Rapid and precise diagnosis of disseminated T.marneffei infection assisted by high-throughput sequencing of multifarious specimens in a HIV-negative patient: a case report
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Talaromyces marneffei, is an opportunistic pathogenic fungus that is most commonly reported in Southeast Asia and disseminated T.marneffei infection predominantly occurs in patients with immunodeficiency. With a potential to invade multiple organs, it can be fatal for patients if diagnosis and treatment are delayed. In current clinical practice, the diagnosis of T.marneffei infection relies heavily on tissue culture and histologic analysis, which may suffer from limited positive rate and is sometimes time consuming. The rapid and accurate diagnosis of disseminated T.marneffei infection remains challenging.
A 22-year-old man gradually developed fever, cough, lower extremities weakness, jaundice and rash, for which a 3-month extensive investigation failed to reach a diagnosis. After admitted into our hospital, laboratory and radiological tests revealed multiple lesions in the patient’s brain, spinal cord, and lungs. We performed next generation sequencing on the patient’s skin tissue, bone marrow, blood and cerebrospinal fluid, which all identified numerous Talaromyces marneffei nucleotide sequences and leaded to the rapid diagnosis and treatment of disseminated T.marneffei infection.
This case underline the clinical significance of T.marneffei as a possible pathogen in immune-competent patients. This successful application of the next generation sequencing assisting the rapid diagnosis of disseminated T.marneffei infection provides a new perspective in the clinical approach to the systematic fungi infections and highlights the potential of this technique in rapid etiological diagnosis.
KeywordsTalaromyces marneffei Fungi Next generation sequencing High throughput sequencing Sterile body fluids
Bronchoalveolar lavage Fluid
Cryptococcal latex agglutination test
Direct bilirubin concentration
Hepatitis B virus
Hepatitis C virus
Human immunodeficiency virus
Magnetic resonance imaging
Next generation sequencing
Polymerase chain reaction
Positron emission tomography/computed tomography
Maximum standardized uptake value
Total bilirubin concentration
Talaromyces marneffei, formerly known as Penicillium marneffei, is a pathogenic, thermal dimorphic fungus that is most commonly reported in Southeast Asia. T.marneffei can cause disseminated infections and invade multiple organ systems such as blood, bone marrow, central nervous system, lungs and skins, therefore can be fatal for patients if not diagnosed and treated in time. Disseminated T.marneffei is known to predominantly occurr in patients with immunosuppression, such as HIV patients and patients receiving monoclonal antibody treatment [1, 2, 3], and is seldomly reported in non-HIV patients. The standard diagnostic method for T.marneffei infection mainly relies on tissue culture and histologic analysis, and previous studies have reported that whole blood nested polymerase chain reaction (PCR) and real-time PCR can provide an identification of the pathogen in HIV-infected patients . Nevertheless, due to the low yield rate of tissue culture, the timely and accurate diagnosis of disseminated T.marneffei infection remains challenging.
After the treatment, no remission of the previous symptoms was observed and the patient developed new discomforts including fever (Tmax 39 °C), productive cough and jaundice in the early April. Repeated blood, sputum and CSF cultures all came back negative. During the next two months, the patient continuously to have intermittent low grade fever, weakness of lower extremities and jaundice and by the end of May, rashes started to appear over his face and back. The local medical facility then transferred the patient to our hospital on June 7th, 2017.
We then conducted sequencing of the isolated strain from the skin lesion culture, and a 97% coverage of T.marneffei was identified (Fig. 3b). Close relationship between our strain and other T.marneffei strains was revealed in phylogenetic analysis (Fig. 3c). Confirmatory nested PCR  was done in patient’s blood, CSF, BALF and bone marrow samples and all PCR results were positive.
Discussion and conclusions
This is the first case report describing the diagnosis of disseminated T.marneffei infection in a HIV-negative patient with the assistance of unbiased NGS, which detected unique sequence reads corresponding to T.marneffei from multiple body fluids and tissues including bone marrow, CSF, BALF, and skin tissue. In this particular case, NGS identified T.marneffei as the causative agent without any prior assumptions from the physicians, leading to a prompt treatment and quick relieve of the disease. The sequencing data, which was in consistent with the patient’s clinical features and skin tissue culture result, finally assisted clinical physicians in approaching the diagnosis of disseminated T.marneffei infection.
Traditionally, the gold standard for infection diagnosis relies heavily on the isolation of pathogenic pathogen and therefore may suffer from drawbacks due to the limited culture positive rate . Traditional PCR (including RT-PCR and nested PCR) or advanced multiplexed PCR assays provides advantages of high sensitivity, low cost and fast detection, however, the former method requires the physician to raise a few suspicious pathogens prior to examination and the latter method is usually restricted to only limited range of pathogens. Therefore, these methods are currently best applicable in certain clinical situations such as sepsis and respiratory infections [6, 7]. Under such circumstances, the implementation of NGS in clinical field enabled a fast and comparably accurate diagnostic tool for physicians, and most importantly, it does not require a predefined range of suspicious pathogens. The principles of NGS in infectious diseases are composed of three main procedures, the identification of the DNA nucleotides in the targeted samples, the comparison of these nucleotides against the catalogue of causative agents, and the decision-making progress whether the acquired sequences points to the possible etiological hypothesis . The first study reporting using NGS in the diagnosis of infectious diseases was published in 2014, in a case of neuroleptospirosis in which 475 of 3,063,784 leptospira sequence reads (0.016%) were detected in the spinal fluid . Since then, multiple studies have reported the use of NGS in central nervous system, bloodstream, and respiratory infections [10, 11, 12, 13]. However, to our knowledge, few study has reported the use of NGS in the clinical fungi diagnosis.
One important limitation of NGS is the interference of the massive human nucleotide sequences during the data analysis. In a study of etiology diagnosis of NGS, the cell-free DNA (cfDNA) is extracted from plasma instead of whole blood for sequencing to maximize the percentage of sequences mapped to the pathogens. However, human reads still takes up around 95% of the total reads in the plasma samples . In CSF, BALF and other body fluids, DNA rather than cfDNA is extracted from the sample and the studies have reported the detection of around 70–90% of the human reads [11, 12]. Hence, the filtration of the human nucleotide sequences during analysis is critical. Another major challenge of clinical use of NGS is how to interpret the results and determine whether the microorganism whose sequences are identified is truly the causative pathogen. The nature of NGS tends to detect all nucleotide sequences not only from the samples but also those acquired from the contamination during clinical procedures or laboratory processing, and thus NGS is limited in discriminating among colonization, infection or contamination. What’s more, identification of any nucleotide sequences of certain pathogens could not draw to the conclusion that this specific agents indeed exists in the sample, as the analysis merely indicate the mapping of a partial DNA fragment to a certain pathogen but could not retrieve its complete genome . This may result in one read matching to multiple microorganisms due to the homology among them, as previous studies have shown . Although some studies have reported the possible correlations between the number of pathogen-matched reads detected and the possibility of infection, few acknowledged explanation criteria of sequencing result has been established. Currently, there still lacks high-quality cohort study to reinforce this point and we now still rely on real-time PCR or other laboratory confirmation tests to assist NGS in the clinical approach.
This study provided a valuable case exploring the potential and possibility of NGS assisting the rapid clinical actionable diagnosis of T.marneffei and possibly other fungi from a multifarious body fluids and tissue types. Complementing the traditional laboratory and imaging tests, NGS may thus facilitate the precise diagnosis and the efficacious antimicrobial treatment in the field of clinical fungi infection.
This study was supported by the New and Advanced Technology Project of Shanghai Municipal Hospital: Application of next generation sequencing technique in precise diagnosis of infectious diseases (SHDC12017104).
Availability of data and materials
The sequencing data of the isolated T.marneffei can be downloaded at “https://github.com/uuashark/Infectious-Diseases-Department-of-Huashan-Hospital/tree/master/Talaromyces_ Marneffei_Huashan_strain”. Other data supporting our findings is contained within the manuscript. The datasets used and/or analysed during the current study are also available from the corresponding author on reasonable request.
JWA, YMZ and BX collected and analyzed medical data of the patient, wrote and revised the manuscript. WHZ, QC, YYQ and XZ participated in the treatment of the patient during hospitalization. PC, HCZ, HLW, LX, RHW and YJL participated in the next generation sequencing and data analysis. WHZ made a critical contribution to the treatment plan of the patient and made a critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
We identified this patient during routine clinical practice and consented to give venous blood samples after elaborate information. Written informed consent was obtained from the patient for publication of this case report and all accompanying images. A copy of the written consent is available for review by the editor of this journal. Involvement of the ethical committee of the Huashan Hospital of Fudan University was considered unnecessary, since the project was not based on a study protocol.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and all accompanying images.
The authors declare that they have no competing interests.
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