Metagenomic Analysis Identifying a Polymicrobial Pulmonary Infection in a Non-HIV Immunocompromised Patient: A Case Report

doi:10.21203/rs.3.rs-5307741/v1

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Case Report

Metagenomic Analysis Identifying a Polymicrobial Pulmonary Infection in a Non-HIV Immunocompromised Patient: A Case Report

https://doi.org/10.21203/rs.3.rs-5307741/v1

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Background

Polymicrobial pulmonary infections, common in immunocompromised patients, often manifest more severe symptoms than monomicrobial infections. Clinical diagnosis delays may lead to mortality, emphasizing the importance of fast and accurate diagnosis for these patients. Metagenomic next-generation sequencing (mNGS), as an unbiased method capable of detecting all microbes, is a valuable tool to identify pathogens, particularly in cases where infections are difficult to diagnosis using conventional methods.

Case presentation:

A 50-year-old male patient was admitted due to cough, expectoration and dyspnea. CT scan revealed diffuse inflammatory and cavernous lung lesion, and blood examination suggested a polymicrobial infection. However, no etiology was found by routine examination. mNGS of bronchoalveolar lavage simultaneously detected the presence of Pneumocystis jirovecii, Aspergillus fumigates, Nocardia farcinica, Salmonella enterica subsp. enterica, and cytomegalovirus (CMV). The patient was successfully treated with compound sulfamethoxazole, cefoperazone/sulbactam, moxifloxacin, voriconazole, and ganciclovir. The patient recovered after two weeks of anti-infection therapy and maintained good health at a six-month follow-up.

Conclusion

For immunocompromised patients with multiple infections and atypical symptoms, mNGS emerged as a reliable approach to pathogen detection and guiding antibiotic therapy.

mNGS

polymicrobial infection

immunocompromised patient

case report

Immunocompromised condition, a common problem in Non-HIV patients who have low immunity, thus results in diarrhea, high fever, cough and other pulmonary infections. Immunosuppression is often caused by solid tumors, hematological malignancies, organ transplantation, and autoimmune inflammatory diseases. These individuals are more susceptible to infections caused by opportunistic pathogens. Opportunistic pathogens, such as Nocardia farcinica, Pneumocystis jirovecii, cytomegalovirus (CMV), and so on, are reported to have a higher infection rate in immunosuppressed individuals, and coexisting infections are common[1, 2]. Prompt and accurate diagnosis is critical for effective treatment and outcomes, but conventional diagnostic methods, such as microbial culture, microscopic smear and nucleic acid hybridization, often fall short due to atypical clinical manifestations, which would lead to a poor prognosis[3].

Metagenomic next-generation sequencing (mNGS), an unbiased method capable of detecting all microbes without a prior hypothesis, is suitable for identifying polymicrobial infections caused by difficult-to-culture pathogens. Herein, we reported a case which a variety of pathogens, such as Salmonellas enterica subsp. enteric, Nocardia farcinica, Pneumocystis jirovecii, Aspergillus fumigatus (A. fumigates) and CMV, were detected in specimens of an immunocompromised patient. And mNGS played an essential role in the diagnosis and appropriate antibiotic treatment of an immunocompromised patient with a rare polymicrobial pulmonary infection.

A 50-year-old male patient was admitted to our hospital on October 17th, 2021. During the 10 days before admission, hard nodules, swollen boils, ulcers, and scabs appeared on several areas of his skin, while 7 days ago the patient began to cough, expectorate and experience dyspnea. Methylprednisolone (48 mg/day, PO) had been administrated for the past three months to treat focal segmental glomerulosclerosis (FSGS). On Admission, the patient’s body temperature was 39.4℃, pulse was 130 beats/min, SpO₂ was 67%, and noninvasive positive pressure ventilation was applied (35 L/min, O₂ 60%). CT scan showed diffuse inflammatory and exudative changes in both lungs with multiple lung nodules. Cavernous lesions could be seen in the upper lobe of the right lung (Fig. 1). Blood samples were taken for laboratory tests and culture for pathogens. The white blood cell count was 13.42×10⁹/L, CRP was > 200 mg/L, SAA was > 300 mg/L, and procalcitonin was 1.80 ng/ml, all of which were above the upper limit of the biological reference interval. Fungal infection markers (1,3)-β-D-glucan (479.48 pg/ml) and galactomannan (1.019 µg/L) were also out of the normal range. The counts of CD3⁺ (73 cells/µl), CD4⁺ (38 cells/µl) and CD8⁺ T cells (30 cells/µl) were all less than 10% of the lower limit, indicating impairment of the immune function (Table 1). Based on the clinical symptoms, imaging results, and laboratory examinations, an infection was suspected. Cefoperazone/sulbactam (3 g q8h, iv, drip), moxifloxacin (0.4 g qd, iv, drip), combined with compound sulfamethoxazole (SMZ 0.8 g-TMP 0.16 g q12 h, PO) were administered to treat gram-negative bacilli, gram-positive cocci, and opportunistic fungi such as P.jirovecii. On the second day of hospitalization, bronchoalveolar lavage was performed. A portion of the BALF was collected for microscopic examination of bacteria, filamentous fungi, mycobacterium tuberculosis, and P.jirovecii. Another part of the BALF was subjected to mNGS and culture. On the third day, Salmonellas enterica subsp. enteric, which was sensitive to moxifloxacin and sulfamethoxazole, was identified by blood culture. P.jirovecii was detected by hexamine silver staining of BALF on the same day. Filamentous gram-positive bacilli resembling Nocardia spp. were also observed under oil lens (Fig. 1). As the antibiotic regiments were effective to these pathogens, the patient’s symptoms improved, and no adjustment of antibiotics was necessary. On the 4th day, N.farcinica (1,089 reads), P.jirovecii (6,825 reads), A.fumigatus (3 reads), and CMV (47 reads) were recovered by mNGS (Fig. 2). Cefoperazone/sulbactam was discontinued, voriconazole (0.4 g q12 h, iv, drip) was added to treat A. fumigates. Ganciclovir (0.25 g q12h, iv.drip) was added to treat CMV, the dosage of sulfamethoxazole was increased (SMZ 1.2 g-TMP 0.24 g q6h, PO) to treat P. jirovecii and N.farcinica. On the 5th day, N.farcinica was isolated from BALF on blood agar by culture, and the presence of CMV in the BALF was confirmed by Real-time PCR (3.09×10³ copies/ml). A. fumigates (2 colonies) was isolated by culture on the 6th day. On the 10th day, the patient`s condition improved significantly, while PCR of CMV was negative, so ganciclovir was discontinued. Moxifloxacin was stopped on the 13th day, as the blood culture was negative. After 14 days of treatment, the patient’s condition improved significantly, so he was discharged from the hospital. To prevent any recurrence of the infection, voriconazole (0.2 g q12h, PO) and SMZ-TMP (1.2 g-0.24 g q6h, PO) were prescribed for 3 months. During the 6 month’s follow-up, the patient’s condition remained normal, confirming the effectiveness of the treatment.

Table 1

Abnormal laboratory results on admission
	Indexes	Results	Normal range	Unit
Inflammation indexes	Leukocyte	13.42×10⁹	3.5×10⁹-9.5×10⁹	/L
	CRP	> 200	0–10	mg/L
	SAA	> 300	0–10	mg/L
	Procalcitionin	1.80	0-0.05	ng/ml
Fungi marker	(1,3)-β-D-glucan	479.48	< 60	pg/ml
	galactomannan	1.019	< 0.75	µg/L
T cell count	CD3⁺ T cell	73	1185–1901	cell/µl
	CD4⁺ T cell	38	561–1137	cell/µl
	CD8⁺ T cell	30	404–754	cell/µl

Low immunity is frequently encountered among patients undergoing long-term radiotherapy and chemotherapy for cancer, patients with autoimmune diseases, patients with kidney diseases, and others[4, 5]. In our case, this patient has a history of three months of methylprednisolone for kidney disease and exhibits a low CD3⁺, CD4⁺ and CD8⁺ T cell counts, which is indicative of a compromised immune status. Immunocompromised patients often suffer from a polymicrobial pulmonary infection, such as P. jirovecii, Nocardia farcinica. P. jirovecii is a severe fungal infection that has a high prevalence in immunocompromised individuals, CD4⁺ lymphocyte counts < 200 cells/ul and corticosteroid use are risk factors for P.jirovecii colonization[6]. Early diagnosis and medication are important for good prognosis, however, diagnosis of P. jirovecii remains challenging due to its atypical clinical manifestations and low sensitivity and specificity of current diagnostic methods[7]. Nocardia farcinica is a weak acidic actinomycete that is widely present in soil, water and air. Inhalation of spores or fragments of hyphae results in pulmonary nocardiosis, about 50 percent of norcardiosis occurs in immunocompromised settings while Nocardia farcinica is more common than other Norcardia species [8–10]. The variable clinical manifestation and the difficulty in pathogen detection often lead to delays in the treatment of norcardiosis patients. Recently, several studies illustrated that compared to conventional methods, mNGS had a satisfactory diagnostic value in P. jirovecii and Norcardia farcinica diagnoses, reducing the detection turnaround time[11–13]. As reported, the application of mNGS corrected misdiagnosed tuberculosis, diagnosed a rare case of multisystem infection and a multi-site infection by Norcardia farcinica, and illustrated the competence of mNGS in the diagnosis of Norcardia spp. [14–16].

The mNGS technique is a promising approach for detecting co-pathogens in mixed pulmonary infection, with key advantages in speed and sensitivity[17]. In cases where multiple pathogens coexist, such as in immunocompromised patients, mNGS can significantly simplify the diagnostic process by identifying bacteria, fungi, parasites, and viruses concurrently. Unlike traditional methods that often rely on prior hypotheses and targeted testing, mNGS is unbiased and can detect even atypical, rare, or emerging pathogens. The high detection sensitivity of mNGS, coupled with its ability to detect unculturable organisms, makes it superior to culture-based methods[18].

In our case, given the history of methylprednisolone usage and the low CD4⁺ T cells count, they are indeed high-risk factors for opportunistic infection caused by P.jirovecii, A.fumigates, N.farcinica, and CMV. These pathogens are commonly found in immunocompromised individuals, but a simultaneous infection with all four of them is extremely rare. To the best of our knowledge, this is the first reported case of all four pathogens being detected in a single BALF sample using the mNGS technology in China. The conventional diagnostic methods, often struggle with detecting multiple pathogens simultaneously due to their limitations in sensitivity, specificity, throughput, and the need for pre-hypotheses. These limitations can lead to delays in diagnosis and poor prognosis, particularly in cases where multiple pathogens are involved. Although the current gold standard for diagnosing invasive pulmonary aspergillus is based on histopathological evidence and a positive tissue aspergillus polymerase chain reaction (PCR), these two diagnostic methods are invasive. Especially considering the severity of this patient's condition, it is challenging to clinically apply of these invasive diagnostic methods.

The rapid and comprehensive pathogen detection capability of mNGS technology can significantly guide the rational use of antibiotics[19, 20]. In this case, the mNGS results assisted clinical doctors in prescribing VCZ for the treatment of Aspergillus fumigatus infection and ganciclovir for the treatment of CMV infection, both of which played a crucial role in the successful management and treatment of the patient’s disease, highlighting the value of mNGS in precision medicine. Given the patient's severe condition and based on clinical experience, the use of VCZ for antifungal treatment is appropriate. The post-treatment microbiological test results also confirm the effectiveness of the medication.

In conclusion, patients in immunocompromised states often suffer from polymicrobial pulmonary infection. Compared to the limitations of traditional diagnostic methods, the mNGS technology has shown significant potential in enhancing the diagnosis and treatment of such complex and challenging infectious cases.

Acknowledgements

We would like to express our gratitude to Zhang ran and Li yanhua for their work on microscopic examination.

Authors’ contributions

Jing Duan and Jie Ding were major contributors in drafting the manuscript, Jing Duan, Zhiqing You and Ding Li participated in the diagnosis of the patient and review of medical records, Yingxin Zhang and Chao Chen participated in revising the manuscript critically. All authors read and approved the final manuscript.

Funding

This research was supported by the Medical Science and Technology Development Foundation of Department of Health of Shandong Province (2019WS113).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

As this manuscript is a case report of clinical care provided to a patient, and it does not include any of the patient’s personal identifiers, ethics approval by the institutional review board was not required.

Consent for publication

The patient provided written informed consent for the publication of the data.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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You are reading this latest preprint version

Metagenomic Analysis Identifying a Polymicrobial Pulmonary Infection in a Non-HIV Immunocompromised Patient: A Case Report

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Abstract

Background

Case presentation:

Conclusion

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Background

Case Presentation

Discussion and Conclusion

Declarations

References

Additional Declarations

Status:

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