Diagnosis of a malayan filariasis case using a shotgun diagnostic metagenomics assay
© Gao et al. 2016
Received: 16 December 2015
Accepted: 5 February 2016
Published: 16 February 2016
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.
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.
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.
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 socioeconomic development . It accounts for about 40 million chronically disabled or incapacitated people . Three filarial nematodes, Brugia malayi, Brugia timori, and Wucheria bancrofti are responsible for lymphatic filariasis, which involves asymptomatic, acute, and chronic conditions .
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 inconvenient 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 peripheral blood. Since the 1990s, a number of techniques including 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 filariasis at the same time. Therefore a doctor easily makes misdiagnosis 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  and bacterial pathogen detection . It has rarely been used in the detection of parasitic infection except in limited related cases [9, 10]. Here we confirmed a case of malayan filariasis in a 34-year-old woman from New York, in the United States with this method.
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 in the deep dermis and subcutaneous tissues of the left little finger, and a calcific body was located in the center of granuloma (shown in Additional file 1: Figure S1). Although these examinations in general revealed a biological pathogen infection, 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 eosinophilia 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 % eosinophils (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 infected with helminthes especially filaria. However, polymerase 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 approach was ultimately used to detect the pathogen infecting 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.
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 collected 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.
Total clean reads report and the subtraction of human reads
No. of reads
No. of reads
The number of non-human reads mapped to each filarial genome
Sample of eye discharge
Number of reads mapped to filaria genomes
Number of unique reads belonging to each species
Unique reads of each species/total filarial reads
The distribution of malayan filariasis is only confined to Asia. Prior to this report, malayan filariasis is rarely reported from the United States. In this case, the patient had not left New York in over a year until her father visited her. Her father reported that he had been living in Shandong Province, which was never a malayan filariasis endemic area historically , 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 presented 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 adenolymphangitis, lymphedema, lymphadenopathy and urogenital lymphangiectasia . 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 . This is the first report about detection of helminthic infections using metagenomic analysis from clinical samples, 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.
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).
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