The Open AIDS Journal




ISSN: 1874-6136 ― Volume 13, 2019
CASE REPORT

Utility of Whole-Genome Next-Generation Sequencing of Plasma in Identifying Opportunistic Infections in HIV/AIDS



Yang Zhou1, Vagish Hemmige2, Sudeb C. Dalai3, 6, David K. Hong3, Kenneth Muldrew4, Mayar Al Mohajer5, *
1 Department of Internal Medicine, Baylor College of Medicine, Houston, TX, USA
2 Department of Infectious Disease, Montefiore Medical Center, Bronx, NY, USA
3 Department of Medical Affairs, Karius, Inc., Redwood City, CA, USA
4 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA
5 Infection Prevention and Antimicrobial Stewardship, Baylor St. Luke's Medical Center, Houston, TX, USA
6 Division of Infectious Diseases, Stanford University Medical Center, Stanford, CA, USA

Abstract

Background:

AIDS-associated Opportunistic Infections (OIs) have significant morbidity and mortality and can be diagnostically challenging, requiring invasive procedures as well as a combination of culture and targeted molecular approaches.

Objective:

We aimed to demonstrate the clinical utility of Next-generation Sequencing (NGS) in pathogen identification; NGS is a maturing technology enabling the detection of miniscule amounts of cell-free microbial DNA from the bloodstream.

Methods:

We utilized a novel Next-generation Sequencing (NGS) test on plasma samples to diagnose a series of HIV-associated OIs that were diagnostically confirmed through conventional microbial testing.

Results:

In all cases, NGS test results were available sooner than conventional testing. This is the first case series demonstrating the utility of whole-genome NGS testing to identify OIs from plasma in HIV/AIDS patients.

Conclusion:

NGS approaches present a clinically-actionable, comprehensive means of diagnosing OIs and other systemic infections while avoiding the labor, expense, and delays of multiple tests and invasive procedures.

Keywords: Human immunodeficiency virus, Acquired Immune Deficiency Syndrome, Opportunistic infections, Next-generation sequencing, Cell-free DNA, Toxoplasma gondii, Toxoplasmosis, Mycobacterium avium complex, Mycobacterium tuberculosis, Cryptosporidium.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 7
Last Page: 11
Publisher Id: TOAIDJ-13-7
DOI: 10.2174/1874613601913010007

Article History:

Received Date: 10/8/2018
Revision Received Date: 18/12/2018
Acceptance Date: 09/01/2019
Electronic publication date: 13/2/2019
Collection year: 2019

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© 2019 Zhou et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Baylor College of Medicine, Infection Prevention and Antimicrobial Stewardship, Baylor St. Luke's Medical Center, Houston, TX, United States; Tel: (832) 355-7848; E-mail: Mayar.AlMohajer@bcm.edu




1. BACKGROUND

Despite advancements in Highly-active Antiretroviral Therapy (HAART) and overall survival in HIV/AIDS, Opportunistic Infections (OIs) remain a significant cause of AIDS-associated morbidity and mortality, particularly in the setting of advanced disease and delayed or limited access to care [1Kaplan JE, Benson C, Holmes KK et al. Guidelines for the prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: Recommendations from the Centers for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America MMWR Recomm Rep 2009; 58(RR-4): 1-207.]. Many OIs remain diagnostically challenging, requiring invasive procedures for tissue/fluid sampling as well as a combination of culture and targeted molecular approaches that can be laborious, expensive and limited by the sensitivity/specificity of individual assays. Next-generation Sequencing (NGS) is a maturing technology enabling detection of miniscule amounts of genetic material [2Metzker ML. Sequencing technologies - The next generation. Nat Rev Genet 2010; 11(1): 31-46.[http://dx.doi.org/10.1038/nrg2626] [PMID: 19997069] ] that has demonstrated clinical utility in the diagnosis of fetal chromosomal anomalies [3Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008; 105(42): 16266-71.[http://dx.doi.org/10.1073/pnas.0808319105] [PMID: 18838674] ], solid organ transplant rejection [4De Vlaminck I, Martin L, Kertesz M, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci USA 2015; 112(43): 13336-41.[http://dx.doi.org/10.1073/pnas.1517494112] [PMID: 26460048] ], and more recently pathogen identification [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press-10Vudatha V, Ranson M, Blair L, Ahmed AA. Rapid detection of BCG-associated mycotic aortic aneurysm utilizing novel cell-free DNA assay. J Vasc Surg in press]. We describe our experience in the diagnosis of AIDS-associated OIs at a single institution with a novel, noninvasive NGS test that detects pathogen cell-free DNA (cfDNA) from plasma samples [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press-7Abril MK, Barnett AS, Wegermann K, et al. Diagnosis of Capnocytophaga canimorsus sepsis by whole-genome next-generation sequencing. Open Forum Infect Dis 2016; 3(3): ofw144.[http://dx.doi.org/10.1093/ofid/ofw144] [PMID: 27704003] ]. Cases included in this series are summarized in the accompanying Table and described below.

Methods for sample preparation and plasma NGS for microbial cfDNA in the present series have been previously described [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press, 6Hong DK, Blauwkamp TA, Kertesz M, Bercovici S, Truong C, Banaei N. Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn Microbiol Infect Dis 2018; 92(3): 210-3.[http://dx.doi.org/10.1016/j.diagmicrobio.2018.06.009] [PMID: 3001 7314] ] and validated in terms of accuracy, precision, bias and robustness [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press]. Briefly, blood was collected in a Plasma Preparation Tube (PPT, BD Biosciences), centrifuged at 1100 RCF for 10 minutes and then shipped to a CLIA-certified, CAP-accredited laboratory for analysis (Karius, Inc., Redwood City, CA), where cfDNA was extracted from plasma. For NGS, sequencing libraries were constructed and control samples were processed alongside test samples in every batch. Batched libraries were multiplexed and sequenced on Illumina NextSeq500 sequencers. Samples generated, on average, approximately 24 million sequencing reads each. After removing human reads, remaining sequences were aligned using BLAST+ [11Camacho C, Coulouris G, Avagyan V, et al. BLAST+: Architecture and applications. BMC Bioinformatics 2009; 10: 421.[http://dx.doi.org/10.1186/1471-2105-10-421] [PMID: 20003500] ] to a proprietary curated microorganism database allowing detection of over 1000 microbial pathogens including DNA viruses, bacteria, fungi and other eukaryotes. Several filtering algorithms were used to mitigate false-positive results, including removal of environmental contaminants and cross-reactivity with other microorganisms [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press]. Subsequently, organisms present above a predefined significance threshold were reported. Analysis methods have been described in detail elsewhere [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press, 6Hong DK, Blauwkamp TA, Kertesz M, Bercovici S, Truong C, Banaei N. Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn Microbiol Infect Dis 2018; 92(3): 210-3.[http://dx.doi.org/10.1016/j.diagmicrobio.2018.06.009] [PMID: 3001 7314] ].

2. CASE PRESENTATIONS

2.1. Case 1

2.1.1. CNS Toxoplasmosis

A 49-year-old man presented with one week of left hand weakness and two days of progressive headache, nausea, and vomiting. Notably, two weeks prior at an outside hospital the patient was found to have an enhancing intra-cerebral mass, with initial pathology from biopsy concerning for high-grade glioma. Repeat head CT on the present admission showed no new findings. On hospital day 4, the patient became more somnolent, and CT showed increased cerebral edema which improved with medical management. On day 10, brain MRI performed to evaluate for primary malignancy revealed ring-enhancing lesions in the left cerebral hemisphere and right cerebellum. Subsequent HIV testing was positive (CD4+ cell count 4 cells/mL, HIV-1 viral load 197,000 copies/mL) and on day 11 empiric trime thoprim-sulfamethoxazole was initiated for possible CNS toxoplasmosis. Chest CT showed bilateral pulmonary infiltrates, with subsequent bronchoscopic workup unrevealing (Table 1). Blood cultures and antigen tests for Strongyloides (stool), Cryptococcus (blood) and Histoplasma (urine) were also negative. Blood Cytomegalovirus (CMV) PCR was positive. Toxoplasma serum IgG was positive with negative IgM. Lumbar Puncture (LP) could not be performed safely due to the risk of cerebral herniation.

Table 1
Summary of HIV-associated opportunistic infections identified by NGS*.


Minimal clinical improvement was seen after one week of hospitalization, and a repeat brain MRI suggested disease progression. Treatment was progressively escalated to pyrimethamine, sulfadiazine, and leucovorin for CNS toxoplasmosis (day 28) in addition to empiric treatment for bacterial infection, CMV, and Tuberculosis (TB). Plasma NGS testing sent on day 28 was positive on day 30 for Toxoplasma gondii, Nocardia farcinica, CMV, Enterococcus faecalis and Enterococcus faecium. Pathology slides obtained from the outside hospital during the patient’s prior admission were stain-positive for Toxoplasma. On day 34, HAART was initiated and on day 39 steroids were escalated and an extraventricular drain (EVD) placed for mental deterioration and possible paradoxical Immune Reconstitution Inflammatory Syndrome. Cerebrospinal Fluid (CSF) obtained from the EVD was subsequently positive for Toxoplasma gondii PCR. The patient gradually improved and was discharged on day 56 with prolonged treatment for CNS toxoplasmosis until his CD4+ count recovered to >200 cells/mL.

2.2. Case 2

2.2.1. Disseminated Mycobacterium Avium (MAC)

A 23-year-old male with intravenous drug use and known HIV/AIDS (CD4+ cell count 9 cells/mL, HIV-1 viral load 809,000 copies/mL) was admitted two weeks after starting HAART with intermittent fever and night sweats without localizing symptoms. Labs demonstrated mild transaminitis and elevated Alkaline Phosphatase (ALP). Initial blood cultures were unrevealing. CT chest, abdomen and pelvis showed multiple prominent mediastinal, abdominal, and pelvic Lymph Nodes (LNs).

After 9 days of persistent fever, the patient was started on empiric MAC therapy. A plasma NGS test was sent, detecting Mycobacterium Avium Complex (MAC) and CMV two days later. Bone marrow biopsy demonstrated granulomas with negative AFB and fungal stains. Blood CMV PCR was negative, but AFB blood cultures grew MAC several days after the NGS result. Bone marrow samples eventually grew MAC as well. The patient defervesced on appropriate MAC therapy and was discharged.

2.3. Case 3

2.3.1. Mycobacterium Tuberculosis (MTB) Lymphadenitis

A 56-year-old male with untreated HIV/AIDS (CD4+ cell count 25 cells/mL, HIV-1 viral load 527,000 copies/ mL) presented with 6 weeks of worsening fatigue, confusion, imbalance, headaches, abdominal pain, diarrhea, and anorexia. On admission, he was febrile, tachycardic, and cachetic, with initial labs showing leukopenia, anemia and elevated creatinine. He denied sick contacts, travel, animal exposures, or new foods, and was last sexually active six months prior with consistent condom use. Chest x-ray was unremarkable and CT abdomen and pelvis showed reactive LNs in the abdomen and retroperitoneum. The patient was started on vancomycin and cefepime but continued having high fevers, so ampicillin and acyclovir were added. Diagnostic workup included negative blood cultures, negative fecal leukocytes, and normal CSF studies (Table 1). Brain imaging showed no acute abnormalities. Empiric treatment was initiated for suspected disseminated MAC infection. CT chest showed multiple pulmonary nodules and patchy airspace disease. Bronchoscopy and tissue pathology revealed granulomas, but multiple diagnostic studies were negative (Table 1).

Plasma NGS testing sent on day 4 was positive on day 10 for MTB and EBV. Retroperitoneal LN biopsy on day 4 showed acute necrotizing lymphadenitis with positive AFB stain. Isoniazid and pyrazinamide were added for TB treatment. The patient improved and was discharged on day 17 with 4-drug TB treatment, HAART and relevant OI prophylaxis. AFB cultures of LN and respiratory samples eventually became positive for MTB following hospital discharge 21 and 26 days after collection, respectively. The patient’s CD4+ count increased from 25 to 93 cells/ mm3 and viral load decreased from 527,000 to 365 copies/mL on appropriate therapy.

2.4. Case 4

2.4.1. Microsporidiosis

A 45-year-old man without significant medical history presented with 6-8 weeks of chronic diarrhea unresponsive to empiric metronidazole treatment. He was newly diagnosed with HIV (CD4+ cell count 339 cells/mL, HIV-1 viral load 221,000 copies/mL) with mild transaminitis and elevated ALP. He experienced intermittent fevers during hospitalization. CT chest, abdomen, and pelvis revealed diffuse lymphadenopathy. LP showed CSF WBC count 160 cells/mm3 with lymphocyte predominance (91%), protein 84 mg/dL, and glucose 37 mg/dL. Multiple CSF studies were negative (Table 1). Stool ova/parasite examination was negative for Cyclospora, Isospora and Cryptosporidia. A gastrointestinal pathogen stool PCR panel tested positive for Norovirus and Cryptosporidium. Plasma NGS testing was positive for Enterocytozoon bieneusi, a cause of microsporidiosis, on day 6. It should be noted that Norovirus is an RNA pathogen and as such, would not be detected by plasma NGS testing for cfDNA. Also, the GI PCR panel used does not test for Enterocytozoon or other Microsporidia. The detection of Enterocytozoon by NGS was felt by the treating physician to be consistent with the detection of Crypto sporidium by stool PCR testing, as Microsporidia/Cryptosporidia co-infections can be seen in up to 81% of HIV/ AIDS patients with chronic diarrhea [12Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, et al. Cryptosporidiosis and microsporidiosis in ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus. Am J Trop Med Hyg 2005; 73(5): 921-5.[http://dx.doi.org/10.4269/ajtmh.2005.73.921] [PMID: 16282304] ]. The patient was started on nitazoxanide to treat both Cryptosporidium and Microsporidium [13Bicart-Sée A, Massip P, Linas MD, Datry A. Successful treatment with nitazoxanide of Enterocytozoon bieneusi microsporidiosis in a patient with AIDS. Antimicrob Agents Chemother 2000; 44(1): 167-8.[http://dx.doi.org/10.1128/AAC.44.1.167-168.2000] [PMID: 10602 740] ] and symptoms resolved with treatment.

DISCUSSION AND CONCLUSION

OIs are often the initial presentation for the estimated 15% of HIV-positive U.S. adults who are unaware of their HIV status [14Satcher Johnson A, Song R, Hall HI. Estimated HIV incidence, prevalence, and undiagnosed infections in US States and Washington, DC, 2010-2014. J Acquir Immune Defic Syndr 2017; 76(2): 116-22.[http://dx.doi.org/10.1097/QAI.0000000000001495] [PMID: 28700 407] ]. Timely diagnosis remains critical to improving survival and preventing long-term sequelae. Diagnosis of CNS OIs can be challenging due to limited availability and/or sensitivity of the array of simultaneous diagnostic tests typically used, including CSF microscopy, antibody/antigen and targeted PCR testing, serologies for specific pathogens, neuroimaging and brain biopsy [15Bowen LN, Smith B, Reich D, Quezado M, Nath A. HIV-associated opportunistic CNS infections: Pathophysiology, diagnosis and treatment. Nat Rev Neurol 2016; 12(11): 662-74.[http://dx.doi.org/10.1038/nrneurol.2016.149] [PMID: 27786246] , 16Tan IL, Smith BR, von Geldern G, Mateen FJ, McArthur JC. HIV-associated opportunistic infections of the CNS. Lancet Neurol 2012; 11(7): 605-17.[http://dx.doi.org/10.1016/S1474-4422(12)70098-4] [PMID: 22710 754] ]. Similarly, diagnosis of mycobacterial infections such as MTB or disseminated MAC relies on a combination of radiologic imaging, skin or blood tests, and tissue analysis including microscopy, AFB culture, and PCR. These methods are time-consuming and costly, have varying sensitivity and specificity, and/or necessitate invasive tissue sampling for definitive diagnosis [17Daley CL. Mycobacterium avium complex disease. Microbiol Spectr 2017; 5(2)[http://dx.doi.org/10.1128/microbiolspec.TNMI7-0045-2017] [PMID: 28 429679] , 18Scott L, da Silva P, Boehme CC, Stevens W, Gilpin CM. Diagnosis of opportunistic infections: HIV co-infections - tuberculosis. Curr Opin HIV AIDS 2017; 12(2): 129-38.[http://dx.doi.org/10.1097/COH.0000000000000345] [PMID: 28059 955] ].

The advantages of NGS approaches to infectious disease diagnosis include rapidity, accuracy, breadth of detection, and avoidance of laborious, costly and/or invasive procedures. We describe 4 cases of HIV-associated OIs where diagnostic uncertainty along with critical illness requiring aggressive management prompted the use of a novel high-sensitivity NGS platform that provided rapid, unbiased detection of cell-free pathogen DNA from a plasma sample. Results from plasma NGS were available faster than conventional diagnostics and were consistent with other test results (Table 1) as well as the clinical scenario.

Nonetheless, a number of challenges must be overcome in order to apply NGS approaches to clinical infectious disease practice. Although strong pathogen nucleic acid signal is present in cell-free plasma, there is a significantly higher abundance of human DNA. To overcome this challenge, an enrichment protocol was developed that allows for efficient detection of pathogen signal relative to the background. Furthermore, a high-quality microorganism database is essential to support a comprehensive and accurate assay. Reference microorganism sequences originating from public databases are known to be both incomplete and inaccurate. To address potential errors stemming from inaccuracies in sequence data, a proprietary database was developed that 1) includes only reference sequences with indicators of high-quality assembly (e.g. completeness, N50 score, etc.); 2) is continuously curated to minimize human cross-reactivity as well as cross-reactivity between pathogens; and 3) is screened to mitigate contamination with sequences from human or other organisms. The clinical challenges of utilizing NGS include distinguishing colonization from true infection which can affect specificity. In the cases presented, polymicrobial results could be difficult to interpret. However, when considering the high pretest probability for particular OIs, the relevant and causative pathogens were readily discerned. Finally, NGS testing for clinical care requires sophisticated instrumentation and personnel expertise alongside robust procedures and protocols that will mitigate the risks of contamination and errors in specimen handling. In the present series, NGS was performed at a CAP-accredited and CLIA-certified reference laboratory which currently provides validated commercial NGS testing for hospitals.

The advantages of using cell-free DNA for non-invasive diagnostic testing are already being realized in prenatal diagnosis of genetic disorders [3Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008; 105(42): 16266-71.[http://dx.doi.org/10.1073/pnas.0808319105] [PMID: 18838674] ], cancer diagnostics [19Aravanis AM, Lee M, Klausner RD. Next-generation sequencing of circulating tumor DNA for early cancer detection. Cell 2017; 168(4): 571-4.[http://dx.doi.org/10.1016/j.cell.2017.01.030] [PMID: 28187279] ], and transplant rejection monitoring [4De Vlaminck I, Martin L, Kertesz M, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci USA 2015; 112(43): 13336-41.[http://dx.doi.org/10.1073/pnas.1517494112] [PMID: 26460048] ], and are more recently being realized in the field of infectious diseases [5Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press-10Vudatha V, Ranson M, Blair L, Ahmed AA. Rapid detection of BCG-associated mycotic aortic aneurysm utilizing novel cell-free DNA assay. J Vasc Surg in press]. This is the first case series describing the utility of NGS on cfDNA for diagnosis of OIs in advanced HIV/AIDS patients. We propose that genome-wide approaches to pathogen identification are now clinically relevant and actionable, and offer distinct advantages over traditional diagnostic methods in feasibility, avoidance of invasive procedures, cost-effectiveness, and clinical outcomes. Further studies will seek to validate and document the cost, time, and impacts on morbidity/ mortality.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors S.C. Dalai and D.K. Hong are employees of Karius, Inc. All other authors have no conflicts of interest to report.

ACKNOWLEDGEMENTS

We would like to thank Sumedha Sinha and David Katzenstein for critical review of the manuscript.

REFERENCES

[1] Kaplan JE, Benson C, Holmes KK et al. Guidelines for the prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: Recommendations from the Centers for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America MMWR Recomm Rep 2009; 58(RR-4): 1-207.
[2] Metzker ML. Sequencing technologies - The next generation. Nat Rev Genet 2010; 11(1): 31-46.[http://dx.doi.org/10.1038/nrg2626] [PMID: 19997069]
[3] Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008; 105(42): 16266-71.[http://dx.doi.org/10.1073/pnas.0808319105] [PMID: 18838674]
[4] De Vlaminck I, Martin L, Kertesz M, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci USA 2015; 112(43): 13336-41.[http://dx.doi.org/10.1073/pnas.1517494112] [PMID: 26460048]
[5] Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free dna sequencing test for infectious disease. Nat Microbiol in press
[6] Hong DK, Blauwkamp TA, Kertesz M, Bercovici S, Truong C, Banaei N. Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn Microbiol Infect Dis 2018; 92(3): 210-3.[http://dx.doi.org/10.1016/j.diagmicrobio.2018.06.009] [PMID: 3001 7314]
[7] Abril MK, Barnett AS, Wegermann K, et al. Diagnosis of Capnocytophaga canimorsus sepsis by whole-genome next-generation sequencing. Open Forum Infect Dis 2016; 3(3): ofw144.[http://dx.doi.org/10.1093/ofid/ofw144] [PMID: 27704003]
[8] Kowarsky M, Camunas-Soler J, Kertesz M, et al. Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA. Proc Natl Acad Sci USA 2017; 114(36): 9623-8.[http://dx.doi.org/10.1073/pnas.1707009114] [PMID: 28830999]
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[10] Vudatha V, Ranson M, Blair L, Ahmed AA. Rapid detection of BCG-associated mycotic aortic aneurysm utilizing novel cell-free DNA assay. J Vasc Surg in press
[11] Camacho C, Coulouris G, Avagyan V, et al. BLAST+: Architecture and applications. BMC Bioinformatics 2009; 10: 421.[http://dx.doi.org/10.1186/1471-2105-10-421] [PMID: 20003500]
[12] Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, et al. Cryptosporidiosis and microsporidiosis in ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus. Am J Trop Med Hyg 2005; 73(5): 921-5.[http://dx.doi.org/10.4269/ajtmh.2005.73.921] [PMID: 16282304]
[13] Bicart-Sée A, Massip P, Linas MD, Datry A. Successful treatment with nitazoxanide of Enterocytozoon bieneusi microsporidiosis in a patient with AIDS. Antimicrob Agents Chemother 2000; 44(1): 167-8.[http://dx.doi.org/10.1128/AAC.44.1.167-168.2000] [PMID: 10602 740]
[14] Satcher Johnson A, Song R, Hall HI. Estimated HIV incidence, prevalence, and undiagnosed infections in US States and Washington, DC, 2010-2014. J Acquir Immune Defic Syndr 2017; 76(2): 116-22.[http://dx.doi.org/10.1097/QAI.0000000000001495] [PMID: 28700 407]
[15] Bowen LN, Smith B, Reich D, Quezado M, Nath A. HIV-associated opportunistic CNS infections: Pathophysiology, diagnosis and treatment. Nat Rev Neurol 2016; 12(11): 662-74.[http://dx.doi.org/10.1038/nrneurol.2016.149] [PMID: 27786246]
[16] Tan IL, Smith BR, von Geldern G, Mateen FJ, McArthur JC. HIV-associated opportunistic infections of the CNS. Lancet Neurol 2012; 11(7): 605-17.[http://dx.doi.org/10.1016/S1474-4422(12)70098-4] [PMID: 22710 754]
[17] Daley CL. Mycobacterium avium complex disease. Microbiol Spectr 2017; 5(2)[http://dx.doi.org/10.1128/microbiolspec.TNMI7-0045-2017] [PMID: 28 429679]
[18] Scott L, da Silva P, Boehme CC, Stevens W, Gilpin CM. Diagnosis of opportunistic infections: HIV co-infections - tuberculosis. Curr Opin HIV AIDS 2017; 12(2): 129-38.[http://dx.doi.org/10.1097/COH.0000000000000345] [PMID: 28059 955]
[19] Aravanis AM, Lee M, Klausner RD. Next-generation sequencing of circulating tumor DNA for early cancer detection. Cell 2017; 168(4): 571-4.[http://dx.doi.org/10.1016/j.cell.2017.01.030] [PMID: 28187279]

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