Typing of blast hits after analyzing subcutaneous tissue sample and details of the phylogenetic MEGAN output. (DOC 198 kb

Dian Gao

Fan Xiong

Guangqiang Wang

Guitang Wang

Qiongfang Yu

Parasites & Vectors

Dian Gao (496377)

Qiongfang Yu (3462968)

Guangqiang Wang (3462971)

Guitang Wang (112107)

Fan Xiong (3324015)

FigShare

Additional file 4: Figure S3. of Diagnosis of a malayan filariasis case using a shotgun diagnostic metagenomics assay

Additional file 2: of Diagnosis of a malayan filariasis case using a shotgun diagnostic metagenomics assay

Springer - Publisher Connector

Diagnosis of a malayan filariasis case using a shotgun diagnostic metagenomics assay

AbstractBackgroundMalayan filariasis is a lymphatic filariasis caused by Brugia malayi. It is easily misdiagnosed in non-endemic areas for atypical symptoms and rare diagnostic experience. A 34-year-old Chinese woman in New York presented with diffuse erythema on her body, swelling of her body, and watery, itchy, red, sore, swollen and stinging of the eyes, and severe night-time itching. No hospital that the patient visited could make a definite diagnosis by conventional diagnostic methods. It is therefore necessary to explore a new effective method to detect the pathogen that infected the patient.FindingsAn unbiased metagenomic approach was used in this study. After DNA was extracted from the patient’s eye discharge sample and subcutaneous tissue sample, extended parallel sequencing was performed. The obtained raw reads were aligned to human genome to filter out the reads of the host, and the remaining reads were aligned to a candidate pathogenic protein database and four filarial genomes. The result showed that the reads of B. malayi accounted for an overwhelming ratio in the two samples, which indicated that the patient suffered from malayan filariasis. The subsequent therapeutic efficacy of anti-filariasis treatment validated the result of metagenomics assay.ConclusionsThe present study proved that metagenomic assay can be an effective approach in the diagnosis of parasitic infection. We report a rare case of malayan filariasis from the United States

Gao,Dian

Yu,Qiongfang

Wang,Guangqiang

Wang,Guitang

Xiong,Fan

Institute of Hydrobiology, Chinese Academy Of Sciences

SHORT REPORT Open Access
Diagnosis of a malayan filariasis case using
a shotgun diagnostic metagenomics assay
Dian Gao1, Qiongfang Yu2, Guangqiang Wang3, Guitang Wang4 and Fan Xiong4*
Abstract
Background: Malayan filariasis is a lymphatic filariasis caused by Brugia malayi. It is easily misdiagnosed in non-endemic
areas for atypical symptoms and rare diagnostic experience. A 34-year-old Chinese woman in New York presented with
diffuse erythema on her body, swelling of her body, and watery, itchy, red, sore, swollen and stinging of the eyes, and
severe night-time itching. No hospital that the patient visited could make a definite diagnosis by conventional
diagnostic methods. It is therefore necessary to explore a new effective method to detect the pathogen that
infected the patient.
Findings: An unbiased metagenomic approach was used in this study. After DNA was extracted from the patient’s eye
discharge sample and subcutaneous tissue sample, extended parallel sequencing was performed. The obtained raw
reads were aligned to human genome to filter out the reads of the host, and the remaining reads were aligned to a
candidate pathogenic protein database and four filarial genomes. The result showed that the reads of B. malayi
accounted for an overwhelming ratio in the two samples, which indicated that the patient suffered from malayan
filariasis. The subsequent therapeutic efficacy of anti-filariasis treatment validated the result of metagenomics assay.
Conclusions: The present study proved that metagenomic assay can be an effective approach in the diagnosis of
parasitic infection. We report a rare case of malayan filariasis from the United States.
Keywords: Filariasis, Parasite, Helminth, Metagenomics, Brugia malayi
Background
Lymphatic filariasis (LF) is a global health problem and
is endemic in 73 countries throughout the tropics and
sub-tropics, where it is a major cause of acute and
chronic morbidity and a significant impediment to so-
cioeconomic development [1]. It accounts for about 40
million chronically disabled or incapacitated people [2].
Three filarial nematodes, Brugia malayi, Brugia timori,
and Wucheria bancrofti are responsible for lymphatic fil-
ariasis, which involves asymptomatic, acute, and chronic
conditions [3].
In the past decades, the standard method to confirm a
filarial infection was the identification of microfilariae in
peripheral blood by microscopic examination. Besides
the low sensitivities of this method, it is also inconveni-
ent as the blood has to be collected at night, for the
most prevalent filarial species (W. bancrofti), to coincide
with the peak appearance of the microfilariae in periph-
eral blood. Since the 1990s, a number of techniques in-
cluding detections of specific antibodies and circulating
antigen and PCR array have been developed to simplify
the diagnosis of filariasis and improve test reliability [4–6].
However, none of these methods have enough sensitivity
and accuracy to detect all three kinds of lymphatic filaria-
sis at the same time. Therefore a doctor easily makes mis-
diagnosis when patient presents with atypical symptoms,
especially in non endemic areas.
Shotgun metagenomics, which is applied to the direct
sequencing of DNA extracted from a sample without
culture or target-specific amplification or capture, has
been widely used in virus discovery [7] and bacterial
pathogen detection [8]. It has rarely been used in the de-
tection of parasitic infection except in limited related
cases [9, 10]. Here we confirmed a case of malayan filar-
iasis in a 34-year-old woman from New York, in the
United States with this method.* Correspondence: xiongcandy@hotmail.com
4Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072,
China
Full list of author information is available at the end of the article
© 2016 Gao et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Gao et al. Parasites & Vectors  (2016) 9:86 
DOI 10.1186/s13071-016-1363-2
Case report
A 34-year-old female Chinese student who used to study
in New York presented to our services in May 2014.
Over the past 12 months she had visited hospitals in the
United States and China but had received no clear diag-
nosis and was considered to be experiencing symptoms
of psychosis. She reported a one year history of diffuse
erythema to her body, and watery, itchy, red, sore, swell-
ing and stinging of the eyes, and six months of some mi-
gratory subcutaneous nodules in her scapular region,
retroauricular region and buttocks region, and three months
of swelling of the body (Fig. 1). She felt aggravated itching
especially between 10 p.m and 3 a.m and was unable to
sleep through the night. Her father showed similar but
milder clinical symptoms. She was treated empirically with
anti-allergy, steroids and anti-worm medication (ivermectin
and mebendazole) in a local hospital in New York after pre-
senting clinical signs. Despite these interventions, the patient
got worse. Seven months ago, the patient and her father
left for China. In some hospitals of China, the patient
was ever suspected with helminthic infections. However,
all the image and etiological examination (detection of
blood-borne microfilariae), serum immunological tests
(detection of IgG or/ and IgG4 or /IgM to Spirometra
mansoni Sparganum, Cysticercus cellulose, echinococcus
granulosus, Clonorchis sinensis, Paragonimus westermani,
Schistosoma japonicum, richinella spiralis, Angiostrongylus
cantonensis, filarial worms and Toxoplasma gondii) failed
to confirm this suspicion. Given continued suspicion for a
helminthic etiology, the patient was treated with more anti-
parasitic agents such as pumpkin seed, pumpkin seed oil
belonging to traditional herbal medicine and praziquantel
under doctor’s instruction. Initially, these drugs effectively
alleviated the clinical manifestations but the effects were
short lived. The patient started to have strong feelings that
something was moving in the subcutaneous tissues such as
finger, arm and leg joints, head, heel, and exacerbation of
the itching at night.
In the following months after she returned to China,
more image and pathological examinations were performed.
The image examination was unremarkable. The pathological
examination showed there was granulomatous inflammation
Fig. 1 Facial and lower body clinical photographs of the patient. a and b Diffuse red rash and erythema involving the face and leg. c and d Some
migratory subcutaneous nodules scattered in the retroauricular region and buttock region. Photographs courtesy of patient
Table 1 Total clean reads report and the subtraction of human
reads
Sample source Human Non-human
No. of reads Ratio No. of reads Ratio
Eye discharge 7955797 78.3 % 2201909 21.7 %
Subcutaneous tissue 14216801 97.8 % 318269 2.2 %
Gao et al. Parasites & Vectors  (2016) 9:86 Page 2 of 5
in the deep dermis and subcutaneous tissues of the left little
finger, and a calcific body was located in the center of granu-
loma (shown in Additional file 1: Figure S1). Although these
examinations in general revealed a biological pathogen in-
fection, it was still unclear as to what kind of creature this
pathogen could be.
In our hospital, Qilu Hospital of Shandong University,
China, an initial blood test showed leukopenia and eo-
sinophilia with the white blood cell count 3.06 × 109/L
(normal range, 4–10 × 109/L) with 35.70 % neutrophils
(normal range 40–60 %), 35.60 % lymphocytes (normal
range, 20–40 %), 16.30 % monocytes, 12.10 % eosino-
phils (normal range, 1 to 4 %), 0.30 % basophils (normal
range, 0.5–1 %). The haemoglobin was 120 g/L (normal
range, 120–150 g/L); the platelet count was 120 × 109/L
(normal range, 150–400 × 109/L). Urinalysis and stool
tests were unremarkable. The granulomas of the skin,
markedly elevated proportion of eosinophilia and night-
time itching indicated that the patient was possibly in-
fected with helminthes especially filaria. However, poly-
merase chain reaction (PCR) testing of conservative
genes of Brugia malayi, Brugia timori, and Wucheria
bancrofti from DNA in the patient’s blood was negative.
These amplified genes include cytochrome c oxidase
subunit 1 (COXI) gene and ribosomal DNA 18S and
ITS2. Given the absence of a diagnosis and an aggravated
itching and body swelling, an unbiased metagenomic ap-
proach was ultimately used to detect the pathogen infect-
ing the patient. The result suggested that the patient had
been infected by B. malayi. Then the patient and her
father were treated with oral diethylcarbamazine. Three
months later, the symptoms of the patient disappeared.
Methods
Ethical approval
Written consent for clinical sample analysis was obtained
from the patient. This study was approved by the Ethics
Committee of Qilu Hospital of Shandong University.
Because the patient’s eyes were itching terribly and
often secreted foreign matter, the first sample was from
the patient’s eye discharge. Another sample was col-
lected from subcutaneous abdominal tissue which was
suspected to contain pathogen. Detailed methods for
nucleic acid extraction, next-generation sequencing and
bioinformatics analysis are provided in Additional file 2.
Results
The raw reads were submitted to the DDBJ Sequence
Read Archive (SRA) under the following accession
numbers: PRJNA296435 and SRX1267325. As we estimated,
Table 2 The number of non-human reads mapped to each filarial genome
Sample of eye discharge B. malayi O. volvulus L. loa W. bancrofti
Number of reads mapped to filaria genomes 94985 1832 78 4384
Number of unique reads belonging to each species 94841 291 31 2757
Unique reads of each species/total filarial reads 96.9 % 0.3 % 0.03 % 2.8 %
Fig. 2 The Venn Diagram of the four sets of reads mapped to filariae of eye discharge sample
Gao et al. Parasites & Vectors  (2016) 9:86 Page 3 of 5
the non-human reads accounted for a small proportion
in the total sample reads (Table 1). When aligned, the
non-human reads mapped to a candidate parasite pro-
tein database, the reads assigned to B. malayi possess
the largest proportion in both samples of eye discharge
(76.66 %) and subcutaneous tissue (36.45 %) (shown in
Additional file 3: Figure S2 and Additional file 4: Figure S3).
To find more evidence, the non-human reads from the eye
discharge sample were mapped to the four available filarial
genomes separately, and then overlap reads were calculated.
The unique reads of B. malayi possess the largest ratio
96.9 % in the four filarial reads (Table 2). Although W.
bancrofti is assigned the second-largest number of
reads, it only possesses 2.8 % ratio of the total reads.
The number of unique and overlapping reads among
those four genomes was shown in Fig. 2. In the eye dis-
charge sample, B. malayi possesses as many as 94841
unique reads. It suggests that B. malayi could be the
most possible pathogen. Then oral diethylcarbamazine
therapy was initiated for the treatment of B. malayi.
Both the patient and her father received 1000 mg daily
dose for three months. Then erythema and swelling
gradually disappeared and nodules formed scars. In
addition, the patient and her father did not feel itching
any more.
Discussion
The distribution of malayan filariasis is only confined to
Asia. Prior to this report, malayan filariasis is rarely re-
ported from the United States. In this case, the patient
had not left New York in over a year until her father vis-
ited her. Her father reported that he had been living in
Shandong Province, which was never a malayan filariasis
endemic area historically [11], and he had never traveled
elsewhere in the past year before he came to the United
States. In addition, the patient and her father almost pre-
sented similar clinical symptoms at the same time. So it
is difficult to speculate that the patient’s father had been
infected in China before he came to the United States
and the patient was infected through contact indirectly
with her father.
The patient presented an atypical clinical manifestation
compared with previous reports, where patients have
some degree of lymphatic abnormality including adeno-
lymphangitis, lymphedema, lymphadenopathy and uro-
genital lymphangiectasia [12]. In this case, the patient
went through almost all the medical examinations in
the United States and China, but almost all of which
failed to draw any meaningful conclusion. Even in some
hospitals, the patient was considered malayan filariasis
as psychosis. So the more sensitive and broad-spectrum
method of diagnostic metagenomics is promoted as a
potentially life-saving gift to explore the pathogen [13].
This is the first report about detection of helminthic
infections using metagenomic analysis from clinical sam-
ples, and we built an appropriate and rapid pipeline for
the sequence analysis. For infection with a pathogen,
metagenomic analysis should be considered when no
other effective proof is presented.
Additional files
Additional file 1: Figure S1. Histopathological findings in the nodules
of the left little finger. (DOC 2022 kb)
Additional file 2: Methods. (DOC 31 kb)
Additional file 3: Figure S2. Typing of blast hits after analyzing the
eye discharge sample and detail of the phylogenetic MEGAN output.
(DOC 117 kb)
Additional file 4: Figure S3. Typing of blast hits after analyzing
subcutaneous tissue sample and details of the phylogenetic MEGAN
output. (DOC 198 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GQW managed the patient. DG, GTW and FX conceived and designed the
experiments. GTW and FX performed the experiments. QFY and FX analyzed
the data. GQW and FX contributed reagents/materials/analysis tools. DG
wrote the article. All authors read and approved the final manuscript.
Acknowledgments
The authors thank Dr. Wirnkar S. Jadwiga for his help in English language
editing. This study was supported by grants from National Natural Science
Foundation of China (No. 31460696 and 81460462) and the Major Scientific
and Technological Innovation Project of Hubei Province (2015ABA045).
Author details
1Department of Pathogen Biology and Immunology, Medical College of
Nanchang University, Nanchang 330006, China. 2Department of
Gastroenterology and Hepatology, Second Affiliated Hospital of Nanchang
University, Nanchang 330006, China. 3Qilu Hospital of Shandong University,
Jinan 250012, China. 4Institute of Hydrobiology, Chinese Academy of
Sciences, Wuhan 430072, China.
Received: 16 December 2015 Accepted: 5 February 2016
References
1. WHO. Sustaining the drive to overcome the global impact of neglected
tropical diseases. 2013. http://www.who.int/neglected_diseases/
9789241564540/en/.
2. Chu BK, Hooper PJ, Bradley MH, McFarland DA, Ottesen EA. The economic
benefits resulting from the first 8 years of the Global Programme to
Eliminate Lymphatic Filariasis (2000–2007). PLoS Negl Trop Dis. 2010;4(6):e708.
doi:10.1371/journal.pntd.0000708.
3. Rebollo MP, Bockarie MJ. Toward the elimination of lymphatic filariasis by
2020: treatment update and impact assessment for the endgame. Expert
Rev Anti Infect Ther. 2013;11(7):723–31. doi:10.1586/14787210.2013.811841.
4. Plichart C, Lemoine A. Monitoring and evaluation of lymphatic filariasis
interventions: an improved PCR-based pool screening method for high
throughput Wuchereria bancrofti detection using dried blood spots. Parasit
Vectors. 2013;6:110. doi:10.1186/1756-3305-6-110. http://parasitesandvectors.
biomedcentral.com/articles/.
5. Ramzy RM, Helmy H, Faris R, Gad AM, Chandrashekar R, Weil GJ. Evaluation
of a recombinant antigen-based antibody assay for diagnosis of bancroftian
filariasis in Egypt. Ann Trop Med Parasitol. 1995;89(4):443–6.
6. Weil GJ, Lammie PJ, Weiss N. The ICT Filariasis Test: A rapid-format antigen
test for diagnosis of bancroftian filariasis. Parasitol Today. 1997;13(10):401–4.
7. Naccache SN, Peggs KS, Mattes FM, Phadke R, Garson JA, Grant P, et al.
Diagnosis of neuroinvasive astrovirus infection in an immunocompromised
Gao et al. Parasites & Vectors  (2016) 9:86 Page 4 of 5
adult with encephalitis by unbiased next-generation sequencing. Clin Infect
Dis. 2015;60(6):919–23. doi:10.1093/cid/ciu912.
8. Bhatt AS, Freeman SS, Herrera AF, Pedamallu CS, Gevers D, Duke F, et al.
Sequence-based discovery of Bradyrhizobium enterica in cord colitis
syndrome. N Engl J Med. 2013;369(6):517–28. doi:10.1056/NEJMoa1211115.
9. Khairat R, Ball M, Chang CC, Bianucci R, Nerlich AG, Trautmann M, et al. First
insights into the metagenome of Egyptian mummies using next-generation
sequencing. J Appl Genet. 2013;54(3):309–25. doi:10.1007/s13353-013-0145-1.
10. McCarthy CB, Diambra LA, Rivera Pomar RV. Metagenomic analysis of taxa
associated with Lutzomyia longipalpis, vector of visceral leishmaniasis, using
an unbiased high-throughput approach. PLoS Negl Trop Dis. 2011;5(9), e1304.
doi:10.1371/journal.pntd.0001304.
11. Jiang F, Pang Z, Yu S, Li Z. Study on the filariasis eradicated in Qingdao. J
Med Pest Contr. 2005;21(12):86–8 (in Chinese with English abstract).
12. Nutman TB. Insights into the Pathogenesis of Disease in Human Lymphatic
Filariasis. Lymphat Res Biol. 2013;11(3):144–8. doi:10.1089/lrb.2013.0021.
13. Nakamura S, Maeda N, Miron IM, Yoh M, Izutsu K, Kataoka C, et al.
Metagenomic diagnosis of bacterial infections. Emerg Infect Dis. 2008;
14(11):1784–6. doi:10.3201/eid1411.080589.
•  We accept pre-submission inquiries 
•  Our selector tool helps you to find the most relevant journal
•  We provide round the clock customer support 
•  Convenient online submission
•  Thorough peer review
•  Inclusion in PubMed and all major indexing services 
•  Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
Submit your next manuscript to BioMed Central 
and we will help you at every step:
Gao et al. Parasites & Vectors  (2016) 9:86 Page 5 of 5


file:///data/core-remote/dit/data/Springer-OA/pdf/3ec/aHR0cDovL2xpbmsuc3ByaW5nZXIuY29tLzEwLjExODYvczEzMDcxLTAxNi0xMzYzLTIucGRm.pdf

Diagnosis of a malayan filariasis case using a shotgun diagnostic metagenomics assay

Abstract

Similar works

Full text

Available Versions

FigShare

FigShare

Springer - Publisher Connector

Institute of Hydrobiology, Chinese Academy Of Sciences

Springer - Publisher Connector

Institute of Hydrobiology, Chinese Academy Of Sciences