Diagnosing Leprosy in Infants:

A Histopathological Challenge using Several Staining Technique

Joanne Natasha

, Rizky L. Prayogo

, Michael H. Angriawan

, Rinadewi Astriningrum

, Melani

Marissa

, Sri Linuwih Menaldi

, Sondang P. Sirait

Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia/

Dr. Cipto Mangunkusumo Central National General Hospital

*Corresponding author

Keywords: Leprosy, Histopathology, Infants, Stains

Abstract: Leprosy in infants is a rare case compared to adults. There is an excellent variety of the clinical cutaneous

finding in leprosy. Leprosy diagnostic is based on the classical cardinal signs, the presence of acid-fast

bacilli which is obtained by slit skin smear and skin biopsy. The gold standard to diagnose leprosy is the

identification of Mycobacterium leprae (M. leprae) bacilli using Hematoxylin and Eosin (H&E) and Fite

Faraco (FF) stains. Periodic acid-Schiff (PAS) and Gomori (Grocott) methenamine-silver (GMS), known to

give the positive result for fungal staining, revealed the other function is becoming the alternative staining

for M. leprae. Hereby, we report a case of a six-month-old male patient who presented unusual clinical

findings that were never considered as leprosy initially. Histopathological examination with several stains

was performed, and a diagnosis of lepromatous leprosy was obtained.

1 INTRODUCTION

The occurrence of leprosy during infancy is

uncommon. Infants are believed to be the most

vulnerable group to infection with M. leprae due to

their immature immunity and exposure to the

member of the family who has leprosy.(Narang T et

al., 2018) WHO data showed 210.671 newly

diagnosed leprosy cases during 2017, 16.979 were

children, represented almost 7,5% of all new cases

(5-14 years old).(Narang T et al., 2018) During

2002, based on the data, Indonesia has 21,2% (95%

CI: 12,5–29,9%) newly detected leprosy cases in

children 6-16 years old.(Bakker et al., 2002) Our

data showed 25 new cases leprosy in children (5-14

years old) from 150 patient’s visit in the

Dermatology Department of Dr. Cipto

Mangunkusumo Central National General Hospital

from 2017 to 2018, but none of them were infants.

The spectrum of leprosy in children or infants

has reported mostly to be tuberculoid (TT),

borderline tuberculoid (BT), mid-borderline (BB),

and indeterminate (I) forms. Lepromatous leprosy

(LL) type is rare in children or infants. The great

variety of presentations made the diagnosis of

leprosy a problematic challenge. The most likely

portal of entry is considered to be the upper

respiratory tract, but other possibilities through

direct contact. (Girdhar et al., 1989) Hematogenous

dissemination occurs in LL.(Sabin et al., 2005).

Leprosy diagnostic is based on the clinical

characteristics, finding acid-fast bacilli (AFB) in slit

skin smear, and histopathology. .(Narang T et al.,

2018). Hematoxylin and Eosin (H&E) and FF stains

are the chosen method for AFB staining. The other

study reported PAS and GMS stains could stain

Mycobacteria besides the previously known main

staining for fungal. Nevertheless, there is still

limited literature concerning the results of these

stains when applied to other Mycobacteria

species(Csillag, 1960;Wright et al., 2017;Schinietz

et al., 2018). We have recently highlighted the AFB

positive result by using PAS and GMS stains in the

case of an infant's LL. Hence, the use of these stains

may be considered to diagnose leprosy.

2 CASE

A six-month-old baby boy was consulted from the

Pediatric Department, on October 2018, with

290

Natasha, J., Prayogo, R., Angriawan, M., Astriningrum, R., Marissa, M., Menaldi, S. and Sirait, S.

Diagnosing Leprosy in Infants: A Histopathological Challenge using Several Staining Technique.

DOI: 10.5220/0009987002900294

In Proceedings of the 2nd International Conference on Tropical Medicine and Infectious Disease (ICTROMI 2019), pages 290-294

ISBN: 978-989-758-469-5

complaints of erythematous collarette fine-scale

papules, plaque, and slightly nodules on the face,

chest, abdomen, arms, legs, buttocks and soles since

three months ago. The lesion was not accompanied

by pain. He was not seen scratching the lesion.

Alloanamnesis with the patient's mother, he had a

recurrent fever before the lesion appeared on the

skin. Pityriasis lichenoides chronica, atypical

pityriasis rosea, and psoriasis Vulgaris were initially

chosen to become the differential diagnosis based on

alloanamnesis and physical examination.

Skin biopsy was performed on November 2018

with a histopathological picture of granulomas with

numerous foamy cells in the lower dermis. There

were many vacuoles containing slightly grey-

basophilic colored material, including

polymorphonuclear (PMN), extravasation of red

blood cells, thickened vessel walls, and visible

granulomas surrounded nerves structures, suggesting

the diagnosis of lepromatous leprosy. Additional

staining using the FF, showed numerous AFB,

mostly in the solid forms. Periodic acid-Schiff and

GMS were also performed, gave a positive result for

bacilli inside the cytoplasm of foamy cells. Acid-fast

bacilli finding in histopathology was followed by slit

skin smears examination. This was obtained positive

on both ears, with 4/6 bacterial index and 0%

morphological index.

The patient was diagnosed with Severe

Combined Immune Deficiency (SCID) and

malnourished by the Pediatric Department since he

was five months old. He was the second child of two

siblings, with full-term delivery. He obtained

incomplete immunization. He lives in one house

together with his father, mother, mother's sibling,

and grandmother.

Figure 1. Erythematous to hyperpigmented papule-plaque-nodule with collarette scale in November 2018

Figure 2. Positive AFB on both ear lobes was revealed from slit skin smear

Figure 3. Histopathology, H&E staining, 100x, granuloma in the lower dermis

Diagnosing Leprosy in Infants: A Histopathological Challenge using Several Staining Technique

291

Figure 4. Histopathology, H&E staining, 400x (A), FF staining, 400x (B), PAS staining, 400x (C), GMS staining, 400x

(D). All shows numerous foamy macrophage containing globi of AFB. GMS and PAS show more bacilli than FF

3 DISCUSSION

Children or infants have a four-fold risk of

developing leprosy in the presence of leprosy close

contact in the neighborhood, and this risk increases

to nine-fold if there is a contact with leprosy patient

within the household. (Jain et al., 2014) The

occurrence of leprosy and its complication are

related to changes in the host’s immune response.

Individuals who are immunosuppressed could be

more at risk of developing leprosy.(Massone, 2012)

Individuals with sufficient exposure to M. leprae

may develop a broad range of clinical

manifestations. The bacillary proliferation and

hematogenous dissemination occur in LL. .(Sabin et

al., 2005).

Initially, the cutaneous lesion was confusing with

the other dermatological ailments. Several

differential diagnoses were chosen, but a primary

diagnosis was not established yet. The clinical

findings were never considered as leprosy, hence the

skin biopsy was done to obtain a definite diagnosis.

Papules, plaque, nodules skin lesions did not appear

as a typical lesion of leprosy, therefore slit skin

smear examination was done considering this infant

was not able having the nerve examination. The

result did not show globi as in the histopathology.

The number of AFB should be in the same quantity

as in the smear according to the bacterial index of

granuloma, such as +6 shows many clumps globi

(>1000 bacilli). (Massone, 2012). Possibly because

of the incision was not deep enough, since the

granuloma is in the deep dermis.

Leprosy diagnostic has generally been performed

based on a cardinal sign of clinical criteria

(anesthetic skin lesions, enlarged or thickened

peripheral nerves) and the presence of AFB from

tissue smears stained by modified Ziehl-Neelsen

staining. These great varieties of presentations in

infants made the diagnosis of leprosy as a challenge.

Histopathology is the gold standard to diagnose

leprosy, particularly in an early stage of leprosy. The

infant’s histopathology determined LL, in

accordance with nodular granuloma in the dermis,

macrophage cytoplasm were loaded with bacilli or

globi with large quantities of lipids, which on

staining give the appearance of foamy cells

(Virchow cells), and the infiltrate was separated

from the epidermis by collagenous fiber bands

(Unna band) giving the appearance of subepidermal

clear zone(Narang T et al., 2018;Weedon, 2012). In

a systematic review examined 24 children

histopathologically, the majority showed

characteristic features of BT leprosy (19/24), three

were BL leprosy, and two were indeterminate

leprosy. (Jain et al., 2014). Histological confirmation

is mandatory in all cases of leprosy because the gold

standard to diagnose leprosy is the identification of

M. leprae bacilli using H&E and FF stains. The gold

standard to diagnose leprosy is strictly related to

three significant points such as well done and deep,

6 mm punch biopsy performed at the right site of the

lesion, well-cut, stained H&E and FF slides, also

detailed clinical information. (.(Massone,

2012;Xavier Jnior JCC et al., 2016)

Job-Chacko modified FF staining for M. leprae

enables the better chance of detecting the bacteria in

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the early leprosy granuloma. Histopathology, carried

out by FF staining, displays the sensitivity of 74,6%

and positive predictive value of 85,9% and negative

predictive value of 56,7%. This study shows that

diagnosis was revealed with FF staining, better than

other stain.(Reja AHH et al., 2013) Based on authors

experience and other literature, FF staining is often

negative even in multibacillary leprosy.(Adiga et al.,

2013;Joshi,2014)

Mycobacteria is challenging to demonstrate by

the Gram technique as they possess a capsule

containing a long-chain fatty acid (mycolic acid)

which makes them hydrophobic. Mycobacteria are

PAS-positive due to the carbohydrate content of

their cell walls. However, this positivity is evident

only when the large concentration of the

microorganisms are present. When these organisms

die, they lose their fatty capsule and consequently

their carbol fuchsin positivity. The carbohydrate can

still be stained by GMS reaction, which may prove

useful when acid-fast procedures fail, particularly if

the patient is already receiving therapy. These

organisms are acid- and alcohol-fast but are usually

easily identified as contaminants by their appearance

as clumps, or floaters.(

Morris GB et al., 2015)

Although GMS and PAS often used to identify

fungal infection, it is important to recognize their

other function of non-fungal staining in order to

obtain the correct diagnosis and guide appropriate

clinical management. Identified non-fungal

organisms, which are stained with GMS, include

parasitic worms, virally infected cells, acid-fast

bacilli, partially acid-fast bacilli, and non-acid-fast

bacteria. Gomori methenamine-silver and PAS

stains have also been reported to stain M.

leprae.(Xavier Junior JCC et al.,2013) In this case

report, GMS and PAS were useful to stain the bacilli

inside the foamy cells. Wright reported three of the

nine cases, which also revealed as non-fungal using

GMS and PAS stains. Organisms were not clinically

suspected as non-fungal. The non-fungal organisms

had silver deposition in varying locations including

the cell walls acid-fast Mycobacteria.(Wright et al.,

2017). In this case, histopathology showed more

numerous AFB in the cytoplasm of macrophage

foamy cells which were stained by GMS and PAS

compared to H&E and FF.

Gomori methenamine-silver is a chromic acid,

sodium bisulfate stain which precipitates silver ions

in fungal polysaccharide walls, producing the

characteristic black stain on light microscopy. This

is helpful to recognize aberrant GMS staining to

avoid misdiagnose of fungal elements. GMS stains

several non-fungal human pathogens which may be

useful diagnostic aid if the infectious condition is

not clinically suspected or the number of organisms

is sparse and having any difficulty to visualize with

routine staining methods.(Csillag, 1960). The other

study also reported the prominent PAS and GMS

positivity in a case of cutaneous Mycobacterium

avium complex (MAC) infection, and organisms

show the higher intensity of staining with GMS

compared to routine AFB staining. Strong staining

of M. tuberculosis and M. leprae has been reported

with GMS with a modified silver stain.

Periodic

acid-Schiff method selectively stains some

carbohydrates and carbohydrate compounds such as

glycogen, starch, mucin, and chitin. Csillag also

reported PAS-positive material, in all strain of

Mycobacteria. PAS-positive smears often contain

clearly distinguishable vacuolated cells, though

these might only be present in small numbers. The

reaction was strong and the material colored

intensely red, although the study did not include M.

leprae. In the challenging cases, as in this case,

clinical manifestation showed some variety of

unusual cutaneous lesion in an infant with

immunocompromised. Leprosy was initially

unexpected as the differential diagnosis. (Csillag,

1960).

4 CONCLUSION

In children, leprosy is dominated by borderline type

to tuberculoid type leprosy. Unusual case leprosy in

infant showed atypical cutaneous lesion. Hence

leprosy was not an unexpected differential diagnosis.

As seen in this case, the diagnosis of lepromatous

leprosy was established by histopathological

examination. Leprosy could be diagnosed by slit

skin smear and histopathology due to the difficulty

in conducting the sensitivity test, primarily in

children under ten years of age, and the need for

knowing other dermatoses commonly found in

childhood as the differential diagnosis. This case

was solved through the histological analyses using

leprosy routine stains and also alternative stains.

This case report showed that GMS and PAS were

also considered choices to stain AFB in leprosy

besides H&E and FF. Although GMS and PAS are

not specific for AFB, it can stain bacteria inside the

cytoplasm of foamy cells in leprosy.

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