Campylobacter jejuni Outbreak Linked to Raw Oysters in Rhode Island, 2021.

Oysters and other shellfish are not a food that is commonly highlighted as high risk for Campylobacter contamination. The Rhode Island Department of Health (RIDOH) conducted a multiagency investigation of a Campylobacter jejuni outbreak that was linked to the consumption of raw oysters; the first such outbreak was detected in Rhode Island. The environmental investigation identified birds as the likely source of contamination of the aquacultured oysters. As a result of this outbreak response, several investigative processes and best practice recommendations are offered. 1) RIDOH will be including exposure to raw shellfish as a question on their case report forms to better identify future oyster-related Campylobacter clusters. 2) It is important that shellfish aquaculture farms be aware of the risks of using floating gear to hold oyster cages and of the importance of using bird abatement to keep birds off floating aquaculture gear to prevent contamination of oysters from bird feces. 3) It should be recognized that fecal coliform water samples collected near an oyster aquaculture farm may not act as an adequate indicator for the presence of Campylobacter. 4) For the first time, Rhode Island has developed guidelines for the closure and reopening of oyster harvest areas due to contamination with Campylobacter. It is hoped that increased awareness and mitigation of the described risk factors will help prevent future similar outbreaks of illness.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

SARS-CoV-2 Variants in Rhode Island; May 2022 Update.

BACKGROUND: Genomic surveillance allows identification of circulating SARS-CoV-2 variants. We provide an update on the evolution of SARS-CoV-2 in Rhode Island (RI). METHODS: All publicly available SARS-CoV-2 RI sequences were retrieved from https://www.gisaid.org. Genomic analyses were conducted to identify variants of concern (VOC), variants being monitored (VBM), or non-VOC/non-VBM, and investigate their evolution. RESULTS: Overall, 17,340 SARS-CoV-2 RI sequences were available between 2/2020-5/2022 across five (globally recognized) major waves, including 1,462 (8%) sequences from 36 non-VOC/non-VBM until 5/2021; 10,565 (61%) sequences from 8 VBM between 5/2021-12/2021, most commonly Delta; and 5,313 (31%) sequences from the VOC Omicron from 12/2021 onwards. Genomic analyses demonstrated 71 Delta and 44 Omicron sub-lineages, with occurrence of variant-defining mutations in other variants. CONCLUSION: Statewide SARS-CoV-2 genomic surveillance allows for continued characterization of circulating variants and monitoring of viral evolution, which inform the local health force and guide public health on mitigation efforts against COVID-19.

A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants.

Next-generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the world; however, it is costly and time-consuming to perform and is not widely available in low-resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)-PCR-pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS-CoV-2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS-CoV-2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%-80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant's respective emergence period, finding that Delta emerged 1.37-2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%-167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1-10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta's enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta's infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta's enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Low Seroprevalence of SARS-CoV-2 in Rhode Island blood donors during may 2020 as determined using multiple serological assay formats.

BACKGROUND: Epidemic projections and public health policies addressing Coronavirus disease (COVID)-19 have been implemented without data reporting on the seroconversion of the population since scalable antibody testing has only recently become available. METHODS: We measured the percentage of severe acute respiratory syndrome- Coronavirus-2 (SARS-CoV-2) seropositive individuals from 2008 blood donors drawn in the state of Rhode Island (RI). We utilized multiple antibody testing platforms, including lateral flow immunoassays (LFAs), enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). To estimate seroprevalence, we utilized the Bayesian statistical method to adjust for sensitivity and specificity of the commercial tests used. RESULTS: We report than an estimated seropositive rate of RI blood donors of approximately 0.6% existed in April-May of 2020. Daily new case rates peaked in RI in late April 2020. We found HTSAs and LFAs were positively correlated with ELISA assays to detect antibodies specific to SARS-CoV-2 in blood donors. CONCLUSIONS: These data imply that seroconversion, and thus infection, is likely not widespread within this population. We conclude that IgG LFAs and HTSAs are suitable to conduct seroprevalence assays in random populations. More studies will be needed using validated serological tests to improve the precision and report the kinetic progression of seroprevalence estimates.

SARS-CoV-2 Variants in Rhode Island.

COVID-19 is a worldwide public health emergency caused by SARS-CoV-2. Genomic surveillance of SARS-CoV-2 emerging variants is important for pandemic monitoring and informing public health responses. Through an interstate academic-public health partnership, we established Rhode Island's capacity to sequence SARS-CoV-2 genomes and created a systematic surveillance program to monitor the prevalence of SARS-CoV-2 variants in the state. We describe circulating SARS-CoV-2 lineages in Rhode Island; provide a timeline for the emerging and expanding contribution of variants of concern (VOC) and variants of interest (VOI), from their first introduction to their eventual predominance over other lineages; and outline the frequent identification of known adaptively beneficial spike protein mutations that appear to have independently arisen in non-VOC/non-VOI lineages. Overall, the described Rhode Island- centric genomic surveillance initiative provides a valuable perspective on SARS-CoV-2 in the state and contributes data of interest for future epidemiological studies and state-to-state comparisons.

Validation and performance comparison of three SARS-CoV-2 antibody assays.

Serology testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly being used during the current pandemic of coronavirus disease 2019 (COVID-19), although its clinical and epidemiologic utilities are still debatable. Characterizing these assays provides scientific basis to best use them. The current study assessed one chemiluminescent assay (Abbott COVID-2 IgG) and two lateral flow assays (STANDARD Q [SQ] IgM/IgG Duo and Wondfo total antibody test) using 113 blood samples from 71 PCR-confirmed COVID-19 hospitalized patients, 119 samples with potential cross-reactions, and 1068 negative controls including 942 pre-pandemic samples. SARS-CoV-2 IgM antibodies became detectable 3-4 days post-symptom onset using SQ IgM test and IgG antibodies were first detected 5-6 days post-onset using SQ IgG. Abbott IgG and Wondfo Total were able to detect antibodies 7 to 8 days post-onset. After 14 days post-symptom onset, the SQ IgG, Abbott IgG and Wondfo Total tests were able to detect antibodies from 100% of the PCR-confirmed patients in this series; 87.5% sensitivity for SQ IgM. Overall agreement was 88.5% between SQ IgM/IgG and Wondfo Total and 94.6% between SQ IgG and Abbott IgG. No cross-reaction due to recent sera with three of the endemic coronaviruses was observed. Viral hepatitis and autoimmune samples were the main source of limited cross-reactions. The specificities were 100% for SQ IgG and Wondfo Total, 99.62% for Abbott IgG, and 98.87% for SQ IgM. These findings demonstrated high sensitivity and specificity of appropriately validated SARS-CoV-2 serologic assays with implications for clinical use and epidemiological seroprevalence studies.

Bordetella holmesii: initial genomic analysis of an emerging opportunist.

Bordetella holmesii is an emerging opportunistic pathogen that causes respiratory disease in healthy individuals and invasive infections among patients lacking splenic function. We used 16S rRNA gene analysis to confirm B. holmesii as the cause of bacteremia in a child with sickle cell disease. Semiconductor-based draft genome sequencing provided insight into B. holmesii phylogeny and potential virulence mechanisms and also identified a toluene-4-monoxygenase locus unique among bordetellae.

Identification of a novel Acinetobacter baumannii clone in a US hospital outbreak by multilocus polymerase chain reaction/electrospray-ionization mass spectrometry.

We investigated the relatedness of multidrug-resistant Acinetobacter baumannii isolates from a burn intensive care unit (BICU) outbreak, control isolates, and isolates from a previous 2007 outbreak by 3 molecular typing methods (repetitive sequence-based polymerase chain reaction [rep-PCR], broad-range PCR and electrospray ionization mass spectrometry [PCR/ESI-MS], and pulsed-field gel electrophoresis [PFGE]). Partial rpoB gene sequencing confirmed all tested isolates as A. baumannii. Molecular typing analysis showed that 17 of 19 outbreak isolates were indistinguishable or closely related to each other. Three of 4 non-BICU outbreak control isolates and 5 of 6 isolates from the previous outbreak closely matched the BICU outbreak genotype. The outbreak strain represented a novel strain type, ST96, on PCR/ESI-MS with a new combination of alleles not previously seen in the United States. The ST96 strain also represented a novel rpoB Seqtype. Results of PCR/ESI-MS and PFGE genotyping were most closely correlated, while rep-PCR and PCR/ESI-MS systems generated rapid results.

Mycobacterium tuberculosis spoligotypes that may derive from mixed strain infections are revealed by a novel computational approach.

Global control of tuberculosis is increasingly dependent on rapid and accurate genetic typing of Mycobacteriumtuberculosis. Spoligotyping is a first-line genotypic fingerprinting method for M.tuberculosis isolates. An international online database (SpolDB4) of spoligotype patterns has been established wherein a clustered pattern (shared by ≥2 isolates) is designated a shared international type (SIT). Dual infections of single patients by distinct strains of M. tuberculosis is increasingly reported in high tuberculosis incidence areas, raising the possibility of false composite spoligotype patterns if performed upon mixed strain samples. A computational approach was applied to SpolDB4 and found that of the reported 1939 SITs, 54% could be a composite of two other SITs. Although many of the spoligotypes listed in SpolDB4 may be the product of admixing, the majority of patterns were reported with a corresponding low case frequency and so the effect of misclassification upon database integrity with these is likely minimal. Phylogenetic analysis of the five SITs most prone to be a composite demonstrated that these patterns designate nodes from which the ramifications of large families T, MANU, LAM, and EAI emerged. We illustrate how geographic context may indicate when an observed pattern could be the product of mixed infection. Importantly, when one of the most composite-prone SITs is obtained, further genetic testing by alternate methods is prudent to rule-out mixed infection, especially in high tuberculosis prevalence areas. These findings have broad practical implications for tuberculosis control and surveillance, as well as highlight the utility of a computational approach in providing solutions to biological questions in which the information can be digitalized.

Distinct clinical and epidemiological features of tuberculosis in New York City caused by the RD(Rio) Mycobacterium tuberculosis sublineage.

BACKGROUND: Genetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RD(Rio) M. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001-2005) to: (1) affirm the position of RD(Rio) strains within the M. tuberculosis phylogenetic structure, (2) determine its prevalence, and (3) define transmission, demographic, and clinical characteristics associated with RD(Rio) TB. METHODS: Isolates classified as RD(Rio) or non-RD(Rio) M. tuberculosis by multiplex PCR were further classified as clustered (≥2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RD(Rio) was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics. RESULTS: RD(Rio) strains were found to: (1) be restricted to the Latin American-Mediterranean family, (2) cause approximately 8% of TB cases in NYC, and (3) be associated with heightened transmission as shown by: (i) a higher cluster proportion compared to other prevalent lineages, (ii) a higher secondary case rate, and (iii) cases in children. Furthermore, RD(Rio) strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance. CONCLUSIONS: The RD(Rio) genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RD(Rio) case and exposure identification could be of benefit due to its association with heightened transmission.

Trends in methicillin-resistant Staphylococcus aureus anovaginal colonization in pregnant women in 2005 versus 2009.

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.

Comparison of the automated Phoenix with the Vitek 2 for the identification of Streptococcus pneumoniae.

Rapid and accurate identification of Streptococcus pneumoniae is a critical component in the optimal management of infected patients. The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was evaluated for identification of S. pneumoniae (n = 311) and was compared to the Vitek 2 (bioMérieux, Marcy l'Etoile, France). Strains with discordant identification between methods were resolved with 16S rRNA gene sequencing as the gold standard. The Phoenix and the Vitek 2 correctly identified 96.8% (n = 301) and 95.2% (n = 296) of S. pneumoniae strains, respectively. Overall, there was no statistically significant difference in the performance of the 2 automated systems for the identification of S. pneumoniae in this study. The Vitek 2 mean time-to-results for all streptococcal identification was 1.5 h faster than that for the Phoenix. We conclude that the automated Phoenix and the Vitek 2 systems are comparable in their ability to identify S. pneumoniae and are preferable to the use of routine biochemical assays, which have delayed time-to-results and are not dependably accurate.

Distinct genotypic profiles of the two major clades of Mycobacterium africanum.

BACKGROUND: Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis (TB) and a member of the M. tuberculosis complex (MTC). Additional MTC species that cause TB in humans and other mammals include Mycobacterium africanum and Mycobacterium bovis. One result of studies interrogating recently identified MTC phylogenetic markers has been the recognition of at least two distinct lineages of M. africanum, known as West African-1 and West African-2. METHODS: We screened a blinded non-random set of MTC strains isolated from TB patients in Ghana (n = 47) for known chromosomal region-of-difference (RD) loci and single nucleotide polymorphisms (SNPs). A MTC PCR-typing panel, single-target standard PCR, multi-primer PCR, PCR-restriction fragment analysis, and sequence analysis of amplified products were among the methods utilized for the comparative evaluation of targets and identification systems. The MTC distributions of novel SNPs were characterized in the both the Ghana collection and two other diverse collections of MTC strains (n = 175 in total). RESULTS: The utility of various polymorphisms as species-, lineage-, and sublineage-defining phylogenetic markers for M. africanum was determined. Novel SNPs were also identified and found to be specific to either M. africanum West African-1 (Rv1332(523); n = 32) or M. africanum West African-2 (nat(751); n = 27). In the final analysis, a strain identification approach that combined multi-primer PCR targeting of the RD loci RD9, RD10, and RD702 was the most simple, straight-forward, and definitive means of distinguishing the two clades of M. africanum from one another and from other MTC species. CONCLUSION: With this study, we have organized a series of consistent phylogenetically-relevant markers for each of the distinct MTC lineages that share the M. africanum designation. A differential distribution of each M. africanum clade in Western Africa is described.