# Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017

Voahangy Andrianaivoarimanana, Cyril Savin, Dawn N Birdsell, Amy J Vogler, Anne-Sophie Le Guern, Soloandry Rahajandraibe, Sylvie Brémont, Soanandrasana Rahelinirina, Jason W Sahl, Beza Ramasindrazana, et al.

► To cite this version:

Voahangy Andrianaivoarimanana, Cyril Savin, Dawn N Birdsell, Amy J Vogler, Anne-Sophie Le Guern, et al.. Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017. Emerging Infectious Diseases, 2024, 30 (2), 10.3201/eid3002.230759. pasteur-04418614

HAL Id: pasteur-04418614

https://pasteur.hal.science/pasteur-04418614

Submitted on 26 Jan 2024

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# Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017

Voahangy Andrianaivoarimanana, Cyril Savin, Dawn N Birdsell, Amy J Vogler, Anne-Sophie Le Guern, Soloandry Rahajandraibe, Sylvie Brémont, Soanandrasana Rahelinirina, Jason W Sahl, Beza Ramasindrazana, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Voahangy Andrianaivoarimanana, Cyril Savin, Dawn N Birdsell, Amy J Vogler, Anne-Sophie Le Guern, et al.. Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017. Emerging Infectious Diseases, 2024, 30 (2), 10.3201/eid3002.230759. pasteur-04418614

HAL Id: pasteur-04418614

https://pasteur.hal.science/pasteur-04418614v1

Submitted on 26 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
# Multiple Introductions of *Yersinia pestis* during Urban Pneumonic Plague Epidemic, Madagascar, 2017

Voahangy Andrianayoarimanana,<fnref n="1" /> Cyril Savin,<fnref n="1" /> Dawn N. Birdsell,<fnref n="1" /> Amy J. Vogler, Anne-Sophie Le Guern, Soloandry Rahajandraibe, Sylvie Brémont, Soanandrasana Rahelinirina, Jason W. Sahl, Beza Ramasindrazana, Rado Jean Luc Rakotonanahary, Fanjasoa Rakotomanana, Rindra Randremanana, Viviane Maheriniaina, Vaoary Razafimbia, Aurelia Kwasiborski, Charlotte Balière, Maherisoa Ratsitorahina, Laurence Baril, Paul Keim, Valérie Caro, Voahangy Rasolofo, André Spiegel, Javier Pizarro-Cerda,<fnref n="2" /> David M. Wagner,<fnref n="2" /> Minoarisoa Rajerison<fnref n="2" />

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and *Yersinia pestis* genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci ≥20 times during August–November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other *Y. pestis* lineages may have caused persistent PP transmission chains in Antananarivo. Multiple *Y. pestis* lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Madagascar reports more human plague (causative agent: *Yersinia pestis*) cases annually than any other country (1), often several hundred each season (typically September–March) (2). *Y. pestis* persists in multiple rural foci in the central and northern highlands of Madagascar (regions >800 m elevation),

wherein it cycles primarily among nonnative rat hosts via nonnative and native flea vectors (3,4). Occurrence and seasonality of human plague is closely tied to rice cultivation in rural areas (3,4), which increases contact between humans and rats carrying *Y. pestis*-infected fleas. Most human cases in Madagascar are bubonic plague (BP), which originates from a flea bite (2). Fleaborne transmission of *Y. pestis* between humans has not been documented in Madagascar, so all BP cases are considered independently acquired from the environment. Pneumonic plague (PP) is not obtained from the environment but results from untreated BP that progresses to secondary PP (SPP), which subsequently can be passed human-to-human as primary PP. Increases in Madagascar in the proportion of BP cases progressing to PP is attributed to the deteriorating healthcare system (2). Human-to-human transmission of PP occurs in Madagascar (4–8) but is less common. Human plague in urban areas of Madagascar is rare because the rodents and fleas in those areas seldom carry *Y. pestis*.

*Y. pestis* was introduced to Madagascar in 1898, during the third plague pandemic (9). Phylogenographic analyses of *Y. pestis* have identified multiple distinct subgroups that occur and persist in specific

Author affiliations: Institut Pasteur de Madagascar, Antananarivo, Madagascar (V. Andrianayoarimanana, S. Rahelinirina, B. Ramasindrazana, R.J.L. Rakotonanahary, F. Rakotomanana, R. Randremanana, M. Ratsitorahina, L. Baril, V. Rasolofo, A. Spiegel, M. Rajerison); Institut Pasteur, Paris, France (C. Savin, A.-S. Le Guern, S. Brémont, A. Kwasiborski, C. Balière, V. Caro, J. Pizarro-Cerda); Northern Arizona University, Flagstaff, Arizona,

USA (D.N. Birdsell, A.J. Vogler, J.W. Sahl, P. Keim, D.M. Wagner); Madagascar Ministry of Public Health, Antananarivo (S. Rahajandraibe, V. Maheriniaina, V. Razafimbia)

DOI: http://doi.org/10.3201/eid3002.230759

<footnote n="1">¹These authors contributed equally to this article.</footnote>

<footnote n="2">²These senior authors contributed equally to this article.</footnote>
# Multiple Introductions of *Yersinia pestis* during Urban Pneumonic Plague Epidemic, Madagascar, 2017

Voahangy Andrianavoarimanana,<fnref n="1" /> Cyril Savin,<fnref n="1" /> Dawn N. Birdsell,<fnref n="1" /> Amy J. Vogler, Anne-Sophie Le Guern, Soloandry Rahajandraibe, Sylvie Brémont, Soanandrasana Rahelinirina, Jason W. Sahl, Beza Ramasindrazana, Rado Jean Luc Rakotonanahary, Fanjasoa Rakotomanana, Rindra Randremanana, Viviane Maheriniaina, Vaoary Razafimbia, Aurelia Kwasiborski, Charlotte Balière, Maherisoa Ratsitorahina, Laurence Baril, Paul Keim, Valérie Caro, Voahangy Rasolofo, André Spiegel, Javier Pizarro-Cerda,<fnref n="2" /> David M. Wagner,<fnref n="2" /> Minoarisoa Rajerison<fnref n="2" />

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and *Yersinia pestis* genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci ≥20 times during August–November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other *Y. pestis* lineages may have caused persistent PP transmission chains in Antananarivo. Multiple *Y. pestis* lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Madagascar reports more human plague (causative agent: *Yersinia pestis*) cases annually than any other country (1), often several hundred each season (typically September–March) (2). *Y. pestis* persists in multiple rural foci in the central and northern highlands of Madagascar (regions >800 m elevation),

wherein it cycles primarily among nonnative rat hosts via nonnative and native flea vectors (3,4). Occurrence and seasonality of human plague is closely tied to rice cultivation in rural areas (3,4), which increases contact between humans and rats carrying *Y. pestis*-infected fleas. Most human cases in Madagascar are bubonic plague (BP), which originates from a flea bite (2). Fleaborne transmission of *Y. pestis* between humans has not been documented in Madagascar, so all BP cases are considered independently acquired from the environment. Pneumonic plague (PP) is not obtained from the environment but results from untreated BP that progresses to secondary PP (SPP), which subsequently can be passed human-to-human as primary PP. Increases in Madagascar in the proportion of BP cases progressing to PP is attributed to the deteriorating healthcare system (2). Human-to-human transmission of PP occurs in Madagascar (4–8) but is less common. Human plague in urban areas of Madagascar is rare because the rodents and fleas in those areas seldom carry *Y. pestis*.

*Y. pestis* was introduced to Madagascar in 1898, during the third plague pandemic (9). Phylogenographic analyses of *Y. pestis* have identified multiple distinct subgroups that occur and persist in specific

Author affiliations: Institut Pasteur de Madagascar, Antananarivo, Madagascar (V. Andrianavoarimanana, S. Rahelinirina, B. Ramasindrazana, R.J.L. Rakotonanahary, F. Rakotomanana, R. Randremanana, M. Ratsitorahina, L. Baril, V. Rasolofo, A. Spiegel, M. Rajerison); Institut Pasteur, Paris, France (C. Savin, A.-S. Le Guern, S. Brémont, A. Kwasiborski, C. Balière, V. Caro, J. Pizarro-Cerda); Northern Arizona University, Flagstaff, Arizona,

USA (D.N. Birdsell, A.J. Vogler, J.W. Sahl, P. Keim, D.M. Wagner); Madagascar Ministry of Public Health, Antananarivo (S. Rahajandraibe, V. Maheriniaina, V. Razafimbia)

DOI: http://doi.org/10.3201/eid3002.230759

<footnote n="1">¹These authors contributed equally to this article.</footnote>

<footnote n="2">²These senior authors contributed equally to this article.</footnote>
geographic locations in Madagascar; subgroups are occasionally dispersed between rural foci but rarely become established (10-12). Given this high fidelity between specific *Y. pestis* molecular subgroups and particular geographic locations in Madagascar, assigning isolates to known molecular subgroups can identify *Y. pestis* dispersal events and likely geographic areas where BP cases were acquired from the environment (10).

The 2017–18 human plague season in Madagascar was characterized by a typical number of suspected BP and PP human cases reported from rural endemic foci but an atypically large number of suspected cases, primarily PP, reported from Antananarivo, the capital city, and Toamasina, the main seaport, which are the largest urban areas in Madagascar. We noted a dramatic increase in notified PP cases starting in September 2017, continuing until this urban PP epidemic was officially declared over on November 27, 2017 (13). The true number of infections associated with this event remains unknown, as well as whether this urban PP epidemic was caused by an extended chain of transmission of a single clone of *Y. pestis* or by multiple independent introduction events from endemic rural foci (14). We prepared detailed case histories for the first documented transmission chain (29 cases) from the PP epidemic and used molecular characterization of *Y. pestis* isolates and human sputum samples obtained from urban areas and rural endemic foci in 2017 to determine the sources of this urban PP epidemic.

## Methods

### Definitions and Investigation

In August–December 2017, plague cases in Madagascar were notified to the plague national surveillance system, which is mandatory; no ethics approval is required to use those public health data. We classified cases as suspect, probable, or confirmed as previously defined (13) and as urban if they occurred within cities with population ≥150,000. We considered disease onset as the first day plague symptoms occurred. We conducted an epidemiologic description for the 29 initial human cases. We have anonymized information for all cases.

### Sample Analysis

*Y. pestis* was isolated from human samples and tested for susceptibility to multiple antimicrobial drugs as previously described (Appendix 1 sections 1, 2, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App1.pdf) (13); we generated whole-genome

sequences (WGSs) for isolates (Appendix 1 section 3, Table 1). We conducted 2 rounds of targeted capture and enrichment of *Y. pestis* DNA from DNA extracted from sputum samples positive for *Y. pestis* via PCR, and then sequenced the enriched samples (Appendix 1 sections 5,6, Table 2). We inferred a maximum-likelihood phylogeny using single nucleotide polymorphisms (SNPs) identified from 36 WGSs from 2017 isolates and 54 other isolates representative of the overall phylogenetic diversity of *Y. pestis* in Madagascar (10) (Appendix 1 section 4, Table 1; Appendix 2, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App2.xlsx). SNPs identified in the phylogeny as specific to 2017 isolates, or to clades containing 2017 isolates, were queried in sequence data from enriched sputum samples (Appendix 1, section 7, Table 2).

### Emergence Events

We determined that multiple 2017 human isolates resulted from the same emergence of *Y. pestis* from environmental reservoirs into humans if they differed by ≤2 SNPs in the phylogeny. We defined an emergence as independent from other emergences if there was no epidemiologic association and if the isolates from that emergence were more closely related to older isolates than to other 2017 emergences or differed from other 2017 emergences by ≥5 SNPs.

## Results

### Cases, Isolates, and Samples

A total of 2,549 suspected plague cases were notified throughout Madagascar during August–December 2017, including 1,347 from urban areas and 1,241 classified as PP. Confirmed or probable PP cases were reported from Antananarivo during August 28–November 20 and from Toamasina during September 12–October 27, 2017. We obtained and sequenced *Y. pestis* isolates from 36 cases from 2017 (Appendix 1 sections 1, 3, Table 1). We identified SNPs specific to 2017 isolates or to clades containing 2017 isolates in 7 enriched sputum samples (Appendix 1 section 7, Table 5).

### Multiple Emergences of Human Plague in Rural Endemic Foci

Starting in Miarinarivo District in August, human plague emerged independently from environmental reservoirs ≥20 times in multiple rural endemic foci in Madagascar during August–November 2017 (Table 1; Appendix 1 section 8). Those events occurred in 19 different communes located in 12 different districts in the central and northern highlands (Table 1; Figure 1). Clinical *Y. pestis* isolates obtained from them
geographic locations in Madagascar; subgroups are occasionally dispersed between rural foci but rarely become established (10-12). Given this high fidelity between specific *Y. pestis* molecular subgroups and particular geographic locations in Madagascar, assigning isolates to known molecular subgroups can identify *Y. pestis* dispersal events and likely geographic areas where BP cases were acquired from the environment (10).

The 2017–18 human plague season in Madagascar was characterized by a typical number of suspected BP and PP human cases reported from rural endemic foci but an atypically large number of suspected cases, primarily PP, reported from Antananarivo, the capital city, and Toamasina, the main seaport, which are the largest urban areas in Madagascar. We noted a dramatic increase in notified PP cases starting in September 2017, continuing until this urban PP epidemic was officially declared over on November 27, 2017 (13). The true number of infections associated with this event remains unknown, as well as whether this urban PP epidemic was caused by an extended chain of transmission of a single clone of *Y. pestis* or by multiple independent introduction events from endemic rural foci (14). We prepared detailed case histories for the first documented transmission chain (29 cases) from the PP epidemic and used molecular characterization of *Y. pestis* isolates and human sputum samples obtained from urban areas and rural endemic foci in 2017 to determine the sources of this urban PP epidemic.

## Methods

### Definitions and Investigation

In August–December 2017, plague cases in Madagascar were notified to the plague national surveillance system, which is mandatory; no ethics approval is required to use those public health data. We classified cases as suspect, probable, or confirmed as previously defined (13) and as urban if they occurred within cities with population ≥150,000. We considered disease onset as the first day plague symptoms occurred. We conducted an epidemiologic description for the 29 initial human cases. We have anonymized information for all cases.

### Sample Analysis

*Y. pestis* was isolated from human samples and tested for susceptibility to multiple antimicrobial drugs as previously described (Appendix 1 sections 1, 2, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App1.pdf) (13); we generated whole-genome

sequences (WGSs) for isolates (Appendix 1 section 3, Table 1). We conducted 2 rounds of targeted capture and enrichment of *Y. pestis* DNA from DNA extracted from sputum samples positive for *Y. pestis* via PCR, and then sequenced the enriched samples (Appendix 1 sections 5,6, Table 2). We inferred a maximum-likelihood phylogeny using single nucleotide polymorphisms (SNPs) identified from 36 WGSs from 2017 isolates and 54 other isolates representative of the overall phylogenetic diversity of *Y. pestis* in Madagascar (10) (Appendix 1 section 4, Table 1; Appendix 2, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App2.xlsx). SNPs identified in the phylogeny as specific to 2017 isolates, or to clades containing 2017 isolates, were queried in sequence data from enriched sputum samples (Appendix 1, section 7, Table 2).

### Emergence Events

We determined that multiple 2017 human isolates resulted from the same emergence of *Y. pestis* from environmental reservoirs into humans if they differed by ≤2 SNPs in the phylogeny. We defined an emergence as independent from other emergences if there was no epidemiologic association and if the isolates from that emergence were more closely related to older isolates than to other 2017 emergences or differed from other 2017 emergences by ≥5 SNPs.

## Results

### Cases, Isolates, and Samples

A total of 2,549 suspected plague cases were notified throughout Madagascar during August–December 2017, including 1,347 from urban areas and 1,241 classified as PP. Confirmed or probable PP cases were reported from Antananarivo during August 28–November 20 and from Toamasina during September 12–October 27, 2017. We obtained and sequenced *Y. pestis* isolates from 36 cases from 2017 (Appendix 1 sections 1, 3, Table 1). We identified SNPs specific to 2017 isolates or to clades containing 2017 isolates in 7 enriched sputum samples (Appendix 1 section 7, Table 5).

### Multiple Emergences of Human Plague in Rural Endemic Foci

Starting in Miarinarivo District in August, human plague emerged independently from environmental reservoirs ≥20 times in multiple rural endemic foci in Madagascar during August–November 2017 (Table 1; Appendix 1 section 8). Those events occurred in 19 different communes located in 12 different districts in the central and northern highlands (Table 1; Figure 1). Clinical *Y. pestis* isolates obtained from them
were closely related to previous isolates from those
locations (Figure 2), confirming *Y. pestis* continues to
persist in the environment in these regions (10). PP
arose from BP in at least 6 of these events; confirmed
human cases originating from 5 events, including 4
PP cases, were documented in urban areas during the
epidemic (Table 1).

Initial 29 Cases Associated with the First Urban
PP Transmission Chain

Case-patient 1 exhibited PP symptoms (fever, gas-
trointestinal and respiratory distress, but no cough)

on August 25, 2017, in Ankazobe District (Tables 1,
2; Figures 1, 3) where he had been living and work-
ing and elected to travel to his permanent home in
Toamasina. His employer drove him by car to An-
tananarivo, where he boarded a shared bush taxi to
Toamasina on August 27; case-patient 2 sat beside
him; case-patient 3 immediately behind; and case-
patient 5, wife of case-patient 3, beside her spouse.
That same day near Moramanga, case-patient 1 expe-
rienced a deteriorated health status, including respi-
ratory distress, and died; case-patients 2 and 3 cared
for him while he was dying. The corpse was removed

**Figure 1.** Plague emergence in Madagascar, August–November 2017. A) Locations of emergences. Gray shading indicates plague-endemic regions in the central and northern highlands; yellow shading indicates 12 districts from which human plague emerged from environmental reservoirs 20 times during August–November 2017. Districts are listed in chronological order of emergences. Multiple numbers in the list correspond to different independent emergences from the same district (Table 1); only the first number is indicated on the map. Black box indicates the area shown in panel B. B) Movements (red arrows) of some of the cases (Table 2) associated with the first urban pneumonic plague transmission chain (emergence 2 in Table 1). Blue polygons indicate districts of origin/destination for travel; blue circles indicate the cities of Moramanga and Toamasina.

<figure><img src="image_2.png" /></figure>
were closely related to previous isolates from those
locations (Figure 2), confirming *Y. pestis* continues to
persist in the environment in these regions (10). PP
arose from BP in at least 6 of these events; confirmed
human cases originating from 5 events, including 4
PP cases, were documented in urban areas during the
epidemic (Table 1).

Initial 29 Cases Associated with the First Urban
PP Transmission Chain

Case-patient 1 exhibited PP symptoms (fever, gas-
trointestinal and respiratory distress, but no cough)

on August 25, 2017, in Ankazobe District (Tables 1,
2; Figures 1, 3) where he had been living and work-
ing and elected to travel to his permanent home in
Toamasina. His employer drove him by car to An-
tananarivo, where he boarded a shared bush taxi to
Toamasina on August 27; case-patient 2 sat beside
him; case-patient 3 immediately behind; and case-
patient 5, wife of case-patient 3, beside her spouse.
That same day near Moramanga, case-patient 1 expe-
rienced a deteriorated health status, including respi-
ratory distress, and died; case-patients 2 and 3 cared
for him while he was dying. The corpse was removed

**Figure 1.** Plague emergence in Madagascar, August–November 2017. A) Locations of emergences. Gray shading indicates plague-endemic regions in the central and northern highlands; yellow shading indicates 12 districts from which human plague emerged from environmental reservoirs 20 times during August–November 2017. Districts are listed in chronological order of emergences. Multiple numbers in the list correspond to different independent emergences from the same district (Table 1); only the first number is indicated on the map. Black box indicates the area shown in panel B. B) Movements (red arrows) of some of the cases (Table 2) associated with the first urban pneumonic plague transmission chain (emergence 2 in Table 1). Blue polygons indicate districts of origin/destination for travel; blue circles indicate the cities of Moramanga and Toamasina.

<figure><img src="image_3.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Maximum-likelihood phylogeny of 90 *Yersinia pestis* isolates obtained in rural endemic foci from Madagascar during August–November 2017 (boldface) and reference sequences. Tree was created using 483 core-genome SNPs discovered from WGSs and rooted using North American strain CO92. Stars indicate 5 isolates obtained within the urban areas of Antananarivo or Mahajanga. Numbers in yellow circles and squares indicate 20 emergence events from environmental reservoirs (Table 1); yellow squares and rectangles along branches indicate phylogenetic position of SNPs that were queried in *Y. pestis* sequence data from enriched sputum samples (Appendix 1, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App1.pdf). Labels for each isolate indicate identification number, year of isolation, host-disease form, and district-commune of isolation; letters on branches and colors of branches indicate known lineages (10). Some known lineages without isolates during August–November 2017 are unlabeled or collapsed. BP, bubonic plague; H, human; F, flea; PP, pneumonic plague; PPP, primary pneumonic plague; R, rat; SNP, single-nucleotide polymorphism; WGS, whole-genome sequencing. An expanded figure is available online (https://wwwnc.cdc.gov/EID/article/30/2/23-0759-F2.pdf).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Maximum-likelihood phylogeny of 90 *Yersinia pestis* isolates obtained in rural endemic foci from Madagascar during August–November 2017 (boldface) and reference sequences. Tree was created using 483 core-genome SNPs discovered from WGSs and rooted using North American strain CO92. Stars indicate 5 isolates obtained within the urban areas of Antananarivo or Mahajanga. Numbers in yellow circles and squares indicate 20 emergence events from environmental reservoirs (Table 1); yellow squares and rectangles along branches indicate phylogenetic position of SNPs that were queried in *Y. pestis* sequence data from enriched sputum samples (Appendix 1, https://wwwnc.cdc.gov/EID/article/30/2/23-0759-App1.pdf). Labels for each isolate indicate identification number, year of isolation, host-disease form, and district-commune of isolation; letters on branches and colors of branches indicate known lineages (10). Some known lineages without isolates during August–November 2017 are unlabeled or collapsed. BP, bubonic plague; H, human; F, flea; PP, pneumonic plague; PPP, primary pneumonic plague; R, rat; SNP, single-nucleotide polymorphism; WGS, whole-genome sequencing. An expanded figure is available online (https://wwwnc.cdc.gov/EID/article/30/2/23-0759-F2.pdf).</figcaption></figure>
<table><caption>**Table 1.** Twenty emergence events of *Yersinia pestis* bacteria from environmental reservoirs into humans in rural plague foci, Madagascar, August–November 2017</caption><thead><tr><th>Event</th><th>Major Y. pestis group</th><th>District/Commune</th><th>Earliest recorded onset date</th><th>Progression from BP to PP?</th><th>Travel</th><th>Spread to urban areas (evidence)</th></tr></thead><tbody><tr><td>1</td><td>s</td><td>Miarinarivo/Ambatomanjaka</td><td>Aug 13</td><td>No</td><td>No</td><td></td></tr><tr><td>2</td><td>s</td><td>Ankazobe/Marondry</td><td>Aug 25</td><td>Yes</td><td>Yes</td><td>Toamasina (sputum 135–2017), Mahajanga (isolate 17/17), Antananarivo (sputum 2093–2017)</td></tr><tr><td>3</td><td>s</td><td>Miarinarivo/Anosibe Ifanja</td><td>Aug 26</td><td>No</td><td>No</td><td></td></tr><tr><td>4</td><td>q</td><td>Moramanga/Ambohibary</td><td>Sep 2</td><td>No</td><td>No</td><td></td></tr><tr><td>5</td><td>s</td><td>Tsiroanomandidy/Tsinjoarivo Imanga</td><td>Sep 16</td><td>No</td><td>No</td><td></td></tr><tr><td>6</td><td>y</td><td>Manandriana/Ambohimahazo</td><td>Sep 17</td><td>No</td><td>No</td><td></td></tr><tr><td>7</td><td>s</td><td>Andramasina/Sabotsy Ambohitromby</td><td>Sep 21</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 21/17)</td></tr><tr><td>8</td><td>j</td><td>Tsiroanomandidy/Bemahatazana</td><td>Sep 26</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 35/17, possibly sputum 819–2017)</td></tr><tr><td>9</td><td>s</td><td>Manjakandriana/Ranovao</td><td>Oct 2</td><td>No</td><td>Yes</td><td>Antananarivo (isolate 22/17)</td></tr><tr><td>10</td><td>j</td><td>Tsiroanomandidy/Ambalanirana</td><td>Oct 3</td><td>No</td><td>No</td><td>Antananarivo (possibly sputum 819–2017)</td></tr><tr><td>11</td><td>s</td><td>Arivonimamo/Arivonimamo II</td><td>Oct 5</td><td>Yes</td><td>No</td><td></td></tr><tr><td>12</td><td>β</td><td>Ambalavao/Miarinarivo</td><td>Oct 7</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 34/17, sputum 1494–2017)</td></tr><tr><td>13</td><td>s</td><td>Ankazobe/Fiadanana</td><td>Oct 8</td><td>No</td><td>No</td><td></td></tr><tr><td>14</td><td>α</td><td>Mandritsara/Andratamarina</td><td>Oct 14</td><td>No</td><td>No</td><td></td></tr><tr><td>15</td><td>s</td><td>Miarinarivo/Anosibe Ifanja</td><td>Oct 17</td><td>No</td><td>No</td><td></td></tr><tr><td>16</td><td>t</td><td>Manandriana/Ambovombe Centre</td><td>Oct 18</td><td>No</td><td>No</td><td></td></tr><tr><td>17</td><td>x</td><td>Arivonimamo/Mahatsinjo Est</td><td>Oct 23</td><td>No</td><td>No</td><td></td></tr><tr><td>18</td><td>q</td><td>Antananarivo Avaradrano/Manandriana</td><td>Oct 30</td><td>Yes</td><td>Yes</td><td></td></tr><tr><td>19</td><td>s</td><td>Faratsiho/Antsampanimahazo</td><td>Nov 7</td><td>No</td><td>No</td><td></td></tr><tr><td>20</td><td>β</td><td>Ambalavao/Anjoma</td><td>Nov 9</td><td>No</td><td>No</td><td></td></tr></tbody></table>

at the Moramanga health center, and the bush taxi continued to Toamasina.

Case-patient 2 experienced PP symptoms in Toamasina on September 1 and died there September 2. His brother (case-patient 22) transferred the corpse by car from Toamasina to Antananarivo, by plane from Antananarivo to Sambava-Vohemar, and then by car and on foot to their native village at Ambodisakoa, Vohemar District, where case-patient 2 was buried on September 6. Case-patient 22 sought care for PP symptoms on September 6 and was admitted to a hospital at Vohemar, where he recovered. Case-patient 23, brother-in-law of case-patient 2, sought care for PP symptoms at Vohemar on September 15 and was successfully treated. No other cases were reported from Vohemar. Case-patient 20, a friend of case-patient 2 who had direct contact with him during his disease, was admitted to a hospital at Toamasina with PP symptoms on September 12; case-patient 2 might have also had contact with case-patient 22 in Toamasina.

Case-patient 3 was admitted to hospital at Toamasina with PP symptoms on September 2 and died on September 3; a wake was held that night at Toamasina. Case-patients 5 and 7 (sister of case-patient 3, resident of Mahajanga) attended the wake. Case-patient 4, a nurse who cared for case-patient 3, exhibited PP

symptoms on September 5 and fully recovered after treatment; no secondary cases were identified from case-patient 4. Case-patient 7 returned to Mahajanga, where she experienced PP symptoms on September 6; she fully recovered after treatment and no additional cases were reported from Mahajanga.

On September 4, case-patients 5 and 6 (another sister of case-patient 3) transferred the corpse of case-patient 3 by car from Toamasina to Antohomadinika-Antananarivo, where another night wake was held from September 4–6, followed by burial in Andramasina on September 6. Case-patients 5, 6, 8, 9, 12, 21, and 28 attended the night wake, funeral, or both; case-patient 5 exhibited PP symptoms on September 6. Case-patient 8 exhibited PP symptoms in Antananarivo on September 9, case-patient 9 in Tsiroanomandidy District on September 5, case-patient 12 in Miarinarivo District on September 11, case-patient 21 in Toamasina on September 12, and case-patient 28 in Ambohidratimo District on September 17. Case-patients 8, 9, 12, 21, and 28 recovered after treatment, and no secondary cases were reported from them.

Case-patient 5 started travel to Faratsiho on September 9 but died on the way. It is unknown who was traveling with her. Her corpse was transported to Alatsinainy Bandroka, Faratsiho District, and buried
<table><caption>**Table 1.** Twenty emergence events of *Yersinia pestis* bacteria from environmental reservoirs into humans in rural plague foci, Madagascar, August–November 2017</caption><thead><tr><th>Event</th><th>Major Y. pestis group</th><th>District/Commune</th><th>Earliest recorded onset date</th><th>Progression from BP to PP?</th><th>Travel</th><th>Spread to urban areas (evidence)</th></tr></thead><tbody><tr><td>1</td><td>s</td><td>Miarinarivo/Ambatomanjaka</td><td>Aug 13</td><td>No</td><td>No</td><td></td></tr><tr><td>2</td><td>s</td><td>Ankazobe/Marondry</td><td>Aug 25</td><td>Yes</td><td>Yes</td><td>Toamasina (sputum 135–2017), Mahajanga (isolate 17/17), Antananarivo (sputum 2093–2017)</td></tr><tr><td>3</td><td>s</td><td>Miarinarivo/Anosibe Ifanja</td><td>Aug 26</td><td>No</td><td>No</td><td></td></tr><tr><td>4</td><td>q</td><td>Moramanga/Ambohibary</td><td>Sep 2</td><td>No</td><td>No</td><td></td></tr><tr><td>5</td><td>s</td><td>Tsiroanomandidy/Tsinjoarivo Imanga</td><td>Sep 16</td><td>No</td><td>No</td><td></td></tr><tr><td>6</td><td>y</td><td>Manandriana/Ambohimahazo</td><td>Sep 17</td><td>No</td><td>No</td><td></td></tr><tr><td>7</td><td>s</td><td>Andramasina/Sabotsy Ambohitromby</td><td>Sep 21</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 21/17)</td></tr><tr><td>8</td><td>j</td><td>Tsiroanomandidy/Bemahatazana</td><td>Sep 26</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 35/17, possibly sputum 819–2017)</td></tr><tr><td>9</td><td>s</td><td>Manjakandriana/Ranovao</td><td>Oct 2</td><td>No</td><td>Yes</td><td>Antananarivo (isolate 22/17)</td></tr><tr><td>10</td><td>j</td><td>Tsiroanomandidy/Ambalanirana</td><td>Oct 3</td><td>No</td><td>No</td><td>Antananarivo (possibly sputum 819–2017)</td></tr><tr><td>11</td><td>s</td><td>Arivonimamo/Arivonimamo II</td><td>Oct 5</td><td>Yes</td><td>No</td><td></td></tr><tr><td>12</td><td>β</td><td>Ambalavao/Miarinarivo</td><td>Oct 7</td><td>Yes</td><td>Yes</td><td>Antananarivo (isolate 34/17, sputum 1494–2017)</td></tr><tr><td>13</td><td>s</td><td>Ankazobe/Fiadanana</td><td>Oct 8</td><td>No</td><td>No</td><td></td></tr><tr><td>14</td><td>α</td><td>Mandritsara/Andratamarina</td><td>Oct 14</td><td>No</td><td>No</td><td></td></tr><tr><td>15</td><td>s</td><td>Miarinarivo/Anosibe Ifanja</td><td>Oct 17</td><td>No</td><td>No</td><td></td></tr><tr><td>16</td><td>t</td><td>Manandriana/Ambovombe Centre</td><td>Oct 18</td><td>No</td><td>No</td><td></td></tr><tr><td>17</td><td>x</td><td>Arivonimamo/Mahatsinjo Est</td><td>Oct 23</td><td>No</td><td>No</td><td></td></tr><tr><td>18</td><td>q</td><td>Antananarivo Avaradrano/Manandriana</td><td>Oct 30</td><td>Yes</td><td>Yes</td><td></td></tr><tr><td>19</td><td>s</td><td>Faratsiho/Antsampanimahazo</td><td>Nov 7</td><td>No</td><td>No</td><td></td></tr><tr><td>20</td><td>β</td><td>Ambalavao/Anjoma</td><td>Nov 9</td><td>No</td><td>No</td><td></td></tr></tbody></table>

at the Moramanga health center, and the bush taxi continued to Toamasina.

Case-patient 2 experienced PP symptoms in Toamasina on September 1 and died there September 2. His brother (case-patient 22) transferred the corpse by car from Toamasina to Antananarivo, by plane from Antananarivo to Sambava-Vohemar, and then by car and on foot to their native village at Ambodisakoa, Vohemar District, where case-patient 2 was buried on September 6. Case-patient 22 sought care for PP symptoms on September 6 and was admitted to a hospital at Vohemar, where he recovered. Case-patient 23, brother-in-law of case-patient 2, sought care for PP symptoms at Vohemar on September 15 and was successfully treated. No other cases were reported from Vohemar. Case-patient 20, a friend of case-patient 2 who had direct contact with him during his disease, was admitted to a hospital at Toamasina with PP symptoms on September 12; case-patient 2 might have also had contact with case-patient 22 in Toamasina.

Case-patient 3 was admitted to hospital at Toamasina with PP symptoms on September 2 and died on September 3; a wake was held that night at Toamasina. Case-patients 5 and 7 (sister of case-patient 3, resident of Mahajanga) attended the wake. Case-patient 4, a nurse who cared for case-patient 3, exhibited PP

symptoms on September 5 and fully recovered after treatment; no secondary cases were identified from case-patient 4. Case-patient 7 returned to Mahajanga, where she experienced PP symptoms on September 6; she fully recovered after treatment and no additional cases were reported from Mahajanga.

On September 4, case-patients 5 and 6 (another sister of case-patient 3) transferred the corpse of case-patient 3 by car from Toamasina to Antohomadinika-Antananarivo, where another night wake was held from September 4–6, followed by burial in Andramasina on September 6. Case-patients 5, 6, 8, 9, 12, 21, and 28 attended the night wake, funeral, or both; case-patient 5 exhibited PP symptoms on September 6. Case-patient 8 exhibited PP symptoms in Antananarivo on September 9, case-patient 9 in Tsiroanomandidy District on September 5, case-patient 12 in Miarinarivo District on September 11, case-patient 21 in Toamasina on September 12, and case-patient 28 in Ambohidratimo District on September 17. Case-patients 8, 9, 12, 21, and 28 recovered after treatment, and no secondary cases were reported from them.

Case-patient 5 started travel to Faratsiho on September 9 but died on the way. It is unknown who was traveling with her. Her corpse was transported to Alatsinainy Bandroka, Faratsiho District, and buried
after a traditional funeral. Case-patients 13–19 and 24, all Faratsiho residents, helped prepare the corpse, participated in the funeral, or both; all exhibited PP

symptoms starting on September 12 (case-patients 13–19) or 13 (case-patient 24). Case-patients 26 and 27 had documented but unspecified contacts with ≥1 of the

<table><caption>**Table 2.** Information on 29 case-patients associated with the first known urban pneumonic plague transmission chain, Madagascar, August–November 2017*</caption><thead><tr><th>Case-patient no.</th><th>Age, y/sex</th><th>Outcome</th><th>Onset date</th><th>Onset district</th><th>Onset location</th><th>Travel</th><th>Documented contact with other case-patients</th><th>Sample/positive Y. pestis result</th><th>Case definition</th></tr></thead><tbody><tr><td>1</td><td>32/M</td><td>Died</td><td>Aug 25</td><td>Ankazobe</td><td>Rural</td><td>Yes</td><td>2, 3, 5</td><td>None collected</td><td>Suspected</td></tr><tr><td>2</td><td>26/M</td><td>Died</td><td>Sep 1</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>1, 3, 5, 20, 22</td><td>None collected</td><td>Suspected</td></tr><tr><td>3</td><td>36/M</td><td>Died</td><td>Sep 2</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>1, 2, 4, 5, 7</td><td>None collected</td><td>Suspected</td></tr><tr><td>4</td><td>23/F</td><td>Recovered</td><td>Sep 5</td><td>Toamasina I</td><td>Urban</td><td>No</td><td>3, 5, 7</td><td>Sputum 135-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>5</td><td>16/F</td><td>Died</td><td>Sep 6</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>Yes</td><td>1, 3, 4, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 21, 24, 28</td><td>None collected</td><td>Suspected</td></tr><tr><td>6</td><td>47/F</td><td>Died</td><td>Sep 9</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>Yes</td><td>5, 8, 9, 10, 11, 12, 21, 25, 28, 29</td><td>Sputum 118-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>7</td><td>40/F</td><td>Recovered</td><td>Sep 6</td><td>Mahajanga I</td><td>Urban</td><td>Yes</td><td>3, 4, 5</td><td>Sputum 121-2017/cultured isolate 17/17</td><td>Confirmed</td></tr><tr><td>8</td><td>45/M</td><td>Recovered</td><td>Sep 9</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>5, 6, 9, 12, 21, 28</td><td>Sputum 150-2017/RDT</td><td>Probable</td></tr><tr><td>9</td><td>30/M</td><td>Recovered</td><td>Sep 5</td><td>Tsiroanomandidy</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 12, 21, 28</td><td>Sputum 143-2017/RDT</td><td>Probable</td></tr><tr><td>10</td><td>21/F</td><td>Recovered</td><td>Sep 11</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 11, 29</td><td>Sputum 119-2017/none</td><td>Suspect</td></tr><tr><td>11</td><td>15/M</td><td>Recovered</td><td>Sep 11</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 10, 29</td><td>Sputum 120-2017/none</td><td>Suspected</td></tr><tr><td>12</td><td>37/M</td><td>Recovered</td><td>Sep 11</td><td>Miarinarivo</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 9, 21, 28</td><td>Sputum 153-2017/RDT</td><td>Probable</td></tr><tr><td>13</td><td>11/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 14, 15, 16, 17, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>14</td><td>52/M</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 15, 16, 17, 18, 19, 24</td><td>Sputum 124-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>15</td><td>48/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 16, 17, 18, 19, 24</td><td>Sputum 125-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>16</td><td>9/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 17, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>17</td><td>39/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>18</td><td>38/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 19, 24</td><td>Sputum 129-2017/RDT</td><td>Probable</td></tr><tr><td>19</td><td>19/M</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 18, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>20</td><td>25/M</td><td>Recovered</td><td>Sep 12</td><td>Toamasina I</td><td>Urban</td><td>No</td><td>2</td><td>Sputum 136-2017/RDT</td><td>Probable</td></tr><tr><td>21</td><td>31/M</td><td>Recovered</td><td>Sep 12</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>5, 6, 8, 9, 12, 28</td><td>Sputum 149-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>22</td><td>30/M</td><td>Recovered</td><td>Sep 6</td><td>Vohemar</td><td>Rural</td><td>Yes</td><td>2, 23</td><td>Sputum 184-2017/RDT</td><td>Probable</td></tr><tr><td>23</td><td>33/M</td><td>Recovered</td><td>Sep 15</td><td>Vohemar</td><td>Rural</td><td>No</td><td>22</td><td>Sputum 185-2017/RDT</td><td>Probable</td></tr><tr><td>24</td><td>UNK/F</td><td>Recovered</td><td>Sep 13</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 18, 19</td><td>Sputum 131-2017/none</td><td>Suspected</td></tr><tr><td>25</td><td>49/M</td><td>Recovered</td><td>Sep 14</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6</td><td>Blood 188-2017/RDT</td><td>Probable</td></tr><tr><td>26</td><td>13/M</td><td>Recovered</td><td>Sep 14</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>≥1 of 13, 14, 15, 16, 17, 18, 19, 24</td><td>Sputum 133-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>27</td><td>39/M</td><td>Recovered</td><td>Sep 14</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>≥1 of 13, 14, 15, 16, 17, 18, 19, 24</td><td>Sputum 134-2017/none</td><td>Suspected</td></tr><tr><td>28</td><td>25/F</td><td>Recovered</td><td>Sep 17</td><td>Ambohidratimo</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 9, 12, 21</td><td>Sputum 164-2017/none</td><td>Suspected</td></tr><tr><td>29</td><td>46/F</td><td>Recovered</td><td>Sep 10</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 10, 11</td><td>Sputum 181-2017/RDT</td><td>Probable</td></tr></tbody></table>

<footnote>*UNK, unknown.</footnote>
after a traditional funeral. Case-patients 13–19 and 24, all Faratsiho residents, helped prepare the corpse, participated in the funeral, or both; all exhibited PP

symptoms starting on September 12 (case-patients 13–19) or 13 (case-patient 24). Case-patients 26 and 27 had documented but unspecified contacts with ≥1 of the

<table><caption>**Table 2.** Information on 29 case-patients associated with the first known urban pneumonic plague transmission chain, Madagascar, August–November 2017*</caption><thead><tr><th>Case-patient no.</th><th>Age, y/sex</th><th>Outcome</th><th>Onset date</th><th>Onset district</th><th>Onset location</th><th>Travel</th><th>Documented contact with other case-patients</th><th>Sample/positive Y. pestis result</th><th>Case definition</th></tr></thead><tbody><tr><td>1</td><td>32/M</td><td>Died</td><td>Aug 25</td><td>Ankazobe</td><td>Rural</td><td>Yes</td><td>2, 3, 5</td><td>None collected</td><td>Suspected</td></tr><tr><td>2</td><td>26/M</td><td>Died</td><td>Sep 1</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>1, 3, 5, 20, 22</td><td>None collected</td><td>Suspected</td></tr><tr><td>3</td><td>36/M</td><td>Died</td><td>Sep 2</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>1, 2, 4, 5, 7</td><td>None collected</td><td>Suspected</td></tr><tr><td>4</td><td>23/F</td><td>Recovered</td><td>Sep 5</td><td>Toamasina I</td><td>Urban</td><td>No</td><td>3, 5, 7</td><td>Sputum 135-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>5</td><td>16/F</td><td>Died</td><td>Sep 6</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>Yes</td><td>1, 3, 4, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 21, 24, 28</td><td>None collected</td><td>Suspected</td></tr><tr><td>6</td><td>47/F</td><td>Died</td><td>Sep 9</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>Yes</td><td>5, 8, 9, 10, 11, 12, 21, 25, 28, 29</td><td>Sputum 118-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>7</td><td>40/F</td><td>Recovered</td><td>Sep 6</td><td>Mahajanga I</td><td>Urban</td><td>Yes</td><td>3, 4, 5</td><td>Sputum 121-2017/cultured isolate 17/17</td><td>Confirmed</td></tr><tr><td>8</td><td>45/M</td><td>Recovered</td><td>Sep 9</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>5, 6, 9, 12, 21, 28</td><td>Sputum 150-2017/RDT</td><td>Probable</td></tr><tr><td>9</td><td>30/M</td><td>Recovered</td><td>Sep 5</td><td>Tsiroanomandidy</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 12, 21, 28</td><td>Sputum 143-2017/RDT</td><td>Probable</td></tr><tr><td>10</td><td>21/F</td><td>Recovered</td><td>Sep 11</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 11, 29</td><td>Sputum 119-2017/none</td><td>Suspect</td></tr><tr><td>11</td><td>15/M</td><td>Recovered</td><td>Sep 11</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 10, 29</td><td>Sputum 120-2017/none</td><td>Suspected</td></tr><tr><td>12</td><td>37/M</td><td>Recovered</td><td>Sep 11</td><td>Miarinarivo</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 9, 21, 28</td><td>Sputum 153-2017/RDT</td><td>Probable</td></tr><tr><td>13</td><td>11/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 14, 15, 16, 17, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>14</td><td>52/M</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 15, 16, 17, 18, 19, 24</td><td>Sputum 124-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>15</td><td>48/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 16, 17, 18, 19, 24</td><td>Sputum 125-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>16</td><td>9/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 17, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>17</td><td>39/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 18, 19, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>18</td><td>38/F</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 19, 24</td><td>Sputum 129-2017/RDT</td><td>Probable</td></tr><tr><td>19</td><td>19/M</td><td>Recovered</td><td>Sep 12</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 18, 24</td><td>None collected</td><td>Suspected</td></tr><tr><td>20</td><td>25/M</td><td>Recovered</td><td>Sep 12</td><td>Toamasina I</td><td>Urban</td><td>No</td><td>2</td><td>Sputum 136-2017/RDT</td><td>Probable</td></tr><tr><td>21</td><td>31/M</td><td>Recovered</td><td>Sep 12</td><td>Toamasina I</td><td>Urban</td><td>Yes</td><td>5, 6, 8, 9, 12, 28</td><td>Sputum 149-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>22</td><td>30/M</td><td>Recovered</td><td>Sep 6</td><td>Vohemar</td><td>Rural</td><td>Yes</td><td>2, 23</td><td>Sputum 184-2017/RDT</td><td>Probable</td></tr><tr><td>23</td><td>33/M</td><td>Recovered</td><td>Sep 15</td><td>Vohemar</td><td>Rural</td><td>No</td><td>22</td><td>Sputum 185-2017/RDT</td><td>Probable</td></tr><tr><td>24</td><td>UNK/F</td><td>Recovered</td><td>Sep 13</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>5, 13, 14, 15, 16, 17, 18, 19</td><td>Sputum 131-2017/none</td><td>Suspected</td></tr><tr><td>25</td><td>49/M</td><td>Recovered</td><td>Sep 14</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6</td><td>Blood 188-2017/RDT</td><td>Probable</td></tr><tr><td>26</td><td>13/M</td><td>Recovered</td><td>Sep 14</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>≥1 of 13, 14, 15, 16, 17, 18, 19, 24</td><td>Sputum 133-2017/RDT, PCR</td><td>Probable</td></tr><tr><td>27</td><td>39/M</td><td>Recovered</td><td>Sep 14</td><td>Faratsiho</td><td>Rural</td><td>No</td><td>≥1 of 13, 14, 15, 16, 17, 18, 19, 24</td><td>Sputum 134-2017/none</td><td>Suspected</td></tr><tr><td>28</td><td>25/F</td><td>Recovered</td><td>Sep 17</td><td>Ambohidratimo</td><td>Rural</td><td>Yes</td><td>5, 6, 8, 9, 12, 21</td><td>Sputum 164-2017/none</td><td>Suspected</td></tr><tr><td>29</td><td>46/F</td><td>Recovered</td><td>Sep 10</td><td>Antananarivo-Renivohitra</td><td>Urban</td><td>No</td><td>6, 10, 11</td><td>Sputum 181-2017/RDT</td><td>Probable</td></tr></tbody></table>

<footnote>*UNK, unknown.</footnote>
above cases and exhibited PP symptoms on Sep-
tember 14. All case-patients in Faratsiho recovered
after treatment.

Case-patient 6 experienced PP symptoms on Sep-
tember 9 in Antananarivo, sought care at a private clin-
ic with a physically altered state on September 11, and
was transferred to a military hospital where she died
on September 11; this case was the first identified case
from the epidemic that triggered the subsequent public
health response (13). Four secondary cases in Antanan-
arivo resulted from contact with case-patient 6: case-
patients 10 and 11 (daughter and son of case patient
6; PP onset September 11), case-patient 29 (daughter-
in-law of case-patient 6; PP onset September 19), and
case-patient 25. Case-patient 25 (onset September 14),
who had septicemic plague, was a health agent and
handled and disinfected the corpse of case-patient 6.

Sample 121-2017, collected in Mahajanga from
case-patient 7, yielded the only *Y. pestis* isolate ob-
tained from this transmission chain, 17/17, which is

most closely related to older isolates from Ankazobe,
Faratsiho, and Betafo districts (Figure 2). This result is
consistent with Ankazobe District as the geographic
source of this transmission chain; case-patient 1 was
living and working there but had no recent travel to
Faratsiho or Betafo Districts. Enrichment and sequenc-
ing of sputum samples obtained from case-patient 4
in Toamasina (135-2017), case-patient 15 in Faratsiho
(125-2017), and case-patient 22 in Vohemar (184-2017)
(Table 2) revealed the presence in those samples of 1-5
SNPs specific to isolate 17/17 (Appendix 1 section 7,
Table 5), documenting that those cases were all part of
a single transmission chain that was spread to multiple
urban areas and regions in Madagascar. On Novem-
ber 8, sputum sample 2093-2017 was collected from an
Antananarivo resident with no recent travel outside
the city (Table 1). After we enriched and sequenced
this sample, we determined that it contained 3 SNPs
specific to isolate 17/17 that were also present in spu-
tum sample 125-2017 from case-patient 15 (Appendix

<figure><img src="image_4.png" /></figure>

**Figure 3.** Transmission patterns among the initial 29 cases associated with the first urban PP transmission chain in Madagascar, August–November 2017 (emergence 2 in Table 1). Dates are illness onset dates. Solid arrows indicate likely infection sources based on known contact (Table 2); dotted arrows indicate hypothetical infection sources inferred from genetic data. Epidemiologic data, or both. Symbols for individual cases indicate male or female sex; red indicates persons who died and blue indicates survivors. Red box indicates the first identified case from the epidemic that triggered the subsequent public health response.
above cases and exhibited PP symptoms on Sep-
tember 14. All case-patients in Faratsiho recovered
after treatment.

Case-patient 6 experienced PP symptoms on Sep-
tember 9 in Antananarivo, sought care at a private clin-
ic with a physically altered state on September 11, and
was transferred to a military hospital where she died
on September 11; this case was the first identified case
from the epidemic that triggered the subsequent public
health response (13). Four secondary cases in Antanan-
arivo resulted from contact with case-patient 6: case-
patients 10 and 11 (daughter and son of case patient
6; PP onset September 11), case-patient 29 (daughter-
in-law of case-patient 6; PP onset September 19), and
case-patient 25. Case-patient 25 (onset September 14),
who had septicemic plague, was a health agent and
handled and disinfected the corpse of case-patient 6.

Sample 121-2017, collected in Mahajanga from
case-patient 7, yielded the only *Y. pestis* isolate ob-
tained from this transmission chain, 17/17, which is

most closely related to older isolates from Ankazobe,
Faratsiho, and Betafo districts (Figure 2). This result is
consistent with Ankazobe District as the geographic
source of this transmission chain; case-patient 1 was
living and working there but had no recent travel to
Faratsiho or Betafo Districts. Enrichment and sequenc-
ing of sputum samples obtained from case-patient 4
in Toamasina (135-2017), case-patient 15 in Faratsiho
(125-2017), and case-patient 22 in Vohemar (184-2017)
(Table 2) revealed the presence in those samples of 1-5
SNPs specific to isolate 17/17 (Appendix 1 section 7,
Table 5), documenting that those cases were all part of
a single transmission chain that was spread to multiple
urban areas and regions in Madagascar. On Novem-
ber 8, sputum sample 2093-2017 was collected from an
Antananarivo resident with no recent travel outside
the city (Table 1). After we enriched and sequenced
this sample, we determined that it contained 3 SNPs
specific to isolate 17/17 that were also present in spu-
tum sample 125-2017 from case-patient 15 (Appendix

<figure><img src="image_5.png" /></figure>

**Figure 3.** Transmission patterns among the initial 29 cases associated with the first urban PP transmission chain in Madagascar, August–November 2017 (emergence 2 in Table 1). Dates are illness onset dates. Solid arrows indicate likely infection sources based on known contact (Table 2); dotted arrows indicate hypothetical infection sources inferred from genetic data. Epidemiologic data, or both. Symbols for individual cases indicate male or female sex; red indicates persons who died and blue indicates survivors. Red box indicates the first identified case from the epidemic that triggered the subsequent public health response.
1 section 7, Table 5), suggesting possible community
spread of this transmission chain in Antananarivo and
persistence there until at least November 2017.

**Introduction of Other *Y. pestis* Lineages to Antananarivo**

Isolate 21/17 was obtained from a sputum sample col-
lected on September 29 from an Antananarivo resident.
It is distinct from isolate 17/17 but identical to isolate
20/17 (Figure 2), which was obtained from a bubo as-
pirate collected on September 22 from a resident of a
rural area of Andramasina District (Table 1; Appendix
1 section 8). There was no known relationship between
those 2 persons, and neither reported travel. The per-
son yielding isolate 20/17 had clinical signs consistent
with SPP, including cough for <5 days. Although un-
known, this person may have initiated an undocu-
mented PP outbreak in Andramasina District; another
person infected from that outbreak may have traveled
to Antananarivo, leading to a transmission chain there
that infected the person who yielded 21/17.

A foreign tourist sought care at a hospital in An-
tananarivo on October 1 for PP symptoms; he had
traveled outside Antananarivo but the specific details
are unknown. His sputum sample yielded isolate
35/17, which is distinct from isolate 17/17 but closely
related to multiple human isolates obtained from BP
cases in Tsiroanomandidy District starting in Septem-
ber (Table 1; Figure 2). This finding suggests plague
activity in this rural focus as the ultimate source of
the infection in the tourist, from whom no known
secondary cases were reported. However, sequenc-
ing of enriched sputum sample 819-2017, collected
on October 12 from an Antananarivo resident with no
recent travel, contained 1 SNP specific to the j phylo-
genetic group of *Y. pestis* in Madagascar (Appendix
1 section 7, Table 5), which suggested the possibility
of additional undocumented cases associated with
this transmission chain in Antananarivo. Emergence
10 was also assigned to phylogenetic group j (Table
1; Figure 2), so it is possible the patient who yielded
sputum sample 819-2017 could have been infected
through a transmission chain from that event involv-
ing undocumented travel to Antananarivo.

Isolate 22/17 (Table 1) was obtained on October 2
from a bubo aspirate collected in Antananarivo from
a girl who died the next day. She resided in a rural re-
region of Manjakandriana District; her family brought
her to Antananarivo for treatment when she became
ill. Upon sequencing, isolate 22/17 was distinct from
isolate 17/17 and most closely related to a 2007 hu-
man isolate from Manjakandriana District (Figure 2).
No secondary cases were reported from this person,
which is not unexpected because it was BP.

Isolate 34/17 was reportedly collected from a
newborn baby in Antananarivo on October 15; his
parents were residents of Antananarivo with no
recent travel. The baby was febrile and transferred
to a children's hospital as a suspect plague case; staff
collected a sputum sample using a bronchoscope. The
sputum sample attributed to this newborn yielded
isolate 34/17, which is distinct from isolate 17/17 but
identical to multiple human isolates obtained from
plague activity in a rural region of Ambalavao Dis-
strict (Table 1; Figure 2; Appendix 1 section 8). The
earliest documented case there (onset date October 7)
yielded isolate 32/17 and was diagnosed as BP, but
PP symptoms were also present. Subsequent PP cases
were reported from this rural area into late Novem-
ber. No secondary cases were reported from the baby,
but SNP data suggest subsequent community spread
of closely related *Y. pestis* in Antananarivo.

On October 24, sputum sample 1494-2017 was
collected from an Antananarivo resident with no
travel history. Enrichment and sequencing of that
sample determined it contained an SNP specific
to human isolates 30/17, 32/17, 33/17, 34/17, and
55/17 obtained in Ambalavao District as part of the
investigation of emergence 12 (Appendix 1 section 8).

**Discussion**

Our epidemiologic and genomic analyses of 29 human plague cases associated with the first emergence event to reach urban areas of Madagascar document that the transmission chain started in late August 2017 in rural Ankazobe District and subsequently spread to ≥8 other districts by mid-September, including the urban areas of Antananarivo, Toamasina, and Mahajanga, as well as multiple rural districts (Table 2; Figure 1). This transmission chain demonstrates that movements of infected persons can rapidly spread PP across large distances. Prompt public health responses prevented subsequent spread of this transmission chain in Mahajanga and Vohemar, but it apparently persisted into mid-November 2017 in Antananarivo. Although we did not identify genetic data to confirm continued presence of this transmission chain in Toamasina, it is located well outside the plague-endemic region in Madagascar; confirmed and probable PP cases continued to be reported there through mid-October, which suggests possible community spread. Similar to previous reports from Madagascar and elsewhere in Africa (4,6-8,15-17), this PP transmission chain was associated with traditional funeral practices.

PP transmission requires close contact with an in-
fected person in the end stage of disease (14,18). On
the day he died, case-patient 1 traveled both in a car
1 section 7, Table 5), suggesting possible community spread of this transmission chain in Antananarivo and persistence there until at least November 2017.

**Introduction of Other *Y. pestis* Lineages to Antananarivo**

Isolate 21/17 was obtained from a sputum sample collected on September 29 from an Antananarivo resident. It is distinct from isolate 17/17 but identical to isolate 20/17 (Figure 2), which was obtained from a bubo aspirate collected on September 22 from a resident of a rural area of Andramasina District (Table 1; Appendix 1 section 8). There was no known relationship between those 2 persons, and neither reported travel. The person yielding isolate 20/17 had clinical signs consistent with SPP, including cough for <5 days. Although unknown, this person may have initiated an undocumented PP outbreak in Andramasina District; another person infected from that outbreak may have traveled to Antananarivo, leading to a transmission chain there that infected the person who yielded 21/17.

A foreign tourist sought care at a hospital in Antananarivo on October 1 for PP symptoms; he had traveled outside Antananarivo but the specific details are unknown. His sputum sample yielded isolate 35/17, which is distinct from isolate 17/17 but closely related to multiple human isolates obtained from BP cases in Tsiroanomandidy District starting in September (Table 1; Figure 2). This finding suggests plague activity in this rural focus as the ultimate source of the infection in the tourist, from whom no known secondary cases were reported. However, sequencing of enriched sputum sample 819-2017, collected on October 12 from an Antananarivo resident with no recent travel, contained 1 SNP specific to the phylogenetic group of *Y. pestis* in Madagascar (Appendix 1 section 7, Table 5), which suggested the possibility of additional undocumented cases associated with this transmission chain in Antananarivo. Emergence 10 was also assigned to phylogenetic group j (Table 1; Figure 2), so it is possible the patient who yielded sputum sample 819-2017 could have been infected through a transmission chain from that event involving undocumented travel to Antananarivo.

Isolate 22/17 (Table 1) was obtained on October 2 from a bubo aspirate collected in Antananarivo from a girl who died the next day. She resided in a rural region of Manjakandriana District; her family brought her to Antananarivo for treatment when she became ill. Upon sequencing, isolate 22/17 was distinct from isolate 17/17 and most closely related to a 2007 human isolate from Manjakandriana District (Figure 2). No secondary cases were reported from this person, which is not unexpected because it was BP.

Isolate 34/17 was reportedly collected from a newborn baby in Antananarivo on October 15; his parents were residents of Antananarivo with no recent travel. The baby was febrile and transferred to a children's hospital as a suspect plague case; staff collected a sputum sample using a bronchoscope. The sputum sample attributed to this newborn yielded isolate 34/17, which is distinct from isolate 17/17 but identical to multiple human isolates obtained from plague activity in a rural region of Ambalavao District (Table 1; Figure 2; Appendix 1 section 8). The earliest documented case there (onset date October 7) yielded isolate 32/17 and was diagnosed as BP, but PP symptoms were also present. Subsequent PP cases were reported from this rural area into late November. No secondary cases were reported from the baby, but SNP data suggest subsequent community spread of closely related *Y. pestis* in Antananarivo.

On October 24, sputum sample 1494-2017 was collected from an Antananarivo resident with no travel history. Enrichment and sequencing of that sample determined it contained an SNP specific to human isolates 30/17, 32/17, 33/17, 34/17, and 55/17 obtained in Ambalavao District as part of the investigation of emergence 12 (Appendix 1 section 8).

**Discussion**

Our epidemiologic and genomic analyses of 29 human plague cases associated with the first emergence event to reach urban areas of Madagascar document that the transmission chain started in late August 2017 in rural Ankazobe District and subsequently spread to ≥8 other districts by mid-September, including the urban areas of Antananarivo, Toamasina, and Mahajanga, as well as multiple rural districts (Table 2; Figure 1). This transmission chain demonstrates that movements of infected persons can rapidly spread PP across large distances. Prompt public health responses prevented subsequent spread of this transmission chain in Mahajanga and Vohemar, but it apparently persisted into mid-November 2017 in Antananarivo. Although we did not identify genetic data to confirm continued presence of this transmission chain in Toamasina, it is located well outside the plague-endemic region in Madagascar; confirmed and probable PP cases continued to be reported there through mid-October, which suggests possible community spread. Similar to previous reports from Madagascar and elsewhere in Africa (4,6-8,15-17), this PP transmission chain was associated with traditional funeral practices.

PP transmission requires close contact with an infected person in the end stage of disease (14,18). On the day he died, case-patient 1 traveled both in a car
with his employer and in a bush taxi. Bush taxis in Madagascar are extremely crowded environments, typically loaded beyond the original capacity of the vehicle and departing only when completely full (19). Despite this close contact, only 2 secondary cases, case-patients 2 and 3 (Figure 1), appear to have resulted directly from the presence of case-patient 1 in the bush taxi and none from travel in the car. This result is likely because of his lack of cough; transmission of PP is thought to occur from either inhalation of respiratory droplets expelled by coughing persons or by direct contact (14,18), and only those 2 secondary cases were known to have had direct contact with case-patient 1 in the bush taxi.

Assessing the true number of plague cases associated with the 2017 urban PP epidemic in Madagascar has been challenging for multiple reasons (13,20). One reason is that sputum is a poor-quality sample type; isolating *Y. pestis* from sputum is always complicated by commensal flora and sample quality, and the large number of sputum samples collected during this epidemic overwhelmed public health laboratories (13). The associated delay in culturing sputum samples, as well as evidence that many patients may have been self-administering antimicrobial drugs effective against *Y. pestis* (13,20), likely further explains why so few *Y. pestis* isolates were obtained from this epidemic. It is also possible that many of the suspected and probable cases were not true infections (14,21). Regardless, only 4 isolates were obtained from sputum samples collected from urban areas during this epidemic: 17/17 from Mahajanga and 35/17, 21/17, and 34/17 from Antananarivo. Isolate 22/17 also was collected in Antananarivo from a BP case. All 5 of the isolates obtained in urban areas are highly distinct from each other (Figure 2) and associated with 5 different independent emergence events in rural foci (Table 1). Those patterns document that *Y. pestis* was introduced to Antananarivo ≥5 times during the epidemic, with evidence that 3 of those introductions may have led to subsequent community transmission. The patterns also suggest there may have been more introductions of *Y. pestis* to Antananarivo, Toamasina, or both that could not be documented because there were no isolates.

Movement of infected persons from rural disease-endemic regions to urban areas may have been caused by panic that arose in the Malagasy population in response to the PP epidemic (1). The number of notified cases associated with this epidemic increased dramatically in late September and early October 2017 (13), coinciding with the timing

of the first situation report from the World Health Organization (22) and widespread coverage in the media; this increase in cases was probably caused in part from fear and panic in urban inhabitants unfamiliar with plague (20). Residents of rural regions are much more aware of plague and perceive it to be rapidly fatal; however, they are less familiar with PP than BP (23). The increased public communications regarding the PP epidemic in urban areas, as well as the increased availability there of public health resources from mobilization of substantial domestic and international public health resources (1), may have led some infected persons from rural areas to travel to Antananarivo to seek treatment. A previous study (11) described evidence of plague-infected persons moving from the central highlands to Mahajanga and from Mahajanga to Antananarivo.

In conclusion, the 2017 urban PP epidemic in Madagascar involved the introduction of multiple independent lineages of *Y. pestis* from several rural foci, which may partly explain why this epidemic was so difficult to control. Because PP spreads person-to-person, control of PP outbreaks and epidemics is focused on identifying cases and their known contacts and providing antimicrobial treatment. Those types of investigations were largely impossible in this instance, given the extent of the epidemic, which likely hampered control efforts. Our results suggest that control efforts also might have been diminished by the presence of multiple independent transmission chains that may have resulted in continuation or expansion of the epidemic. Our findings highlight the importance of using existing genotyping tools (24) and developing genomics capabilities in Madagascar, elsewhere in Africa, and other global locations (25) so they can be used during outbreaks of plague and other diseases to promptly identify multiple sources and transmission chains to better inform control efforts.

Funding for this work was provided by LabEx

Integrative Biology of Emerging Infectious Diseases (grant no. ANR LBX-62 IBEID), the US Department of Defense's Defense Threat Reduction Agency (contract no. HDTRA1-20-C-0007), the Department of International Affairs of the Institut Pasteur via a cooperative agreement with the Office of the Assistant Secretary for Preparedness and Response in the US Department of Health and Human Services (grant no. IDSEP140020-01-00), the Institut Pasteur via the ANR Investissement d'Avenir Program (grant no. 10-LABX-62-IBEID), and the US Agency for International Development (grant no. AID-687-G-13-0003). The World Health Organization assisted with transport of biological samples from healthcare facilities.
with his employer and in a bush taxi. Bush taxis in Madagascar are extremely crowded environments, typically loaded beyond the original capacity of the vehicle and departing only when completely full (19). Despite this close contact, only 2 secondary cases, case-patients 2 and 3 (Figure 1), appear to have resulted directly from the presence of case-patient 1 in the bush taxi and none from travel in the car. This result is likely because of his lack of cough; transmission of PP is thought to occur from either inhalation of respiratory droplets expelled by coughing persons or by direct contact (14,18), and only those 2 secondary cases were known to have had direct contact with case-patient 1 in the bush taxi.

Assessing the true number of plague cases associated with the 2017 urban PP epidemic in Madagascar has been challenging for multiple reasons (13,20). One reason is that sputum is a poor-quality sample type; isolating *Y. pestis* from sputum is always complicated by commensal flora and sample quality, and the large number of sputum samples collected during this epidemic overwhelmed public health laboratories (13). The associated delay in culturing sputum samples, as well as evidence that many patients may have been self-administering antimicrobial drugs effective against *Y. pestis* (13,20), likely further explains why so few *Y. pestis* isolates were obtained from this epidemic. It is also possible that many of the suspected and probable cases were not true infections (14,21). Regardless, only 4 isolates were obtained from sputum samples collected from urban areas during this epidemic: 17/17 from Mahajanga and 35/17, 21/17, and 34/17 from Antananarivo. Isolate 22/17 also was collected in Antananarivo from a BP case. All 5 of the isolates obtained in urban areas are highly distinct from each other (Figure 2) and associated with 5 different independent emergence events in rural foci (Table 1). Those patterns document that *Y. pestis* was introduced to Antananarivo ≥5 times during the epidemic, with evidence that 3 of those introductions may have led to subsequent community transmission. The patterns also suggest there may have been more introductions of *Y. pestis* to Antananarivo, Toamasina, or both that could not be documented because there were no isolates.

Movement of infected persons from rural disease-endemic regions to urban areas may have been caused by panic that arose in the Malagasy population in response to the PP epidemic (1). The number of notified cases associated with this epidemic increased dramatically in late September and early October 2017 (13), coinciding with the timing

of the first situation report from the World Health Organization (22) and widespread coverage in the media; this increase in cases was probably caused in part from fear and panic in urban inhabitants unfamiliar with plague (20). Residents of rural regions are much more aware of plague and perceive it to be rapidly fatal; however, they are less familiar with PP than BP (23). The increased public communications regarding the PP epidemic in urban areas, as well as the increased availability there of public health resources from mobilization of substantial domestic and international public health resources (1), may have led some infected persons from rural areas to travel to Antananarivo to seek treatment. A previous study (11) described evidence of plague-infected persons moving from the central highlands to Mahajanga and from Mahajanga to Antananarivo.

In conclusion, the 2017 urban PP epidemic in Madagascar involved the introduction of multiple independent lineages of *Y. pestis* from several rural foci, which may partly explain why this epidemic was so difficult to control. Because PP spreads person-to-person, control of PP outbreaks and epidemics is focused on identifying cases and their known contacts and providing antimicrobial treatment. Those types of investigations were largely impossible in this instance, given the extent of the epidemic, which likely hampered control efforts. Our results suggest that control efforts also might have been diminished by the presence of multiple independent transmission chains that may have resulted in continuation or expansion of the epidemic. Our findings highlight the importance of using existing genotyping tools (24) and developing genomics capabilities in Madagascar, elsewhere in Africa, and other global locations (25) so they can be used during outbreaks of plague and other diseases to promptly identify multiple sources and transmission chains to better inform control efforts.

Funding for this work was provided by LabEx Integrative Biology of Emerging Infectious Diseases (grant no. ANR LBX-62 IBEID), the US Department of Defense's Defense Threat Reduction Agency (contract no. HDTRA1-20-C-0007), the Department of International Affairs of the Institut Pasteur via a cooperative agreement with the Office of the Assistant Secretary for Preparedness and Response in the US Department of Health and Human Services (grant no. IDSEP140020-01-00), the Institut Pasteur via the ANR Investissement d'Avenir Program (grant no. 10-LABX-62-IBEID), and the US Agency for International Development (grant no. AID-687-G-13-0003). The World Health Organization assisted with transport of biological samples from healthcare facilities.
## About the Author

Dr. Andrianayoarimanana is a researcher in the Plague Unit at the Institut Pasteur de Madagascar. Her research interests are immune response between *Y. pestis* and its hosts and plague epidemiology.

## References

1. Bertherat E. World Health Organization. Plague around the world in 2019. Wkly Epidemiol Rec. 2019;94:289-92.

2. Andrianayoarimanana V, Piola P, Wagner DM, Rakotomanana F, Maheriniaina V, Andrianalimanana S, et al. Trends of human plague, Madagascar, 1998–2016. Emerg Infect Dis. 2019;25:220–8. https://doi.org/10.3201/eid2502.171974

3. Andrianayoarimanana V, Kreppel K, Elissa N, Duplantier JM, Carniel E, Rajerison M, et al. Understanding the persistence of plague foci in Madagascar. PLoS Negl Trop Dis. 2013;7:e2382. https://doi.org/10.1371/journal.pntd.0002382

4. Duplantier J-M, Duchemin J-B, Chanteau S, Carniel E. From the recent lessons of the Malagasy foci towards a global understanding of the factors involved in plague reemergence. Vet Res. 2005;36:437–53. https://doi.org/10.1051/vetres:2005007

5. Chanteau S, Ratsitorahina M, Rahalison L, Rasoamanana B, Chan F, Boisier P, et al. Current epidemiology of human plague in Madagascar. Microbes Infect. 2000;2:25–31. https://doi.org/10.1016/S1286-4579(00)00289-6

6. Migliani R, Chanteau S, Rahalison L, Ratsitorahina M, Boutin JP, Ratsifasoamanana L, et al. Epidemiological trends for human plague in Madagascar during the second half of the 20th century: a survey of 20,900 notified cases. Trop Med Int Health. 2006;11:1228–37. https://doi.org/10.1111/j.1365-3156.2006.01677.x

7. Ratsitorahina M, Chanteau S, Rahalison L, Ratsifasoamanana L, Boisier P. Epidemiological and diagnostic aspects of the outbreak of pneumonic plague in Madagascar. Lancet. 2000; 355:111–3. https://doi.org/10.1016/S0140-6736(99)05163-6

8. Andrianayoarimanana V, Wagner DM, Birdsell DN, Nikolay B, Rakotoarimanana F, Randriantseheno LN, et al. Transmission of antimicrobial resistant *Yersinia pestis* during a pneumonic plague outbreak. Clin Infect Dis. 2022;74:695–702. https://doi.org/10.1093/cid/ciab606

9. Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, Wagner DM, et al. *Yersinia pestis* genome sequencing identifies patterns of global phylogenetic diversity. Nat Genet. 2010;42:1140–3. https://doi.org/10.1038/ng.705

10. Vogler AJ, Andrianayoarimanana V, Telfer S, Hall CM, Sahl JW, Hepp CM, et al. Temporal phylogeography of *Yersinia pestis* in Madagascar: insights into the long-term maintenance of plague. PLoS Negl Trop Dis. 2017; 11:e0005887. https://doi.org/10.1371/journal.pntd.0005887

11. Vogler AJ, Chan F, Nottingham R, Andersen G, Drees K, Beckstrom-Sternberg SM, et al. A decade of plague in Mahajanga, Madagascar: insights into the global maritime spread of pandemic plague. MBio. 2013;4:e00623–12. https://doi.org/10.1128/mBio.00623-12

12. Vogler AJ, Chan F, Wagner DM, Roumagnac P, Lee J, Nera R, et al. Phylogeography and molecular epidemiology of *Yersinia pestis* in Madagascar. PLoS Negl Trop Dis. 2011;5:e1319. https://doi.org/10.1371/journal.pntd.0001319

13. Randremanana R, Andrianayoarimanana V, Nikolay B, Ramasindrazana B, Paireau J, Ten Bosch QA, et al.

Epidemiological characteristics of an urban plague epidemic in Madagascar, August–November, 2017: an outbreak report. Lancet Infect Dis. 2019;19:537–45. https://doi.org/10.1016/S1473-3099(18)30730-8

14. Mead P. Epidemics of plague past, present, and future. Lancet Infect Dis. 2019;19:459–60. https://doi.org/10.1016/S1473-3099(18)30794-1

15. Apangu T, Acayo S, Atiku LA, Apio H, Candini G, Okoth F, et al. Intervention to stop transmission of imported pneumonic plague—Uganda, 2019. MMWR Morb Mortal Wkly Rep. 2020;69:241–4. https://doi.org/10.15585/mmwr.mm6909a5

16. Begier EM, Asiki G, Anywaine Z, Yockey B, Schriefer ME, Aleti P, et al. Pneumonic plague cluster, Uganda, 2004. Emerg Infect Dis. 2006;12:460–7. https://doi.org/10.3201/eid1203.051051

17. Jullien S, de Silva NL, Garner P. Plague transmission from corpses and carcasses. Emerg Infect Dis. 2021;27:2033–41. https://doi.org/10.3201/eid2708.200136

18. Kool JL, Weinstein RA. Risk of person-to-person transmission of pneumonic plague. Clin Infect Dis. 2005;40:1166–72. https://doi.org/10.1086/428617

19. Wulf V, Misaki K, Randall D, Rohde M. Travelling by taxi brousse in Madagascar. An investigation into practices of overland transportation. Media in Action Interdisciplinary Journal on Cooperative Media. 2018;1:57–97.

20. Salam AP, Raberahona M, Andriantsalama P, Read L, Andrianarintsiferantsoa F, Razafinambintsoa T, et al. Factors influencing atypical clinical presentations during the 2017 Madagascar pneumonic plague outbreak: a prospective cohort study. Am J Trop Med Hyg. 2020;102:1309–15. https://doi.org/10.4269/ajtmh.19-0576

21. Ten Bosch Q, Andrianayoarimanana V, Ramasindrazana B, Mikaty G, Rakotonanahary RJL, Nikolay B, et al. Analytical framework to evaluate and optimize the use of imperfect diagnostics to inform outbreak response: application to the 2017 plague epidemic in Madagascar. PLoS Biol. 2022; 20:e3001736. https://doi.org/10.1371/journal.pbio.3001736

22. World Health Organization. Plague outbreak in Madagascar: external situation report 01. 2017 [cited 2023 May 20], https://iris.who.int/bitstream/handle/10665/259181/Ext-PlagueMadagascar4102017.pdf

23. Rakotosamimanana S, Rakotoarimanana FJ, Raharimanga V, Taglioni F, Ramamonjisoa J, Randremanana RV, et al. Influence of sociospatial determinants on knowledge, attitudes and practices related to the plague in a population living in endemic areas in the central highlands, Madagascar. BMC Public Health. 2021;21:1102. https://doi.org/10.1186/s12889-021-11101-3

24. Mitchell CL, Andrianayoarimanana V, Colman RE, Busch J, Hornstra-O'Neill H, Keim PS, et al. Low cost, low tech SNP genotyping tools for resource-limited areas: plague in Madagascar as a model. PLoS Negl Trop Dis. 2017;11:e0006077. https://doi.org/10.1371/journal.pntd.0006077

25. Inzaule SC, Tessema SK, Kebede Y, Ogwell Ouma AE, Nkengasong JN. Genomic-informed pathogen surveillance in Africa: opportunities and challenges. Lancet Infect Dis. 2021;21:e281–9. https://doi.org/10.1016/S1473-3099(20)30939-7

Address for correspondence: Minoarisoa Rajerison, Plague Unit, Institut Pasteur de Madagascar, BP 1274 Ambatofotsikely, Antananarivo 101, Madagascar; email: mino@pasteur.mg
## About the Author

Dr. Andrianaitvoarimanana is a researcher in the Plague Unit at the Institut Pasteur de Madagascar. Her research interests are immune response between *Y. pestis* and its hosts and plague epidemiology.

## References

1. Bertherat E. World Health Organization. Plague around the world in 2019. Wkly Epidemiol Rec. 2019;94:289-92.

2. Andrianaitvoarimanana V, Piola P, Wagner DM, Rakotomanana F, Maheriniaina V, Andrianalimanana S, et al. Trends of human plague, Madagascar, 1998–2016. Emerg Infect Dis. 2019;25:220–8. https://doi.org/10.3201/eid2502.171974

3. Andrianaitvoarimanana V, Kreppel K, Elissa N, Duplantier JM, Carniel E, Rajerison M, et al. Understanding the persistence of plague foci in Madagascar. PLoS Negl Trop Dis. 2013;7:e2382. https://doi.org/10.1371/journal.pntd.0002382

4. Duplantier J-M, Duchemin J-B, Chanteau S, Carniel E. From the recent lessons of the Malagasy foci towards a global understanding of the factors involved in plague reemergence. Vet Res. 2005;36:437–53. https://doi.org/10.1051/vetres:2005007

5. Chanteau S, Ratsitorahina M, Rahalison L, Rasoamanana B, Chan F, Boisier P, et al. Current epidemiology of human plague in Madagascar. Microbes Infect. 2000;2:25–31. https://doi.org/10.1016/S1286-4579(00)00289-6

6. Migliani R, Chanteau S, Rahalison L, Ratsitorahina M, Boutin JP, Ratsifasoamanana L, et al. Epidemiological trends for human plague in Madagascar during the second half of the 20th century: a survey of 20,900 notified cases. Trop Med Int Health. 2006;11:1228–37. https://doi.org/10.1111/j.1365-3156.2006.01677.x

7. Ratsitorahina M, Chanteau S, Rahalison L, Ratsifasoamanana L, Boisier P. Epidemiological and diagnostic aspects of the outbreak of pneumonic plague in Madagascar. Lancet. 2000; 355:111–3. https://doi.org/10.1016/S0140-6736(99)05163-6

8. Andrianaitvoarimanana V, Wagner DM, Birdsell DN, Nikolay B, Rakotoarimanana F, Randriantseheno LN, et al. Transmission of antimicrobial resistant *Yersinia pestis* during a pneumonic plague outbreak. Clin Infect Dis. 2022;74:695–702. https://doi.org/10.1093/cid/ciab606

9. Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, Wagner DM, et al. Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nat Genet. 2010;42:1140–3. https://doi.org/10.1038/ng.705

10. Vogler AJ, Andrianaitvoarimanana V, Telfer S, Hall CM, Sahl JW, Hepp CM, et al. Temporal phylogeography of *Yersinia pestis* in Madagascar: insights into the long-term maintenance of plague. PLoS Negl Trop Dis. 2017; 11:e0005887. https://doi.org/10.1371/journal.pntd.0005887

11. Vogler AJ, Chan F, Nottingham R, Andersen G, Drees K, Beckstrom-Sternberg SM, et al. A decade of plague in Mahajanga, Madagascar: insights into the global maritime spread of pandemic plague. MBio. 2013;4:e00623–12. https://doi.org/10.1128/mBio.00623-12

12. Vogler AJ, Chan F, Wagner DM, Roumagnac P, Lee J, Nera R, et al. Phylogeography and molecular epidemiology of *Yersinia pestis* in Madagascar. PLoS Negl Trop Dis. 2011;5:e1319. https://doi.org/10.1371/journal.pntd.0001319

13. Randremanana R, Andrianaitvoarimanana V, Nikolay B, Ramasindrazana B, Paireau J, Ten Bosch QA, et al.

Epidemiological characteristics of an urban plague epidemic in Madagascar, August–November, 2017: an outbreak report. Lancet Infect Dis. 2019;19:537–45. https://doi.org/10.1016/S1473-3099(18)30730-8

14. Mead P. Epidemics of plague past, present, and future. Lancet Infect Dis. 2019;19:459–60. https://doi.org/10.1016/S1473-3099(18)30794-1

15. Apangu T, Acayo S, Atiku LA, Apio H, Candini G, Okoth F, et al. Intervention to stop transmission of imported pneumonic plague—Uganda, 2019. MMWR Morb Mortal Wkly Rep. 2020;69:241–4. https://doi.org/10.15585/mmwr.mm6909a5

16. Begier EM, Asiki G, Anywaine Z, Yockey B, Schriefer ME, Aleti P, et al. Pneumonic plague cluster, Uganda, 2004. Emerg Infect Dis. 2006;12:460–7. https://doi.org/10.3201/eid1203.051051

17. Jullien S, de Silva NL, Garner P. Plague transmission from corpses and carcasses. Emerg Infect Dis. 2021;27:2033–41. https://doi.org/10.3201/eid2708.200136

18. Kool JL, Weinstein RA. Risk of person-to-person transmission of pneumonic plague. Clin Infect Dis. 2005;40:1166–72. https://doi.org/10.1086/428617

19. Wulf V, Misaki K, Randall D, Rohde M. Travelling by taxi brousse in Madagascar. An investigation into practices of overland transportation. Media in Action Interdisciplinary Journal on Cooperative Media. 2018;1:57–97.

20. Salam AP, Raberahona M, Andriantsalama P, Read L, Andrianarintsiferantsoa F, Razafinambintsoa T, et al. Factors influencing atypical clinical presentations during the 2017 Madagascar pneumonic plague outbreak: a prospective cohort study. Am J Trop Med Hyg. 2020;102:1309–15. https://doi.org/10.4269/ajtmh.19-0576

21. Ten Bosch Q, Andrianaitvoarimanana V, Ramasindrazana B, Mikaty G, Rakotonanahary RJL, Nikolay B, et al. Analytical framework to evaluate and optimize the use of imperfect diagnostics to inform outbreak response: application to the 2017 plague epidemic in Madagascar. PLoS Biol. 2022; 20:e3001736. https://doi.org/10.1371/journal.pbio.3001736

22. World Health Organization. Plague outbreak in Madagascar: external situation report 01. 2017 [cited 2023 May 20], https://iris.who.int/bitstream/handle/10665/259181/Ext-PlagueMadagascar4102017.pdf

23. Rakotosamimanana S, Rakotoarimanana FJ, Raharimanga V, Taglioni F, Ramamonjisoa J, Randremanana RV, et al. Influence of sociospatial determinants on knowledge, attitudes and practices related to the plague in a population living in endemic areas in the central highlands, Madagascar. BMC Public Health. 2021;21:1102. https://doi.org/10.1186/s12889-021-11101-3

24. Mitchell CL, Andrianaitvoarimanana V, Colman RE, Busch J, Hornstra-O'Neill H, Keim PS, et al. Low cost, low tech SNP genotyping tools for resource-limited areas: plague in Madagascar as a model. PLoS Negl Trop Dis. 2017;11:e0006077. https://doi.org/10.1371/journal.pntd.0006077

25. Inzaule SC, Tessema SK, Kebede Y, Ogwell Ouma AE, Nkengasong JN. Genomic-informed pathogen surveillance in Africa: opportunities and challenges. Lancet Infect Dis. 2021;21:e281–9. https://doi.org/10.1016/S1473-3099(20)30939-7
Article DOI: http://doi.org/10.3201/eid3002.230759

EID cannot ensure accessibility for supplementary materials supplied by authors. Readers who have difficulty accessing supplementary content should contact the authors for assistance.

# Multiple Introductions of *Yersinia pestis* during Urban Pneumonic Plague Epidemic, Madagascar, 2017

## Appendix

### 1. Collection and Testing of Clinical Samples

Biologic samples (bubo, sputum, blood, liver, and lung biopsies) from clinically suspected plague patients were sent to the Malagasy Central Laboratory for Plague (CLP) housed at the Plague Unit of the Institut Pasteur de Madagascar (IPM) for biologic confirmation as part of Madagascar's national plague surveillance program overseen by the Malagasy Ministry of Health. This program requires declaration of all suspected human plague cases and collection of biologic samples from those cases. These samples and any cultures or DNA derived from those samples are all delinked from the patients from whom they originated and are analyzed anonymously if used in any research study, such as this one. As such, additional review of this research was not required due to the anonymous nature of the samples. Storage conditions for samples varied before receipt at IPM, but once at IPM all samples were at -20°C. A rapid diagnostic test (RDT) against F1-antigen (1) (produced by IPM) was performed on samples from most suspect cases, and DNA was extracted from many samples and tested with PCR, as previously described (2). Each sample also was streaked on selective medium Cefsulodin Irgasan Novobiocin (CIN, Oxoid) agar plates and cultured in peptone broth before incubation at 26°C for more than 48h. In parallel, amplification by intraperitoneal injection of the sample was also performed in two laboratory mice (*Mus musculus*) that were subsequently observed for 10 days; this work was performed in a BSL2 animal facility at IPM. These animal studies were performed in accordance with the directive 2010/63/EU of the European Parliament (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF). If a mouse died between 2–10 days post injection, the spleen was removed, macerated, and cultured on CIN
Article DOI: http://doi.org/10.3201/eid3002.230759

EID cannot ensure accessibility for supplementary materials supplied by authors. Readers who have difficulty accessing supplementary content should contact the authors for assistance.

# Multiple Introductions of *Yersinia pestis* during Urban Pneumonic Plague Epidemic, Madagascar, 2017

## Appendix

### 1. Collection and Testing of Clinical Samples

Biologic samples (bubo, sputum, blood, liver, and lung biopsies) from clinically suspected plague patients were sent to the Malagasy Central Laboratory for Plague (CLP) housed at the Plague Unit of the Institut Pasteur de Madagascar (IPM) for biologic confirmation as part of Madagascar's national plague surveillance program overseen by the Malagasy Ministry of Health. This program requires declaration of all suspected human plague cases and collection of biologic samples from those cases. These samples and any cultures or DNA derived from those samples are all delinked from the patients from whom they originated and are analyzed anonymously if used in any research study, such as this one. As such, additional review of this research was not required due to the anonymous nature of the samples. Storage conditions for samples varied before receipt at IPM, but once at IPM all samples were at -20°C. A rapid diagnostic test (RDT) against F1-antigen (1) (produced by IPM) was performed on samples from most suspect cases, and DNA was extracted from many samples and tested with PCR, as previously described (2). Each sample also was streaked on selective medium Cefsulodin Irgasan Novobiocin (CIN, Oxoid) agar plates and cultured in peptone broth before incubation at 26°C for more than 48h. In parallel, amplification by intraperitoneal injection of the sample was also performed in two laboratory mice (*Mus musculus*) that were subsequently observed for 10 days; this work was performed in a BSL2 animal facility at IPM. These animal studies were performed in accordance with the directive 2010/63/EU of the European Parliament (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF). If a mouse died between 2–10 days post injection, the spleen was removed, macerated, and cultured on CIN
agar plate for isolation of *Y. pestis* (3). Suspected *Y. pestis* colonies were then enriched on Brain Heart Infusion slant agar media for 24h before biochemical identification using API20E (Biomérieux) strips and bacteriophage lysis test. Isolated *Y. pestis* strains were stored in glycerol conservation media at -80°C and sent on dry ice to the Yersinia Research Unit of the Institut Pasteur in Paris, France for genomic sequencing and analysis.

**2. Antimicrobial Testing of Isolates**

Each *Y. pestis* isolate was tested for susceptibility to gentamycin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, and streptomycin using a disk diffusion method as per the Clinical Laboratory Standards Institute (CSLI) guidelines, as previously described (4). All were susceptible to all tested antimicrobials.

**3. Whole-Genome Sequencing of Isolates**

Isolates were subcultured in Luria-Bertani broth at 28°C for 48h. Two ml of culture was centrifuged at 6,000 g for 5 minutes. The pellet was then subjected to DNA extraction with PureLinkTM Genomic DNA minikit (K1820-02, Invitrogen) following the manufacturer's instructions except that DNA was eluted using 150 µL of nuclease-free H2O (AM9937, Ambion). Preparation of the libraries for genomic sequencing was performed with TruSeq Nano DNA library HT Prep kit (Illumina, San Diego, California, USA) following the manufacturer's instructions. Library quality was evaluated with Agilent High sensitivity DNA chips (2100 Bioanalyzer, Agilent) and libraries were quantified with KAPA library Quantification kit (Lightcycler®480, Roche). The resulting libraries were strategically clustered to achieve at least 1.5 M of reads per sample and sequenced on an Illumina MiSeq platform for 250bp paired-end sequencing with MiSeq Reagent Kit v2 (500-cycles). Raw sequence data for all strains have been deposited at NCBI under BioProject PRJNA504742; accession numbers for individual strains are provided in Appendix 1 Table 1.

**4. SNP Discovery from Isolates and Phylogenetic Methods**

Single nucleotide polymorphisms (SNP) were discovered from whole genome sequences of 90 *Y. pestis* isolates from Madagascar; accession numbers and other information are provided
agar plate for isolation of *Y. pestis* (3). Suspected *Y. pestis* colonies were then enriched on Brain Heart Infusion slant agar media for 24h before biochemical identification using API20E (Biomérieux) strips and bacteriophage lysis test. Isolated *Y. pestis* strains were stored in glycerol conservation media at -80°C and sent on dry ice to the Yersinia Research Unit of the Institut Pasteur in Paris, France for genomic sequencing and analysis.

## 2. Antimicrobial Testing of Isolates

Each *Y. pestis* isolate was tested for susceptibility to gentamycin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, and streptomycin using a disk diffusion method as per the Clinical Laboratory Standards Institute (CSLI) guidelines, as previously described (4). All were susceptible to all tested antimicrobials.

## 3. Whole-Genome Sequencing of Isolates

Isolates were subcultured in Luria-Bertani broth at 28°C for 48h. Two ml of culture was centrifuged at 6,000 g for 5 minutes. The pellet was then subjected to DNA extraction with PureLinkTM Genomic DNA minikit (K1820-02, Invitrogen) following the manufacturer's instructions except that DNA was eluted using 150 µL of nuclease-free H2O (AM9937, Ambion). Preparation of the libraries for genomic sequencing was performed with TruSeq Nano DNA library HT Prep kit (Illumina, San Diego, California, USA) following the manufacturer's instructions. Library quality was evaluated with Agilent High sensitivity DNA chips (2100 Bioanalyzer, Agilent) and libraries were quantified with KAPA library Quantification kit (Lightcycler®480, Roche). The resulting libraries were strategically clustered to achieve at least 1.5 M of reads per sample and sequenced on an Illumina MiSeq platform for 250bp paired-end sequencing with MiSeq Reagent Kit v2 (500-cycles). Raw sequence data for all strains have been deposited at NCBI under BioProject PRJNA504742; accession numbers for individual strains are provided in Appendix 1 Table 1.

## 4. SNP Discovery from Isolates and Phylogenetic Methods

Single nucleotide polymorphisms (SNP) were discovered from whole genome sequences of 90 *Y. pestis* isolates from Madagascar; accession numbers and other information are provided
in Appendix 1 Table 1. Raw reads were aligned against reference genome *Y. pestis* CO92 (GCF_000009065.1) (5) with minimap2 v2.24 (6). SNPs were called with GATK v4.2.6.1 (7). SNPs with a depth of coverage <10 or a proportion <0.90 were filtered from downstream analyses. Any position that fell within a duplicated region, based on a reference self-alignment with NUCmer v3.1 (8), was also filtered; all methods were wrapped by NASP v1.2.0 (9). Additional positions were filtered from the matrix if they were associated with known CO92 errors (9), were in close proximity to other SNPs (<5 nt), or were found in tandem repeats annotated with Tandem Repeats Finder v4.10.0 (10). A maximum likelihood phylogeny was inferred from a concatenated SNP alignment with IQ-TREE v2.2.0.3 (11) using the integrated ModelFinder method (12). The final phylogeny was constructed using 483 SNPs (see separate supplemental Excel file [Appendix 2], derived SNP allele states are highlighted in blue in that file), including 455 SNPs that varied among the 90 *Y. pestis* isolates from Madagascar and an additional 28 SNPs that differed between CO92 and all *Y. pestis* isolates from Madagascar.

**5. Design of *Y. pestis* DNA Capture and Enrichment System**

The pan-genome of a set of 373 *Y. pestis* genomes was determined with LS-BSR v1.2.3 (13), resulting in 16,171 unique coding region sequences (CDSs). CDSs shorter than 120nts were filtered from the analysis, as well as any genes containing ambiguous nucleotide characters, leaving 13,290 CDSs. CDSs were sliced into 120nt fragments, overlapping by 60nts, resulting in 126,938 potential capture probes. These probes were clustered with USEARCH v11 (14) at 80% ID, resulting in 111,329 remaining probes. Probes were then screened against *Y. pestis* rRNA genes (5S, 16S, 23S) with LS-BSR, and probes with a blast score ratio (BSR) value >0.8 (15) to any rRNA gene were removed. Remaining probes were aligned back against the set of 373 *Y. pestis* genomes with LS-BSR and BSR values were calculated. Probes conserved only in a single *Y. pestis* genome (BSR value >0.8) were removed as they could represent contamination, resulting in a set of 107,048 potential probes. Remaining probes were screened with LS-BSR against a set of >125,000 non-*Y. pestis* bacterial genomes in GenBank and those with a BSR of >0.8 were removed. Because we enriched from human sputum samples, the probes also were aligned against the human genome and any probes with significant alignments were removed from the design, resulting in a final set of 99,353 probes that covered ~88.8% of the CO92 reference genome. Regions with extremely high GC content (>50% GC) or extremely low GC
in Appendix 1 Table 1. Raw reads were aligned against reference genome *Y. pestis* CO92 (GCF_000009065.1) (5) with minimap2 v2.24 (6). SNPs were called with GATK v4.2.6.1 (7). SNPs with a depth of coverage <10 or a proportion <0.90 were filtered from downstream analyses. Any position that fell within a duplicated region, based on a reference self-alignment with NUCmer v3.1 (8), was also filtered; all methods were wrapped by NASP v1.2.0 (9). Additional positions were filtered from the matrix if they were associated with known CO92 errors (9), were in close proximity to other SNPs (<5 nt), or were found in tandem repeats annotated with Tandem Repeats Finder v4.10.0 (10). A maximum likelihood phylogeny was inferred from a concatenated SNP alignment with IQ-TREE v2.2.0.3 (11) using the integrated ModelFinder method (12). The final phylogeny was constructed using 483 SNPs (see separate supplemental Excel file [Appendix 2], derived SNP allele states are highlighted in blue in that file), including 455 SNPs that varied among the 90 *Y. pestis* isolates from Madagascar and an additional 28 SNPs that differed between CO92 and all *Y. pestis* isolates from Madagascar.

**5. Design of *Y. pestis* DNA Capture and Enrichment System**

The pan-genome of a set of 373 *Y. pestis* genomes was determined with LS-BSR v1.2.3 (13), resulting in 16,171 unique coding region sequences (CDSs). CDSs shorter than 120nts were filtered from the analysis, as well as any genes containing ambiguous nucleotide characters, leaving 13,290 CDSs. CDSs were sliced into 120nt fragments, overlapping by 60nts, resulting in 126,938 potential capture probes. These probes were clustered with USEARCH v11 (14) at 80% ID, resulting in 111,329 remaining probes. Probes were then screened against *Y. pestis* rRNA genes (5S, 16S, 23S) with LS-BSR, and probes with a blast score ratio (BSR) value >0.8 (15) to any rRNA gene were removed. Remaining probes were aligned back against the set of 373 *Y. pestis* genomes with LS-BSR and BSR values were calculated. Probes conserved only in a single *Y. pestis* genome (BSR value >0.8) were removed as they could represent contamination, resulting in a set of 107,048 potential probes. Remaining probes were screened with LS-BSR against a set of >125,000 non-*Y. pestis* bacterial genomes in GenBank and those with a BSR of >0.8 were removed. Because we enriched from human sputum samples, the probes also were aligned against the human genome and any probes with significant alignments were removed from the design, resulting in a final set of 99,353 probes that covered ~88.8% of the CO92 reference genome. Regions with extremely high GC content (>50% GC) or extremely low GC
content (<22%) are considered difficult to hybridize using probes. To increase the likelihood of capturing these regions, our library design included a boosting strategy wherein probes corresponding to these types of regions were multiplied by 2X-10X copies, assigning higher redundancy to the most extreme regions (>70% and <15% GC). The final set of probes was ordered from Agilent Technologies.

## 6. *Y. pestis* DNA Capture, Enrichment, and Sequencing from Select Sputum Samples

**DNA extraction of clinical samples and selection process for enrichment.** *Y. pestis* DNA capture, enrichment, and sequencing was attempted on 42 residual sputum samples (Appendix 1 Table 2). The original 42 sputum samples were obtained from cases categorized as probable or confirmed based upon the testing procedures detailed in Section 1 above. These 42 samples were selected for *Y. pestis* DNA capture and enrichment because they were obtained from the urban areas of Antananarivo or Toamasina, or were associated with the first urban transmission chain (Table 2). DNA was extracted from remaining residuals of these 42 sputum samples using the Qiagen QIAmp kit following manufacture protocol (Qiagen, Valencia, CA) and the resulting extracts were shipped from IPM to NAU. At NAU, an additional PCR was performed using a published *pla*-specific TaqMan assay (16); all 42 samples yielded positive results (Appendix 1 Table 2).

**Library preparation.** The complex DNA extracts (<1 ng-100 ng) from the 42 human residual sputum samples were sheared to ~250 bp using a QSonica Q800 Sonicator (QSonica, Newtown, CT) at 60% amplitude, with 15 sec on/off settings. Size of fragments was evaluated using a Fragment Analyzer (Agilent, Santa Clara, CA). Sheared samples were end-repaired, A-tailed, indexed with adaptor ligation, and amplified using the SureSelect XT-low input (LI) sample kit (Agilent Technologies, Santa Clara, CA). All DNA purification steps were carried out using Agencourt AMPure XP beads (0.8X bead ratio; Beckman Coulter, Brea, CA). Briefly, end repair and A-tailing of the sheared DNA were completed using the SureSelectXT-LI End Repair/A-tailing Reaction Mix (Agilent Technologies, Santa Clara, CA). A single index adaptor was ligated to the ends of the DNA fragments using the SureSelectXT Ligation Master Mix, followed by bead purification. Each ligated fragment was uniquely indexed through PCR-
content (<22%) are considered difficult to hybridize using probes. To increase the likelihood of capturing these regions, our library design included a boosting strategy wherein probes corresponding to these types of regions were multiplied by 2X-10X copies, assigning higher redundancy to the most extreme regions (>70% and <15% GC). The final set of probes was ordered from Agilent Technologies.

## 6. *Y. pestis* DNA Capture, Enrichment, and Sequencing from Select Sputum Samples

**DNA extraction of clinical samples and selection process for enrichment.** *Y. pestis* DNA capture, enrichment, and sequencing was attempted on 42 residual sputum samples (Appendix 1 Table 2). The original 42 sputum samples were obtained from cases categorized as probable or confirmed based upon the testing procedures detailed in Section 1 above. These 42 samples were selected for *Y. pestis* DNA capture and enrichment because they were obtained from the urban areas of Antananarivo or Toamasina, or were associated with the first urban transmission chain (Table 2). DNA was extracted from remaining residuals of these 42 sputum samples using the Qiagen QIAmp kit following manufacture protocol (Qiagen, Valencia, CA) and the resulting extracts were shipped from IPM to NAU. At NAU, an additional PCR was performed using a published *pla*-specific TaqMan assay (16); all 42 samples yielded positive results (Appendix 1 Table 2).

**Library preparation.** The complex DNA extracts (<1 ng-100 ng) from the 42 human residual sputum samples were sheared to ~250 bp using a QSonica Q800 Sonicator (QSonica, Newtown, CT) at 60% amplitude, with 15 sec on/off settings. Size of fragments was evaluated using a Fragment Analyzer (Agilent, Santa Clara, CA). Sheared samples were end-repaired, A-tailed, indexed with adaptor ligation, and amplified using the SureSelect XT-low input (LI) sample kit (Agilent Technologies, Santa Clara, CA). All DNA purification steps were carried out using Agencourt AMPure XP beads (0.8X bead ratio; Beckman Coulter, Brea, CA). Briefly, end repair and A-tailing of the sheared DNA were completed using the SureSelectXT-LI End Repair/A-tailing Reaction Mix (Agilent Technologies, Santa Clara, CA). A single index adaptor was ligated to the ends of the DNA fragments using the SureSelectXT Ligation Master Mix, followed by bead purification. Each ligated fragment was uniquely indexed through PCR-
amplification for 9 cycles (2 min at 98°C, 9 cycles for 30 s at 98°C, 30 s at 60°C, 1 min at 72°C, and a final extension of 5 min at 72°C), followed by bead purification. Final library size and quantity were assessed by Fragment Analyzer and Qubit Br dsDNA (ThermoFisher Scientific, Waltham, MA), respectively.

**Probe-hybridization.** To reduce the probability of capturing off-targets, each sample library was enriched in a single reaction instead of pooling multiple samples. Due to the ~50% GC content of the *Y. pestis* genome, the fast-hybridization protocol using hybridization reagents from SureSelect (Agilent Technologies, Santa Clara, CA) was used. Approximately 2000 ng of the total library was hybridized at 65°C for 3 hours (following manufacturer's protocol) with probes. Hybridized libraries were recovered using 50 uL Dynabeads MyOne Streptavidin T1 Kit (Thermo Fisher Scientific, Waltham, MA). To remove off target fragments, the beads underwent two different washing procedures according to the manufacturer's directions. PCR amplification occurred directly from washed beads with the SureSelect XT-LI Primer Mix (Agilent Technologies, Santa Clara, CA) using the same PCR conditions as the library preparation but with a 14-cycle parameter to increase the concentration of captured library. The amplicons were separated from the beads using a magnetic plate and transferred to a new tube. To amplify the residual capture library remaining on the beads, a second PCR was performed directly on the beads using KAPA HiFi PCR ready mix (Roche KAPA Biosystems, Wilmington, DE Cat# KK2612). The captured libraries from both PCR events were combined and purified. To further enrich for *Y. pestis* signal, we performed a second round of capture enrichment on the captured libraries following the same method as the first enrichment. Library quantity was assessed by Qubit Br dsDNA (Thermo Fisher Scientific, Waltham, MA). The size and quality were assessed by Fragment Analyzer.

**Sequencing.** Final libraries were quantified using the KAPA Library Quantification Kit (ROX Low, Roche KAPA Biosystems, Wilmington, DE) and pooled in equimolar concentrations. Sequencing of the 42 enriched libraries was performed using the Illumina NextSeq 550 platform with the 500/550 High Output KT v2.3 (300 cycles) and 2 × 150 bp read lengths or MiSeq V3 (600 cycles) and 2 × 300 bp read lengths (Illumina, San Diego, CA).
amplification for 9 cycles (2 min at 98°C, 9 cycles for 30 s at 98°C, 30 s at 60°C, 1 min at 72°C, and a final extension of 5 min at 72°C), followed by bead purification. Final library size and quantity were assessed by Fragment Analyzer and Qubit Br dsDNA (ThermoFisher Scientific, Waltham, MA), respectively.

**Probe-hybridization.** To reduce the probability of capturing off-targets, each sample library was enriched in a single reaction instead of pooling multiple samples. Due to the ~50% GC content of the *Y. pestis* genome, the fast-hybridization protocol using hybridization reagents from SureSelect (Agilent Technologies, Santa Clara, CA) was used. Approximately 2000 ng of the total library was hybridized at 65°C for 3 hours (following manufacturer's protocol) with probes. Hybridized libraries were recovered using 50 uL Dynabeads MyOne Streptavidin T1 Kit (Thermo Fisher Scientific, Waltham, MA). To remove off target fragments, the beads underwent two different washing procedures according to the manufacturer's directions. PCR amplification occurred directly from washed beads with the SureSelect XT-LI Primer Mix (Agilent Technologies, Santa Clara, CA) using the same PCR conditions as the library preparation but with a 14-cycle parameter to increase the concentration of captured library. The amplicons were separated from the beads using a magnetic plate and transferred to a new tube. To amplify the residual capture library remaining on the beads, a second PCR was performed directly on the beads using KAPA HiFi PCR ready mix (Roche KAPA Biosystems, Wilmington, DE Cat# KK2612). The captured libraries from both PCR events were combined and purified. To further enrich for *Y. pestis* signal, we performed a second round of capture enrichment on the captured libraries following the same method as the first enrichment. Library quantity was assessed by Qubit Br dsDNA (Thermo Fisher Scientific, Waltham, MA). The size and quality were assessed by Fragment Analyzer.

**Sequencing.** Final libraries were quantified using the KAPA Library Quantification Kit (ROX Low, Roche KAPA Biosystems, Wilmington, DE) and pooled in equimolar concentrations. Sequencing of the 42 enriched libraries was performed using the Illumina NextSeq 550 platform with the 500/550 High Output KT v2.3 (300 cycles) and 2 × 150 bp read lengths or MiSeq V3 (600 cycles) and 2 × 300 bp read lengths (Illumina, San Diego, CA).
# 7. Analysis of Enriched *Y. pestis* DNA from Select Sputum Samples

*In silico* SNP analysis of enriched samples. Raw reads for the 42 enriched sputum samples (Appendix 1 Table 2) were aligned to the CO92 reference genome (GCF_000009065.1) with minimap2 v2.24 (6). PCR duplicates were removed with Samtools v1.6 (17) and SNPs were called with GATK v4.2.6 (7) (7); SNPs were compiled across all samples with NASP v1.2.0 (9). Specific SNP positions used to construct the maximum likelihood phylogeny (Figure 2 in the main text) were then manually queried from the resulting NASP matrix; novel *Y. pestis* SNPs that may have been present in these enriched samples were not called. Known SNPs were called if there was at least 3x depth of coverage and >80% agreement among the reads. Known SNPs were identified in seven of the 42 enriched samples; raw reads for these seven enriched samples are available at NCBI under BioProject PRJNA1018588. Coverage of the reference varied across the seven enriched samples with identified known SNPs (Appendix 1 Table 3).

Confirmation of SNP calls with TaqMan assays. To confirm the genotypes of the known SNPs from the maximum likelihood phylogeny that were called from the sequencing data generated from the seven analyzed sputum samples (Appendix 1 Table 3), six of those SNPs were incorporated into Dual Probe TaqMan real-time PCR canSNP assays (Appendix 1 Tables 4 and 5). Primers (Integrated DNA Technologies, San Diego, CA) and allele-specific TaqMan-minor groove binding (MGB) probes (Life Technologies, Applied Biosystems, Foster City, CA) for each assay were designed using Primer Express software (Applied Biosystems) and the CO92 *Y. pestis* reference genome. PCR conditions and probe concentration for different assays varied and are described in Appendix 1 Table 4.

PCR amplification per assay was carried out in 10 µL volume with either 1× TaqMan Universal PCR master mix (Life Technologies, Applied Biosystems, Foster City, CA) or an in-house custom master mix. The in-house custom master mix comprised 1x PCR buffer without MgCl₂, MgCl₂ 5 mM, 0.2 mM deoxynucleoside triphosphate, 0.4 units of platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 6.36 µL molecular grade water. Each assay contained 0.2 µM of probes (derived and ancestral allele-specific) and forward and reverse primers at 0.6 µM to achieve 10 µL total volume. Two negative controls per SNP assay and two allele positive controls (representing ancestral and derived SNP states) were included with each experiment. Ancestral allele templates were genomic DNA extracts from North American *Y*.
# 7. Analysis of Enriched *Y. pestis* DNA from Select Sputum Samples

*In silico* SNP analysis of enriched samples. Raw reads for the 42 enriched sputum samples (Appendix 1 Table 2) were aligned to the CO92 reference genome (GCF_000009065.1) with minimap2 v2.24 (6). PCR duplicates were removed with Samtools v1.6 (17) and SNPs were called with GATK v4.2.6 (7) (7); SNPs were compiled across all samples with NASP v1.2.0 (9). Specific SNP positions used to construct the maximum likelihood phylogeny (Figure 2 in the main text) were then manually queried from the resulting NASP matrix; novel *Y. pestis* SNPs that may have been present in these enriched samples were not called. Known SNPs were called if there was at least 3x depth of coverage and >80% agreement among the reads. Known SNPs were identified in seven of the 42 enriched samples; raw reads for these seven enriched samples are available at NCBI under BioProject PRJNA1018588. Coverage of the reference varied across the seven enriched samples with identified known SNPs (Appendix 1 Table 3).

Confirmation of SNP calls with TaqMan assays. To confirm the genotypes of the known SNPs from the maximum likelihood phylogeny that were called from the sequencing data generated from the seven analyzed sputum samples (Appendix 1 Table 3), six of those SNPs were incorporated into Dual Probe TaqMan real-time PCR canSNP assays (Appendix 1 Tables 4 and 5). Primers (Integrated DNA Technologies, San Diego, CA) and allele-specific TaqMan-minor groove binding (MGB) probes (Life Technologies, Applied Biosystems, Foster City, CA) for each assay were designed using Primer Express software (Applied Biosystems) and the CO92 *Y. pestis* reference genome. PCR conditions and probe concentration for different assays varied and are described in Appendix 1 Table 4.

PCR amplification per assay was carried out in 10 µL volume with either 1× TaqMan Universal PCR master mix (Life Technologies, Applied Biosystems, Foster City, CA) or an in-house custom master mix. The in-house custom master mix comprised 1x PCR buffer without MgCl₂, MgCl₂ 5 mM, 0.2 mM deoxynucleoside triphosphate, 0.4 units of platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 6.36 µL molecular grade water. Each assay contained 0.2 µM of probes (derived and ancestral allele-specific) and forward and reverse primers at 0.6 µM to achieve 10 µL total volume. Two negative controls per SNP assay and two allele positive controls (representing ancestral and derived SNP states) were included with each experiment. Ancestral allele templates were genomic DNA extracts from North American *Y*.
*pestis* isolates A1122 and CO92. Derived allele templates for all five TaqMan assays were synthetically designed and printed on Genomic-block gene fragment (Integrated DNA Technologies, San Diego, CA). Thermal cycling parameters were as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 15 s and 60 - 63°C (Appendix 1 Table 4) for 60 sec. All PCR amplifications were performed with the Applied Biosystems QuantStudio Flex Real-Time PCR System (Invitrogen, ThermoFisher).

**SNP genotyping data and interpretation.** Partial *Y. pestis* SNP genotyping calls were made for 14 of the SNPs used to construct the maximum likelihood phylogeny (Figure 2 in the main text) in seven enriched sputum samples (Appendix 1 Table 5). Complete genotyping data were not available for these samples due to the poor quality of the sputum samples (see main text and Appendix 1 Table 2). For this same reason, genotyping data were not available for the other 35 enriched sputum samples. The SNP genotypes that were determined were based upon sequence data generated from enriched sputum samples, TaqMan assays performed on the enriched sequencing libraries, or both (Appendix 1 Table 5); in all cases there was agreement between TaqMan and sequencing data when both data types were generated for a particular SNP in a given sample.

Just one isolate, 17/17, was obtained from the transmission chain described in detail in the main text, the transmission chain associated with Emergence 2. SNP calls were made in four sputum samples collected from human cases known to be associated with this transmission chain and one additional sputum sample not previously known to be associated with this transmission chain (Appendix 1 Table 5). One SNP specific to isolate 17/17 in the maximum likelihood phylogeny and two SNPs specific to phylogenetic group “s” or a subclade within this group in the maximum likelihood phylogeny were identified in sputum sample 135–2017; this sample was obtained on 11 September 2017 from Case 4 in this transmission chain. Five SNPs specific to isolate 17/17 in the maximum likelihood phylogeny and seven SNPs specific to phylogenetic group “s” or a subclade within this group in the maximum likelihood phylogeny were identified in sputum sample 121–2017; this sample was obtained on 11 September 2017 from Case 7 in this transmission chain. Note that sputum sample 121–2017 from Case 7 was also the source of isolate 17/17 (see main text); all SNPs genotyped in the enrichment of this sputum sample were identical in the whole genome sequence of isolate 17/17, providing confidence in the overall enrichment approach. Five SNPs specific to isolate 17/17 in the maximum likelihood phylogeny
*pestis* isolates A1122 and CO92. Derived allele templates for all five TaqMan assays were synthetically designed and printed on Genomic-block gene fragment (Integrated DNA Technologies, San Diego, CA). Thermal cycling parameters were as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 15 s and 60 - 63°C (Appendix 1 Table 4) for 60 sec. All PCR amplifications were performed with the Applied Biosystems QuantStudio Flex Real-Time PCR System (Invitrogen, ThermoFisher).

**SNP genotyping data and interpretation.** Partial *Y. pestis* SNP genotyping calls were made for 14 of the SNPs used to construct the maximum likelihood phylogeny (Figure 2 in the main text) in seven enriched sputum samples (Appendix 1 Table 5). Complete genotyping data were not available for these samples due to the poor quality of the sputum samples (see main text and Appendix 1 Table 2). For this same reason, genotyping data were not available for the other 35 enriched sputum samples. The SNP genotypes that were determined were based upon sequence data generated from enriched sputum samples, TaqMan assays performed on the enriched sequencing libraries, or both (Appendix 1 Table 5); in all cases there was agreement between TaqMan and sequencing data when both data types were generated for a particular SNP in a given sample.

Just one isolate, 17/17, was obtained from the transmission chain described in detail in the main text, the transmission chain associated with Emergence 2. SNP calls were made in four sputum samples collected from human cases known to be associated with this transmission chain and one additional sputum sample not previously known to be associated with this transmission chain (Appendix 1 Table 5). One SNP specific to isolate 17/17 in the maximum likelihood phylogeny and two SNPs specific to phylogenetic group “s” or a subclade within this group in the maximum likelihood phylogeny were identified in sputum sample 135–2017; this sample was obtained on 11 September 2017 from Case 4 in this transmission chain. Five SNPs specific to isolate 17/17 in the maximum likelihood phylogeny and seven SNPs specific to phylogenetic group “s” or a subclade within this group in the maximum likelihood phylogeny were identified in sputum sample 121–2017; this sample was obtained on 11 September 2017 from Case 7 in this transmission chain. Note that sputum sample 121–2017 from Case 7 was also the source of isolate 17/17 (see main text); all SNPs genotyped in the enrichment of this sputum sample were identical in the whole genome sequence of isolate 17/17, providing confidence in the overall enrichment approach. Five SNPs specific to isolate 17/17 in the maximum likelihood phylogeny
and seven SNPs specific to phylogenetic group “s” or a subclade within this group in the
maximum likelihood phylogeny were identified in sputum sample 125–2017; this sample was
obtained on 12 September 2017 from Case 15 in this transmission chain. One SNP specific to
isolate 17/17 in the maximum likelihood phylogeny was identified in sputum sample 184–2017;
this sample was obtained on 12 September 2017 from Case 22 in this transmission chain. Finally,
three SNPs specific to isolate 17/17 in the maximum likelihood phylogeny were identified in
sputum sample 2093–2017. This sample was obtained from an Antananarivo resident on 8
November 2017 with no recent travel outside the city and no known contact with the cases
above, suggesting community spread of this transmission chain in Antananarivo and persistence
there until at least November 2017.

A single derived SNP call was made from sputum sample 819–2017 (Appendix 1 Table
5), in a SNP that is specific to all of the “j” phylogenetic group isolates in the maximum
likelihood phylogeny (Figure 2 in the main text). This sputum sample was collected on 12
October 2017 from a resident of Antananarivo with no recent travel history. Two separate
emergence events were associated with the j phylogenetic group (Table 1 in main text):
Emergence 8 (first onset date: 26 September 2017, see below) and Emergence 10 (first onset
date: 3 October 2017, see below). There was documented travel to Antananarivo by a foreign
tourist that presented at hospital in Antananarivo 1 October with PP symptoms; he had traveled
outside Antananarivo, but the specific details are unknown. His sputum sample yielded isolate
35/17, which is closely related to multiple human isolates obtained from BP cases in
Tsiroanomandidy District starting in September that are assigned to the phylogenetic group
(Figure 2 in the main text), suggesting plague activity in this rural focus as the ultimate source of
the infection in the tourist, from whom no known secondary cases were reported. Thus, it is
possible that the infection in the Antananarivo resident that yielded sputum sample 819–2017
was the result of additional, undocumented cases associated with this transmission chain in
Antananarivo. Alternatively, Emergence 10 was also assigned to phylogenetic group j (Figure 2
in the main text), so it is also possible that the individual that yielded sputum sample 819–2017
could have been infected via a transmission chain from that other event that involved
undocumented travel to Antananarivo. Finally, since the single SNP called in sputum sample
819–2017 is specific to all of the phylogenetic group isolates in the maximum likelihood
phylogeny (Figure 2 in the main text), the infection in this individual could have resulted from
and seven SNPs specific to phylogenetic group “s” or a subclade within this group in the
maximum likelihood phylogeny were identified in sputum sample 125–2017; this sample was
obtained on 12 September 2017 from Case 15 in this transmission chain. One SNP specific to
isolate 17/17 in the maximum likelihood phylogeny was identified in sputum sample 184–2017;
this sample was obtained on 12 September 2017 from Case 22 in this transmission chain. Finally,
three SNPs specific to isolate 17/17 in the maximum likelihood phylogeny were identified in
sputum sample 2093–2017. This sample was obtained from an Antananarivo resident on 8
November 2017 with no recent travel outside the city and no known contact with the cases
above, suggesting community spread of this transmission chain in Antananarivo and persistence
there until at least November 2017.

A single derived SNP call was made from sputum sample 819–2017 (Appendix 1 Table
5), in a SNP that is specific to all of the “j” phylogenetic group isolates in the maximum
likelihood phylogeny (Figure 2 in the main text). This sputum sample was collected on 12
October 2017 from a resident of Antananarivo with no recent travel history. Two separate
emergence events were associated with the j phylogenetic group (Table 1 in main text):
Emergence 8 (first onset date: 26 September 2017, see below) and Emergence 10 (first onset
date: 3 October 2017, see below). There was documented travel to Antananarivo by a foreign
tourist that presented at hospital in Antananarivo 1 October with PP symptoms; he had traveled
outside Antananarivo, but the specific details are unknown. His sputum sample yielded isolate
35/17, which is closely related to multiple human isolates obtained from BP cases in
Tsiroanomandidy District starting in September that are assigned to the phylogenetic group
(Figure 2 in the main text), suggesting plague activity in this rural focus as the ultimate source of
the infection in the tourist, from whom no known secondary cases were reported. Thus, it is
possible that the infection in the Antananarivo resident that yielded sputum sample 819–2017
was the result of additional, undocumented cases associated with this transmission chain in
Antananarivo. Alternatively, Emergence 10 was also assigned to phylogenetic group j (Figure 2
in the main text), so it is also possible that the individual that yielded sputum sample 819–2017
could have been infected via a transmission chain from that other event that involved
undocumented travel to Antananarivo. Finally, since the single SNP called in sputum sample
819–2017 is specific to all of the phylogenetic group isolates in the maximum likelihood
phylogeny (Figure 2 in the main text), the infection in this individual could have resulted from
yet another, undocumented emergence event associated with the *j* phylogenetic group.

Regardless, this documents that a transmission chain associated with the *j* phylogenetic group was present in Antananarivo and it was distinct from the other transmission chains documented there, which were associated with Emergences 2, 7, and 12 (Table 1 in the main text).

A single derived SNP call was made from sputum sample 1494–2017 (Appendix 1 Table 5), in a SNP that is specific to human isolates 30/17, 32/17, 33/17, 34/17, 48/17, and 55/17 from the “β” phylogenetic group (Figure 2 in the main text, below); all of these isolates except isolate 34/17 were obtained in Ambalavao District as part of the investigation of Emergences 12 and 20 (see below). Sputum sample 1494–2017 was collected on 24 October 2017 from an Antananarivo resident with no travel history. Isolate 34/17 was reportedly collected from a newborn baby in Antananarivo 15 October; his parents were residents of Antananarivo with no recent travel. The baby was febrile and transferred to a children’s hospital as a suspect plague case, where a bronchoscope was used to collect a sputum sample from him. The sputum sample attributed to this newborn yielded isolate 34/17, which is identical to isolates 30/17, 32/17, 33/17, and 55/17 (Figure 2 in the main text). No secondary cases were reported from the baby, but the SNP call in sputum sample 1494–2017 (Appendix 1 Table 5) suggests subsequent community spread of closely related *Y. pestis* in Antananarivo.

**8. Detailed Information on Multiple Emergences of *Y. pestis* from Environmental Reservoirs into Humans**

Human plague emerged independently from environmental reservoirs into humans at least 20 times in multiple rural endemic foci in Madagascar from August-November 2017 (Table 1 in the main text). These events occurred in 19 different communes located in 12 different districts in the central and northern highlands (Figure 1, panel A in the main text) and clinical *Y. pestis* isolates obtained from them were closely related to previous isolates from those locations (Figure 2 in the main text).

The following provides more information on the 20 emergence events. All dates are from 2017.

* Emergence 1
yet another, undocumented emergence event associated with the *j* phylogenetic group.

Regardless, this documents that a transmission chain associated with the *j* phylogenetic group was present in Antananarivo and it was distinct from the other transmission chains documented there, which were associated with Emergences 2, 7, and 12 (Table 1 in the main text).

A single derived SNP call was made from sputum sample 1494–2017 (Appendix 1 Table 5), in a SNP that is specific to human isolates 30/17, 32/17, 33/17, 34/17, 48/17, and 55/17 from the “β” phylogenetic group (Figure 2 in the main text, below); all of these isolates except isolate 34/17 were obtained in Ambalavao District as part of the investigation of Emergences 12 and 20 (see below). Sputum sample 1494–2017 was collected on 24 October 2017 from an Antananarivo resident with no travel history. Isolate 34/17 was reportedly collected from a newborn baby in Antananarivo 15 October; his parents were residents of Antananarivo with no recent travel. The baby was febrile and transferred to a children’s hospital as a suspect plague case, where a bronchoscope was used to collect a sputum sample from him. The sputum sample attributed to this newborn yielded isolate 34/17, which is identical to isolates 30/17, 32/17, 33/17, and 55/17 (Figure 2 in the main text). No secondary cases were reported from the baby, but the SNP call in sputum sample 1494–2017 (Appendix 1 Table 5) suggests subsequent community spread of closely related *Y. pestis* in Antananarivo.

**8. Detailed Information on Multiple Emergences of *Y. pestis* from Environmental Reservoirs into Humans**

Human plague emerged independently from environmental reservoirs into humans at least 20 times in multiple rural endemic foci in Madagascar from August-November 2017 (Table 1 in the main text). These events occurred in 19 different communes located in 12 different districts in the central and northern highlands (Figure 1, panel A in the main text) and clinical *Y. pestis* isolates obtained from them were closely related to previous isolates from those locations (Figure 2 in the main text).

The following provides more information on the 20 emergence events. All dates are from
2017.

* Emergence 1
o Human BP isolate 14/17 assigned to phylogenetic subclade s04 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Ambatomanjaka

o BP isolates: 14/17 (from sample 110-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated with these isolates/cases: 14/17 (110-2017): 13 August

o Travel: No

o Note: 14/17 is the earliest culture confirmed BP isolate from 2017 and the earliest 2017 isolate overall in our WGS phylogeny

## Emergence 2

o Human primary PP isolate 17/17 assigned to phylogenetic subclade s13 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Ankazobe/Marondry

o BP isolates: None

o PP isolates: 17/17 (121-2017)

o Other cases: Yes - see Table 2 in the main text

o Onset dates associated with these isolates/cases: 17/17 (121-2017): 6 September for this case (Case 7 in the main text), but onset date for Case 1 from this transmission chain (see main text) was 25 August

o Travel: Yes - extensive. See Figure 1, panel B and Table 2 in main text.

## Emergence 3

o Human BP isolates 15/17 and 19/17 assigned to phylogenetic subclade s03 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Anosibe Ifanja

o BP isolates: 15/17 (113-2017), 19/17 (114-2017)
o Human BP isolate 14/17 assigned to phylogenetic subclade s04 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Ambatomanjaka

o BP isolates: 14/17 (from sample 110-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated with these isolates/cases: 14/17 (110-2017): 13 August

o Travel: No

o Note: 14/17 is the earliest culture confirmed BP isolate from 2017 and the earliest 2017 isolate overall in our WGS phylogeny

## Emergence 2

o Human primary PP isolate 17/17 assigned to phylogenetic subclade s13 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Ankazobe/Marondry

o BP isolates: None

o PP isolates: 17/17 (121-2017)

o Other cases: Yes - see Table 2 in the main text

o Onset dates associated with these isolates/cases: 17/17 (121-2017): 6 September for this case (Case 7 in the main text), but onset date for Case 1 from this transmission chain (see main text) was 25 August

o Travel: Yes - extensive. See Figure 1, panel B and Table 2 in main text.

## Emergence 3

o Human BP isolates 15/17 and 19/17 assigned to phylogenetic subclade s03 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Anosibe Ifanja

o BP isolates: 15/17 (113-2017), 19/17 (114-2017)
* PP isolates: None

* Other cases: Yes – see notes

* Onset dates associated w/ these isolates/cases: 19/17 (114–2017): 26 August; 15/17 (113–2017): 27 August

* Travel: No

* Notes: The individuals that yielded isolates 15/17 and 19/17 came from the same hamlet (Soanafindra), Fokontany Ambatolampy, Commune Anosibe Ifanja, and District Miarinarivo. Two other suspected cases were notified from the same location at almost the same time, but they did not yield isolates. These two other cases were also BP. They are probably all contacts of each other. Notification forms of all these cases mentioned the presence of dead rats either inside houses or in the village.

* Emergence 4

* Human BP isolate 16/17 assigned to phylogenetic subclade q15 within major phylogenetic group “q” in the phylogeny

* District/commune of origin: Moramanga/Ambohibary

* BP isolates: 16/17 (117–2017)

* PP isolates: None

* Other cases: Yes – see notes

* Onset dates associated w/ these isolates/cases: Contact #1 (see below): 2 September; Contact #2 (see below): 3 September; 16/17 (117–2017): 4 September

* Travel: No

* Notes: The individual that yielded isolate 16/17 had two other BP contacts, but there were no notification forms for these other contacts. These two contacts were found dead in the same kitchen with the individual that yielded
* PP isolates: None

* Other cases: Yes – see notes

* Onset dates associated w/ these isolates/cases: 19/17 (114–2017): 26 August; 15/17 (113–2017): 27 August

* Travel: No

* Notes: The individuals that yielded isolates 15/17 and 19/17 came from the same hamlet (Soanafindra), Fokontany Ambatolampy, Commune Anosibe Ifanja, and District Miarinarivo. Two other suspected cases were notified from the same location at almost the same time, but they did not yield isolates. These two other cases were also BP. They are probably all contacts of each other. Notification forms of all these cases mentioned the presence of dead rats either inside houses or in the village.

* Emergence 4

* Human BP isolate 16/17 assigned to phylogenetic subclade q15 within major phylogenetic group “q” in the phylogeny

* District/commune of origin: Moramanga/Ambohibary

* BP isolates: 16/17 (117–2017)

* PP isolates: None

* Other cases: Yes – see notes

* Onset dates associated w/ these isolates/cases: Contact #1 (see below): 2 September; Contact #2 (see below): 3 September; 16/17 (117–2017): 4 September

* Travel: No

* Notes: The individual that yielded isolate 16/17 had two other BP contacts, but there were no notification forms for these other contacts. These two contacts were found dead in the same kitchen with the individual that yielded
16/17. Contact 1: 13 yo, presence of sub-maxillary bubo. Contact 2: 6 yo, presence of sub-maxillary bubo.

* Emergence 5

o Human BP isolates 18/17, 29/17, 31/17, and 40/17 assigned to phylogenetic subclade s04 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Tsiroanomandidy/Tsinjoarivo Imanga (18/17, 31/17, 40/17) and Ambararatabe (29/17)

o BP isolates: 18/17 (176-2017), 29/17 (724-2017), 31/17 (1228-2017), 40/17 (1875-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 18/17 (176-2017): 16 September; 29/17 (724-2017): 7 October; 31/17 (1228-2017): 12 October; 40/17 (1875-2017): 21 October

o Travel: No

o Notes: No relationship among these cases, except the individuals that yielded isolates 18/17, 31/17, and 40/17 all live in the same commune of Tsinjoarivo Imanga. Notification of dead rats on the form for the individual that yielded isolate 31/17.

* Emergence 6

o Human BP isolates 27/17 and 50/17 assigned to phylogenetic subclade y03 within major phylogenetic group "y" in the phylogeny

o District/commune of origin: Manandriana/Ambohimahazo (27/17), Ambositra/Ivato (50/17)

o BP isolates: 27/17 (591-2017), 50/17 (2299-2017)

o PP isolates: None

o Other cases: None
16/17. Contact 1: 13 yo, presence of sub-maxillary bubo. Contact 2: 6 yo, presence of sub-maxillary bubo.

* Emergence 5

o Human BP isolates 18/17, 29/17, 31/17, and 40/17 assigned to phylogenetic subclade s04 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Tsiroanomandidy/Tsinjoarivo Imanga (18/17, 31/17, 40/17) and Ambararatabe (29/17)

o BP isolates: 18/17 (176-2017), 29/17 (724-2017), 31/17 (1228-2017), 40/17 (1875-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 18/17 (176-2017): 16 September; 29/17 (724-2017): 7 October; 31/17 (1228-2017): 12 October; 40/17 (1875-2017): 21 October

o Travel: No

o Notes: No relationship among these cases, except the individuals that yielded isolates 18/17, 31/17, and 40/17 all live in the same commune of Tsinjoarivo Imanga. Notification of dead rats on the form for the individual that yielded isolate 31/17.

* Emergence 6

o Human BP isolates 27/17 and 50/17 assigned to phylogenetic subclade y03 within major phylogenetic group "y" in the phylogeny

o District/commune of origin: Manandriana/Ambohimahazo (27/17), Ambositra/Ivato (50/17)

o BP isolates: 27/17 (591-2017), 50/17 (2299-2017)

o PP isolates: None

o Other cases: None
o Onset dates associated w/ these isolates/cases: 27/17 (591-2017): 17 September; 50/17 (2299-2017): 12 November

o Travel: No

## Emergence 7

o Human BP isolate 20/17 and primary PP isolate 21/17 assigned to phylogenetic subclade s14 within major phylogenetic group “s” in the phylogeny and are identical to each other (i.e., no SNP differences)

o District/commune of origin: Andramasina/Sabotsy Ambohitromby

o BP isolates: 20/17 (189-2017)

o PP isolates: 21/17 (212-2017)

o Other cases: None recorded – see notes

o Onset dates associated w/ these isolates/cases: 20/17 (189-2017): 21 September; 21/17 (212-2017): 25 September

o Travel: Yes, but unclear by whom. Isolate 21/17 was obtained from an individual in Antananarivo with no reported travel.

o Notes: No known relationship between the individuals that yielded these two isolates and no reported travel by either of them. The individual that yielded 20/17 had BP that was progressing to SPP but also had no known travel. Clinical signs: 39.6°C, cervical bubo (bean size), cough <5 days; bubo sample collected on 22 September 2017. The likely explanation here is that this individual – or someone else infected from the same environmental source – initiated a PP outbreak in this district and then someone infected from that outbreak traveled to Antananarivo leading to a transmission chain there that infected the individual that yielded 21/17.

## Emergence 8

o Human BP isolates 23/17, 24/17, 25/17, 41/17, 44/17, and 46/17 and primary PP isolate 35/17 assigned to phylogenetic subclade j02 within major phylogenetic group “j” in the phylogeny
* Onset dates associated w/ these isolates/cases: 27/17 (591-2017): 17 September; 50/17 (2299-2017): 12 November

* Travel: No

* Emergence 7

* Human BP isolate 20/17 and primary PP isolate 21/17 assigned to phylogenetic subclade s14 within major phylogenetic group "s" in the phylogeny and are identical to each other (i.e., no SNP differences)

* District/commune of origin: Andramasina/Sabotsy Ambohitromby

* BP isolates: 20/17 (189-2017)

* PP isolates: 21/17 (212-2017)

* Other cases: None recorded - see notes

* Onset dates associated w/ these isolates/cases: 20/17 (189-2017): 21 September; 21/17 (212-2017): 25 September

* Travel: Yes, but unclear by whom. Isolate 21/17 was obtained from an individual in Antananarivo with no reported travel.

* Notes: No known relationship between the individuals that yielded these two isolates and no reported travel by either of them. The individual that yielded 20/17 had BP that was progressing to SPP but also had no known travel. Clinical signs: 39.6°C, cervical bubo (bean size), cough <5 days; bubo sample collected on 22 September 2017. The likely explanation here is that this individual - or someone else infected from the same environmental source - initiated a PP outbreak in this district and then someone infected from that outbreak traveled to Antananarivo leading to a transmission chain there that infected the individual that yielded 21/17.

* Emergence 8

* Human BP isolates 23/17, 24/17, 25/17, 41/17, 44/17, and 46/17 and primary PP isolate 35/17 assigned to phylogenetic subclade j02 within major phylogenetic group "j" in the phylogeny
* District/commune of origin: Tsiroanomandidy/Bemahatazana

* BP isolates: 23/17 (284-2017), 24/17 (283-2017), 25/17 (282-2017), 41/17 (722-2017), 44/17 (2098-2017), 46/17 (2097-2017)

* PP isolates: Yes. Isolate 35/17 (309-2017) differs by just one SNP from BP isolates 23/17, 25/17, and 41/17.

* Other cases: None

* Onset dates associated w/ these isolates/cases: 23/17 (284-2017): 26 September; 24/17 (283-2017): 30 September; 25/17 (282-2017): 30 September; 35/17 (309-2017): 1 October; 41/17 (722-2017): 6 October; 44/17 (2098-2017): 7 November; 46/17 (2097-2017): 7 November

* Travel: Yes. Isolate 35/17 was from a tourist who traveled around Madagascar, but the details of his travel are unknown. He was treated in Antananarivo, which is where isolate 35/17 was obtained from him.

* Notes: Multiple BP cases and multiple dates, so multiple emergences of BP from the environment in this location. BP cases arose for >1 month.

## Emergence 9

* Human BP isolate 22/17 assigned to phylogenetic subclade s05 within major phylogenetic group "s" in the phylogeny

* District/commune of origin: Manjakandriana/Ranovao

* BP isolates: 22/17 (281-2017)

* PP isolates: None

* Other cases: None

* Onset dates associated w/ these isolates/cases: 22/17 (281-2017): 2 October (probably not 100% accurate as she died the next day and this was BP)

* Travel: Yes. The family of this individual took her to Antananarivo for treatment where she died on 3 October.

## Emergence 10
* District/commune of origin: Tsiroanomandidy/Bemahatazana

* BP isolates: 23/17 (284-2017), 24/17 (283-2017), 25/17 (282-2017), 41/17 (722-2017), 44/17 (2098-2017), 46/17 (2097-2017)

* PP isolates: Yes. Isolate 35/17 (309-2017) differs by just one SNP from BP isolates 23/17, 25/17, and 41/17.

* Other cases: None

* Onset dates associated w/ these isolates/cases: 23/17 (284-2017): 26 September; 24/17 (283-2017): 30 September; 25/17 (282-2017): 30 September; 35/17 (309-2017): 1 October; 41/17 (722-2017): 6 October; 44/17 (2098-2017): 7 November; 46/17 (2097-2017): 7 November

* Travel: Yes. Isolate 35/17 was from a tourist who traveled around Madagascar, but the details of his travel are unknown. He was treated in Antananarivo, which is where isolate 35/17 was obtained from him.

* Notes: Multiple BP cases and multiple dates, so multiple emergences of BP from the environment in this location. BP cases arose for >1 month.

## Emergence 9

* Human BP isolate 22/17 assigned to phylogenetic subclade s05 within major phylogenetic group "s" in the phylogeny

* District/commune of origin: Manjakandriana/Ranovao

* BP isolates: 22/17 (281-2017)

* PP isolates: None

* Other cases: None

* Onset dates associated w/ these isolates/cases: 22/17 (281-2017): 2 October (probably not 100% accurate as she died the next day and this was BP)

* Travel: Yes. The family of this individual took her to Antananarivo for treatment where she died on 3 October.

## Emergence 10
o Human BP isolate 45/17 assigned to phylogenetic subclade j02 within major phylogenetic group “j” in the phylogeny

o District/commune of origin: Tsiroanomandidy/Ambalanirana

o BP isolates: 45/17 (1230–2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 45/17 (1230–2017): 3 October

o Travel: No

## Emergence 11

o Human primary PP isolate 28/17 assigned to phylogenetic subclade s03 within major phylogenetic group “s” in the phylogeny

o District/commune of origin: Arivonimamo/Arivonimamo II

o BP isolates: None

o PP isolates: 28/17 (594–2017)

o Other cases: None recorded – see notes

o Onset dates associated w/ these isolates/cases: 28/17 (594–2017): 5 October

o Travel: No

o Notes: Obviously there were other cases associated with this one as it is a primary PP case, so it had to have been obtained from another individual with PP. But there is no information on those other cases.

## Emergence 12

o Human BP isolates 30/17, 32/17, and 33/17 and primary PP isolates 34/17 and 55/17 assigned to major phylogenetic group “β” in the phylogeny and are identical to each other (i.e., no SNP differences)

o District/commune of origin: Ambalavao/Miarinarivo

o BP isolates: 30/17 (1099–2017), 32/17 (1095–2017), 33/17 (1097–2017)
o Human BP isolate 45/17 assigned to phylogenetic subclade j02 within major phylogenetic group "j" in the phylogeny

o District/commune of origin: Tsiroanomandidy/Ambalanirana

o BP isolates: 45/17 (1230-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated with these isolates/cases: 45/17 (1230-2017): 3 October

o Travel: No

### Emergence 11

o Human primary PP isolate 28/17 assigned to phylogenetic subclade s03 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Arivonimamo/Arivonimamo II

o BP isolates: None

o PP isolates: 28/17 (594-2017)

o Other cases: None recorded - see notes

o Onset dates associated with these isolates/cases: 28/17 (594-2017): 5 October

o Travel: No

o Notes: Obviously there were other cases associated with this one as it is a primary PP case, so it had to have been obtained from another individual with PP. But there is no information on those other cases.

### Emergence 12

o Human BP isolates 30/17, 32/17, and 33/17 and primary PP isolates 34/17 and 55/17 assigned to major phylogenetic group "β" in the phylogeny and are identical to each other (i.e., no SNP differences)

o District/commune of origin: Ambalavao/Miarinarivo

o BP isolates: 30/17 (1099-2017), 32/17 (1095-2017), 33/17 (1097-2017)
* PP isolates: 34/17 (1024–2017) and 55/17 (2394–2017)

* Other cases: Yes – but did not yield isolates.

* Onset dates associated w/ these isolates/cases: 32/17 (1095–2017): 7 October; 1096–2017 (no isolate): 7 October; 33/17 (1097–2017): 8 October; 30/17 (1099–2017): 8 October; 34/17 (1024–2017): 14 October; 55/17 (2394–2017): 22 November

* Travel: Yes. Somehow the identical strain got from Ambalavao/Miarinarivo to Antananarivo (see notes).

* Notes: The individual that yielded isolate 32/17 had symptoms consistent with BP that progressed to secondary PP. Primary PP isolates 34/17 and 55/17 are both 100% identical to BP isolates 30/17, 32/17, and 33/17 in the phylogeny. Isolate 34/17 (onset date = 14 October) is reportedly from the newborn baby who was tested in Antananarivo (see main text). 55/17 (onset date = 22 November) is from an individual from Ambalavao/Miarinarivo with no known travel. So, this *Y. pestis* lineage was somehow transferred to Antananarivo but the details of this transfer are unknown.

## Emergence 13

* Human BP isolate 39/17 assigned to phylogenetic subclade s05 within major phylogenetic group “s” in the phylogeny

* District/commune of origin: Ankazobe/Fiadanana

* BP isolates: 39/17 (1780–2017)

* PP isolates: None

* Other cases: None

* Onset dates associated w/ these isolates/cases: 39/17 (1780–2017): 8 October

* Travel: No

## Emergence 14
* PP isolates: 34/17 (1024–2017) and 55/17 (2394–2017)

* Other cases: Yes – but did not yield isolates.

* Onset dates associated w/ these isolates/cases: 32/17 (1095–2017): 7 October; 1096–2017 (no isolate): 7 October; 33/17 (1097–2017): 8 October; 30/17 (1099–2017): 8 October; 34/17 (1024–2017): 14 October; 55/17 (2394–2017): 22 November

* Travel: Yes. Somehow the identical strain got from Ambalavao/Miarinarivo to Antananarivo (see notes).

* Notes: The individual that yielded isolate 32/17 had symptoms consistent with BP that progressed to secondary PP. Primary PP isolates 34/17 and 55/17 are both 100% identical to BP isolates 30/17, 32/17, and 33/17 in the phylogeny. Isolate 34/17 (onset date = 14 October) is reportedly from the newborn baby who was tested in Antananarivo (see main text). 55/17 (onset date = 22 November) is from an individual from Ambalavao/Miarinarivo with no known travel. So, this *Y. pestis* lineage was somehow transferred to Antananarivo but the details of this transfer are unknown.

* Emergence 13

* Human BP isolate 39/17 assigned to phylogenetic subclade s05 within major phylogenetic group “s” in the phylogeny

* District/commune of origin: Ankazobe/Fiadanana

* BP isolates: 39/17 (1780–2017)

* PP isolates: None

* Other cases: None

* Onset dates associated w/ these isolates/cases: 39/17 (1780–2017): 8 October

* Travel: No

* Emergence 14
o Human BP isolate 38/17 assigned to major phylogenetic group "α" in the phylogeny

o District/commune of origin: Mandritsara/Andratamarina

o BP isolates: 38/17 (2095-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 38/17 (2095-2017): 14 October

o Travel: No

o Note: This location is in the northern highlands.

### Emergence 15

o Human BP isolate 37/17 assigned to phylogenetic subclade s03 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Anosibe Ifanja

o BP isolates: 37/17 (1542-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 37/17 (1542-2017): 17 October

o Travel: No

### Emergence 16

o Human BP isolate 49/17 assigned to phylogenetic subclade t11 within major phylogenetic group "t" in the phylogeny

o District/commune of origin: Manandriana/Ambovombe Centre

o BP isolates: 49/17 (2255-2017)

o PP isolates: None

o Other cases: None
o Human BP isolate 38/17 assigned to major phylogenetic group "α" in the phylogeny

o District/commune of origin: Mandritsara/Andratamarina

o BP isolates: 38/17 (2095-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 38/17 (2095-2017): 14 October

o Travel: No

o Note: This location is in the northern highlands.

### Emergence 15

o Human BP isolate 37/17 assigned to phylogenetic subclade s03 within major phylogenetic group "s" in the phylogeny

o District/commune of origin: Miarinarivo/Anosibe Ifanja

o BP isolates: 37/17 (1542-2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 37/17 (1542-2017): 17 October

o Travel: No

### Emergence 16

o Human BP isolate 49/17 assigned to phylogenetic subclade t11 within major phylogenetic group "t" in the phylogeny

o District/commune of origin: Manandriana/Ambovombe Centre

o BP isolates: 49/17 (2255-2017)

o PP isolates: None

o Other cases: None
* Onset dates associated with these isolates/cases: 49/17 (2255-2017): 18 October

* Travel: No

* Emergence 17

* Human BP isolate 36/17 assigned to phylogenetic subclade x01 within major phylogenetic group "x" in the phylogeny

* District/commune of origin: Arivonimamo/Mahatsinjo Est

* BP isolates: 36/17 (1700-2017)

* PP isolates: None

* Other cases: None

* Onset dates associated with these isolates/cases: 36/17 (1700-2017): 23 October

* Travel: No

* Emergence 18

* Human primary PP isolate 43/17 assigned to phylogenetic subclade q04 within major phylogenetic group "q" in the phylogeny

* District/commune of origin of infection (see below): Antananarivo-Avaradrano/Manandriana

* BP isolates: None

* PP isolates: 43/17 (2048-2017)

* Other cases: Yes - see notes

* Onset dates associated with these isolates/cases: 43/17 (2048-2017): 30 October

* Travel: Yes - see notes

* Notes: The individual that yielded isolate 43/17 lived in Andramasina/Tankafatra but traveled to Antananarivo-Avaradrano/Manandriana (Imerimandroso) before becoming sick. He had a contact at Imerimandroso who had a cough <5 days and who also died. This contact was not evaluated but mentioned in the notification form for the
* Onset dates associated with these isolates/cases: 49/17 (2255-2017): 18 October

* Travel: No

* **Emergence 17**

* Human BP isolate 36/17 assigned to phylogenetic subclade x01 within major phylogenetic group "x" in the phylogeny

* District/commune of origin: Arivonimamo/Mahatsinjo Est

* BP isolates: 36/17 (1700-2017)

* PP isolates: None

* Other cases: None

* Onset dates associated with these isolates/cases: 36/17 (1700-2017): 23 October

* Travel: No

* **Emergence 18**

* Human primary PP isolate 43/17 assigned to phylogenetic subclade q04 within major phylogenetic group "q" in the phylogeny

* District/commune of origin of infection (see below): Antananarivo-Avaradrano/Manandriana

* BP isolates: None

* PP isolates: 43/17 (2048-2017)

* Other cases: Yes - see notes

* Onset dates associated with these isolates/cases: 43/17 (2048-2017): 30 October

* Travel: Yes - see notes

* Notes: The individual that yielded isolate 43/17 lived in Andramasina/Tankafatra but traveled to Antananarivo-Avaradrano/Manandriana (Imerimandroso) before becoming sick. He had a contact at Imerimandroso who had a cough <5 days and who also died. This contact was not evaluated but mentioned in the notification form for the
individual that yielded 43/17. Thus, the environmental source of this isolate is
listed as Antananarivo-Avaradrano/Manandriana, not
Andramasina/Tankafatra, as no other cases were reported from
Andramasina/Tankafatra.

* Emergence 19

o Human BP isolate 47/17 assigned to phylogenetic subclade s03 within major phylogenetic group “s” in the phylogeny

o District/commune of origin: Faratsiho/Antsampanimahazo

o BP isolates: 47/17 (2154–2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 47/17 (2154–2017): 7 November

o Travel: No

* Emergence 20

o Human BP isolate 48/17 assigned to major phylogenetic group “β” in the phylogeny

o District/commune of origin: Ambalavao/Anjoma

o BP isolates: 48/17 (2167–2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 48/17 (2167–2017): 9 November

o Travel: No

o Note: Dead rats were present in the house of the individual that yielded this isolate.
individual that yielded 43/17. Thus, the environmental source of this isolate is
listed as Antananarivo-Avaradrano/Manandriana, not
Andramasina/Tankafatra, as no other cases were reported from
Andramasina/Tankafatra.

* Emergence 19

o Human BP isolate 47/17 assigned to phylogenetic subclade s03 within major phylogenetic group “s” in the phylogeny

o District/commune of origin: Faratsiho/Antsampanimahazo

o BP isolates: 47/17 (2154–2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 47/17 (2154–2017): 7 November

o Travel: No

* Emergence 20

o Human BP isolate 48/17 assigned to major phylogenetic group “β” in the phylogeny

o District/commune of origin: Ambalavao/Anjoma

o BP isolates: 48/17 (2167–2017)

o PP isolates: None

o Other cases: None

o Onset dates associated w/ these isolates/cases: 48/17 (2167–2017): 9 November

o Travel: No

o Note: Dead rats were present in the house of the individual that yielded this isolate.
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7. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303. PubMed https://doi.org/10.1101/gr.107524.110

8. Delcher AL, Salzberg SL, Phillippy AM. Using MUMmer to identify similar regions in large sequence sets. Curr Protoc Bioinformatics. 2003 Feb;Chapter 10:Unit 10 3.

9. Sahl JW, Lemmer D, Travis J, Schupp JM, Gillece JD, Aziz M, et al. NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats. Microb Genom. 2016;2:e000074. PubMed https://doi.org/10.1099/mgen.0.000074

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7. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303. PubMed https://doi.org/10.1101/gr.107524.110

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9. Sahl JW, Lemmer D, Travis J, Schupp JM, Gillece JD, Aziz M, et al. NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats. Microb Genom. 2016;2:e000074. PubMed https://doi.org/10.1099/mgen.0.000074

10. Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999;27:573–80. PubMed https://doi.org/10.1093/nar/27.2.573
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12. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14:587–9. PubMed https://doi.org/10.1038/nmeth.4285

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16. Iqbal SS, Chambers JP, Goode MT, Valdes JJ, Brubaker RR. Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR. Mol Cell Probes. 2000;14:109–14. PubMed https://doi.org/10.1006/mcpr.2000.0295

17. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al.; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9. PubMed https://doi.org/10.1093/bioinformatics/btp352
11. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37:1530–4. PubMed https://doi.org/10.1093/molbev/msaa015

12. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14:587–9. PubMed https://doi.org/10.1038/nmeth.4285

13. Sahl JW, Caporaso JG, Rasko DA, Keim P. The large-scale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes. PeerJ. 2014;2:e332. PubMed https://doi.org/10.7717/peerj.332

14. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1. PubMed https://doi.org/10.1093/bioinformatics/btq461

15. Rasko DA, Myers GS, Ravel J. Visualization of comparative genomic analyses by BLAST score ratio. BMC Bioinformatics. 2005;6:2. PubMed https://doi.org/10.1186/1471-2105-6-2

16. Iqbal SS, Chambers JP, Goode MT, Valdes JJ, Brubaker RR. Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR. Mol Cell Probes. 2000;14:109–14. PubMed https://doi.org/10.1006/mcpr.2000.0295

17. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al.; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9. PubMed https://doi.org/10.1093/bioinformatics/btp352
<table><caption>Appendix Table 1. 91 Y. pestis whole genomes sequences used to construct the maximum likelihood phylogeny*</caption><thead><tr><th>Strain</th><th>Accession#</th><th>Year</th><th>Source</th><th>District</th><th>Commune</th><th>Lineage</th></tr></thead><tbody><tr><td>20/17</td><td>SRS4032725</td><td>2017</td><td>Human</td><td>Andramasina</td><td>Sabotsy Ambohitromby</td><td>s14</td></tr><tr><td>21/17</td><td>SRS4032726</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Ankadikely Ilafy</td><td>s14</td></tr><tr><td>56/13</td><td>SRR11676332</td><td>2013</td><td>Human</td><td>Faratsiho</td><td>Faratsiho</td><td>s14</td></tr><tr><td>160/07</td><td>SRS4032764</td><td>2007</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s15</td></tr><tr><td>42/08</td><td>SRR4175426</td><td>2008</td><td>Human</td><td>Faratsiho</td><td>Faratsiho</td><td>s16</td></tr><tr><td>78/06</td><td>SRR4175415</td><td>2006</td><td>Rat</td><td>Betafo</td><td>Ambohimanambola</td><td>s13</td></tr><tr><td>17/17</td><td>SRS4032722</td><td>2017</td><td>Human</td><td>Mahajanga I</td><td>Mahabibo</td><td>s13</td></tr><tr><td>207/11</td><td>SRS4032760</td><td>2011</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>65/16</td><td>SRR12199758</td><td>2016</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s13</td></tr><tr><td>25/09</td><td>SRS4032763</td><td>2009</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>178/12</td><td>SRS4032749</td><td>2012</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s13</td></tr><tr><td>67/05</td><td>SRS4032748</td><td>2005</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>24/14</td><td>SRR12199765</td><td>2014</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>155/13</td><td>SRS4032767</td><td>2013</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>174/11</td><td>SRS4032751</td><td>2011</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>214/08</td><td>SRR12199763</td><td>2008</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>25/16</td><td>SRR12199759</td><td>2016</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>13/14</td><td>SRR12199764</td><td>2014</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>146/14</td><td>SRR12199760</td><td>2014</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>39/17</td><td>SRS4032758</td><td>2017</td><td>Human</td><td>Ankazobe</td><td>Fiadanana</td><td>s05</td></tr><tr><td>79/15</td><td>SRS4032770</td><td>2015</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>02/09</td><td>SRR4175439</td><td>2009</td><td>Human</td><td>Manjakandriana</td><td>Ambatomena</td><td>s22</td></tr><tr><td>154/98B</td><td>SRR651089</td><td>1998</td><td>Human</td><td>Mahajanga I</td><td>Mahabibo</td><td>s09</td></tr><tr><td>296/03</td><td>SRR12199766</td><td>2003</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>324/04</td><td>SRR12199761</td><td>2004</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>409/04</td><td>SRR12199762</td><td>2004</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>193/07</td><td>SRR4175437</td><td>2007</td><td>Human</td><td>Manjakandriana</td><td>Ambatolaona</td><td>s17</td></tr><tr><td>22/17</td><td>SRS4032729</td><td>2017</td><td>Human</td><td>Manjakandriana</td><td>Ranovao</td><td>s05</td></tr><tr><td>18/17</td><td>SRS4032727</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>29/17</td><td>SRS4032737</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambararatabe</td><td>s04</td></tr><tr><td>31/17</td><td>SRS4032738</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>40/17</td><td>SRS4032761</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>14/17</td><td>SRS4032723</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Ambatomanjaka</td><td>s04</td></tr><tr><td>15/17</td><td>SRS4032724</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>19/17</td><td>SRS4032728</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>37/17</td><td>SRS4032755</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>89/14</td><td>SRS4032766</td><td>2014</td><td>Rat</td><td>Mahajanga I</td><td>Mahajanga</td><td>s03</td></tr><tr><td>28/17</td><td>SRS4032735</td><td>2017</td><td>Human</td><td>Arivonimamo</td><td>Arivonimamo II</td><td>s03</td></tr><tr><td>47/17</td><td>SRS4032762</td><td>2017</td><td>Human</td><td>Faratsiho</td><td>Antsampanimahazo</td><td>s03</td></tr><tr><td>24/08</td><td>SRR12199767</td><td>2008</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s03</td></tr><tr><td>39/07</td><td>SRR4175414</td><td>2007</td><td>Flea</td><td>Betafo</td><td>Inanantonana</td><td>s12</td></tr><tr><td>35/15</td><td>SRS4032765</td><td>2015</td><td>Human</td><td>Moramanga</td><td>Ampasimpotsy Gara</td><td>q12</td></tr><tr><td>139/06</td><td>SRR4175441</td><td>2006</td><td>Human</td><td>Betafo</td><td>Anosiarivo Manapa</td><td>q13</td></tr><tr><td>01/08</td><td>SRR4175443</td><td>2008</td><td>Human</td><td>Manjakandriana</td><td>Ranovao</td><td>q06</td></tr><tr><td>43/17</td><td>SRS4032753</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Manandriana</td><td>q04</td></tr><tr><td>16/17</td><td>SRS4032721</td><td>2017</td><td>Human</td><td>Moramanga</td><td>Ambohibary</td><td>q15</td></tr><tr><td>11/09</td><td>SRR4175442</td><td>2009</td><td>Human</td><td>Moramanga</td><td>Ambohibary</td><td>q14</td></tr><tr><td>135/11</td><td>SRR4175440</td><td>2011</td><td>Human</td><td>Moramanga</td><td>Lakato</td><td>q11</td></tr><tr><td>172/03</td><td>SRR4175416</td><td>2003</td><td>Human</td><td>Soavinandriana</td><td>Tamponala</td><td>j06</td></tr><tr><td>357/04</td><td>SRR4175417</td><td>2004</td><td>Human</td><td>Mandoto</td><td>Mandoto</td><td>j04</td></tr><tr><td>74/98</td><td>SRS4032745</td><td>1998</td><td>Rat</td><td>Brickaville</td><td>Ranomafana Est</td><td>j07</td></tr><tr><td>88/11</td><td>SRR4175419</td><td>2011</td><td>Human</td><td>Mandoto</td><td>Antanambao Ambary</td><td>j10</td></tr><tr><td>43/07</td><td>SRR4175418</td><td>2007</td><td>Flea</td><td>Betafo</td><td>Inanantonana</td><td>j02</td></tr><tr><td>45/17</td><td>SRS4032741</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambalanirana</td><td>j02</td></tr><tr><td>24/17</td><td>SRS4032731</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>41/17</td><td>SRS4032759</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>23/17</td><td>SRS4032730</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>25/17</td><td>SRS4032732</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambatolampy</td><td>j02</td></tr><tr><td>35/17</td><td>SRS4032752</td><td>2017</td><td>Human</td><td>Antananarivo Renivohitra</td><td>1er Arrondissement</td><td>j02</td></tr><tr><td>46/17</td><td>SRS4032742</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>44/17</td><td>SRS4032750</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>IP275</td><td>GCA_000168235</td><td>1995</td><td>Human</td><td>Ambalavao</td><td>Besoa</td><td>j02</td></tr><tr><td>48/17</td><td>SRS4032769</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Anjoma</td><td>β</td></tr><tr><td>32/17</td><td>SRS4032739</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>55/17</td><td>SRS4032743</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>34/17</td><td>SRS4032754</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Ankadikely Ilafy</td><td>β</td></tr><tr><td>30/17</td><td>SRS4032736</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>33/17</td><td>SRS4032740</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr></tbody></table>
<table><caption>Appendix Table 1. 91 Y. pestis whole genomes sequences used to construct the maximum likelihood phylogeny*</caption><thead><tr><th>Strain</th><th>Accession#</th><th>Year</th><th>Source</th><th>District</th><th>Commune</th><th>Lineage</th></tr></thead><tbody><tr><td>20/17</td><td>SRS4032725</td><td>2017</td><td>Human</td><td>Andramasina</td><td>Sabotsy Ambohitromby</td><td>s14</td></tr><tr><td>21/17</td><td>SRS4032726</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Ankadikely Ilafy</td><td>s14</td></tr><tr><td>56/13</td><td>SRR11676332</td><td>2013</td><td>Human</td><td>Faratsiho</td><td>Faratsiho</td><td>s14</td></tr><tr><td>160/07</td><td>SRS4032764</td><td>2007</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s15</td></tr><tr><td>42/08</td><td>SRR4175426</td><td>2008</td><td>Human</td><td>Faratsiho</td><td>Faratsiho</td><td>s16</td></tr><tr><td>78/06</td><td>SRR4175415</td><td>2006</td><td>Rat</td><td>Betafo</td><td>Ambohimanambola</td><td>s13</td></tr><tr><td>17/17</td><td>SRS4032722</td><td>2017</td><td>Human</td><td>Mahajanga I</td><td>Mahabibo</td><td>s13</td></tr><tr><td>207/11</td><td>SRS4032760</td><td>2011</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>65/16</td><td>SRR12199758</td><td>2016</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s13</td></tr><tr><td>25/09</td><td>SRS4032763</td><td>2009</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>178/12</td><td>SRS4032749</td><td>2012</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s13</td></tr><tr><td>67/05</td><td>SRS4032748</td><td>2005</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>s13</td></tr><tr><td>24/14</td><td>SRR12199765</td><td>2014</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>155/13</td><td>SRS4032767</td><td>2013</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>174/11</td><td>SRS4032751</td><td>2011</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>214/08</td><td>SRR12199763</td><td>2008</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>25/16</td><td>SRR12199759</td><td>2016</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>13/14</td><td>SRR12199764</td><td>2014</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>146/14</td><td>SRR12199760</td><td>2014</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>39/17</td><td>SRS4032758</td><td>2017</td><td>Human</td><td>Ankazobe</td><td>Fiadanana</td><td>s05</td></tr><tr><td>79/15</td><td>SRS4032770</td><td>2015</td><td>Human</td><td>Ankazobe</td><td>Marondry</td><td>s05</td></tr><tr><td>02/09</td><td>SRR4175439</td><td>2009</td><td>Human</td><td>Manjakandriana</td><td>Ambatomena</td><td>s22</td></tr><tr><td>154/98B</td><td>SRR651089</td><td>1998</td><td>Human</td><td>Mahajanga I</td><td>Mahabibo</td><td>s09</td></tr><tr><td>296/03</td><td>SRR12199766</td><td>2003</td><td>Rat</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>324/04</td><td>SRR12199761</td><td>2004</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>409/04</td><td>SRR12199762</td><td>2004</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s05</td></tr><tr><td>193/07</td><td>SRR4175437</td><td>2007</td><td>Human</td><td>Manjakandriana</td><td>Ambatolaona</td><td>s17</td></tr><tr><td>22/17</td><td>SRS4032729</td><td>2017</td><td>Human</td><td>Manjakandriana</td><td>Ranovao</td><td>s05</td></tr><tr><td>18/17</td><td>SRS4032727</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>29/17</td><td>SRS4032737</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambararatabe</td><td>s04</td></tr><tr><td>31/17</td><td>SRS4032738</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>40/17</td><td>SRS4032761</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Tsinjoarivo Imanga</td><td>s04</td></tr><tr><td>14/17</td><td>SRS4032723</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Ambatomanjaka</td><td>s04</td></tr><tr><td>15/17</td><td>SRS4032724</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>19/17</td><td>SRS4032728</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>37/17</td><td>SRS4032755</td><td>2017</td><td>Human</td><td>Miarinarivo</td><td>Anosibe Ifanja</td><td>s03</td></tr><tr><td>89/14</td><td>SRS4032766</td><td>2014</td><td>Rat</td><td>Mahajanga I</td><td>Mahajanga</td><td>s03</td></tr><tr><td>28/17</td><td>SRS4032735</td><td>2017</td><td>Human</td><td>Arivonimamo</td><td>Arivonimamo II</td><td>s03</td></tr><tr><td>47/17</td><td>SRS4032762</td><td>2017</td><td>Human</td><td>Faratsiho</td><td>Antsampanimahazo</td><td>s03</td></tr><tr><td>24/08</td><td>SRR12199767</td><td>2008</td><td>Human</td><td>Ankazobe</td><td>Ambohitromby</td><td>s03</td></tr><tr><td>39/07</td><td>SRR4175414</td><td>2007</td><td>Flea</td><td>Betafo</td><td>Inanantonana</td><td>s12</td></tr><tr><td>35/15</td><td>SRS4032765</td><td>2015</td><td>Human</td><td>Moramanga</td><td>Ampasimpotsy Gara</td><td>q12</td></tr><tr><td>139/06</td><td>SRR4175441</td><td>2006</td><td>Human</td><td>Betafo</td><td>Anosiarivo Manapa</td><td>q13</td></tr><tr><td>01/08</td><td>SRR4175443</td><td>2008</td><td>Human</td><td>Manjakandriana</td><td>Ranovao</td><td>q06</td></tr><tr><td>43/17</td><td>SRS4032753</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Manandriana</td><td>q04</td></tr><tr><td>16/17</td><td>SRS4032721</td><td>2017</td><td>Human</td><td>Moramanga</td><td>Ambohibary</td><td>q15</td></tr><tr><td>11/09</td><td>SRR4175442</td><td>2009</td><td>Human</td><td>Moramanga</td><td>Ambohibary</td><td>q14</td></tr><tr><td>135/11</td><td>SRR4175440</td><td>2011</td><td>Human</td><td>Moramanga</td><td>Lakato</td><td>q11</td></tr><tr><td>172/03</td><td>SRR4175416</td><td>2003</td><td>Human</td><td>Soavinandriana</td><td>Tamponala</td><td>j06</td></tr><tr><td>357/04</td><td>SRR4175417</td><td>2004</td><td>Human</td><td>Mandoto</td><td>Mandoto</td><td>j04</td></tr><tr><td>74/98</td><td>SRS4032745</td><td>1998</td><td>Rat</td><td>Brickaville</td><td>Ranomafana Est</td><td>j07</td></tr><tr><td>88/11</td><td>SRR4175419</td><td>2011</td><td>Human</td><td>Mandoto</td><td>Antanambao Ambary</td><td>j10</td></tr><tr><td>43/07</td><td>SRR4175418</td><td>2007</td><td>Flea</td><td>Betafo</td><td>Inanantonana</td><td>j02</td></tr><tr><td>45/17</td><td>SRS4032741</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambalanirana</td><td>j02</td></tr><tr><td>24/17</td><td>SRS4032731</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>41/17</td><td>SRS4032759</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>23/17</td><td>SRS4032730</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>25/17</td><td>SRS4032732</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Ambatolampy</td><td>j02</td></tr><tr><td>35/17</td><td>SRS4032752</td><td>2017</td><td>Human</td><td>Antananarivo Renivohitra</td><td>1er Arrondissement</td><td>j02</td></tr><tr><td>46/17</td><td>SRS4032742</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>44/17</td><td>SRS4032750</td><td>2017</td><td>Human</td><td>Tsiroanomandidy</td><td>Bemahatazana</td><td>j02</td></tr><tr><td>IP275</td><td>GCA_000168235</td><td>1995</td><td>Human</td><td>Ambalavao</td><td>Besoa</td><td>j02</td></tr><tr><td>48/17</td><td>SRS4032769</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Anjoma</td><td>β</td></tr><tr><td>32/17</td><td>SRS4032739</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>55/17</td><td>SRS4032743</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>34/17</td><td>SRS4032754</td><td>2017</td><td>Human</td><td>Antananarivo Avaradrano</td><td>Ankadikely Ilafy</td><td>β</td></tr><tr><td>30/17</td><td>SRS4032736</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr><tr><td>33/17</td><td>SRS4032740</td><td>2017</td><td>Human</td><td>Ambalavao</td><td>Miarinarivo</td><td>β</td></tr></tbody></table>
<table><thead><tr><th>Strain</th><th>Accession#</th><th>Year</th><th>Source</th><th>District</th><th>Commune</th><th>Lineage</th></tr></thead><tbody><tr><td>201/07</td><td>SRR4175444</td><td>2007</td><td>Human</td><td>Ambatofinandrahana</td><td>Ambatofinandrahana</td><td>y05</td></tr><tr><td>27/17</td><td>SRS4032734</td><td>2017</td><td>Human</td><td>Manandriana</td><td>Ambohimahazo</td><td>y03</td></tr><tr><td>50/17</td><td>SRS4032746</td><td>2017</td><td>Human</td><td>Ambositra</td><td>Ivato</td><td>y03</td></tr><tr><td>62/09</td><td>SRR4175420</td><td>2009</td><td>Human</td><td>Antanifotsy</td><td>Ambohitompina</td><td>z06</td></tr><tr><td>38/17</td><td>SRS4032757</td><td>2017</td><td>Human</td><td>Mandritsara</td><td>Andratamarina</td><td>α</td></tr><tr><td>55/08</td><td>SRR4175435</td><td>2008</td><td>Human</td><td>Antsirabe II</td><td>Tsarahonenana Sahanivotry</td><td>w02</td></tr><tr><td>67/09</td><td>SRR4175423</td><td>2009</td><td>Human</td><td>Antsirabe II</td><td>Manandona</td><td>t02</td></tr><tr><td>49/17</td><td>SRS4032744</td><td>2017</td><td>Human</td><td>Manandriana</td><td>Ambovombe Centre</td><td>t11</td></tr><tr><td>45/04</td><td>SRR4175421</td><td>2004</td><td>Human</td><td>Antsirabe II</td><td>Manandona</td><td>t11</td></tr><tr><td>12/09</td><td>SRR4175436</td><td>2009</td><td>Human</td><td>Fandriana</td><td>Miarinavaratra</td><td>u03</td></tr><tr><td>145/08</td><td>SRR4175428</td><td>2008</td><td>Human</td><td>Ambatofinandrahana</td><td>Soavina</td><td>h04</td></tr><tr><td>18/06</td><td>SRR4175425</td><td>2006</td><td>Human</td><td>Antsirabe II</td><td>Mangarano</td><td>h10</td></tr><tr><td>46/12</td><td>SRR4175429</td><td>2012</td><td>Human</td><td>Betafo</td><td>Alakamisy Marososona</td><td>h06</td></tr><tr><td>214/07</td><td>SRR4175427</td><td>2007</td><td>Human</td><td>Antsirabe II</td><td>Ambohitsimanova</td><td>h16</td></tr><tr><td>36/17</td><td>SRS4032756</td><td>2017</td><td>Human</td><td>Arivonimamo</td><td>Mahatsinjo Est</td><td>x01</td></tr><tr><td>24/09</td><td>SRR4175438</td><td>2009</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>x03</td></tr><tr><td>102/06</td><td>SRS4032747</td><td>2006</td><td>Human</td><td>Faratsiho</td><td>Ramainandro</td><td>x03</td></tr><tr><td>09/12</td><td>SRR4175434</td><td>2012</td><td>Human</td><td>Betafo</td><td>Mahaiza</td><td>v03</td></tr><tr><td>30/10</td><td>SRR4175432</td><td>2010</td><td>Human</td><td>Antsirabe II</td><td>Ambohitsimanova</td><td>v06</td></tr><tr><td>95/07</td><td>SRR4175431</td><td>2007</td><td>Human</td><td>Betafo</td><td>Manohisoa</td><td>v13</td></tr><tr><td>197/11</td><td>SRR4175433</td><td>2011</td><td>Human</td><td>Betafo</td><td>Ambohimanambola</td><td>v09</td></tr><tr><td>69/04</td><td>SRR4175430</td><td>2004</td><td>Human</td><td>Mandoto</td><td>Ankazomiriotra</td><td>v10</td></tr><tr><td>CO92<br>(root)</td><td>GCA_001293415.1</td><td>1992</td><td>Human</td><td>N/A</td><td>N/A</td><td>N/A</td></tr></tbody></table>

<table><caption>**Appendix Table 2.** Residual sputum samples from 42 probable or confirmed cases used for *Y. pestis* DNA capture, enrichment, and sequencing*</caption><thead><tr><th>Sputum sample</th><th>District</th><th>Collection date</th><th>RDT result</th><th>Culture result</th><th>PCR (IPM) result</th><th>Case classification</th><th>PCR (NAU) result (Ct)</th></tr></thead><tbody><tr><td>121–2017</td><td>Mahajanga I</td><td>14-Sep-17</td><td>POS</td><td>POS</td><td>POS</td><td>Confirmed</td><td>27.3</td></tr><tr><td>135–2017</td><td>Toamasina I</td><td>15-Sep-17</td><td>POS</td><td>NEG</td><td>POS</td><td>Confirmed</td><td>35.0</td></tr><tr><td>125–2017</td><td>Faratsiho</td><td>16-Sep-17</td><td>POS</td><td>NEG</td><td>POS</td><td>Confirmed</td><td>29.6</td></tr><tr><td>133–2017</td><td>Faratsiho</td><td>16-Sep-17</td><td>POS</td><td>NEG</td><td>POS</td><td>Confirmed</td><td>38.8</td></tr><tr><td>129–2017</td><td>Faratsiho</td><td>16-Sep-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.7</td></tr><tr><td>184–2017</td><td>Vohemar</td><td>22-Sep-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.4</td></tr><tr><td>597–2017</td><td>Antananarivo Renivohitra</td><td>10-Oct-17</td><td>NEG</td><td>-</td><td>POS</td><td>Probable</td><td>37.0</td></tr><tr><td>630–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.3</td></tr><tr><td>647–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.8</td></tr><tr><td>653–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.2</td></tr><tr><td>663–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>46.5</td></tr><tr><td>632–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.9</td></tr><tr><td>636–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.3</td></tr><tr><td>641–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.5</td></tr><tr><td>666–2017</td><td>Toamasina I</td><td>11-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>36.8</td></tr><tr><td>819–2017</td><td>Antananarivo Renivohitra</td><td>12-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>40.8</td></tr><tr><td>841–2017</td><td>Antananarivo Renivohitra</td><td>12-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.0</td></tr><tr><td>843–2017</td><td>Antananarivo Renivohitra</td><td>12-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.2</td></tr><tr><td>1100–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>POS</td><td>Confirmed</td><td>36.1</td></tr><tr><td>1074–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.7</td></tr><tr><td>1078–2017</td><td>Antananarivo Atsimondrano</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.5</td></tr><tr><td>1115–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.8</td></tr><tr><td>1002–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.9</td></tr><tr><td>1006–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.4</td></tr><tr><td>1015–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.8</td></tr><tr><td>1028–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.6</td></tr><tr><td>1040–2017</td><td>Antananarivo Renivohitra</td><td>16-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.7</td></tr><tr><td>1164–2017</td><td>Antananarivo Renivohitra</td><td>17-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.6</td></tr><tr><td>1147–2017</td><td>Antananarivo Renivohitra</td><td>17-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.4</td></tr><tr><td>1237–2017</td><td>Antananarivo Renivohitra</td><td>18-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.9</td></tr><tr><td>1307–2017</td><td>Antananarivo Renivohitra</td><td>20-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.3</td></tr><tr><td>1470–2017</td><td>Antananarivo Renivohitra</td><td>23-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>37.7</td></tr><tr><td>1468–2017</td><td>Antananarivo Renivohitra</td><td>23-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.0</td></tr><tr><td>1494–2017</td><td>Antananarivo Renivohitra</td><td>24-Oct-17</td><td>NEG</td><td>-</td><td>POS</td><td>Probable</td><td>36.3</td></tr><tr><td>1633–2017</td><td>Antananarivo Renivohitra</td><td>26-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>38.2</td></tr><tr><td>1659–2017</td><td>Antananarivo Renivohitra</td><td>26-Oct-17</td><td>POS</td><td>NEG</td><td>NEG</td><td>Probable</td><td>39.7</td></tr></tbody></table>

<footnote>*Sequences are ordered as they appear in the phylogeny (Figure 2 in main text) from top to bottom.</footnote>