Anthrax cases in humans in the Zhambyl region in 2023 were identified through the Zhambyl epidemiological surveillance system. Suspected anthrax reported cases further investigated a group of clinical, epidemiological and laboratory personnel with visit of the outbreak location to conduct investigations and implement disease control interventions. The epidemiologists conducted case-finding for suspected anthrax cases in affected areas, epidemiologically unfavorable area and analyzed their exposure history. The study included cases presenting with clinical signs and/or symptoms of cutaneous anthrax in combination with history of exposure and laboratory testing. All human anthrax cases were diagnosed according to the standardized case definitions established by the Clinical protocol for diagnosis and treatment of anthrax Kazakhstan Ministry of Health in 2016. A suspected case of the cutaneous anthrax is diagnosed in the presence of an acute illness characterized by high fever, a painless primary skin lesion with perifocal or widespread edema, at one of the following stages of development: papule, pustule (hemorrhagic), ulcer (flat, dry, with a black, dense eschar at the base, on an infiltrated foundation, surrounded by a hyperemic blister rim), black, dense eschar. A probable case of anthrax is diagnosed when the criteria for a suspected case are met, with at least one of the following: the patient has resided in or visited an anthrax-endemic area (where cases of anthrax in humans or animals have been reported) within 2 weeks prior to illness onset and has at least one of the following risk factors:

A confirmed case of anthrax is diagnosed when an appropriate clinical specimen (such as pustular exudate, tissue sample from beneath the eschar) is tested and at least one of the following results:

In all suspected anthrax cases, routine diagnostic testing including PCR and bacterial culture was performed at local public health laboratories in accordance with national protocols. Culture-positive samples were subsequently transported under appropriate biosafety conditions to the Aikimbayev’s National Scientific Center for Especially Dangerous Infections (NSCEDI) for confirmatory culture testing and advanced molecular genotyping. This additional analysis was conducted to characterize the B. anthracis isolates and support epidemiological investigations.

Samples collected for laboratory testing included serum and cutaneous lesion samples, such as blister fluid obtained from skin lesions characteristic of cutaneous anthrax.

For culture method was used sheep blood agar 5%. The phenotypic properties of B. anthracis strains were studied according to methodological guidelines “Laboratory diagnostics of anthrax in humans and animals” (based on order of the Ministry of Health of the RK 01.10.2004 and Order of the Ministry of Agriculture of the RK 07.10.2004 No. 725/575 “On strengthening measures to prevent anthrax in the Republic of Kazakhstan”).

Antibiotic susceptibility testing was conducted using the disk diffusion method. Zone diameters were recorded in millimeters and categorized as Susceptible (S), Intermediate (I), or Resistant (R) based on Guideline Determination of Antimicrobial Susceptibility of Causative Agents (11).

DNA was isolated from culture positive samples using the QIAamp DNA Mini Kit according (QIAGEN) manual. The concentration and purity of the extracted DNA were assessed spectrophotometrically using a NanoDrop One instrument (Thermo Fisher Scientific, USA). Samples with A260/A280 ratios in the range of 1.8–2.0 were selected for further analysis. The samples were stored at −20°C.

PCR amplification was carried out in a reaction volume of 25 μL, containing 1 × buffer, 1.5–2.5 mM MgCl₂ (optimized for each locus), 200 μM of each dNTP, 0.25 μM of a fluorescently labeled forward primer and a conventional reverse primer, 1.25 U of Taq DNA polymerase (Thermo Scientific), and 5 μL of template DNA. Amplification was performed using a QuantStudio 5 thermal cycler (Applied Biosystems, USA). Optimized amplification conditions included an initial denaturation at 94°C for 5 min (1 cycle), followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 55–65°C for 35 s, and extension at 72°C for 35 s, with a final extension at 72°C for 7 min. Amplification products were verified by electrophoresis on a 1.2% agarose gel followed by fluorescent detection.

The bacterial culture and DNA extraction were performed in a Biosafety level 3 (BSL-3) laboratory.

Phylogenetic analysis of B. anthracis isolates was performed using Multiple-Locus Variable-Number Tandem Repeat Analysis-31 (MLVA-31), which is based on the amplification of eight VNTR loci: seven classical loci (vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, pXO1, and pXO2) and 24 additional loci, including BAMS and Bavntr markers (Supplementary Table 1) (12, 13). Primers for amplification were selected according to published protocols (14–16) and were synthesized with fluorescent labels (FAM, HEX, NED, ROX, TAMRA, VIC, Cy3). For each isolate, a numerical matrix was generated from the allelic profiles, reflecting the number of repeats at each locus. Only the numeric repeat values were used in subsequent analyses.

Based on the resulting distance matrix, a phylogenetic tree was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method, implemented in the (Phylogenetic Analysis Using Parsimony, version 4.0) PAUP v4.0 software (17). The UPGMA method is widely used for clustering isolates with presumed clonal origin, as it is based on the average distance between all pairs of elements from different clusters.

The constructed tree was exported in Nexus format and visualized using FigTree v1.4.3 (18). The resulting dendrogram served as the basis for cluster analysis, enabling the identification of genetic relationships among the isolates and the delineation of stable genotypic groupings.

Statistical analyses were performed using STATA version 17 (StataCorp, College Station, TX, USA). Descriptive statistics were used to summarize demographic and clinical data. Bivariate associations between risk factors and confirmed cases were assessed using Fisher’s exact test, as appropriate. Stratified analyses by gender, age group, and exposure route were performed to identify potential differences in risk. Incidence rates were calculated with 95% confidence intervals using Poisson distribution.

All procedures in this study adhered to the ethical standards of the local ethical committee. Samples (blood, cutaneous lesion samples) were collected from patients after obtaining their informed consent. The attending physician at the local hospital documented these consents. Positive samples patients’ data were anonymized. The informed consent and the study were approved by the local ethics committee of NSCEDI (protocol 1, 03.02.2023).

In 2023, 41 suspected cases of anthrax were registered in the Zhambyl region, 19 cases of cutaneous anthrax were confirmed by culture and PCR methods. Confirmed cases were registered in four districts and one city; Zhuali district—9 cases, Talas district—6 cases, Sarysu district—1 cases, Baizak district—1 case and in the city of Taraz—2 cases were associated with 11 exposure events (Table 2). The incidence rate of cutaneous anthrax in the Zhambyl region in 2023 was 1.55 (95% CI: 0.92–2.37) cases per 100,000 population. A total of 19 cases of cutaneous anthrax were confirmed by laboratory testing. PCR (targeting pXO1 and pXO2) confirmed Bacillus anthracis in all 19 cases. Culture-based identification was successful in 12 of these cases. The 12 culture-positive isolates were further analyzed by molecular genotyping.

Of the 19 confirmed cutaneus anthrax cases, 84.2% were male and 15.8% female. The mean age was 42.4 years (range: 16–68), with most cases between 36 and 55 years old. Participation in slaughter activities was the most frequently reported exposure route (68.4%), while 26.3% reported handling or consuming processed meat. Sheeps (42.1%) and cattle (31.6%) were the most commonly identified sources of infection. Occupations of registered cases were mainly unemployed 68.4% people. Most patients (84.2%) presented through self-referral to medical facilities, while 15.8% were detected via active surveillance (Table 3).

Bivariate analysis using Fisher’s exact test revealed that individuals who participated in animal slaughter had 2.6 times higher odds of being confirmed anthrax cases compared to those who did not, although this result was not statistically significant (p = 0.21). No significant association was found between processed meat consumption and case confirmation (p = 1.00) (Table 4).

The study identified five newly identified endemic foci of anthrax in the Zhambyl region in 2023, where the disease had not been previously reported (Figure 1). A retrospective analysis of the 2023 cutaneous anthrax cases in humans in the Zhambyl region was conducted to investigate the causes and contributing factors of the epidemiological situation. A chronological list of confirmed cutaneous anthrax cases was compiled (Table 5).

In 2023, human anthrax cases in the Zhambyl region exhibited seasonality, with multiple peaks observed in the end of May and August, the latter having the highest number of cases. From May to August, 63.2% of all reported anthrax cases occurred. Weather conditions played a significant role in shaping the epizootic and epidemiological patterns of anthrax. Human anthrax cases were recorded during the spring–summer period, with a peak in August, mirroring the seasonal patterns observed in livestock.

Laboratory testing confirmed Bacillus anthracis in clinical samples from 12 cases through culture method, while PCR (pXO1+/pXO2+) confirmed the diagnosis in all cases. None of the affected individuals had been vaccinated against anthrax. All 19 patients received antibiotic therapy with ciprofloxacin administered orally or IV for duration of 10–14 days, depending on clinical response according to national guidelines and antibiotic susceptibility. All patients were discharged with a fully recovery.

Among the 19 patients diagnosed with anthrax, bacterial cultures were positive in 12 cases. Antibiotic susceptibility testing was performed on all culture-positive samples. In the remaining 7 cases, cultures did not yield growth of B. anthracis. This is due to the fact that some patients had already begun antibiotic treatment before being admitted to the hospital, which can suppress bacterial growth and lead to negative culture results. These findings underline the importance of collecting clinical samples as early as possible, ideally before starting antimicrobial therapy.

The susceptibility of culture positive B. anthracis strains to antibiotics were assessed using the disk diffusion method. For determining B. anthracis susceptibility, antibacterial agents with specific concentrations recommended for the urgent prevention and treatment of anthrax were selected. The first-line antibiotics included benzylpenicillin, ampicillin, doxycycline, tetracycline, ciprofloxacin, and rifampicin.

The study results indicate that B. anthracis strains exhibit sensitivity to a broad spectrum of antibiotics, including benzylpenicillin, tetracycline, ampicillin, ciprofloxacin, rifampicin, kanamycin, erythromycin, gentamycin, streptomycin and doxycycline (Tables 5, 6).

Among the 12 positive culture samples, MLVA-31 genotyping was successfully performed 11 Bacillus anthracis isolates. The resulting profiles were compared with previously published regional genotypes from Kazakhstan. The Zhambyl isolates clustered into four distinct genotypes, grouped into two major clades. The first clade comprised eight isolates representing three genotypes, which differed at three VNTR loci. The second clade included a single genotype, which differed from the first clade by six VNTR loci.

Genotype 1 (Zham-15, Zham-18, Zham-20, Zham-23) included isolates obtained from patients 10, 13, 14, and 18, who resided in the villages of Tasbastau (Zhualy District), Karatau (Talas District), the city of Taraz, and the village of Koltogan (Zhuali District), respectively. Although these individuals had no known direct contact with each other, all were exposed to potentially contaminated animal products. Specifically, Patient 10 (Zham-15) participated in the slaughter of a sheep; Patient 14 (Zham-18) slaughtered a horse; Patient 13 (Zham-20) handled meat purchased from a local store; and Patient 18 (Zham-23) was involved in the slaughter of a horse. Despite the lack of direct epidemiological links and the geographic spread of the outbreak across approximately 70 to 168 km, the isolates were genetically identical according to MLVA-31 typing.

Genotype 2 (Zham-13, Zham-14) comprised isolates from Patients 7 and 8, who resided in the village of Kyzylzhuldyz (Baizak District) and the city of Taraz, respectively. Both patients were involved in the slaughter of clinically ill sheep as part of a single exposure event, which occurred in two locations approximately 6 km apart.

Genotype 3 (Zham-19, Zham-24) included isolates from Patients 16 and 19, residing in the villages of Kaskabulak (Talas District) and Zhanaryk (Sarysu District), respectively. Both individuals had slaughtered sheep prior to the onset of illness, though the exposures occurred at different times. Although these cases were not epidemiologically linked, the similarity in the nature of exposure despite differences in timing may suggest common environmental risk factors.

Genotype 4 (Zham-1, Zham-11, Zham-12) included isolates from patients 2, 3, and 4, all of whom were residents of the village of Tamdy (Talas District). These cases were directly linked to a single exposure event on May 20, involving the slaughter of a cow owned by patient 2. Patients 3 and 4 participated in this activity and subsequently developed symptoms (Supplementary Figure 1; Supplementary Tables 2, 3).