Metagenomic Next-generation Nequencing Aids the Diagnosis of Co-infection In HIV-infected Patients

Background HIV-infected patients are easy to combine with various opportunistic infections due to their low immunity. Improving microbiological diagnosis in these patients is of paramount clinical importance. Methods Thirty-six HIV-infected patients with suspected infection were retrospectively reviewed from April 2018 to December 2019. The diagnostic performance of pathogens was compared between traditional microbiological methodology and mNGS. Results The sensitivity of mNGS for diagnosing infectious disease was outperformed of culture, especially for virus (mNGS only; P < 0.001), fungus (odds ratio [OR], 1.1[95% condence interval {CI}, 0.9– 1.5]; P < 0.05), parasite (mNGS only; P < 0.01) and Mycobacterium tuberculosis (OR, 1.7 [95% CI, 1.0 − 2.8]; P < 0.05). Moreover, for mNGS-positive cases where the conventional method was inconclusive, 27 (69.2%) cases led to diagnosis modication. Conclusions mNGS was more sensitive and comprehensive for pathogen identication therefore a promising method for microbiological diagnosis in AIDS patients with infection.


Introduction
Globally, 36.9 million people are living with human immunode ciency virus (HIV) infection/acquired immunode ciency syndrome (AIDS), with 1.7 million new infections in 2018 [1]. HIV virus infection reduces the number and functionality Of CD4 helper lymphocytes that direct and coordinate acquired immunity against most pathogens [2]. The gradual decrease in CD4 T lymphocytes cells ultimately results in a loss of control over immune response and the development of various opportunistic infections [3,4], including bacteria, virus, parasites and fungi [5]. Although, the incidence of opportunistic infections has decreased after the discovery of antiretroviral drugs, it continued to be a serious issue which may have a signi cant impact on their well-being, quality of life, health care costs and their survival [6][7][8]. Pathogen identi cation of infectious disease is always di cult, leading precision diagnosis challenging in these patients. The low positivity rate and long time-consuming of conventional culture methodology increased di culty of accurate and timely diagnosis, while the culture-independent techniques such as nucleic acid ampli cation tests needed for target speci c primers. By combining unbiased sequencing, rapid data analysis and comprehensive reference databases, Metagenomic nextgeneration sequencing (mNGS) can be applied for hypothesis-free, universal pathogen detection, promising to improve diagnostic yield for syndromic testing of these infections [5,[9][10][11][12]. It is revolutionizing the eld of medical diagnosis and paves the way for personalized medicine [13]. The technology of mNGS is rapidly becoming routine and shared between diagnostic specialties [14]. as well as regular clinical microbiological assay to evaluate if they were co-infected with other pathogens.

Metagenomic Next-generation Sequencing and Analysis
The protocol of Metagenomic Next-generation Sequencing and criteria for a positive mNGS result refer to previous publications [15]. Samples, including blood, bronchoalveolar lavage uid (BALF) and cerebrospinal uid (CSF), were analyzed using a commercially available mNGS assay, which sequences

Patient Characteristics and Sample
Demographic features of the patients in the current study are provided in Table 1. These patients were either diagnosed with HIV-infected previously or after admission to the hospital. The patients had a median age (range) of 37.5 (26-59) years, with 83.3% (30/36) males. Five of them were previously diagnosed with hepatitis B, nephritis syndrome or epilepsy, and the rest were healthy in the past. Of those, twenty-three contracted HIV through sexual transmission, only one through intravenous drug addiction, and for the remaining patients, the route of HIV infection were unknown. The median frequency of CD4 + T cells in the peripheral blood of 97.5 (ranging from 2 to 549/ul), while the HIV viral load was ranging from undetectable to 1.0E + 07 copies/ml. Of the 36 patients, 25 were hospitalized for once, 7 for twice, 2 for three times, 1 for four times and 1 for ve times, bringing the total number of hospitalization persontimes to 54. In the 54 hospitalizations, BALF was collected from 17, blood from 15, CSF from 6, both BALF and blood from 6, both BALF and CSF from 2, for further detection. Therefore, specimens of 23 BALF, 23 blood and 8 CSF were subjected to standard culture and mNGS testing in a pairwise manner respectively.

Comparison of mNGS and Culture Testing by Samples
The positivity rates of mNGS and culture tests of the BALF, blood, and CSF are illustrated in Fig. 2.
Detection by mNGS and culture of BALF was compared in a pairwise manner, showing that the positivity rates of mNGS and culture of BALF were 21/23 (91.3%) and 5/23 (21.8%), respectively, with the statistical difference was signi cant (P < 0.001) (Fig. 1). As expected, mNGS increased the sensitivity rate by approximately 69.5% in comparison with that of culture. The similar situation goes for blood and CSF, suggesting the sensitivity of mNGS did not differ among sample types.

Comparison of mNGS and Culture Testing by Pathogens
HIV-infected patients are usually associated with multiple pathogens, which increased the di culty of clinical diagnosis and treatment. Detections by mNGS and culture were compared in a pairwise manner, showing that the percentage of mNGS-positive samples was signi cantly higher than that of culturepositive samples in terms of virus (mNGS only; P < 0.001), including Human herpesvirus, (mNGS only; P < 0.001), which was the most commonly detected pathogen, Alphatorquevirus (mNGS only; P < 0.001), and Polyoma virus (mNGS only; P < 0.05). mNGS also showed an increased sensitivity in detecting fungus (odds ratio [OR], 1.1[95% con dence interval {CI}, 0.9-1.5]; P < 0.05) and parasite (mNGS only; P < 0.01), of which was pneumocystis jiroveci (mNGS only; P < 0.01) (Fig. 3). Cryptococcus Neoformans and Cyanobacterium marneffei were also observed to have a higher yield rate by mNGS than that by culture, although the difference was not signi cant due to the small sample size. There is no signi cant difference between mNGS and culture in the positivity rate of common bacteria, while in terms of MTB (OR, 1.7 [95% CI, 1.0 − 2.8]; P < 0.05), a higher positivity rate in mNGS can be seen. It's worth noting that Coxiella burnetii was culture negative and it was detectable in mNGS.

Discussion
This retrospective study evaluated the clinical relevance of mNGS for the investigation of co-infection in a cohort of 36 HIV-infected patients. In our study, we compared detection by mNGS and traditional detection methods and found mNGS to be advantageous in three aspects. Firstly, in general, mNGS showed a higher diagnostic e ciency of infectious disease than culture and smear, although the number of samples is varied. Secondly, in BALF, blood and CSF sample types, we found a remarkably higher sensitivity in detection by mNGS vs culture compared in a pairwise manner. More importantly, mNGS is noted for its superior feasibility in detecting virus, fungus, parasite and MTB in general. Also, mNGS has proved to be a better choice when detecting those special microbes such as Coxiella burnetii. In addition, we have identi ed that a considerable percentage of infection diagnoses were con rmed and modi ed according to mNGS.
The positivity rate of mNGS was consistent with the expectation, which have reported a variety of sensitivities from 36% [17] to 100% [18]. And interestingly, our results indicated that in recognizing common bacteria (excluding MTB), the sensitivity of mNGS is not superior to that of culture, which is consistent with a previous report that, comparing the results obtained by mNGS, a majority (74%) of bacterial pathogen is identi ed by standard culture in bacterium-associated pneumonia [19]. Therefore, we concluded that unlike other microbes like virus, fungus and parasite, mNGS might not have the signi cant advantage in identifying common bacteria. Although mNGS (3000 Ren Min Bi [RMB]) cost more than any other regular methodology, the low rate of positivity, long time consuming and lack of accuracy making pathogen screening of traditional techniques less cost-effective. In conclusion, mNGS could emerge as a promising technology for precision diagnosis and tailored therapy for HIV-infected patients with suspected infection.
For HIV-affected persons, a low CD4 + T-cell count exposes them to a higher risk of opportunistic infections and even worse, multiple infections may co-exist. However, conventional diagnostic assays lack breadth of detection and sensitivity, therefore unable to discover multiple infections, which make it ineffective in microbiological diagnosis. For example, 90-95% of blood cultures remain negative in immunocompromised patients suspected of infection, even in the cases where bacterial or fungal sepsis is likely, negative results are recorded for 50% of the blood samples [20]. A better microbiological test is urgently needed to break this situation. mNGS, with its non-targeted identi cation of microbes, through deep sequencing of biological samples, data mining, and identi cation of pathogen sequences in the absence of a priori assumption, constitutes a paradigm shift in microbiological [21,22], which might contribute greatly to HIV-infected patients with suspected infection. To our knowledge, this is the rst study to evaluate the contributions of mNGS in microbial testing in a cohort of HIV-infected patients.
Our research also has its limitations. As a small-scale cohort retrospective study of 36 patients, different samples from the same patient, including BALF, blood and CSF were treated as independent samples.
Among these 36 patients, some of them have been hospitalized for several times due to different infectious diseases and each hospitalization was counted as an independent individual. Nevertheless, we believe that varied infectious diseases lead to different condition of patients, making each hospitalization analyzed independently practicable and having minimal disturbance to the nal judgment. More prospective studies of mNGS testing with broader samples in real-life clinical practice remain to be documented. For HIV-infected patients with suspected infection, a vast amount of sequence data from a single clinical sample makes interpretation complicated. Can we refer to the conventional standardization? Whether the positive pathogen is causative pathogen or not. Whether treatment is needed. The added value of mNGS in clinical managements of these patients will have to be evaluated, and additional work to associate typologies of microbiota with patient status needed to be further studied. The progressive awareness of physicians to the patients' bene t will certainly help mNGS to become standard in the practice of conducting microbiological diagnosis in HIV-affected patients.

Conclusion
HIV-infected patients are are susceptible to various opportunistic infections due to their low immunity. mNGS can be an important tool to improve the diagnostic yield of pathogens to better guide clinical treatment.

Declarations Ethical approval and Consent to participate
This study was approved by the institutional review board of the Fifth A liated Hospital of Sun Yat-sen University (Zhuhai, P.R. China). Waiver of consent was obtained given the observational nature of the project.

Consent for publication
Not applicable.

Availability of data and materials
Data are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.

Authors , contributions
Xi Liu and Jinyu Xia conceived the study. Jiehua Chen collected data, analyzed and interpreted clinical data. Guangying Deng, Ruihua Zhong, Hongqiong Zhu, Xinghua Li helped to collect data. Jiehua Chen and Xi Liu wrote the manuscript. All authors approved the nal submitted version. Figure 1 Positivity rate comparison among different detection methods. The number of positive samples (y-axis) for different detection methods (x-axis) is plotted against, showing that positivity rate was remarkably increased by metagenomic next-generation sequencing (mNGS) compared with culture and smear.

Figure 2
Positivity rate comparison between mNGS and culture in a pairwise manner. The sensitivity was increased by approximately 69.5% in mNGS in terms of bronchoalveolar lavage uid (BALF) (91.3% vs 21.8%, P <0 .001), blood and cerebrospinal uid (CSF) were similar situation. Interestingly, the overall positivity of mNGS and culture were unaffected by sample types.

Figure 3
The overlap of positivity between mNGS and culture. By pairwise mNGS and culture testing, a total of 23 pathogens were detected and the corresponding frequencies are plotted in histograms. In general, compared to culture, mNGS was signi cantly more sensitive to detect fungi, virus, and parasite, but not bacteria (excluding MTB/ NTM). Besides, MTB demonstrated a higher positivity rate in mNGS in comparison to culture (P < 0.05). MTB: Mycobacterium tuberculosis; NTM: nontuberculous mycobacteria; OR: odds ratio.