Immunocompetent and infected with the lungs of Basket Marnifia

preface

Talaromycesmarneffei (TM) is a highly pathogenic endemic conditionally pathogenic bacterium that infects people
with low immunity in Southeast Asia and southern China.

This article reports a case of immunocompetent elderly male with acute respiratory symptoms, respiratory failure, rapid advancement of imaging, clinical consideration of "severe pneumonia", not excluding "rapidly progressing interstitial lung disease" caused by connective tissue disease, and initially using a larger dose of systemic glucocorticoids
on the basis of conventional anti-infection.

Case passed

The patient, male, 63 years old, was admitted to hospital
on December 30, 2021 due to "fever for 11 days, cough and sputum production for 1 week, chest tightness for 4 days".

Past and personal medical history: history of "cardiac radiofrequency ablation surgery", denial of special drugs, drug abuse, HIV, organ transplantation and other medical history, 40 years of smoking, an average of 10 sticks per day
.

Admission examination: T36.

Ancillary tests:

2021-12-19Chest CT scan of Xianju County People's Hospital, Zhejiang Province: bronchiolitis of both lungs, two lungs scattered in fiber proliferation calcification foci, paratracheal diverticulum (Figure 1a).

2021-12-29Chest CT scan of Xianju County People's Hospital, Zhejiang Province: diffuse infection of both lungs, new compared with 2021-12-19, two lungs scattered in fiber proliferation calcification foci (Figure 1b).

2021-12-29Blood routine of Xianju County People's Hospital of Zhejiang Province: WBC14.

2021-12-30Blood gas analysis: PH7.

Figure 1a CT of the lungs (20211219); Figure 1b CT of the lungs (20211229)

Admission diagnosis: severe pneumonia, type I respiratory failure, interstitial lung disease? After cardiac radiofrequency ablation

Diagnosis and treatment

2022-12-30After admission, RICU was admitted, the relevant examination was improved, high-flow humidified oxygen therapy through the nose, empiric piperacillin tazobactam 4.

2021-12-3100:15 Patients with obvious chest tightness and shortness of breath, a little cough and sputum production, no fever, poor treatment of multiple antibiotics in the hospital, low oxygen saturation, considering the more serious condition, adjust the oxygen concentration, stop piperacillin sodium tazobactam sodium injection, change to imipenemcistatin sodium injection (Taineng) 0.

Related laboratory results returns: Mycoplasma pneumoniae antibody, chlamydia antibody, Legionella antibody, sputum culture, respiratory virus 18 items, respiratory pathogen nucleic acid detection, EBV-DNA, cytomegalovirus DNA blood + urine are negative, TORCH series, antinuclear antibody series + ANCA, serum ferritin, IgE, anti-myositis spectrum 12 items are negative, TBNK test: CD4 absolute value 351/ul, CD427%, CD829%, CD4 / CD80.

2021-12-31Exclusion of contraindications to bedside tracheoscopy alveolar lavage: patency of both sides of the airway, less secretion, left B9, left B10 line alveolar lavage 100ml, recovery of 35ml of light blood lavage solution for examination related examination
.

Fig.

2022-01-02Alveolar lavage solution mNGS (Figure 3): Marnifi Basket Bacteria: Detection of 9494 sequences, Staphylococcus aureus: detection sequence number 503, Acinetobacter baumannii: detection sequence number 286, combined with clinical consideration of "Marnifi Basket pneumonia", not except for the combination of Staphylococcus aureus and Acinetobacter baumannii infection, the treatment plan was adjusted on 2022-01-02: cessimide penisestadine injection, moxifloxacin injection, Cefoperazone sulbactam injection 2.

Figure 3 Alveolar lavage fluid mNGS results

2022-01-08Fungal culture of alveolar lavage fluid: Marnifosa non-basket growth (Figure 4), consistent
with macrogene results.

Figure 4a Culture result: Sapao 25 °C incubation for 7 days
.
Figure 4b Lactophenol cotton blue staining (×100); Figure 4c Fluorescence staining (×100
).

After treatment, the patient's symptoms and oxygenation improved significantly, 2022-01-09 re-examination chest CT showed obvious absorption of the two pneumonia, 2022-01-10 transferred out to the general ward for continued treatment, 2022-01-13 discharged for follow-up
.
Follow-up outpatient treatment with voriconazole capsules was continued, and imaging absorption recovered well (Figure 5
).

Figure 5a CT of the lungs (20220109); Figure 5b CT of the lungs (20220209); Figure 5c CT of the lungs (20220330); Figure 5d CT (20220601)
of the lungs.

Case studies

Inspection case studies

How can I help clinically confirm the causative agent of lung infection?

Clinical examination of the pathogen of lung infection includes traditional culture, smear, serological examination, and molecular diagnosis
.
After the patient was admitted to the hospital, he was sent to the relevant tests that can be tested by our hospital at present, and the testing items were relatively complete
.
However, a series of tests, including traditional sputum culture, serology, and molecular methods such as respiratory pathogens, were negative, and there was no significant improvement
in clinical symptoms after empiric anti-infection.
How to think about it at this time?

Off-hospital long-term broad-spectrum antibiotics resist infection and inhibit bacterial growth, resulting in negative
results in routine cultures.

It is recommended to send alveolar lavage fluid
for testing when there is a condition for lower respiratory tract infection.
Alveolar lavage fluid effectively reduces or avoids oropharyngeal contamination when collecting sputum samples, so that the detection rate of pathogenic bacteria and the pass rate of specimens are significantly improved[2].

In this case, the patient sent sputum for smear, culture and fungal culture results were negative
three times.
Fungal culture of alveolar lavage fluid was sent for examination to see the growth
of Marni non-basket fungus.

Special pathogens such as Nocardia, Mycobacterium, Filamentous Fungi and other pathogens grow slowly and need to be extended for incubation
.
In this case, no bacterial growth was seen in the 2-day culture of alveolar lavage fluid, but a small yellow-brown yeast-like colony was seen after 4 days of incubation at 25 °C of the salfarinal fungus, and the typical soluble burgundy pigment diffusion into the agar was seen after 7 days of incubation (Figure 4a).

Therefore, if there is a clinical suspicion of a target pathogen with a long culture time, it is necessary to communicate with the microbiology chamber in time to extend the culture time and improve the positive detection rate
.

For unexplained fever, repeated negatives of routine tests, and acute and critical infections, mNGS can quickly identify the pathogen of infection and guide the use of clinically targeted antibiotics, which is of great significance for achieving accurate diagnosis of infection

Metagenomenext-generation sequencing (mNGS) enables rapid and unbiased one-time detection of tens of thousands of bacteria, fungi, viruses, parasites, mycobacteria, and chlamydia/mycoplasma, greatly expanding the pathogen spectrum
.
For example, in this case, alveolar lavage fluid mNGS detected the clinically rare Marnificus spp.
, which provided help
for early diagnosis and early treatment.
Later fungal cultures of alveolar lavage fluid were also cultured into typical Marni non-basket colonies, which corroborated
with mNGS results.
With precise treatment of the pathogen, the patient's symptoms and lung imaging improved rapidly, and the prognosis was good
.

Clinical case studies

The patient was acutely ill with acute respiratory symptoms, and was found to have rapid advancement of imaging and respiratory failure in the local hospital for treatment, and the initial imaging was a relatively symmetrical distribution of the lower lobes of the two lungs with a more extensive exudative consolidation, and it was difficult to clinically distinguish infectious or non-infectious lesions of the lungs, and it was clinically considered "severe pneumonia", and it was not excluded that connective tissue disease caused by "rapid-progressed Interstitial Lung Disease, RP-ILD)", so the initial treatment on the basis of high-flow oxygen therapy and broad-spectrum antibiotics, using a larger dose of systemic glucocorticoids, while treatment, we actively searched for the pathogenology of the suscitation, and performed bedside bronchoscopic alveolar lavage on the second day of admission, and sent alveolar lavage fluid-related pathogenesis assays and mNGS
.

mNGS in the postoperative 48h that is, to give us a return "Marnifi Basket", while the patient's other laboratory tests did not indicate the relevant "collagen vascular disease or connective tissue disease" basis, so the clinical consideration of lung infectious lesions, after precise treatment of pathogens, the patient's symptoms improved rapidly, the whole body glucocorticoids were discontinued in time, the imaging absorption was satisfied, and the patient's 2022-01-08 alveolar lavage solution culture results showed the growth of Marnifi basket fungus, and mNGS results corroborated each other, diagnosis" Marnifi Basket Pneumonia" is conclusive
.
Basket Marnifia is more likely to occur in immunodeficiency hosts such as HIV, and the patient's CD4 cell count decreased slightly, and the patient was sent to test "anti-IFN-γ autoantibody negative" (Figure 6a) and "peripheral blood whole exon sequencing did not find immunodeficiency-related genes" (Figure 6b).

The basic judgment is that the immune function is relatively sound of the host, and the clinical is relatively rare
.

Figure 6a anti-interferon-γ autoantibodies; Figure 6b Results
of gene screening for total exon immunodeficiency in peripheral blood.

Knowledge development

1.
High-throughput sequencing of pathogen metagenome (mNGS)

mNGS is a technology based on metagenomics and high-throughput sequencing, which can directly perform high-throughput sequencing
of nucleic acids in various clinical samples without relying on traditional microbial culture.
The traditional pathogenic diagnosis is that the clinician selects the corresponding detection method according to the clinical manifestations of the patient, and there may be adverse consequences
of differential diagnosis error or limited pathogenic detection methodology that eventually leads to delay or even death.
mNGS detection can cover a wider range of pathogens and is not affected by antibiotic use, so mNGS is very suitable for patients with severe illness, critical illness or immunosuppression and immunodeficiency who are clinically suspected of infection; The mNGS guidelines recommend that while improving traditional laboratory and molecular biology testing, samples from suspected sites of infection be collected for second-generation sequencing (B, II.
) [3
].

For patients with respiratory infection, mNGS guidelines state that if a clear etiology is not obtained by traditional laboratory tests within 3 days and empiric anti-infection therapy is ineffective, it is recommended to retain respiratory specimens for second-generation sequencing (A, II.
) [3].

Of course, because the respiratory tract is open, when the second-generation sequencing detects pathogens such as bacteria and fungi, it is not yet possible to accurately determine whether the bacteria are colonized or pathogenic, and it is still necessary to rely on clinicians and laboratory physicians to jointly interpret the mNGS results (A, II.
) according to the patient's condition [3
].

2.
HIV-negative Marnifi Basket infection

The higher case fatality rate in HIV-negative Basket Marnifia infections may be related
to their lack of early diagnostic capacity.
Basket Marnifia is mainly parasitic intracellular and can proliferate in macrophages and spread
through the reticuloendothelial system.
The severity of Basketella Marnifia infection varies depending on the immune status of the host, and HIV-positive patients with Basketella Marnifia infection often presents as disseminated and invades multiple organ systems[4]; HIV-negative infections with Basket Marnifia tend to be localized, with a small proportion being disseminated [5].

Chan[6] et al.
retrospectively summarized the clinical features of 119 cases of HIV-negative Basket Marnefes infection reported in the literature, including fever, weight loss, skin and soft tissue lesions, hepatosplenomegaly, lymphadenopathy, cough and dyspnea, and some patients had osteoarticular involvement and abdominal symptoms; Lung involvement is more common in infected people, mainly manifested as mediastinal and hilar lymphadenopathy, unilateral or bilateral lung consolidation, cavitation formation, interstitial changes in the lungs, pleural effusion, pericardial effusion, etc.
; Laboratory tests often reveal increased or decreased leukocytosis, anemia, thrombocytosis, abnormal liver function, and elevated rates of C-reactive protein and erythrocyte sedimentation in infected people
.

Diseases associated with Basket Marnifia infection include autoimmune diseases such as anti-IFN-γ autoantibodies[7], systemic lupus erythematosus (SLE), mixed connective tissue disease, as well as organ transplant patients, hematologic malignancies, and patients with
novel targeted anticancer therapy 。 There are also sporadic reports of Basket infections in patients with idiopathic CD4+ T lymphopenia, Job's syndrome, diabetes, post-splenectomy, colon, breast, and thyroid cancers[5], but it is difficult to assess the relationship between
these diseases and Basket Marnifia infection due to the low number of cases.
For example, the reasons for infection with Marniffi Basket in patients with basically normal immune function in this case need further study
.

Case summary

Marnifi Basket is a rare or rare pathogen in clinical practice, with the improvement of detection technology, especially mNGS in clinical use, the detection rate of this bacterium has increased in recent years, in the past it was believed that the bacterium is more likely to occur in HIV population, mostly in Guangxi or Southeast Asia in China, and there are few reports
in Jiangsu and Zhejiang.
In the past two years, since the local testing of mNGS in our hospital, a total of four cases of Marnifi Basket have been detected, three of which have HIV-based evidence, and this case is a relatively healthy immunocompetent male, which is relatively rare in clinical practice
.
The pathogenic diagnosis mNGS in this case played a huge role, identifying the pathogen in the early stages of severe pulmonary infection, paving the way for subsequent treatment, and also playing a decisive role in reversing the disease; While mNGS provides guidance, the close communication between clinical and laboratory is also an example in this case, and the successful cultivation of Marnifi in subsequent cultures confirms the results of
mNGS.

Expert reviews

Infectious diseases are common clinical diseases, while respiratory infections are the type of infection
that causes the highest number of deaths worldwide.
There are many types of infectious agents, but 25% to 60% of infectious diseases still do not identify their pathogens due to limitations of testing methods or the lack of selection of appropriate testing methods [8
].
Only by quickly and accurately identifying pathogens early and carrying out targeted anti-infection therapy can the best clinical prognosis
be achieved.

In this case, the pathogen tests of other conventional detection techniques are negative, and mNGS has guided the clinical diagnosis and treatment by virtue of the superiority of its technology to detect suspected pathogens
.
Clinical combination with patient immunological examination, timely elimination of connective tissue disease possibilities, for mNGS detected Marniffy basket bacteria for precise medication, so that the patient's symptoms and imaging rapid improvement
.
In the later stage, the microbial chamber also cultured Marniffy Basket Fungus, which cross-corroborated with mNGS results; After the patient was discharged from the hospital, he continued to take voriconazole capsules for the pathogenic bacteria, and the imaging absorption recovery was good
after outpatient review.
The diagnosis and treatment of the patient's etiology has formed a very good closed loop, which has accumulated good experience
in using mNGS as a new methodology to find pathogens in the clinical and laboratory stages.

(Yu Sufei, Chief Technician, Department of Clinical Laboratory, Taizhou Hospital, Zhejiang Province)

【References】

[1] "13th Five-Year Plan" National Science and Technology Major Special Project AIDS Opportunistic Infection Research Group, Chen Yaokai, Wu Hao, et al.
Expert consensus on the clinical diagnosis and treatment of AIDS combined with Marnifi basket disease[J].
Journal of Southwest University:Natural Science Edition,2020, 42(7):15.

[2] Li Xiuwen,Wang Yunduo,Zhang Yihua.
Diagnostic value of alveolar lavage fluid, fiber bristle brush and sputum pathogen culture on lower respiratory tract infection[J].
Chinese Journal of Experimental Diagnostics, 2018, 22(12):4.

Editorial Board of Chinese Journal of Infectious Diseases.
Expert consensus on the clinical application of Chinese metagenomics second-generation sequencing technology for the detection of infectious pathogens[with corrections herein][J].
Chinese Journal of Infectious Diseases, 2020, 38(11):681-689.

[4] "13th Five-Year Plan" National Science and Technology Major Special Project AIDS Opportunistic Infection Research Group, Chen Yaokai, Wu Hao, et al.
Expert consensus on the clinical diagnosis and treatment of AIDS combined with Marnifi basket disease[J].
Journal of Southwest University:Natural Science Edition,2020, 42(7):15.

[HU Jiaguang,JIANG Zhongsheng,LI Xu,et al.
Research progress of Marniffi Basket infection in HIV-negative patients[J].
Internal Medicine,2021.

[6] Chan J F ， Lau S K ， Yuen K Y ， et al.
Talaromyces (Penicillium)marneffei infection in non-HIV-infected patients[J].
EmergingMicrobes and Infections， 2016， 5(3)：e19.

[7] Tang S F ， Chan F W ， Chen M ， et al.
Disseminated penicilliosis，recurrent bacteremic nontyphoidal salmonellosis， and burkholderiosisassociated with acquired immunodeficiency due to autoantibody againstgamma interferon .
[J].
Clinical & Vaccine Immunology Cvi， 2010，17(7)：1132.

[8] Zhou G ， Zhou Y ， Zhong C ， et al.
Retrospective analysis of 1，641cases of classic fever of unknown origin[J].
Annals of TranslationalMedicine， 2020， 8(11)：690-690.