Mycobacterium africanum cases, California.

Five Mycobacterium tuberculosis complex isolates in California were identified as M. africanum by spoligotyping, single nucleotide polymorphisms, a deletion mutation, and phenotypic traits, confirming it as a cause of tuberculosis in the United States. Three of the five patients from whom M. africanum was isolated had lived in Africa.

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Mycobacterium africanum is a member of the M. tuberculosis complex, which has been isolated from humans in equatorial Africa. The disease produced by M. africanum is similar to that caused by M. tuberculosis or bovis, and like M. tuberculosis, this organism is likely spread by aerosol transmission (1). Human tuberculosis caused by M. africanum has been reported in Europe (2,3). However, we are unaware of previous reports of disease caused by M. africanum in the United States.

The Study

M. africanum may be identified by spoligotyping (4), by specific deletion mutations (5), DNA fingerprinting by IS6110 restriction fragment length polymorphisms (RFLP) (4), or a combination of these methods. Isolates were initially identified as M. tuberculosis complex by using the AccuProbe system (Gen-Probe; San Diego, CA). The isolates then underwent IS6110-based RFLP fingerprinting. The RFLP analyses were performed according to the method of van Embden et al. (6). In addition to providing genotyping results, RFLP fingerprinting confirmed the identification obtained with Accuprobe.

All strain typing was performed in-house at the Microbial Diseases Laboratory, California Department of Health Services. This laboratory has compiled a database (Genomic Solutions BioImage) of approximately 7,000 DNA fingerprints, typed by IS6110 RFLP (6,7) from throughout California; most are from the San Francisco Bay area. Three isolates (from patients A, B, and C) were initially suspected of being M. africanum because of an epidemiologic association with Africa. These isolates were fingerprinted by IS6110 RFLP and by spoligotyping (8). All three were found to have the "signature" spoligotype described by Viana-Niero et al. as being characteristic of M. africanum (4), namely, they were missing spacers 8, 9, and 39 but had spacers 40-43. Using BioImage software, we searched the laboratory's database fur IS6110 fingerprints that matched those of the three cases with an African connection. This search yielded an additional two matches, cases D and E. Isolates from cases D and E were then genotyped by using spoligotyping and found to have the M. africanum signature spoligotype.

The five M. africanum isolates were further characterized by performing standard biochemical identification tests and testing for susceptibility to pyrazinamide (PZA). Niacin production and nitrate reduction were detected as described by Kent and Kubica (9). Susceptibility to PZA was determined by using the BACTEC radiometrie assay performed according to the method of Salfinger et al. (10). The M. africanum isolates were then examined to determine whether they had the RD9 deletion and specific oxyR and katG sequence mutations.

Brosch et al. had reported that isolates of M. tuberculosis do not have the RD9 deletion, whereas other members of the M. tuberculosis complex, including M. bovis, microti, and M. africanum have this deletion (5). Two sets of primers for detecting the RD9 deletion, designed as described by Brosch et al., were obtained from Alex Pym at Stanford University. A [approximately equal to] 480-bp region was amplified by polymerase chain reaction (PCR) from M. africanum by using the flanking primers, and a [approximately equal to] 375-bp region was amplified from M. tuberculosis by using the internal primers. After an initial denaturation step of 10 min at 95[degrees]C, amplification was performed for 35 cycles at 95[degrees]C for 30 s, 55[degrees]C for 1 min, and 72[degrees]C for 4 min, and a final step of elongation at 72[degrees]C for 10 min. The final concentration of each component in the 50-[micro]L PCR reaction tube was 1x PCR buffer, 2.5 mmol/L of Mg[Cl.sub.2], and 1.5 U of AmpliTaq Gold polymerase using AmpliTaq Gold kit with GeneAmp 10x PCR buffer II and Mg[Cl.sub.2] solution (Applied Biosystems, Foster City, CA), 1.25 mmol/L of deoxynucleotide triphosphate mix (Applied Biosystems), 0.2 [micro]mol/L of each primer, 5 [micro]L of DNA template. A M. tuberculosis strain, H37Rv, and M. africanum strain ATCC 25420 were included in PCR runs as reference strains.

A 548-bp region of the oxyR locus was amplified by PCR using the primer sequences described by Sreevatsen et al. (11). After an initial denaturation step of 4 min at 95[degrees]C, amplification was performed for 30 cycles with the following parameters: 95[degrees]C for 30 s, 55[degrees]C for 30 s, and 72[degrees]C for 45 s. A final elongation step was performed for 5 min at 72[degrees]C. For katG, a primer set, 5'-TGCTGGCGCTTGGCAATACA and 5'-GCCGCGCTTGTCGCTACC, was designed to amplify a 429-bp region encompassing codon 463. With the exception of an annealing temperature of 60[degrees]C, the amplification parameters for katG were identical to those described for oxyR. The amplified products were purified by using QIAquick spin columns (Qiagen Inc., Valencia, CA) and subjected to dRhodamine Terminator Cycle Sequencing (Applied Biosystems, Foster City, CA) as recommended by the manufacturer. The cycle sequencing reactions were analyzed on an ABI 377 DNA sequencer, and the sequences aligned with those derived from M. tuberculosis strain H37Rv and M. africanum strain ATCC 25420.

For case histories, see Table 1. The five isolates were identified as M. tuberculosis complex by a positive result with the Gen-Probe AccuProbe system and by the presence of IS6110 insertion sequences. When spoligotyping was performed on isolates from cases A through E, all were found to have the M. africanum signature spoligotype, as described by Viana-Niero et al. (4). The results of spoligotyping are shown in the Figure.

[FIGURE OMITTED]

The five isolates were M. africanum, based on several criteria shown in Table 2. Susceptibility of the strains to PZA, nitrate reduction, production of niacin, and the presence of guanosine at oxyR285 were incompatible with identification of the isolates as M. bovis. The RD9 deletion was shown for all five isolates and the M. africanum reference strain, ATCC 25420, by 1) the presence of a [approximately equal to] 480-bp band on the agarose gel when the PCR was performed with the flanking primers and 2) the absence of a [approximately equal to] 375-bp band when the PCR was performed with the internal primers. The RD9 deletion was incompatible with identification of the isolates as M. tuberculosis, and CTG at katG463 suggested that the isolates were not M. tuberculosis, since most isolates of this species have CGG at this site (7).

Conclusions

David et al. (12) and Frothingham et al. (13) reported that some isolates of M. africanum are resistant to PZA and fail to accumulate niacin, distinguishing them from most strains of M. tuberculosis, while other isolates are susceptible to PZA and produce niacin. These five isolates of M. africanum were susceptible to PZA, and three out of the five tested positive for niacin production and nitrate reduction (the remaining two cultures had lost viability and were not available for this testing). This rendered the cultures phenotypically indistinguishable from M. tuberculosis (12-14). For each of the reported cases, the signature M. africanum spoligotype pattern described by Viana-Niero et al. (4) was supported by the single nucleotide polymorphisms characteristic of M. africanum and by the RD9 mutation that characterizes members of the tuberculosis complex other than M. tuberculosis.

At present, the Microbial Diseases Laboratory plans to spoligotype all strains of M. tuberculosis complex. Clinical features and drug susceptibility were not distinctive for M. africanum isolates, nor were all associated with patients who came from Africa. No connection with a source case from Africa was found for patients D or E. However, since spoligotyping will be routine, noting the signature africanum spoligotype may be worthwhile. This spoligotype may indicate an increased chance that the patient may have acquired the M. tuberculosis complex infection in Africa or possibly from an African source case.

Table 1. Clinical characteristics of patients with Mycobacterium
africanum disease

              Demographic                                 Clinical
Patient     characteristics     Signs and symptoms    characteristics

A         25-y-old black        3 months' produc-    Large left upper
          South African man     tive cough, weight   lobe infiltrate on
          with recent travel    loss, and low-       chest radiograph
          to West Africa        grade fever

B         34-y-old black        2 months' cough,     Right lower lobe
          West African woman    fever, night         cavitation,
          recently arrived in   sweats, fatigue      streaky infil-
          U.S. with metasta-                         trates bilaterally
          tic carcinoma,                             on chest radio-
          treated with chemo-                        graph, normocytic
          therapy 1 year                             anemia
          before presen-
          tation.

C         26-y-old black        Unknown              Cavitary lesion on
          South African,                             chest radiograph
          immigrated to U.S.
          2 y before diagno-
          sis

D         26-y-old Vietnamese   2 weeks' produc-     Unknown
          man, immigrated to    tive cough,
          U.S. 6 y before       positive PPD
          diagnosis. No his-
          tory of travel to
          Africa or African
          contacts with
          tuberculosis

E         27-y-old U.S.-born    2 months' anorexia   Normal chest
          black man with no     and weight loss; 1   radiograph re-
          known risk factors    month of cough and   sults. Pancreatic
          for tuberculosis      night sweats; 1      cyst and fullness
          infection. No         week of fever and    of left psoas
          history of travel     abdominal pain       muscle on CT scan
          to Africa or
          African contacts
          with tuberculosis

Patient        Treatment (a)                  Outcome

A         INH, rifampin, PZA, and    Resolution of symptoms
          ethambutol by directly     and improvement of chest
          observed therapy for 6     radiograph
          months

B         INH, rifampin, PZA, and    Sputum culture negative
          ethambutol by DOT          after 2 weeks of therapy.
                                     Only modest
                                     improvement in chest
                                     radiograph. Returned to
                                     West Africa after 4
                                     months DOT

C         INH, rifampin, PZA, and    Sputum culture and smear
          ethambutol by DOT for 6    negative, chest
          months                     radiograph improved

D         Capreomycin, ofloxacin,    Sputum smear and culture
          PAS, clofazamine, and      converted to negative,
          cycloserine for 2 y by     symptoms resolved. Free
          DOT                        of disease 2 y after
                                     therapy completed

E         INH, rifampin, PZA, and    Symptoms resolved. Free
          ethambutol for 10 months   of disease 1 y after
          by DOT                     therapy completed

(a) INH, izoniazid; PZA, pyrazinamide; DOT. directly observed
therapy; CT, computerized tomography, PPD, purified protein
derivative; PAS, para-aminosalicylic acid.

Table 2. Phenotypic and molecular characteristics of Mycobacterium
africanum isolates and country of birth for five patients

           No. of
          copies of   Spoligo-typing                 Codon
           IS6110     pattern missing    OxyR       katG463
Patient    (bands)    spacers 8,9,39?   285 (a)   sequence (b)

A            10             Yes            G          CTG
B             3             Yes            G          CTG
C           9 (f)           Yes            G          CTG
D            11             Yes            G          CTG
E             9             Yes            G          CTG

          Presence of
              RD9       PZA Suscep-      Niacin        Nitrate
Patient    deletion?    tibility (c)   production   reduction (d)

A             Yes            S             +             3+
B             Yes            S           ND (e)          ND
C             Yes            S             ND            ND
D             Yes            S             +             4+
E             Yes            S             +             3+

          Major site
Patient   of disease   Birth country

A            Lung      South Africa
B            Lung       Ivory Coast
C            Lung      South Africa
D            Lung         Vietnam
E          Pancreas         USA

(a) Isolates of M. bovis do not have G to this site.

(b) CTG is found in M bovis and M. africanum, but not
in most strains of M. tuberculosis.

(c) S, susceptible; PZA, pyrazinamide.

(d) 3+ and 4+ reactions are considered positive.

(e) ND, not done (because the culture lost viability).

(f) Restriction fragment length polymorphism patterns
for isolates from patients C and E were identical.

References

(1.) Adler JJ, Rose DN. Transmission and pathogenesis of tuberculosis. In: Rom WN, Garay SM, editors. Tuberculosis. Boston: Little, Brown, and Co.; 1996. p. 129-40.

(2.) Grange JM, Yates MD. Incidence and nature of human tuberculosis due to Mycobacterium africanum in southeast England. Epidemiol Infect 1989;103:127-32.

(3.) Grosset J, Decroix G, Sors C. Tuberculosis due to Mycobacterium africanum in African Negroes in the Paris area. Revue de Tuberculose et de Pneumologie (Paris) 1971;35:430 6.

(4.) Viana-Niero C, Gutierrez C, Sola C, Filliol I, Boulahbal F, Vincent V, et al Genetic diversity of Mycobacterium africanum clinical isolates based on IS6110-restriction fragment length polymorphism analysis, spoligotyping, and variable number of tandem repeats. J Clin Microbiol 2001;39:57-5.

(5.) Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, Eiglmeier K, et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 2002;99:3684-9.

(6.) van Embden JDA, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993;31:40-9.

(7.) Mazurek, GH, Cave MD, Eisenach KD, Wallace RJ, Bates JH, Crawford JT Chromosomal DNA fingerprint patterns produced with IS6110 as strain-specific markers for epidemiologic study of tuberculosis. J Clin Microbiol 1991;29:2030-3.

(8.) Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35:907 14.

(9.) Kent P, Kubica G. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: Centers for Disease Control; 1985.

(10.) Salfinger M, Reller LB, Demchuk B, Johnson ZT. Rapid radiometric method for pyrazinamide susceptibility testing of Mycobacterium tuberculosis. Res Microbiol 1989;140:301-9.

(11.) Sreevatsen S, Escalante E Pan X, Gillies DA II, Siddiqui S, Khalaf CN, et al. Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis. J Clin Microbiol 1996;34:2007-10.

(12.) David HL, Jahan M-T, Jumin A, Grandry J, Lehman EH. Numerical taxonomy analysis of Mycobacterium africanum. Int J Syst Bacteriol 1978;28:467-72.

(13.) Frothingham R, Strickland PL, Bretzel G, Ramaswamy S, Musser JM, Williams DL. Phenotypic and genotypic characterization of Mycobacterium africanum isolates from West Africa. J Clin Microbiol 1999;37:1921-5.

(14.) Niemann S, Richter E, Rusch-Gerdes S. Differentiation among members of the Mycobacterium tuberculosis complex by molecular and biochemical features: evidence for two pyrazinamide-susceptible subtypes of M. bovis. J Clin Microbiol 2000;38:152-7.

Dr. Desmond has worked at the Microbial Diseases Laboratory, California Department of Health Services, since 1990. His research interests include laboratory methods for detecting drug resistance in Mycobacterium tuberculosis and applications for molecular strain typing in the mycobacteriology laboratory.

Address for correspondence: Edward Desmond, Microbial Diseases Laboratory, California Department of Health Services, 850 Marina Bay Parkway, Richmond, CA 93804, USA; fax: [phone]; email: [e-mail]

Edward Desmond, * Ameena T. Ahmed, * William S. Probert, * Janet Ely, * Yvonne Jang, * Cynthia A. Sanders, * Shou-Yean Lin, * and Jennifer Flood *

* California Department of Health Services, Richmond, California, USA