Acid-fast bacilli smear test of a blood culture sample for the diagnosis of disseminated Mycobacterium genavense infection: A case report

Introduction

Mycobacterium genavense is rarely detected in patients infected with disseminated nontuberculous mycobacteria (NTM). Mycobacterium genavense was first described in a patient with acquired immunodeficiency syndrome (AIDS) as an unidentified Mycobacterium in 1990 ¹ and later identified by polymerase chain reaction (PCR). ² Disseminated M. genavense infection is less frequently observed in immunosuppressed patients. ³ Mycobacterium genavense is a slow-growing organism; therefore, it is challenging to identify the bacteria using routine culture methods.

Herein, we report the case of a patient living with HIV presenting with disseminated M. genavense infection. Mycobacterium genavense was identified in the patient’s blood culture samples by sequencing analysis targeting the heat shock protein 65 (hsp65) gene, although analysis with an automated blood culture system did not show a positive signal during a 6-week incubation period.

Case report

A 40-year-old Japanese man, who was diagnosed with HIV infection 10 years prior to admission and was administered with antiretroviral therapy (ART) (lopinavir/ritonavir/abacavir/lamivudine) for the first five years and discontinued it for the next five years, was admitted to our tertiary hospital because of Pneumocystis jirovecii pneumonia (PJP). In laboratory tests, the CD4⁺ lymphocyte count was 4 cells/μL, and HIV-RNA viral load was 210 000 copies/mL. We commenced treatment for PJP with a trimethoprim–sulfamethoxazole combination of PJP treatment doses as well as 80 mg of prednisolone per day with an improvement in dyspnea and fever. He was also administered with 1200 mg of azithromycin per week to prevent disseminated mycobacterium avium complex (MAC) infection. However, on day 12, febrile illness was observed again.

Apart from PJP, other reasons of fever were explored. Diarrhea, aphtha on the patient’s lips, and cervical lymphadenopathy with tenderness were observed. The laboratory test results were unremarkable besides a C-reactive protein level of 8.45 mg/dL and slightly elevated liver enzyme levels. The results of Gram staining and Ziehl–Neelsen staining of the blood sample were negative. A contrast-enhanced CT scan showed cervical, mesenteric, and para-aortic lymphadenopathy and small intestine extension. On day 21, cytomegalovirus enteritis treatment was commenced using valganciclovir because the cytomegalovirus–antigenemia test (C7HRP) revealed 6 cells/50 000 cells. The fever gradually improved, and the patient commenced ART (darunavir/ritonavir/tenofovir/alafenamide/emtricitabine) on day 25. However, cervical lymphadenopathy was exacerbated, and on day 36, we performed a cervical lymph node biopsy. Ziehl–Neelsen staining of the sample was positive, but Mycobacterium tuberculosis and MAC were negative using PCR analysis. We ceased the administration of azithromycin and commenced treatment with 800 mg of clarithromycin, 750 mg of ethambutol, 150 mg of rifabutin, and 300 mg of isoniazid. Symptoms gradually improved following treatment, and the patient was discharged on day 59. We discontinued incubation of the blood culture since the automated MicroScan WalkAway system (Beckman Coulter, Brea, CA, USA) showed no positive signals during 6 weeks of incubation. The cervical lymph node sample cultured on Ogawa agar (25°C and 37°C) and Mycobacteria Growth Indicator Tube also showed negative results over the 6-week period. We performed Ziehl–Neelsen staining with the blood sample which showed negative result after incubation for 6 weeks; the result was positive (Figure 1). Mycobacterium genavense was identified using PCR fragment length polymorphism analysis and the sequencing of hsp65.OPEN IN VIEWER

The antimycobacterial susceptibility of M. genavense was determined using a broth-based minimum inhibitory concentration (MIC) NTM (Kyokuto Pharmaceutical Industrial. Co., Ltd., Tokyo, Japan). Following the MICs of the antibiotics (Table 1), we changed the antibiotic regimen of the patient at 4 months post-admission to 800 mg clarithromycin, 150 mg rifabutin, and 500 mg levofloxacin. Antibiotic treatment was completed at 18 months following admission. HIV-RNA was undetectable and the CD4 count was found to be >200 cells/μL for 7 months with no symptom recurrence.

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Table 1.

Minimum inhibitory concentration of Mycobacterium genavense isolated from blood culture.

Drug	Minimum inhibitory concentration
Streptomycin	1 μg/mL
Ethambutol	4 μg/mL
Rifabutin	0.06 μg/mL
Levofloxacin	0.5 μg/mL
Clarithromycin	0.06 μg/mL
Amikacin	2 μg/mL

Discussion

Our patient developed disseminated M. genavense infection associated with an immune reconstitution inflammatory syndrome, following ART initiation. Although the automated blood culture system did not show a positive signal during the 6-week incubation period, an acid-fast bacilli smear test using a blood sample from the blood culture bottle led to the diagnosis of M. genavense.

Disseminated M. genavense infection is difficult to diagnose since the optimal conditions required for incubating the organism in blood culture bottles have not been established. Previous studies have shown that the rate of positive blood culture is 20%–33%, while the median time required to detect the positive blood culture is 42–46 days.^4–7 Furthermore, molecular techniques are typically necessary to identify this species directly in broth cultures. Our case indicates that an automated blood culture system cannot detect any mycobacterium in routine 6-week culture, and an acid-fast bacilli smear test using a blood sample from the blood culture bottle should be performed even if the automated blood culture system does not show a positive signal in immunocompromised patients suspected of disseminated infection or patients with positive blood smear test in samples obtained from other sites.

In conclusion, the incubation of M. genavense is challenging and time-consuming; therefore, a diagnosis is difficult. Our method of identifying M. genavense in negative blood culture samples is useful for achieving an earlier diagnosis of the infection.