The Value of Metagenomic Next -Generation Sequence In Acquired Immunodeciency Syndrome With Opportunistic Infections

Background: Metagenomic next-generation sequence (mNGS) is an emerging powerful pan-pathogen test for the diagnosis of infectious diseases. However, the application of mNGS in acquired immunodeciency syndrome with opportunistic infections is limited to clinical cases reports and central nervous system infection.In this study, we evaluated the diagnostic value of mNGS in acquired immunodeciency syndrome(AIDS) with opportunistic infections(OIs). Methods: From January 2018 to February 2021,86 cases were enrolled in this retrospective analysis. All patients underwent mNGS. Clinical data were recorded. Result: In the present study, mNGS identied 76 of 86 infection cases (88.37%).Human betaherpesvirus 5 (CMV) (40.70%), Human gammaherpesvirus 4 (EBV) (40.70%),pneumocystis (31.40%) were the most common pathogens detected. The sensitivity of mNGS (88.37%,76/86) was higher than that of culture (22.10%, 19/86),smear(7%,6/86) and PCR(46.51%,40/86).In the detection of viruses such as (CMV and EBV), the consistency between PCR and mNGS of CMV and EBV was 100%,73.33% respectively. All PCP cases were detected by mNGS. The consistency in detection of talaromyces between culture and mNGS was 75%.mNGS is superior to the common methods such as culture and smear in the detection of mycobacterium tuberculosis and non-mycobacterium tuberculosis. The mNGS ndings led to changes in treatment strategies in 47/86 (54.65%) cases. Compared with the patients’treatment before mNGS, patients had lower rate of broad-spectrum antibiotic drugs use during clinical treatment after mNGS 78/86(90.70%) vs 34/86(39.53%)(cid:0)P<0.0001). Conclusion: mNGS showed a satisfying diagnostic performance in acquired immunodeciency syndrome with opportunistic infections. mNGS may lead to a more precise antimicrobial treatment and reduced the of antibiotic


Introduction
Human Immunode ciency Virus (HIV) infection is still a great public health threat around the world. Since the epidemic of the acquired immunode ciency syndrome, approximately 78 million people have infected HIV, and 35 million people have died because of acquired immunode ciency syndrome(AIDS)-related diseases. The increasing use of antiretroviral treatment can effectively suppress the HIV viral load to undetectable level,so the morbidity and mortality of AIDS is sharply decreased [1][2][3].However,opportunistic infections often occur in AIDS patients even under the antiretroviral therapy(ART).It has a signi cant in uence on patients'well-being, life quality, health care costs and their survival [4][5][6].What is more, opportunistic infections is the major cause of morbidity and mortality among AIDS patients [7,8].
The di culty of diagnosis of opportunistic infections in AIDS patients is a great challenge to clinicians because of its broad spectrum of potential aetiologies. The diagnosis of opportunistic infections with AIDS relies on a detailed history, clinical examination, radiological ndings and conventional tests before mNGS availability. However, conventional tests often result in delayed and missed diagnosis because current microbiological tests such as culture-based methods are time consuming and have low yields rates of positive detection. At same time, the overlap and co-infection of etiologies are often missed which may increase AIDS-related death, and prompt identi cation of causative agents is critical for the timely application of effective treatment.
However, mNGS in AIDS with opportunistic infections is limited to clinical cases reports and central nervous system infection [18][19][20][21].Its utility in AIDS patients with opportunistic infections is still not well established. Therefore, we conducted a retrospective observational study in mainland China to analyze the diagnostic value of mNGS for diagnosis of opportunistic infections in adult AIDS patients.

Procedure
Samples were collected, and then sent to BGI-Huada Genomics Institute (Wuhan, China) for pathogens detection as described previously [17].Brie y,the mNGS procedure for samples includes nucleic acid extraction,library construction,sequencing,information analysis and quality-controlled.Low-quality and short (length < 35bp) reads were removed.Then the remaining sequence were aligned to the current virus, bacterial, fungal, and protozoan databases (NCBI; ftp://ftp.ncbi. nlm.nih.gov/genomes),which is comprised of whole genome sequences of 4061 viral taxa,2473 bacterial genomes or scaffolds,and genomic sequences for 199 fungi related to human infection and 135 parasites associated with human diseases.

Statistical analysis
Continuous variables were expressed as the medians.Categorical variables were summarized as the counts and percentages in each category.Statistical analyses were performed using SPSS version 20.0 (IBM Corporation, Armonk, NY, USA).Figures were conducted using GraphPad Prism 5 software.A p value less than 0.05 was considered statistically signi cant.

Population characteristics
There were 86 patients,who presenting with acquired immunode ciency syndrome with opportunistic infections underwent mNGS between January 2018 and February 2021.The baseline characteristics of patients are shown in Table 1.

Summary of pathogens detected by mNGS
The main sample types of mNGS in our patients were bronchoalveolar lavage uid(BALF) (n = 31), while other sample types included blood(n = 30), bone marrow(n = 5),cerebrospinal uid(n = 18),pleural(n = 2). in total (Fig. 5).Then we compared the consistency between the mNGS and conventional methods in pathogens detection.In the detection of viruses such as CMV and EBV, the consistency between PCR and mNGS of CMV and EBV was 100%,73.33% respectively.All PCP cases were detected by mNGS.The consistency in detection of talaromyces between culture and mNGS was 75%.mNGS is superior to the common methods such as culture and smear in the detection of mycobacterium tuberculosis and nonmycobacterium tuberculosis (Fig. 6).

Impact on treatment decisions by mNGS
Empiric antimicrobial of 86 cases were prescribed early in their course for initial consideration of possible infections.On the admission, before performing mNGS,78/86(90.70%) patients received broad -spectrum antimicrobial(more than two different types antimicrobial often cover Gram-negative, Gram-positive bacteria ,atypic pathogens, virus and fungi) therapy compared with 34/86(39.53%) after-mNGS (P < 0.0001 ) (Fig. 7).
Meanwhile mNGS provided positive identi cation of the pathogens leading to prompt changes in the antiinfection treatment.Antiviral treatment (ganciclovir or aciclovir) was added as VZV infection in two cases was established based on mNGS results.Anti-Toxoplasma gondii treatment was also initiated in one patient based on mNGS results.For patients mNGS provided positive identi cation of mycobacterium avium infection,leading to precise changes in the anti-infection treatment.For patients with PJP con rmed by mNGS, sulphanilamide dose increased from prophylactic medication to therapeutic medication.For patients with talaromyces infection,we added amphotericin B,discontinued to use antimicrobial treatment.

Outcomes
In the present study,15 patients died,the overall death rate was 17.44% (15/86).12 patients died of respiratory failure caused by severe pneumonia,two patients died of sepsis shock,one patient died of Secondary Hemophilic syndrome.The remaining patients continued to improve greatly after targeted treatment according with mNGS(see image 1and 2).

Discussion
We retrospectively enrolled patients diagnosed with acquired immunode ciency syndrome with opportunistic infections during the period from January 2018 to February 2021, and attempted to identify the causal pathogens.We accordingly found that CMV, EBV,pneumocystis were the most common pathogens detected.It is different from previous studies.Louie found that TB, cryptococcosis and pneumocystis pneumonia (PCP) are among the major causes of hospitalisation and mortality among adults living with HIV [22].In Bin Luo's study, bacterial pneumonia was the most prevalent opportunistic infections in HIV-infected patients,followed by candida infection,PCP and TB [23].We assumed possible explanations for this divergence are due to the different enrolled cohorts,different sample types,as well as the different type of infectious diseases.
Mixed infection and pneumocystis jirovecii pneumonia(PJP) are common in immunosuppressed patients.mNGS has its potential to detect multiple pathogens simultaneously in one test.So immunosuppressed patients are more likely to be the largest bene ciaries.In this study,mNGS detected 34 patients who had mixed infection of multiple pathogens but conventional methods only detected 14 cases(P < 0.0001).Our data indicated that mNGS was valuable for the early detection of mixed infection in AIDS patients.It has similar ndings in other studies.More mixed pathogens were found in immunocompromised than immunocompetent patients [24].mNGS exhibited apparent advantages in detecting viruses and identifying mixed infections [25].Yi-Yi Qian recommend timely use of mNGS when infection of mixed or rare pathogens is suspected,especially in immunocompromised individuals and or individuals with severe conditions that require urgent treatment [26].
Meanwhile we found all PJP cases were detected by mNGS,no matter BALF or blood.It indicated mNGS has exellent performance in detecting PJP.There were 6 cases were detected in blood.Jie Chen has reported a case of pneumocystis jirovecii pneumonia in a kidney transplant recipient,which was diagnosed by mNGS of blood [14].Accordingly,if PJP is clinically suspected and bronchoscopy is not appropriate,blood is a good choice.
The undiagnosed cases usually force physicians to prescribe empirical broad-spectrum antibiotic treatments,which will greatly increase the risk of emergence of multidrug-resistant bacteria.So early aetiological diagnosis is necessary and critical for acquired immunode ciency syndrome with opportunistic infections which always present severe manifestations and high mortality.In this study, mNGS provided with de nite pathogens of 76/86(88.37%) patients within 72 hours for us.It also support the clinical decision including prompt changes and discontinuation of unnecessary antimicrobial therapy.In the present study,antibiotic treatment decreased sharply after mNGS.Meanwhile there were ajustments of treatment in 47 patients after mNGS, they had improve greatly after precise treatment according with mNGS.These ndings strongly indicate that mNGS may assist in clinical decision making,especially for reducing unnecessary antibiotic usage and improving prognosis in the treatment of acquired immunode ciency syndrome with opportunistic infections.Ting Sun had same conclusion.Their study showed mNGS may offers the probability for targeted therapy and improved clinical antibiotic overuse in immunocompromised patients [24].Zhang also found that the detection of mNGS can signi cantly shorten the ICU stay, the ventilation time and reduce the ICU cost in immunosuppressed patients with acute respiratory distress syndrome (ARDS) caused by severe pneumonia [27].
As we know, due to the lower sensitivity of conventional methods,mNGS is appealing.Many studies reported that mNGS is sensitive,fast and superior to the common methods [28,29].In our study,mNGS detected pathogens in (88.37%, 76/86) patients but culture,smear and PCR' sensitivity were22.10%, 7%, 46.51% respectively.mNGS has excellent advantages compared with conventional methods in total.It is superior to the common methods such as smear and culture in detection of PJP,mycobacterium tuberculosis and non-mycobacterium tuberculosis.Meanwhile mNGS can identi ed VZV,human herpesvirus that were not detected using conventional PCR.However,mNGS diagnostic performance has limitations.When we compared the consistency and the full turnaround time between mNGS and PCR in detecting the viruses such as CMV and EBV, the full turnaround time of PCR was signi cantly shorter than mNGS.No superiority was noted between mNGS and culture when detected talaromyces.Other studies have had similar ndings [30,31].
Our study has some limitations.First,this was a retrospective study.It included small cases.It needs more futher observation to investigate the diagnostic value of mNGS in AIDS with opportunistic infections.
Second,we used preliminary data for analysis but still lack of control group simultaneously in our cohort. Further,the mNGS results and identi cation may be easily in uenced by many factors.Besides,lack of a gold standard comparator for diagnostics, and classi cation bias were also the limitations of this study.
In our study,we diagnosed acquired immunode ciency syndrome with opportunistic infections by mNGS.It demonstrated that in AIDS with opportunistic infections patients, mNGS had a superior advantage in detecting potential pathogens than conventional methods and mNGS can enable earlier initiation of targeted antimicrobial therapy.
Declarations publication of the Pan American Society for Clinical Virology 2015, 69:96-100. Figure 1 Viruses detected by mNGS   Sensitivity of mNGS and conventional methods Figure 6 consistency between mNGS and conventional methods

Figure 7
The use proportion of broad-specrum antiotic