Amyloidosis in major body organs examined during autopsies at Mbarara Regional Referral Hospital

Background Amyloidosis is an infrequent disease that occurs when an abnormal protein, called amyloid, deposits in body organs and disrupts their normal function. Amyloid is not normally found in the body but it can be formed from several different types of protein. Commonly affected organs include the heart, kidneys, gastrointestinal (GI) tract/liver or the peripheral or autonomic nervous system (NS). Amyloidosis can lead to diseases such as Alzheimer’s disease, spongiform encephalopathies and diabetes mellitus type 2. To establish major examined autopsies at Mbarara referral and to correlate death amyloidosis. and Eosin stain and Congo red staining method. The Haematoxylin and Eosin

each of the above organ samples. These slides were then stained using H&E and Congo red staining protocols and then examined for amyloidosis under the light microscope.

Results
The mean age of the bodies was 61.26 of which 5 (21.7%) were female while 18 (78.3%) were male. 3 bodies (13.0%) tested positive for amyloidosis in the kidney; 2 males (8.70) and 1 female (4.35). All the other organs tested negative.

Conclusion
In our study, body organ investigation revealed amyloid in the kidney of 3 bodies. The prevalence of amyloidosis was 13% therefore amyloidosis diagnosis ought to be put into consideration at Mbarara Regional Referral Hospital.

INTRODUCTION
Amyloidosis is a diverse group of disorders in which there is extracellular deposition of abnormal protein fibrils in different body organs depending on the type of protein (1). Amyloid fibrils penetrate organs and tissues and deposit there leading to swelling and progressive loss of function (2). In naming amyloidoses, the letter A is used to designate amyloid followed by an abbreviation of the name of the protein fibril (3). The amyloidoses vary depending on the type of protein undergoing aggregation, the organs in which they deposit and in their clinical features (4).
The global burden of amyloidosis is difficult to define due to the fact that in many places around the world, the disease is misdiagnosed or underdiagnosed and there are few studies that have reliable data (5).
A cross sectional study in Egypt found the prevalence of amyloidosis in Rheumatoid Arthritis patients to be 4 out of 30 patients (13.3%). This study was carried out by obtaining abdominal fat aspiration biopsies from these patients and confirming amyloid presence by Congo red staining (6).
In Uganda, an autopsy study revealed the incidence of amyloidosis to be 0.57%. This study was done by re-examining haematoxylin and eosin sections from autopsy archives at Mulago hospital, Kampala over a 23-year period. Amyloid presence was then confirmed by Congo red staining (7).
Amyloidosis diagnosis is based on confirmation of presence of deposits of these abnormal proteins in tissues. Biochemical markers such as Brain Natriuretic Peptide (BNP) type B, N-terminal-proBNP and troponins are available but not routinely used and are still under study (8,9).
Congo red stain is the gold standard for detection of the presence of amyloid fibrils.
Under a light microscope, deposits of amyloid stained with Congo red typically have an orange to red colour. Congo red stain should be used to confirm a suspicion and to rule out the possible presence of early amyloid deposits, which are not easily seen in hematoxylin-eosin. (9).
This study also aimed at knowing the burden of amyloidosis in south-western Uganda which will increase its clinical suspicion, diagnosis and treatment.

Setting
The study was carried out in the pathology department of Mbarara Regional Referral Hospital (MRRH). MRRH is located in Mbarara district, south western Uganda. It is approximately 260km from Kampala, the capital city of Uganda and 2km from the town centre on the Mbarara-Kabale highway. MRRH is a public institution and is the regional referral hospital of south western Uganda with a capacity of 1200 beds. The MRRH mortuary and histopathology laboratory were chosen because they receive clients from the entire western region of Uganda; hence the sample size was representative of a big number of Ugandans.

Data Collection
Any dead body of 40 years and above received at a MRRH mortuary in Western Uganda for which an autopsy was requested was included in the study unless it had started decomposing, it was missing critical information such as age, death was due to a highly infectious disease or the relatives did not consent to an autopsy. 23 bodies which suited our inclusion criteria were dissected to obtain brain, liver, kidney and heart tissues which were immersed in 10% neutral buffered formalin in a 1:20 ratio to preserve them. When the tissues were ready to be processed, they were macroscopically examined and small pieces were cut out and placed in tissue cassettes.
Microtomy was performed and tissue sections of 3µm were obtained, and stained with the Haematoxylin and Eosin stain and Congo red staining method. The Haematoxylin and Eosin staining method involved dewaxing and hydrating sections, staining in haematoxylin for 1 minute, blueing for 3 minutes, staining in Eosin for 1 minute, dehydrating through increasing concentrations of alcohol, clearing in xylene and mounting with DPX. The Congo red staining method involved dewaxing and then hydrating sections, staining for 1-5 minutes in 50% alcoholic Congo red, differentiating 1-3 minutes in 0.2% KOH in 80% alcohol, rinsing in tap water, counter staining for 2-8 minutes with alum haematoxylin, washing in tap water, dehydrating quickly through alcohol, clearing in xylene and mounting with DPX.
Information such as age, sex, address and cause of death were obtained as well and filled in a data collection form.

RESULTS
The study enrolled 23 bodies, of which the age group of 60 -69 years had the highest number of bodies constituting 47.8% (11/23). Male bodies were more than female bodies constituting 78.3% (18/23) and 21.7% (5/23) respectively. Most of the bodies were from people residing in the urban areas of Mbarara municipality, and these constituted 30.4% (7/23) of the whole study population. Other areas where bodies came from included; Masaka district, Rukungiri district, Kiruhura district and Ibanda district, each constituting 4.3% (1/23). Table 1.
From our study, we found that only 3 bodies provided tissues which were positive with the Congo red stain. This made the general prevalence of amyloidosis to be 13% (3/23). Table 2 From all the harvested tissues, all those found to be positive with Congo red stain were kidneys.
This meant that the kidney had the highest prevalence by organ (100%). On further analysis, two of the positive kidney samples had been harvested from a male body, while only one had been harvested from a female body. This put our prevalence of amyloidosis by gender to be 66.7% and 33.3% respectively. Table 2.
We also found out that the bodies whose causes of death were Secondary peritonitis, Asphyxia secondary to aspiration pneumonitis and Bronchopneumonia each had one case of Congo red positive kidney tissue. There was no significant correlation between cause of death and amyloidosis. Bronchopneumonia showed a positive but non-significant correlation with

Prevalence by organ and gender
According to James & Owor, (1975 showed that 59% of the 1,430 amyloidosis patients identified between 1995 and 2013 were men. However, Seo et al., (2017) reported that the prevalence of amyloidosis is the same among men and women.

Correlation between amyloidosis and cause of death
We did not find any significant correlation between cause of death and amyloidosis. However,

Limitations
Few post mortems were done at the Regional Referral hospital mortuary during the study period.
Due to COVID-19 pandemic that led to closure of the university in March, our time frame was affected.

CONCLUSION
In our study, all the investigated body organs revealed amyloid in the kidney. We determined the prevalence of amyloidosis which was found to be 13%. The prevalence of amyloidosis was more in men than in women.
There was no significant correlation between cause of death and amyloidosis.

Ethics approval and consent to participate
Approval to carry out the study was given by the Department of Medical Laboratory Science and the Faculty Research Committee of Mbarara University of Science and Technology.
Clearance was also sought from the Department of Pathology of Mbarara Regional Referral Hospital before commencement of the study.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. There is no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

Authors' contributions
KJM, MJB, MFB, MK, GI: Perceived the concept, drafted a proposal, and designed the study protocol and data collection tool. Wrote the analysis plan and implemented the protocol. Took part in quality assurance of the study and laboratory work. Cleaned and analyzed data, and drafted and revised the paper. Met all the financial requirements of the study.

RA:
Provided clearance for the study to be carried out in the pathology department of MRRH and technical guidance on slide examination. Contributed to drafting of the final paper.
BA: Provided guidance in concept development laboratory methods employed in Amyloidosis testing. Coordinated study processes within the pathology department.
BP: Provided technical guidance on logistics and laboratory methods employed in Amyloidosis testing. Provided supervision and technical support that was required for recruitment of study participants as well as collection of specimens in the post mortem of MRRH.