next 10 days). It was observed that, with the initiation of Linezolid, the total white blood cell count started dropping and the fever spikes came down slowly and the child was discharged. By the 5th day after discharge, the child had normal blood counts and no fever spikes.


  Discussion Top


In this case of FUO, when all the high-end tests such as multiplex PCR, MRI brain, 2D ECHO and USG abdomen were negative, only a simple, inexpensive Gram stain of bone marrow aspirate could help in identifying the aetiology of FUO. Furthermore, bone marrow culture did not yield any growth, maybe because of prior antibiotic therapy. This helped the child to recover and unnecessary antibiotics were stopped. The clinical response of the child to treatment with Linezolid and the presence of intracellular Gram-positive cocci in singles, pairs and clusters, in the bone marrow aspirate, proves the aetiological agent of FUO in this case as staphylococci.

We searched PubMed, Google Scholar, Science Direct, Ovid, Medknow and MD Consult published on 'any date' with search terms such as gram stain of bone marrow and bone marrow smear study. We did not fi nd any articles highlighting the importance of Gram's stain smear study of bone marrow aspirate throwing light in the diagnosis of a case of Fever of Unknown origin.

So far, no literature on the usefulness of Gram's stain on bone marrow is available, especially in the absence of any significant changes by cytology/histopathology. Volk et al. in their retrospective study have concluded that bone marrow histopathology and special stains for microorganisms in the absence of granuloma were non-contributory.[2] Although most of the protocols do not mention about Gram's stain of bone marrow, it is an inexpensive, indispensable and simple tool saving a life in this case. This report on the usefulness of Gram's stain on bone marrow aspirate may aid other cases of FUO to detect the aetiological agent.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgement

The authors would like to express their sincere thanks to the parents of the child who are both doctors and the treating paediatricians.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tille PM, editor. Normally Sterile Body Fluids, Bone and Bone Marrow, and Solid Tissues. In: Bailey and Scott's Diagnostic Microbiology. 13th ed. St. Louis: Mosby Inc.; 2014. p. 977-80.  Back to cited text no. 1
    
2.
Volk EE, Miller ML, Kirkley BA, Washington JA. The diagnostic usefulness of bone marrow cultures in patients with fever of unknown origin. Am J Clin Pathol 1998;110:150-3.  Back to cited text no. 2
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]



 

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