MN45 Mini-blotter for Spoligotyping

Spoligotyping is a PCR- based Reverse Line Hybridization method to simultaneously detect and type Mycobacterium tuberculosis complex bacteria in clinical samples, exploiting polymorphism in the “Direct Repeat” (DR) region, which is uniquely present in Mycobacterium tuberculosis complex bacteria which includes Mycobacterium tuberculosis, M. africanum, M. bovis, M. canettii, M. microti, M. caprae, and M. pinnipedii. The presence or absence of the strains can be detected by hybridization of PCR amplified sample to a set of immobilized oligonucleotides on the membranes.

Foam Cushions (PC200) :

Foam Cushions are intended for use with, Mini-blotter which enables you to perform multiple antibody or probe incubation on a single membrane, using microliter volumes of the reagent. Typical applications include monoclonal screening on western blots, animal and human screening, and DNA hybridization. Sample processing is streamlined through the use of washing manifold, which enables washing of all sample lanes, simultaneously. Reactions can be detected by all conventional methods, including enzyme conjugates, chemiluminescence, and radiolabels.

Abstract

Background

Genotyping of Mycobacterium tuberculosis helps to understand the molecular epidemiology of tuberculosis and to address evolutionary questions about the disease spread. Certain genotypes also have implications for the spread of infection and treatment. Indonesia is a very diverse country with a population with multiple ethnicities and cultures and a history of many trade and tourism routes. This study describes the first attempt to map the molecular epidemiology of TB in the Indonesian archipelago.

Mini-blotter
Mini-blotter

Method

From 2008 to 2011, 404 clinical specimens from sputum-smear (SS+) TB patients, age ≥15 years, were collected from 16 TB referral primary health centers (PHC) in 16 provincial capitals in Indonesia. Susceptibility testing to first line drugs was conducted for 262 samples using the agar proportion method as per WHO guidelines. Spoligotyping was done on all samples.

Results

Ninety-three of the 404 samples (23 %) were from the Beijing family, making it the predominant family in the country. However, the geographic distribution of the family varied by region with 86/294 (29.3 %) in the western region, 6/72 (8.3 %) in the central region, and 2/72 (2.8 %) in the eastern region (p < 0.001). The predominant genotype in the central and eastern regions was from the East-African-Indian (EAI) family, comprising 15.3 % (11/72), and 26.3 % (10/38) of the isolates, respectively. Drug susceptibility to first-line anti-TB drugs was tested in 262 isolates. 162 (61.8 %) isolates were susceptible to all TB drugs, 70 (26.7 %) were mono-resistant 16 (6.1 %) were poly-resistant, and 14 (5.4 %) were multi-drug resistant (MDR). The proportion of Beijing family isolates in the susceptible, mono-resistant, poly-resistant, and MDR groups was 33/162 (20.4 %), 28/70 (40.0 %), 6/16 (37.5 %), and 3/14 (21.4 %), respectively. Overall, resistance of the Beijing family isolates to any of the first line TB drugs was significantly higher than non-Beijing families [37/71 (52.1 %) vs. 63/191 (33.0 %) (p-value = 0.003)].

Conclusion

The distribution of Mycobacterium tuberculosis genotypes in Indonesia showed high genetic diversity and tended to vary by geographic regions. Drug susceptibility testing confirmed that the Beijing family of M.tb in Indonesia exhibited greater resistance to first line anti-TB drugs than did other families.

Background

Tuberculosis (TB) remains a major global health problem and ranks as the second leading cause of death from an infectious disease worldwide. There were 9 million new TB cases and 1.5 million TB related deaths in 2013. Among these deaths, it was estimated that more than 200,000 were due to MDR-TB, a high proportion of the 480,000 incident cases of MDR-TB [1]. A serious problem has been the emergence of extensively-drug resistance TB (XDR-TB) comprising 9 % of the MDR cases. The treatment of MDR- and XDR-TB is substantially more costly and difficult to treat, with higher rates of treatment failure and mortality, as compared to drug-susceptible tuberculosis [2, 3].

Indonesia is considered a high-burden country for TB, ranked fifth in TB incidence (460,000 new TB patients each year) worldwide [1]. An Indonesian National Basic Health Survey in 2010 reported 289 cases/100,000 population [4]. According to the Indonesian TB prevalence survey in 2004, differences in TB prevalence among different regions was observed. For example, 82 cases/100,000 population were reported in the Java-Bali region, compared with 343 cases/100,000 population in Eastern Indonesia and 217 cases/100,000 population in the Sumatra-Kalimantan region [5]. The differences in the incidences is likely multifactorial and may include such factors such as income level, hygiene, education and availability of health care facilities [2, 4, 6].

In addition to the high prevalence of TB cases, drug-resistant DR-TB is also an important problem in Indonesia. WHO estimated 1.9 % of new TB cases and 12 % of previously treated TB cases in Indonesia have MDR-TB [1]. Several studies reveal that MDR-TB occurs more frequently in patients with a previous history of pulmonary TB treatment, particularly those with inadequate treatments (including monotherapy, insufficient duration of therapy or inconsistency of adherence), and in patients with co-morbidities, including human immunodeficiency virus (HIV) infection or diabetes mellitus (DM) [3, 4]. There are risk factors for the occurrence of mutations in the specific genes of the bacteria that may lead to drug-resistance. The prevalence of MDR-TB in Indonesia also varies geographically. According to the TB resistance survey conducted between 2004 and 2007, the prevalence of MDR-TB in Central Java, Makassar and Papua was 1.9 %, 4.1 % and 2.0 %, respectively [2]. Therefore, it is important to understand both the geographic distribution of bacterial genotypes as well as the susceptibility of individuals within different regions according to the possibility of genetic susceptibility mutations [7].

Although MDR-TB is emerging throughout Indonesia, there is only one study of nearly 900 patients that evaluates drug resistance and M. tb genotypes. This study did not find drug resistance associated with certain genotypes. However, it did find that the Beijing genotype, found more frequently in West Java (Bandung) than in West Timor (Kupang), was associated with treatment failure [8, 9].

Here we describe the initial mapping of 404 M. tb bacterial genotypes using spoligotyping, the sensitivity of M. tb isolates to first line anti-TB drugs, and the association between the genotypes and drug sensitivity, which will complement the previous publication of this study [10].

Methods

A cross-sectional study was conducted in 16 TB referral primary health centers (PHC) that had the ability to perform microscopic sputum examination. The study was conducted in 16 provincial capitals in Indonesia, including five provincial capitals in Sumatra (Bandar Lampung, Palembang, Padang, Medan and Pekan Baru), four in Java (Serang, Jakarta, Bandung and Surabaya) two in Kalimantan (Banjarmasin and Pontianak), two in Sulawesi (Makassar and Menado) and three in Eastern Indonesia (Mataram, Ambon and Sorong) between 2008 and 2010. Ethical clearance was obtained from the Ethics Review Committee of NIHRD of Indonesia. Laboratory procedures were performed according to the algorithm in Fig. 1.

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