Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014-2018 national laboratory surveillance.

OBJECTIVES: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. METHODS: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). RESULTS: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. CONCLUSIONS: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.

Monitoring the spread of meticillin-resistant Staphylococcus aureus in The Netherlands from a reference laboratory perspective.

BACKGROUND: In The Netherlands, efforts to control meticillin-resistant Staphylococcus aureus (MRSA) in hospitals have been largely successful due to stringent screening of patients on admission and isolation of those that fall into defined risk categories. However, Dutch hospitals are not free of MRSA, and a considerable number of cases are found that do not belong to any of the risk categories. Some of these may be due to undetected nosocomial transmission, whereas others may be introduced from unknown reservoirs. AIM: Identifying multi-institutional clusters of MRSA isolates to estimate the contribution of potential unobserved reservoirs in The Netherlands. METHODS: We applied a clustering algorithm that combines time, place, and genetics to routine data available for all MRSA isolates submitted to the Dutch Staphylococcal Reference Laboratory between 2008 and 2011 in order to map the geo-temporal distribution of MRSA clonal lineages in The Netherlands. FINDINGS: Of the 2966 isolates lacking obvious risk factors, 579 were part of geo-temporal clusters, whereas 2387 were classified as MRSA of unknown origin (MUOs). We also observed marked differences in the proportion of isolates that belonged to geo-temporal clusters between specific multi-locus variable number of tandem repeat analysis (MLVA) clonal complexes, indicating lineage-specific transmissibility. The majority of clustered isolates (74%) were present in multi-institutional clusters. CONCLUSION: The frequency of MRSA of unknown origin among patients lacking obvious risk factors is an indication of a largely undefined extra-institutional but genetically highly diverse reservoir. Efforts to understand the emergence and spread of high-risk clones require the pooling of routine epidemiological information and typing data into central databases.

The population structure of Staphylococcus aureus in China and Europe assessed by multiple-locus variable number tandem repeat analysis; clues to geographical origins of emergence and dissemination.

To compare the genetic population structure of Staphylococcus aureus from China and Europe, 1294 human isolates were characterized by multiple-locus variable number tandem repeat analysis (MLVA). In total, MLVA identified 17 MLVA complexes (MCs), comprising 260 MLVA types (MTs) among the Chinese isolates and 372 MTs among the European isolates. The five most frequent MCs among the Chinese isolates belonged to MC398, MC5 subclade a, MC8, MC437 and MC7 and made up 55% of the sample. For the European isolates, the five most frequent MCs consisted of MC5 subclade a, MC45, MC8, MC30 and MC22, which accounted for 64% of the sample. Phylogeographic analysis of the major MCs shared between China and Europe points to a European origin of MC8 but cannot provide a consistent signal for MC5 subclade a, probably indicating a different origin. Diversity and frequency distributions of other lineages were also compared. Altogether, this study provides the first snapshot of two extant populations of S. aureus from Europe and China, and important clues on the emergence and dissemination of different lineages of S. aureus.

Bacteraemic and non-bacteraemic/urinary antigen-positive pneumococcal community-acquired pneumonia compared.

The diagnosis of invasive pneumococcal pneumonia is based mainly on bacteraemia. Episodes without bacteraemia, but with a positive urinary antigen test (UAT), are considered non-invasive. We determined differences in outcome between patients with bacteraemic and non-bacteraemic/UAT-positive pneumococcal community-acquired pneumonia (CAP). Adult patients with clinical and radiological evidence of CAP with blood cultures and UAT tests performed at presentation in three Dutch laboratories between June 2008 and May 2010 were included. Clinical characteristics were retrospectively extracted from hospital records. Overall, 168 patients had non-bacteraemic/UAT-positive pneumococcal CAP and 123 had bacteraemic pneumococcal CAP. The day-30 mortality was 9% and 13% for non-bacteraemic/UAT-positive and bacteraemic pneumococcal CAP patients, respectively [risk difference -4%, 95% confidence interval (CI) -11% to +3%, p = 0.28]. In a multivariable logistic regression model, age ≥ 65 years, admission to the intensive care unit/coronary care unit (ICU/CCU) and presence of an immunocompromising condition were associated with day-30 mortality. A non-significant association with mortality was found for bacteraemia [odds ratio (OR) 2.21, 95% CI 0.94-5.21, p = 0.07). No such trend was found for UAT positivity. The median lengths of hospital stay were 8 [interquartile range (IQR) 5-14] and 10 (IQR 6-18) days for non-bacteraemic/UAT-positive and bacteraemic pneumococcal CAP patients, respectively (p = 0.05). As compared to non-bacteraemic/UAT-positive pneumococcal CAP, bacteraemic pneumococcal CAP has a stronger association with day-30 mortality.

Transmission and persistence of livestock-associated methicillin-resistant Staphylococcus aureus among veterinarians and their household members.

After the first isolation of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in 2003, this MRSA variant quickly became the predominant MRSA obtained from humans as part of the Dutch national MRSA surveillance. Previous studies have suggested that human-to-human transmission of LA-MRSA, compared to that of other MRSA lineages, rarely occurs. However, these reports describe the transmission of LA-MRSA based on epidemiology and limited molecular characterization of isolates, making it difficult to assess whether transmission actually occurred. In this study, we used whole-genome maps (WGMs) to identify possible transmission of LA-MRSA between humans. For this, we used LA-MRSA isolates originating from a 2-year prospective longitudinal cohort study in which livestock veterinarians and their household members were repeatedly sampled for the presence of S. aureus. A considerable degree of genotypic variation among LA-MRSA strains was observed. However, there was very limited variability between the maps of the isolates originating from the same veterinarian, indicating that each of the veterinarians persistently carried or had reacquired the same LA-MRSA strain. Comparison of WGMs revealed that LA-MRSA transmission had likely occurred within virtually every veterinarian household. Yet only a single LA-MRSA strain per household appeared to be involved in transmission. The results corroborate our previous finding that LA-MRSA is genetically diverse. Furthermore, this study shows that transmission of LA-MRSA between humans occurs and that carriage of LA-MRSA can be persistent, thus posing a potential risk for spread of this highly resistant pathogen in the community.

The dynamic changes of dominant clones of Staphylococcus aureus causing bloodstream infections in the European region: results of a second structured survey.

Staphylococcus aureus is one of the most important human pathogens and meticillin-resistant S. aureus (MRSA) presents a major cause of healthcare- and community-acquired infections. This study investigated the spatial and temporal changes of S. aureus causing bacteraemia in Europe over a five-year interval and explored the possibility of integrating pathogen-based typing data with epidemiological and clinical information at a European level. Between January 2011 and July 2011, 350 laboratories serving 453 hospitals in 25 countries collected 3,753 isolates (meticillin-sensitive S. aureus (MSSA) and MRSA) from patients with S. aureus bloodstream infections. All isolates were sent to the national staphylococcal reference laboratories and characterised by quality-controlled spa typing. Data were uploaded to an interactive web-based mapping tool. A wide geographical distribution of spa types was found, with some prevalent in all European countries. MSSA was more diverse than MRSA. MRSA differed considerably between countries with major international clones expanding or receding when compared to a 2006 survey. We provide evidence that a network approach of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of S. aureus populations such as early signalling of emerging strains, cross-border spread and importation by travel.

Dynamics of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus carriage in pig farmers: a prospective cohort study.

Our purpose was to determine the dynamics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage and its determinants in persons working at pig farms, in order to identify targets for interventions. This prospective cohort study surveyed 49 pig farms in the Netherlands on six sampling dates in 1 year (2010-11). Nasal and oropharyngeal swabs were collected, as well as environmental surface samples from stables and house. Of 110 pig farmers, 38% were persistent MRSA nasal carriers. The average cross-sectional MRSA prevalence was 63%. Methicillin-susceptible S. aureus (MSSA) nasal carriage was associated with fewer MRSA acquisitions (prevalence rate (PR) = 0.47, p 0.02). In multivariate analysis, an age of 40-49 years (PR = 2.13, p 0.01), a working week of ≥40 h (PR=1.89, p 0.01), giving birth assistance to sows (PR=2.26, p 0.03), removing manure of finisher pigs (PR=0.48, p 0.02), and wearing a facemask (PR = 0.13, p 0.02) were significantly related with persistent MRSA nasal carriage. A higher MRSA exposure in stables was associated with MRSA in pig farmers (p <0.0001). This study describes a very high prevalence of LA-MRSA carriage in pig farmers, reflecting extensive exposure during work. We identified the possible protective effects of MSSA carriage and of continuously wearing a facemask during work.

Increased incidence of serotype-1 invasive pneumococcal disease in young female adults in The Netherlands.

Analysis of the Dutch national invasive pneumococcal disease (IPD) surveillance data by sex reveals an increase in the incidence of serotype-1 disease in young female adults in The Netherlands after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the national immunization schedule. This has led to an overall increase in IPD in women aged 20-45 years, which was not observed in men of the same age. No other differences in serotype shifts possibly induced by the introduction of PCV7 were observed between the sexes in this age group. Serotype 1 is a naturally fluctuating serotype in Europe and it has been associated with disease in young healthy adults before. It remains uncertain whether or not there is an association between the observed increase in serotype-1 disease in young female adults and the implementation of PCV7 in The Netherlands.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Seroprevalence of IgG antibodies against 13 vaccine Streptococcus pneumoniae serotypes in the Netherlands.

In this study the seroprevalence of IgG antibodies against 13 vaccine serotypes of the pneumococcus was assessed in the Netherlands. Sera from 7904 persons obtained in a cross-sectional population-based study were analysed. The 13 serotype specific IgG concentrations were assessed simultaneously using a fluorescent bead-based multiplex immuno assay (MIA). Overall, the geometric mean IgG concentrations (GMCs) against the 13 serotypes in unvaccinated individuals increased with age up to 5 years and remained at a plateau thereafter. The data also show that individuals develop antibodies against an increasing number of different serotypes with increasing age. The highest GMCs were found for antibodies directed against serotype 14 and 19F, whereas antibodies against serotypes 4 and 5 had the lowest GMCs. There was no uniform relationship between the occurrence of serotypes causing invasive pneumococcal disease (IPD) and the GMCs against these serotypes. Increased IPD incidence in the elderly did not seem to be the result of a decline in the concentration of IgG antibodies.

Validation of PCR-reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples.

A dermatophyte-specific PCR-reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.

Pre-admission clinical course of meningococcal disease and opportunities for the earlier start of appropriate intervention: a prospective epidemiological study on 752 patients in the Netherlands, 2003-2005.

To improve the timeliness of health care delivery to patients with meningococcal disease, the early disease evolution and clinical manifestation at admission were studied in all 752 patients with invasive meningococcal disease in the Netherlands in 2003-2005. Eighty-eight percent (88%) had serogroup B disease. The case fatality rate (CFR) was 6.7% overall, but reached 16% among adults over 50 years of age. The CFR was similar for serogroups B (6.3%) and C (5.2%). Admission followed 17 h (median) after the onset of symptoms. The CFR in patients admitted within 12 h, 12-18 h, 18-36 h or >36 h after the first symptoms was 10.2, 7.8, 3.5 and 2.2%, respectively. Only 60% of patients had skin lesions, and admission followed 2 h (median) later. Earlier recognition can be achieved when non-petechial clues are included in the diagnosis. A short duration of disease before admission is a simple tool in the recognition of patients with severe disease.

[Two pneumococcal vaccines: the 7-valent conjugate vaccine (Prevenar) for children up to the age of 5 years and the 23-valent polysaccharide vaccine (Pneumo 23) for the elderly and specific groups at risk]. / Twee pneumokokkenvaccins: voor kinderen tot 5 jaar het 7-valente conjugaatvaccin (Prevenar) en voor ouderen en specifieke risicogroepen het 23-valente polysacharidevaccin (Pneumo 23).

Despite the availability of antibiotics and the care offered by intensive-care units, invasive pneumococcal disease still causes serious illness with high morbidity and mortality. Currently, two vaccines are available in the Netherlands that offer protection against invasive pneumococcal disease: a 7-valent conjugated vaccine and a 23-valent polysaccharide vaccine. Case reports and pharmaceutical use as registered by the Foundation for Pharmaceutical Statistics (SFK) show that general practitioners and pharmacists are not always aware of the appropriate use of both vaccines. For vaccination against pneumococci in children aged 0-5 years, general practitioners should prescribe only the conjugated vaccine. The non-conjugated polysaccharide vaccine is intended especially for the elderly and specific groups at risk. Pharmacists should verify that the pneumococcal vaccines they hand out on prescription are recommended for the age-group of the individuals to whom the vaccine will be administered.

Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease.

BACKGROUND: The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. METHODS: In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. RESULTS: Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. CONCLUSIONS: Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/or host factors.

Analysis of Bordetella pertussis populations in European countries with different vaccination policies.

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.

Long-term surveillance of invasive group A streptococcal disease in The Netherlands, 1994-2003.

A nationwide laboratory-based surveillance study of invasive group A streptococcal (GAS) infections was conducted in The Netherlands from May 1994 until December 2003 (average population during this period was 15 729 704). Microbiologically invasive isolates were obtained from 1504 patients, with most (70%) isolates cultured from blood. There was a clear seasonal pattern in invasive streptococcal infections, with an estimated annual incidence that peaked in 1996 (4.0 cases/100 000 individuals/year) and was at its lowest in 1999 (2.0 cases/100 000 individuals/year). Twenty-eight different M-types were identified, of which the most frequent were M1 (339/1504, 23%), M3 (187/1504, 12%), M89 (174/1504, 12%), M28 (164/1504, 11%), M12 (109/1504, 7%) and M6 (55/1504, 4%). There was a high degree of variation in the relative annual contributions of the predominant M-types, but variations in M1 and M3 combined correlated with overall changes in the annual incidence. The contribution of the patient group aged > or = 56 years to all cases of invasive GAS disease increased during the study period, whereas that of the group aged 0-20 years decreased. A peak in the incidence of invasive GAS disease among the patient group aged 30-34 years did not vary during the study period, indicating that the high incidence of invasive GAS disease in this age group was age-specific rather than cohort-related.

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.

[Doubling of the number of cases of tick bites and lyme borreliosis seen by general practitioners in the Netherlands]. / Verdubbeling van het aantal consulten voor tekenbeten en Lyme-borreliose in de huisartsenpraktijk in Nederland.

OBJECTIVE: To obtain detailed information about the current geographical distribution and incidence of tick bites and Lyme borreliosis in the Netherlands and to identify regional differences in the ecological risk factors that might be involved. DESIGN: Retrospective questionnaire study. SETTING: All Dutch general practitioner's (GP) practices. METHODS: In April 2002, all GPs in the Netherlands were asked to complete a short questionnaire on the number of cases of tick bites and erythema migrans seen in 2001 and the size of their practice. Associations with possible risk factors were determined at the municipal level. Results were compared with the results of a similar study done in 1995. RESULTS: The response of the GPs was 64.5% (4730/7330). Together, all GPs reported seeing approximately 61,000 patients in 2001 with tick bites and 12,000 patients with erythema migrans. The incidence of erythema migrans was estimated at 73 per 100,000 inhabitants. There were obvious risk areas. At the municipal level, tick bites and erythema migrans were positively associated with the area covered by forest, sandy soil, the number of roe deer and tourism. There was a negative association with the degree of urbanisation. Increases in tourism in areas with many ticks, new forests in urban regions and an increased number of horses were positively associated with the increase in tick bites and erythema migrans since 1994. CONCLUSION: The number of patients with tick bites and erythema migrans seen by GPs in the Netherlands had doubled between 1994 and 2001. This increase may be attributed partly to changes in ecological risk factors and human behaviour. The number of cases of Lyme borreliosis may be reduced by giving prophylactic information annually about ticks and ways to remove them, plus additional education of patients about the recognition of erythema migrans.

Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis.

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the rpoB gene using reverse line blot hybridization and DNA sequencing. Analysis of the rpoB gene revealed that samples containing rifampin-resistant Mycobacterium tuberculosis carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the rpoB gene was more accurate in identifying mutations in the core region of the rpoB gene. Sequence analysis of the rpoB gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.