A 40-year-old man who had been treated for recurrent oral candidiasis came to our hospital because of general malaise and progressive body weight loss, losing 10 kg in a half year. He was bisexual, having no habit of intravenous drug use. While he had no history of any sexually transmitted diseases, he had experienced herpes zoster 5 years before, but he had never taken any human immunodeficiency virus (HIV) antibody tests.
At presentation, he had low grade fever, gum bleeding and tinglingness in the posterior cervical region. He complained of no particular respiratory and gastrointestinal symptoms. Physical examination revealed oral candidiasis, gingivitis and slight lymphadenopathy involving the cervical, submandibular, left postauricular and inguinal nodes (measuring 4-5 mm in diameter, without tenderness). The liver and spleen were not palpable. Folliculitis was seen on the trunk and extremities. An HIV screening test was positive and a Western blot test was done, and it proved to be positive. Blood examination results were as follows (Table 1); HIV-RNA 1,400,000 copies/mL, CD4 count 10/muL, slight pancytopenia, and hypergloburinemia were seen. The serum alkaline phosphatase (ALP) level and lactate dehydrogenase (LDH) level were slightly elevated. With an extremely low CD4 count and wasting syndrome, he was diagnosed as having AIDS. Therefore, a thorough examination regarding opportunistic infections and sexually transmitted diseases was done. Plasma (1->3) beta-D-glucan, cytomegalovirus antigenemia, HBs-antigen, hepatitis C virus (HCV) antibody, and Treponema pallidum latex agglutination test, and rapid plasma reagin test were negative. Blood, fecal, sputum, and urine cultures were done, but no particular microbes including bacteria, mycobacteria, fungi, and protozoa were detected. A computed tomography (CT) scan showed slight splenomegaly and lymph node swelling in the neck areas. Lymphadenopathy was not detected in the thorax or abdomen. No lung lesions were found in a CT scan. Brain MRI, esophagogastroduodenoscopy and total colonoscopy showed no abnormalities. Pneumocystis pneumonia (PCP) was not suspected, and sulfamethoxazole 800 mg/trimethoprim 160 mg was administered as PCP prophylaxis. Oral azithromycin 1,200 mg weekly was also started as prophylaxis for the MAC infection. At that time, no respiratory symptoms were observed, and mycobacterium was not detected in a smear. However, after four weeks of incubation mycobacterium was detected in a sputum culture, but polymerase chain reaction (PCR) tests for M. tuberculosis and MAC were negative.
After the completion of fluconazole therapy for oral candidiasis, cART was started with abacavir, lamivudine and darunavir/ritonavir. On day 11 (of cART), fever, tender submental lymph node swelling and trismus developed. On day 18, his temperature rose to 40 C and cervical lymph node (LN) swelling became more conspicuous with severe tenderness (Fig. 1). The serum ALP level was 618 U/L and MAC-IRS was suspected. On day 28, we performed a biopsy of the submental lymph node. At this time, the CD4 count was 94/muL and the serum HIV-RNA level had declined to 1,700 copies/mL.
The sampled lymph node had swollen to 2 cm in diameter and the surface of section was gray-white with focal yellowish area. Histologically, non-caseous granuloma was seen (Fig. 2a), and relatively thin bacilli were detected in Ziehl-Neelsen staining (Fig. 2b). Mycobacterium was isolated after 8.8 days in supplemented Middlebrook 7H9 broth liquid culture (BacT/ALERT(R) MP for mycobacterium, Biomerieux, USA) and after 11 days in 7H11 agar culture. In 2% Ogawa egg slant medium (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), it was scotochromogenic with an intense yellow color in both light and dark conditions (Fig. 3). Therefore, the strain was thought to belong to the Runyon II group. DNA-DNA hybridization (DDH Mycobacteria(R) Kyokuto Pharmaceutical Industrial) was done, but there were no matches with any of the 18 mycobacterial species.
To identify this isolate, sequence analyses were performed, targeting fragments of the 16S ribosomal RNA (16S rRNA, 269 bp), RNA polymerase B (rpoB, 315 bp), and heat shock protein 65 (hsp65, 401 bp) genes. In a 16S rRNA sequence, although this isolate best matched with M. simiae with 99.7% homology, it also matched several reference strains with more than 98.5% homology (M. lentiflavum, M. triplex, M. genavense, M. intermedium, M. scrofulaceum, M. kansasii and M. gastri). The sequence analysis showed a high homology rate with Mycobacterium sp. UN-152, (GeneBank accession no. AB547401) reported as M. shigaense; 100% in 16S rRNA, 100% in rpoB, and 94% in hsp65. This strain was thus identified to be M. shigaense. Later the strain was proven to grow at 25 C, 30 C and 37 C, but not at 42 C. A niacin test was negative. Table 2 shows the results of antimicrobial susceptibility tests (BrothMIC NTM(R); Kyokuto Pharmaceutical Industrial). Clarithromycin, ethanbutol and rifabutin seemed to be moderately susceptible, while the others were not susceptible.
An antimycobacterial regimen was started on day 25 of cART. The regimen contained rifabutin (RBT) 150 mg, ethanbutol (EB) 750 mg, and clarithromycin (CAM) 800 mg. Isoniazid (INH) 300 mg was also added until the subspecies was identified.
Soon after initiation of the regimens, the fever subsided, but lymph node enlargement continued. A subcutaneous abscess with a protruding erythematous lesion appeared on the sternum and grew to 5 cm in diameter within a week (Fig. 4). Drainage was performed, and again, mycobacterium was found in Ziehl-Neelsen stain, and it was later identified to be M. shigaense. Thus, the final diagnosis was immune reconstitution syndrome caused by the novel mycobacterium, M. shigaense. Though we could find mycobacterium in the biopsy or drainage specimens, it took over 4 months to correctly identify this strain. Meanwhile, INH was continued.
The serum ALP level normalized at 12 weeks, HIV-RNA turned to negative at 16 weeks, and the CD4 count recovered to over 100/muL at 27 weeks after the initiation of cART (Fig. 5). Blood, urine and stool cultures for Mycobacterial were repeatedly done, but those were all negative. The skin lesions and lymphadenopathy all gradually improved, and the antimycobacterial regimen was stopped after 6 months. Since then, the patient has been followed up without any recurrence of NTM disease for more than a year.