Genotypic and phenotypic prevalence of Nocardia species in Iran: First systematic review and meta-analysis of data accumulated over years 1992-2021

Background Nocardia species belong to the aerobic actinomycetes group of bacteria which are gram-positive and partially acid-fast Bacilli. These bacteria may sometimes be associated with nosocomial infections. Nocardia diseases are not required to be reported to public health authorities in Iran. Hence, the present study was designed to determine the prevalence of human Nocardia spp. in Iran by using a systematic review and meta-analysis according to the preferred reporting items for systematic reviews and meta-Analyses statement. Methods The data of the prevalence of Nocardia species were collected from databases such as Embase, PubMed/MEDLINE via Ovid, Web of Science, Scopus and Google Scholar as well as national Iranian databases, including SID, Magiran. Analyses were conducted by STATA 14.0. Results The meta-analyses showed that the proportion of Nocardia spp. in Iranian studies varied from 1.71(1.17, 2.24) to 0.46(0.09, 0.83). N. asteroides (21% [95% CI 1.17, 2.24]), N. cyriacigeorgica (17% [95% CI 0.99, 1.77]), N. facanica (10% [95% CI 0.75, 1.00]) were considered to be common causative agents. Conclusions Our study presents that despite the fact that Nocardia spp. are normally are saprophytic organisms, are currently accounts as emerging pathogens due to an increase in immunocompromised patients among Iranian populations. Considering our results, the establishment of advanced diagnostic facilities for the rapid detection of Nocardia infections are required for optimal therapeutic strategies of Nocardia spp. in Iran. Our findings could help the programmatic management of the disease within the context of Nocardia control programs.


Literature search
A systematic review of available literature was searched using the electronic database such as: Embase, PubMed/MEDLINE via Ovid, Web of Science, Scopus and Google Scholar as well as national Iranian databases, including SID, Magiran, with medical subject headings (MeSH) terms and a proper use of keywords. The search strategy was as follows: " Nocardia ", "nocardiosis", "Nocardia and human infection", "actinomycete" and "Iran". Original articles on nocardia and a time filter (from August 1992 to January 2021) applied including Persian and English articles were considered. Likewise, the full texts of potentially relevant articles were assessed for eligibility independently and in duplicate by two investigators. In addition to articles published in English, we also looked for relevant articles in Persian.

Inclusion and exclusion criteria
After the search results were merged into Endnote (X7; Thomson Reuters), the resultant was de-duplicated and screened by applying a Rayyan Qatar Computing Research Institute online application. Criteria considered for inclusion were cross-sectional surveys assessing the frequency or prevalence of Nocardia spp. in Iran. Based on the research protocol and the eligibility criteria, the titles and abstracts were separately retrieved by two independent researchers. Following the elimination of repetitive studies, the full text of the papers in terms of eligibility criteria and the required extracted necessary information were studied. Disagreements between the two researchers were resolved by a consensus method. The final data extracted from the search results included corresponding author, year, place, research design, sample size, location, study period, individual Nocardia spp., and detection method. The exclusion criteria were including the papers with the following features: review articles, meta-analyses or systematic reviews, case reports and letter to editor studies, congress abstracts, and the duplication papers, as well as articles in languages other than English or Persian and those available only in abstract form. To evaluate the eligibility of the articles with inadequate information, we made a contact with the corresponding author. Culture as well as biochemical and molecular testes were the standard methods for detection.
To conduct phenotypic methods, the paraffin baiting technique was used and samples were cultured on various cyclohexamide-containing agars (i.e. blood, nutrient, and Sabouraud Dextrose) and were incubated at 35˚C. Kinyoun acid-fast stain and Gram stain were used for initial investigation of colonies grown on culture media. The partially acid-fast and Gram-stained organisms showing that colonies bore a resemblance to the genus Nocardia. Stereomicroscopy was employed to assess the morphology of colonies. Numerous biochemical tests employed on the grown colonies in the present work were as follows: decomposition of L-tyrosine, growth in lysozyme broth and also at 45˚C, hydrolysis of casein, esculin, gelatin, urea, xanthine, and hypoxanthine, utilization of citrate, and production of nitrate reductase, as well as acid production of sorbitol, rhamnose, glucose, L-arabinose, D-xylose, galactose, mannitol, lactose, maltose, sucrose, raffinose, and salicin.

Data extraction
Two reviewers independently extracted the data from eligible studies. According to inclusion and exclusion criteria, all collected data from the selected studies were tabulated as follows: (1) First author, (2) publication date, (3) enrollment time, (4) province of study, (5) all patients included in study, and (6) prevalence of Nocardia human infections. Two authors extracted data from involved studies independently. Inconsistency between the reviewers was resolved through discussion.

Quality assessment
The quality of papers was evaluated using the Strengthening the Reporting of Observational studies in epidemiology (PRISMA) checklist and the guidelines of the Cochrane Handbook for Systematic Reviews and Interventions [9]. This checklist has 8 parts which covers different sections of reports. If necessary, the authors were contacted for further information.

Statistical analysis
In this study, the prevalence of Nocardia in the country was collected and then the variance of each study was determined by Double arcsine conversion method. The point estimates of effect size, the prevalence of Nocardia spp., and its 95% confidence interval (95% CI) were estimated for each study. Random effects models were used, taking into account the possibility of heterogeneity between studies, which was tested with the Cochran's Q-and the I2 statistics. In order to assess possible publication bias, Egger weighted regression methods were used. Value of P < 0.05 was considered indicative of statistically significant publication bias. Analyses were conducted by STATA 14.0 (StataCorp, College Station, TX, US).

Characteristics of the included studies
A total of 93 articles were obtained by a literature search with a combination of keywords from the databases as shown in Fig 1. In secondary screening and after duplication, 18 articles were identified and were removed due to the irrelevant titles. Then based on the abstract evaluation, 55 articles were excluded (3 review articles, 27 case reports, 8 letters to the editor, and 17 were related to non-clinical Nocardia specimens). So, according to quality assessment criteria and inclusion/exclusion criteria data, a remaining 20 most-related articles were included in present study [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Among 20 articles involving a total of 338 Nocardia isolates, the prevalence of Nocardia spp. were recorded. The articles were published between years 1994 to 2021. The characteristics of the selected articles are summarized in Table 1.

The prevalence of different Nocardia spp.
In total, 338 different Nocardia spp. were identified in the studied Iranian articles, with the varied proportion from 1.71(1.17, 2.24) to 0.46 (0.09, 0.83) using 90% confidence interval

The prevalence of different Nocardia spp. in provinces of Iran
Out of these 20 articles, 15 were belong to reports from Tehran, center of Iran. The rest were as follows: 5 from southwest of Iran (4 of them from Khuzestan and 1 from Kermanshah provinces), Isfahan, Yazd, Central (Arak), and Golestan provinces one report each.  (Table 3).

Discussion
Due to the low probability of transmission among people, Nocardia infection was not taken into account as a public health problem, and its incidence still remains unknown [30]. Literature review have revealed that the number of clinical isolations of Nocardia infection is escalating globally, possibly in relation to the increased number of immunocompromised patients and improved laboratory techniques for nocardiosis detection [31,32]. To our knowledge, this is the first study in Iran which focuses on the incidence of Nocardia spp. among Iranian population.
The analysis has revealed a relatively high prevalence of Nocardia spp. among Iranian patients. The overall prevalence of this species was also greater when the study was performed after the year 2000 with the rate of 54% compared to the rate before year 2000 as 38%. The higher number of reports of Nocardia spp. appears to be due to the significance of the disease identified by microbiologists and physicians, advancement of laboratory facilities, and increasing the incidence of immunocompromised hosts, which gives rise to a growth in Nocardia spp. in the entire population. In general, the relatively high incidence (0.49%) of Nocardia spp. in our country may have an adverse impact on public health. Despite the implementing of national control programs, tuberculosis (TB) is still among the highest health hazard in Iran.
Owing to the clinical similarity of Nocardiosis to many other infections, TB in particular, Nocardia infections are commonly missed/ or not suspected and delay in diagnosis [33]. The preliminary and rapid method for suspected lung infections is acid fast staining on prepared sputum smears for the screening of acid fast bacilli (AFB), however this method alone is unable to differentiate M. tuberculosis from non-tuberculous mycobacteria (NTM) and Nocardia spp. Thus, failure in the characterization of Nocardia lung infections that are positive in acid fast staining, contributes to the misclassification of these infections, thereby leading to a failure in the treatment of pulmonary Nocardiosis [34]. Since Nocardia spp. is abundant in different areas of the country, including the central and southwest parts, performing nucleic acid amplification tests (NAAT) is routinely required for differentiating between Nocardia and other AFB smear-positive sputum samples.
In our study, N. asteroides was the most frequent isolate species, which supports the findings of other research works [3,35]. The pathological importance of Nocardia infections lies on the pathogenesis process in human body. The pathogenesis mechanism of Nocardia related to its ability to survive and grow in a variety of human cells including phagocytic cells by mechanisms including production of catalase and superoxide dismutase and inhibition of phagosome-lysosome fusion [36]. In brief, after the organism enters the body, reticuloendothelial system represents the initial response by mobilization of neutrophils, leads to limiting the dissemination of infection. Later, by the action of cell-mediated immunity macrophage activation occurs, i.e. T-cell population, giving rise to direct lymphocyte-mediated toxicity to the organism. The interplay between phagocytic cells and Nocardia hinges on the virulence of the strain  and the growth phase of the nocardial cells. Virulent Nocardia can be explained by the complexity of cell wall glycolipids that prevents the fusion of phagosome-lysosome, declines the activity of lysosomal enzyme in macrophages, neutralizes phagosomal acidification, and even withstand the oxidative killing mechanisms of phagocytes. In chronic granulomatous disease, neutrophils and macrophages are unable to produce a burst of oxidative metabolism in the course of phagocytosis, which induces and impairment in the intracellular killing of catalasepositive bacteria viz Nocardia species [37]. Ultimately, the host have to enhance a lymphocyte response and then release antibody and/or lymphocyte signals, allowing the phagocytic cells to destroy N. asteroides. Pulmonary nocardiosis is the most frequent clinical symptoms of infection because the main route of bacterial exposure is inhalation [37,38]. In some occasions, the gastrointestinal tract, especially the appendix, is penetrated. In rare conditions, pulmonary infection leads to a dental or periodontal infection. Patients on immunosuppressive drugs, as well as cases with chronic granulomatous disease, chronic alcoholism, diabetes mellitus, and human immunodeficiency virus infection are also more susceptible to pulmonary infections with Nocardia [39]. The commencement of symptoms might be acute, subacute, or chronic, and pulmonary nocardiosis, if untreated, can have multiple attributes to the same as tuberculosis, comprising fever, weight loss, nonproductive cough, anorexia, night sweats, dyspnea, hemoptysis. Acute forms of the disease are peculiarly observed in the compromised host [37]. A vast majority of surveys have been performed in the central and southwest regions, but not northern part, of Iran. These data denote that Tehran, the capital city of Iran with many healthcare centers, has a referral role for the whole areas of the country. Therefore, patients, especially those with complicated conditions, are referred to Tehran from all over the country for better management. Considering these data, it seems that the main reason for the isolation of most Nocardia spp. in the central provinces is accessibility of the commercial methodologies for detecting this species. Thus, conducting continuous DNA sequencing of homologous genes with a maximum resolution is considerably recommended for areas having a high incidence of Nocardia spp. [40].
For the ultimate bacteriological diagnosis of nocardiosis, it is required to isolate and identify the agent from clinical material and from the laboratory where the samples were analyzed [41]. The common diagnostic strategies for Nocardia spp. are mostly conventional and molecular methods [42]. In culture media, the growth of Nocardia spp. is slow, and at least two-week incubation is needed. Initial culture discontinuation will diminish the susceptibility of recovery and even may understate the real prevalence of Nocardiosis [37]. Consequently, in view of the clinical presentation and imaging of nocardiosis, which is typically comparable with TB, numerous recent investigations uncovered that the speciation of Nocardia may need to be affirmed by molecular methods. So, different molecular techniques have been put forward to precisely identify Nocardia spp. In Iran, and a large number of studies have utilized molecular analysis to identify Nocardia spp. [10, 12, 13, 18, 19, 24-26, 28, 29]. In cited surveys conducted   Table 3. The prevalence of Nocardia species in certain provinces of Iran (n = 338).

Kermanshah Tehran
before years 2000, the identification system was on the basis of biochemical tests and culture, while after this year, PCR was the method that could differentiate recently identified Nocardia spp. Since the first step in controlling of the spread of Nocardia spp. and the related infections, is precise detection of species by employing sophisticated laboratory methods, so evidence has reflected that the identification of almost all Nocardia isolates were carried out by molecular analysis based on the interconnected gyrB-16S rRNA gene sequences; thus, PCR remains the gold standard in this regard (Table 6). Besides, though the outbreak of Nocardiosis outbreak is still rare in Iran, however, hospitals should maintain strong infection control practices to avoid outbreaks of Nocardiosis. In a systematic review, the limitations related to possible publication bias should be taken into consideration. Likewise, the present study has its own shortcomings. First, the Nocardia spp. prevalence among the Iranian population cannot be fully represented as the magnitude of this species has not yet studied in many areas of the country. Second, the probable influence of age, sex, and immigration could not be examined due to the limitation of information achieved from the studied articles. Third, although the number of articles studied was enormous, after several screenings, there were quite small eligible studies. This limitation in the number of articles could lessen the statistical power for the detection of funnel plot asymmetry. Fourth, in some studies, detection of Nocardia isolates in many cities of Iran are limited to the application of phenotypic methods, while the use of molecular techniques is more sensitive and specific than conventional methods for diagnosis of Nocardia spp. Fifth, in three studies, the source of samples (pulmonary or extra pulmonary) was not known; therefore, we could not report the exact rate based on sample source.

Conclusions
In overall, our study presents that despite the fact that Nocardia spp. are normally are saprophytic organisms, are currently accounts as emerging pathogens due to an increase in immunocompromised patients among Iranian populations. Therefore, the findings of the present survey could help the programmatic management of the disease within the context of Nocardiosis control programs. Moreover, this review emphasizes on the Nocardia spp. as neglected pathogens and related infections should be takes into account more seriously in future. The distinction of Nocardia infection from other pulmonary infections such as tuberculosis has significant practical importance. Considering our results, the establishment of advanced diagnostic facilities for the rapid detection of Nocardia spp. are necessary for optimal therapeutic strategies of Nocardia infections in Iran.

Acknowledgments
This work is part of a research project which was approved in Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. We are grateful to research affairs of the university for their encouragement and support.