Isolation, biochemical and molecular identification of Nocardia species among TB suspects in northeastern, Tanzania; a forgotten or neglected threat?

Members of genus Nocardia are characteristically gram-positive, weakly acid-fast, strictly aerobic, filamentous branching bacilli that fragment into rod or coccoid shaped forms. Nocardia species are ubiquitous environmental bacteria capable of causing opportunistic infections in both human and animals [1, 2]. Nocardia species are increasingly isolated as infectious agents in immunocompromised patients, and at times, even among healthy individuals [3], causing infections ranging from pulmonary, cutaneous and subcutaneous human diseases [4].

The most commonly isolated species in human include N. asteroides, N. farcinica, N. cyiriacigeorgica, N.nova, N. brasiliensis [5–7]. Pulmonary nocardiosis has been reported in patients with debilitating conditions, such as those with organ transplants, diabetes mellitus, leukemia, and other malignancies [8].

The incidence of nocardiosis varies geographically according to a number of factors, like the prevalence of HIV infections, transplants, cancer, climate as well as socio-economic status, and laboratory capacity for Nocardia species detection and identification.

Increased incidence of human nocardiosis may be attributed to the wide use of immunosuppressive drugs, improved diagnostic tests, and increased awareness among microbiologists and health professionals. Nonetheless, in many developing countries where other chronic lung diseases, particularly TB, are prevalent, Nocardia species are either missed or misidentified during diagnosis [9, 10].

Accurate detection and identification of Nocardia species have become increasingly important for prediction of antimicrobial susceptibility since different species have emerged in terms of their virulence and epidemiology. Furthermore, prompt and timely species identification can significantly influence the choice of therapy.

Apart from Gram and modified acid-fast staining, identification of Nocardia species depends largely on biochemical tests and cellular fatty acid analysis, which have proved to be laborious with long turnaround time, and less definitive.

Several molecular identification methods have been successfully employed to identify and characterize Nocardia species. Multilocus sequence analysis (MLSA) of 16S rRNA, the 65-kDa heat shock protein (hsp65), gyrase B of the ß subunit of DNA topoisomerase (gyrB), subunit A of Sec A preprotein translocase (secA1) and RNA polymerase (rpoB) have previously been used to identify Nocardia species [11–13]. However, 16S rRNA gene sequencing, which is the most often used system of bacterial identification, cannot discriminate many species [14], therefore identification based on the basis of the DNA sequence of a single housekeeping gene is hindered by stochastic genetic variation as well as horizontal gene transfer and recombination [15].

Pulmonary nocardiosis mimics pulmonary tuberculosis in both clinical manifestations, being chronic in nature and radiological characteristics makes it difficult to differentiate from M. tuberculosis and may as well be often wrongly treated with anti-TB drugs [16, 17]. Patients might also be confused with other chronic lung infections such as invasive fungal infection [18, 19].

In Tanzania, where tuberculosis is one of the major public health threats and a third leading cause of adult morbidity and mortality after Malaria and HIV/AIDS; clinical reports of Nocardia are rare if found at all. Moreover, the knowledge, as well as the clinical impact of nocardiosis, is unknown, suggesting that infections due to this genus may be underdiagnosed and/or neglected as a cause of human diseases.

Due to the lack of information on pulmonary nocardiosis in Tanzania, the objective of the present study was to isolate and identify Nocardia isolates recovered from TB suspects in Northeastern, Tanzania by using biochemical and molecular methods.

Nocardia infections causing both human and animal diseases are increasingly reported owing to improved diagnostics especially in developed settings which have emphasized the need for rapid characterization of clinically isolated Nocardia [4, 8, 10, 24, 25]. However, data regarding nocardiosis from resource-poor settings like Tanzania heavily stricken by HIV and TB pandemic are rare if available at all. Such diseases are either underdiagnosed or neglected due to the similarity of clinical and radiological features between pulmonary nocardiosis and pulmonary tuberculosis on one hand and on the other due to poor diagnostic capabilities. In Tanzania where TB is still a major public health threat, anti-TB treatment is initiated based on clinical symptoms, direct smear microscopy and radiological diagnosis where available.

In this study, all 372 TB suspects self-presented at four health facilities were diagnosed based on clinical symptoms and direct smear microscopy by acid-fast staining method at the respective clinics. Eighty-one (n = 81, 21.8%) patients were positive for M. tuberculosis by culture and confirmed by GenoType® MTBC and 36 (9.7%) patients had NTM infections confirmed by GenoType® Mycobacterium CM/AS. 202/372 patients sputum samples were negative by culture and 33 other samples were contaminated.

Twenty (5.4%) isolates out of 372 TB suspects were presumptively diagnosed as Nocardia species based on their colony morphology on different culture media and by microscopic appearance using modified ZN for weak acid-fast bacilli.

Isolates of Nocardia species in this study showed good growth on LJ, BA and chocolate agar (Fig. 1). Decontamination of sputum samples with NALC-NaOH in this study did not seem to affect the growth of Nocardia species as observed in a previous study [26].

Samples from 17 suspects were presumptively diagnosed as having Nocardia infection and three patients had infection with other Actinomycetales. Findings that majority of Nocardia species grow well on LJ in this study correspond with findings of other authors [25, 27]. In this scenario, such growth may be confused with that of mycobacteria species, making the diagnosis more complicated. Furthermore, careful microscopic analysis should be thoroughly performed, since these two genera may present difficulties for an inexperienced microscopist [24]. As observed in the previous study [25], where Nocardia species were detected by the modified ZN-method for weak acid-fast bacteria, we also detected Nocardia species by the ZN-method employed for mycobacteria in this study.

All the Nocardia species identified in this study have been reported to be associated with pulmonary nocardiosis in many parts of the world [24, 26–29].

The finding that the biomèrieux ID 32C yeast identification system identified correctly five Nocardia isolates to species level and two isolates to least genus level as Streptomyces (Table 1) when compared to 16S rRNA and hsp65 sequencing results is in agreement with those in a study conducted in Brazil [13]. In the Brazilian study, seven isolates were identified by 31 different phenotypic tests using six identification systems with only two isolates correctly identified by the phenotypic method, compared to multilocus sequence analysis (MLSA) results. However, the biomèrieux ID 32C yeast identification system used in this study appears to be useful in identifying Nocardia species and other aerobic actinomycetes as previously determined [23].

The discrepancies between the ID 32C yeast identification system and sequencing results suggest that a careful interpretation of results indicating different Nocardia species and other Actinomycetales is needed since such methods are not accurate as shown in Table 2. Moreover, although the ID32C system has been used for identification of many aerobic actinomycetes, it does not, however, give consistent results within a species. There is a greater potential for errors than with 16S rRNA or with hsp65 gene sequencing. For example ID32C system present a lot of difficulties in identification of Nocardia asteroides complex, this method uses eight-digit profile as those used for identification of yeast, whereas the last four-digits show a consistent correlation between isolates within species, the first four-digits show poor correlation within a species and between species. There is no single profile for each particular isolate and species with this method, hence this variation. Therefore, results of 16S rRNA and hsp65 gene sequencing are more reliable than those of the ID32C system. Therefore, molecular identification is necessary for definitive identification of Nocardia species [10, 30–33].

Important to note is that the following species: N. cyriacigeorgica, N. farcinica, N. brevicatena and N. nova identified by both 16S rRNA and hsp65 sequencing in this study were separated from the originally referred Nocardia asteroides and later found to be a group of bacteria with a heterozygous pattern of antimicrobial drug susceptibilities [34]. N asteroides complex was further separated and reorganized into different species on the basis of drug susceptibility patterns: Nocardia abscessus, Nocardia brevicatena-paucivorans complex, Nocardia nova complex (includes N nova, Nocardia veterana, Nocardia africana, Nocardia kruczakiae), Nocardia transvalensis complex, Nocardia farcinica, and N asteroides [5]. Moreover, Nocardia cyriacigeorgica, which constitute the majority of Nocardia species in this study (n = 4, 23.5%) was also differentiated from N asteroides and is becoming a more frequently identified clinically significant pathogen [35].

This separation may explain the observed discrepancy between the two methods. While phenotypic identification leads to misidentification, molecular identification can improve the diagnostic accuracy since some molecular targets can present high sequence similarity [2].

Looking into the demographic data of all 17 Nocardia cases in this study, it was observed that nine (52.9%) were males and eight (47.1%) were females. Seven (41.1%) were ≤35 years and ten (58.8%) were ≥35 years. This incidence suggests that nocardiosis can occur in both sexes and in different age groups with more or less the same frequency. However, of importance is that nearly all cases of nocardiosis identified in this study reported similar clinical symptoms as those of TB suspects with chronic coughing featuring in all cases (Table 3). This is not surprising since pulmonary nocardiosis is the most common clinical presentation acquired primarily by inhalation. The onset of symptoms can be subacute to more chronic and can include productive or a non-productive cough, shortness of breath, chest pain, hemoptysis, fever, night sweats, weight loss, and progressive fatigue [4].

In this study, 12 patients had Nocardia alone; two had co-infection with TB and were AFB positive, whereas three had co-infection with HIV. Similar findings have been reported in other studies [8, 36]. It is important that irrespective of a patient’s immunologic status, the isolation of Nocardia from the respiratory tract or another body source should not be regarded as a contaminant or commensal organism [4].

In conclusion, our study reveals that Nocardia species are important causes of pulmonary disease and that may have been underdiagnosed and/or ignored altogether. It is also clear that mycobacterial infections and nocardiosis treatment differ, correct identification of the causative agent is, therefore, critical to avoid treatment failure. Nocardia species identified in this study, underscore the need to consider pulmonary nocardiosis as differential diagnosis especially when there is treatment failure with the standard anti-TB therapy, and as a possible cause of human infections.