Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 96-102 DOI: https://doi.org/10.3126/njb.v7i1.26949

©NJB, Biotechnology Society of Nepal 96 Nepjol.info/index.php/njb.

REVIEW ARTICLE

Infectious Sources of Histoplasmosis and Molecular
Techniques for its Identification

Sudip Bhandari1, Binod Rayamajhee2, Laxmi Dhungel1, Sami Poudel1, Bhagwati Gaire1,

Sunil Shrestha1, Niranjan Parajuli3*

1Department of Biotechnology, National College, Tribhuvan University, Kathmandu, Nepal
2Department of Infectious Diseases and Immunology, Kathmandu Research Institute for Biological

Sciences (KRIBS), Lalitpur, Nepal
3Central Department of Chemistry, Tribhuvan University, Kathmandu, Nepal

Abstract
Histoplasmosis, a fungal infection caused by Histoplasma capsulatum (H. capsulatum), acquired
from contaminated soil with droppings of chicken or birds and found to be distributed in many
parts of the world. The prevalence of histoplasmosis has not well studied in Nepal. The common
symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and
weight loss. Most of the infections associated with histoplasmosis are asymptomatic. People with
compromised immune systems such as HIV/AIDS (PLWHA), cancer, and organ transplant
recipients are at risk of developing this disease. In this review, we have summarised the current
status of histoplasmosis in Nepal and molecular techniques available for its identification. To date,
the significant outbreak is not reported in Nepal, but the risk of infection for the vulnerable
population cannot be undermined. Appropriate preventive measures and treatment on time can
reduce the burden of this fungal disease. Further, this review is also focused on molecular
identification of H. capsulatum. Hence, careful considerations by concerned stakeholders for
national surveillance programs and the treatment of patients on time after proper diagnosis is
highly recommended.
Keywords: Histoplasmosis, asymptomatic, vulnerable, treatment

*Corresponding author:

Email: nparajuli@cdctu.edu.np

Introduction
Histoplasmosis is acquired by inhalation of

spores of H. capsulatum, which is usually found

in the soil contaminated by bird droppings,

which reveals the higher risk of disease in

farmers, gardeners, poultry keepers,

construction workers, pest control workers and

in some instances travelers visiting caves and

tunnels[1]. Histoplasma is a dimorphic fungus,

which produces mycelial form in the soil

environment and converts to the yeast form at

host body temperature (37 °C), and it usually

does not develop the symptomatic infection. So

people need not concern about the infection [2].

However, for immune-compromised people,

the fungus can result in severe infection. Very

young children and elderly people who have a

weak immune system; are more likely to get

histoplasmosis disease [3].

The development of infection and the

dissemination of H. capsulatum are initially

dependent on the condition of the host body[4].

The majority of infected persons could have

either no symptoms or a very less mild sickness,

which is hard to recognize as the cause of

histoplasmosis [5]. The clinical symptoms

generally occur only in a small number (around

1%) of the population when exposed with H.

capsulatum spores [6]. Persons who are usually

immuno-compromised and are unable to

develop effective cell-mediated response are

likely to develop symptomatic disease during

the period of acute dissemination in body [5],

which includes infant child, patients with

HIV/AIDS, organ transplant recipients, and

those with hematologic deficiencies, and also

those patients, who are on corticosteroids drugs

treatment [7]. An individual is under the risk of

developing symptoms even after years leaving

the endemic vicinity of histoplasmosis if the

man or woman was in an immuno-suppressive

situation at that time [8]. Histoplasmosis is of

four major types: pulmonary histoplasmosis,

progressive disseminated histoplasmosis,

Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 96-102 Bhandari et al. 2019

©NJB, Biotechnology Society of Nepal 97 Nepjol.info/index.php/njb.

cutaneous histoplasmosis, and African

histoplasmosis [9, 10].

Pathogenesis of Histoplasmosis
The fungal infection starts after the inhalation of

spores produced by the mycelial form of H.

capsulatum and the spores deposited inside the

alveoli of the lungs [11]. After some time, the

spores start to germinate inside the alveoli at

normal body temperature to become dimorphic

form. Then pulmonary macrophages engulf that

yeast form of dimorphic fungus [12]. After the

engulfment, the yeasts become a parasite and

multiply within the alveolar cells, then travel to

hilar and mediastinal lymph nodes [13]. When

yeast form fungus gets access to the blood

circulation system, then disseminates more

rapidly and swiftly across various organs of the

body. About after two weeks of exposure, the

macrophages become fully fungicidal, then the

cellular immunity starts a defense against the

fungal particles [14]. It leads to necrosis at the

site of infection like in the lungs, liver, spleen,

lymph nodes, and on bone marrow, adrenal

glands, and mucocutaneous membranes, which

results in progressive disseminated

histoplasmosis [4] as shown in Figure 1. The

progressive type of histoplasmosis is much

more lethal and severe as compared to other

kinds of histoplasmosis [15].

Epidemiology
Histoplasmosis is distributed worldwide, but

incidence cases are often reported around the

periphery of river valleys [16]. The majority of

infections in humans are reported from the

central United States, whereas the small number

of cases are also reported from Brazil,

Argentina, India, and South Africa [17].

Currently, due to an increase in the occurrence

of histoplasmosis worldwide, scientists have

been developing several diagnostic strategies.

Histoplasmosis endemicity can be evaluated by

population-based use of a histoplasmosis skin

test [18]. The skin test was found to be useful

when a major endemic area of histoplasmosis

identified in the north-eastern and Midwestern

United States [19].

Increased number of infectious diseases like

histoplasmosis is being reported in areas where

it was previously not thought to be prevalent,

and the changing distribution of infectious

diseases can become an important step to guide

diagnostic workup and direct public health

awareness [17]. Notably, in tuberculosis-

endemic regions, disseminated histoplasmosis

can easily be misdiagnosed as tuberculosis

because of its similar clinical symptoms and

must be considered as histoplasmosis infection

in patients who do not respond to empiric anti-

tubercular therapy [20].

An estimated discern indicates that nearly 3,000

people develop kidney failure yearly in Nepal

[21]. Similarly, there are additionally a high

number of the population in Nepal with a

diabetic condition; the 2016 Diabetes Profile has

shown that 9.1 %of the Nepali people are

dwellings with diabetes [22]. Likewise, the cases

of HIV/AIDS is also in excessive number inside

the country [23]. According to National Centre

for AIDS and STD Control (Nepal), there

are around 31,020 HIV/AIDS sufferers in the

country, and it is estimated to upward thrust at

a rate of 2 patients per day. Those persons

having conditions like a diabetic, organ

transplant, and the person with HIV/AIDS have

a weak immune system, which means that they

are at high risk of developing other secondary

infections like histoplasmosis during their

lifetime.

According to the published reports, the

geographical distribution of histoplasmosis has

not been clearly understood [4]. The occurrence

of histoplasmosis in Asia has not fully

appreciated until recently. Malaysia is the first

country in Asia, where H. capsulatum was

isolated from soil samples [24]. India, a close

neighboring of Nepal, has also reported a high

number of histoplasmosis cases in West Bengal

and Assam till 2018[24]. From, 1995 to 2018,

about 388 cases of H. capsulatum has been

reported, and the number is in increasing order

in India [24]. In recent years, an increasing

number of histoplasmosis cases have been

recognized among HIV-positive and/or

diabetic patients in India [1]. In case of Nepal,

histoplasmosis has only been described four

times, Amatya et al., (2014) reported that the

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cases of histoplasmosis in an Indian national

who traveled to Nepal for medical care and once

in a Nepali citizen, making this the second case

described in the literature by Subedi et al., (2016)

[25]. Recently, another case has been published

by Sharma and Adhikari (2019). According to

them, a 52 years old man with diabetes shows

adrenal involvement in histoplasmosis [26].

Additionally, one case has reported in a

Nepalese migrant to the USA with evidence of

infection being acquired in Nepal by Gandi et

al., in 2015 [27]. Nepal and India have an open

border, and a large number of Nepalese

migrants is directly dependent on Indian

economic and cultural aspects such as

employment, education, social relationship, and

trade among other elements. Such socio-

economic relationship directly has an impact on

the dissemination of the diseases, including

histoplasmosis.

Identification of H. Capsulatum and diagnosis

of histoplasmosis

Traditionally, identification of causative agent

of histoplasmosis is carried out through tissue

culture or body fluids at 25°C on Sabouraud’s

dextrose agar to allow the growth of mycelial

phase of H. capsulatum. For the proper growth of

mold, it takes about six weeks. After the

incubation period, two different types of conidia

are formed. The tuberculate or macroconidia are

of about 8 – 15 µm in diameter and have a thick

wall. Similarly, the microconidia are tiny about

2 -4 µm in diameter and are more infectious

when compared to the more abundant conidia.

However, the procedure of culturing is time-

consuming [28].

Likewise, histopathological examination is

another technique for detection of the

Histoplasma. It is generally done for severely ill

patients [29]. The method includes tissue

biopsy. During the histopathological

observation, other organisms also could mimic

the appearance like that of H. capsulatum. So, this

could be eliminated by using another stain like

methenamine silver or periodic acid- Schiff

stains, which visualize H. capsulatum [30].

Detection of circulating Histoplasma associated

antigen in urine and serum is another diagnostic

option to detect the presence of the fungi using

an immunological technique like sandwich

enzyme immunoassay (EIA), and it was first

described in 1986 [31]. This technique generally

uses H. capsulatum antigen, which is a

polysaccharide, a polyclonal rabbit anti-

Histoplasma immunoglobulin G linked to biotin,

and horseradish peroxidase [4, 18]. The assay

also shows the highest sensitivity against AIDS

patients who had disseminated histoplasmosis

[9].

Antibody tests are also performed to detect

several forms of Histoplasma cases. There are

two standard assays for antibody detection they

are, (i) complement fixation (CF) test, which is

based on the use of two separate antigens- yeast

and mycelial (or histoplasmosis)- and (ii)

immunodiffusion (ID) assay [32]. The ID assay

tests generally detect the presence of M and H

precipitin bands. An M

band often existing in persistent types of

histoplasmosis and lasts for many months to

years after the infection. An H band is also

indicative of the severe form of histoplasmosis

[17]. The ID assay is approximately only 80%

sensitive but is more specific than CF assay

because in CF assay, cross-reaction may occur

with other fungal infections like sarcoidosis [33].

Molecular identification of Histo-
plasmosis
In recent days, the molecular approach is widely

used as a reliable and rapid technique for the

detection of diseases with higher sensitivity and

specificity. At present, the use of chemo-

luminescence labeled DNA probe for the

detection of specific sequences of ribosomal

RNA (rRNA) has been able to detect and

confirm all the H. capsulatum isolates from the

culture [32]. AccuProbe is one of them, which is

based on the principle of nucleic acid

hybridization. A single-stranded

chemiluminescent labeled DNA probe is

complementary to the sequence of ribosomal

RNA of fungus. After the rRNA released from

fungi, the steady DNA-RNA hybrid formed and

that hybrids are detected and measured by

using Hologicluminometer. A positive result is

given by reading equal to or higher than the

predetermined cut-off values, and an adverse

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effect is the cost less than that of cut-off values

[39].

Polymerase chain reaction (PCR)
Assay
PCR is based on the amplification of the fungal

gene for identifying mycoses. In2003, Guedes et

al. designed a nested PCR based technique for

the rapid detection of fungi. The

oligonucleotides used in this method is based on

the M - antigens of H. capsulatum var. capsulatum.

The oligonucleotidesMsp1F - Msp1R (pair 1) and

Msp2F - Msp2R (pair 2) results amplicon of size

111 bp and 279 bp, respectively[34]; (Msp1F: 5′ -

ACA AGA GAC GAC GGT AGC TTC ACG - 3′

andMsp1R: 5′ - GCG TTG GGG ATC AAG CGA

TGA GCC - 3′), (Msp2F: 5′ - CGG GCC GCG TTT

AAC AGC GCC - 3′ and Msp2R: 5′ - ACC AGC

GGC CAT AAG GAC GTC - 3′). The PCR

reaction was performed with 100ng DNA

amplified in a 25 µl reaction using 20 pmol of

oligonucleotides(35 cycles of denaturation,

annealing, and chain extension) [34].

By using this protocol,100% specificity and

accuracy were mentioned in the 31 examined

strains from the animal, soil, and human [34].

The results also confirmed the absence of other

fungal strains such as H. capsulatum var.

farciminosum, Paracoccidioides brasiliensis,

Candida spp., Sporothrix schenckii, Cryptococcus

neoformans,etc in the amplified products[34].

Real-time PCR (RT- PCR)
The real-time RT-PCR or qPCR shows

promising results in the diagnosis of

histoplasmosis infection, which currently

identified the H. capsulatum among several other

fungi [35]. Simon et al., (2010) carried out

research for the detection of H. capsulatum based

on real-time PCR using the TaqMan probe [36].

For this study, specific primers and probe

(TaqManlabeled with fluorescent dye 6-

carboxyfluorescein at 5′ end and a

nonfluorescent quencher at the 3′ end) were

used for the analysis of the internal transcribed

spacer (ITS) region of the rRNA. The

oligonucleotides HcITS-167F (5′-

AACGATTGGCGTCTGAGCAT-3′) and HcITS-

229R (5′-GAGATCCGTTGTTGAAAGTTTTGA-

3′) were used. The following probeHcITS-188P

5′-6- FAM- AGAGCGATAATAATCC- MGB -3′)

was used. The specificity of RT- PCR was

calculated to be 95.4% in comparison to the

culture method. During the process, there is no

PCR inhibitor detected. Among the 275 samples

which were previously reported to be negative

in culture method, 11 samples were reported as

positive by RT-PCR with a specificity of 96.0%.

Similarly, among the 341 samples tested, zero

nonspecific signals were recorded [36].

Despite this promising research result, for the

detection of H. capsulatum in clinical samples,

there are no currently FDA-approved molecular

assays available. Moreover, to date, the

molecular approach for the detection of

histoplasmosis is in a developing stage.

Similarly, an improved and reliable technique

has to be designed as a dependable approach for

the detection of the disease. Regarding the

future accessibility of molecular techniques for

the detection of histoplasmosis is not unclouded

in resource-limited countries like Nepal [37].

Treatment of Histoplasmosis
Based on the current status, most patients with

mild acute histoplasmosis limited to lungs will

be resolved after a month without specific

treatment. However, the disseminated or the

severe form of histoplasmosis should be treated

with antifungal agents[17].

The treatment of chronic and severe form of

pulmonary histoplasmosis is generally done by

prescribing amphotericin B (3.0–5.0 mg/kg

daily for 1–2 weeks, intravenously), followed by

itraconazole (200 mg 3 times per day for 3 days

and after that 200 mg for two times a day, for a

total of 3 months) is required and

recommended[38]. Similarly, for progressive

disseminated histoplasmosis amphotericin B

(3.0 mg/kg daily) is recommended for about 7-

15days, which is followed by oral drug

itraconazole having 200 mg concentration for

daily three times for three days and after that

200 mg daily for two times for a total of at least

a year). Likewise, for the patients with the

immunosuppressed condition, itraconazole

daily (200 mg) is recommended as

prophylaxis[19,38].

Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 96-102 Bhandari et al. 2019

Research status of histoplasmosis in
Nepal
In Nepal, there is poor hygienic practices and

sanitation in major parts of the country and is

offering the home for the continuous emergence

and re-emergence of several life-threatening

infectious diseases. So, Histoplasmosis could be

a serious issue of public health in days to come.

In Nepal, the majority of the population lives in

rural areas with minimal health care facilities.

The burden of the disease is much higher in

rural settings compared to the urban areas.

Although the pattern of diseases might change

regularly, infectious diseases remain the leading

cause of mortality and morbidity in Nepal. In

many rural settings, cases of some diseases are

often identified only through their clinical signs

and symptoms. Lack of proper public health

awareness and treatment of infectious diseases

made people prone to several fungal infections,

including Histoplasmosis, which may be a

severe issue [40]. As the pervasiveness of

HIV/AIDS and Tuberculosis are budding

swiftly, the infection from the Histoplasma may

lead to a significant effect [41]. Thus, careful

diagnosis and treatment on time are paramount

to control the future outbreaks of

histoplasmosis. Moreover, the probability of

undiagnosed histoplasmosis dissemination also

urges for the need of proper diagnostic

strategies of Histoplasma spp. infection in Nepal.

As per institutional information, there is no

routine examination and treatment of

histoplasmosis infection in Tribhuvan

UniversityTeaching Hospital and National

Public Health Laboratory (NPHL-Nepal), while

both of them are considered as reference health

service centres of the country.

Challenges in Nepal
As Nepal is under the way of development, the

budgeting for the health and its related sector is

very less. Lack of proper investment in the

health sector for the diagnosis and treatment of

diseases is a significant problem in Nepal. Death

of hundreds of people by unknown disease is

also prevalent. Lack of proper government

strategies and operative ventures in the health

sector has been a cardinal cause of those strange

deaths. Allocation of the budget on the health

sector is only attentive to the treatment of some

severe disease, which shows that the country

has less interest in disease diagnosis. It is one of

the significant consequences of the death of

people from unknown conditions. Therefore,

early diagnosis of histoplasmosis is mandatory

to reduce the burden of illness.

Conclusion
The clear and accurate picture of the

dissemination of histoplasmosis is complicated

to understand in the resource-limited clinical

sector of Nepal. Many factors could contribute

to this, like lack of proper lab facilities, lack of

expertise on fungal infections, etc. Additionally,

not much reliable data are available from the

government and academia due to incompetence

in the diagnosis strategies. The published data

are also in the form of case reports based on a

hospital visit. It is already emphasized that the

pockets of endemicity do exist in Nepal, which

could lead to a significant impact on public

health. Hence, careful considerations and the

molecular approach of disease identification

may be a suitable alternative. To address this

problem, further research should be done

immediately in Nepal

Conflict of Interest
The authors declare no conflict of interest.

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